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123
Fungal Infections of the Central
Nervous System
Mehmet Turgut • Sundaram Challa
Ali Akhaddar
Editors
This Springer imprint is published by the registered company Springer Nature Switzerland AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Preface I
Fungal infections of the central nervous system, once considered rare, have
become more frequent and pose a diagnostic and therapeutic challenge in the
day-to-day practice. Better awareness of the epidemiological features and
elucidation of the risk factors along with advancements in technology in
imaging and molecular diagnostics contributed to better understanding of the
disease mechanisms and diagnosis. However, geographic variations due to
environmental factors, emerging fungi in different clinical scenarios, and
genetic factors influence the incidence of fungal infections. Though there is a
wealth of information on fungal infections of the central nervous system,
textbook like this provides a comprehensive and rapid access to the various
aspects of these diseases and serves as a ready reference for the trainee and
practicing neuroscientists.
The book has six sections with each section dedicated to one aspect of the
disease. The authors were chosen from various parts of the world, based on
their contributions and special interest in that subspecialty. Each chapter was
edited by an expert in the field to provide concise and up-to-date information
on the subject. The chapters were well-illustrated with tables and figures and
provided with extensive references to guide further reading for residents,
neurologists, internists, and neurological surgeons.
We are grateful to all the authors for their contributions and support to
complete this book project in time. We especially wish to thank Springer
Nature for their support in ensuring quality publication of the book. We are
truly humbled by this experience. We hope this book will be a unique and
important addition to the existing books on this subject.
v
Preface II
Fungi are ubiquitous organisms found in the soil, water, and environment.
Infections of humans are uncommon with only a few species being patho-
genic. However, with changes in the environmental factors and immune sta-
tus of individuals, fungal infections are on the rise. The incidence of fungal
infections of the central nervous system (CNS) parallels that of the systemic
fungal infections. Fungal infections of the CNS are being increasingly diag-
nosed in the past few decades due to steady increase in the number of immu-
nosuppressed individuals, better awareness, and improved diagnostic
modalities. Infections of the CNS are associated with high morbidity and
mortality, but the diagnosis and treatment remain a challenge. Understanding
the pathogenesis and host pathogen interactions helps in devising new diag-
nostic modalities and therapeutic interventions.
The etiologic agents include yeasts, filamentous fungi, and dimorphic
fungi. The common yeast fungi include Cryptococcus and Candida, whereas
filamentous fungi with hyaline septate hyphae include Aspergillus, Fusarium,
and Mucorales and the pigmented fungi include dematiaceous fungi. The
dimorphic fungi include Blastomyces, Histoplasma, Coccidioides, and
Paracoccidioides. Aspergillus and Mucorales are usually opportunistic, but
Aspergillus can cause infections in immunocompetent hosts in certain geo-
graphical regions. Dematiaceous fungi are neurotropic and cause infection in
immunocompetent hosts, and dimorphic fungi cause infections which are
geographically restricted.
The portal of entry is usually by inhalation and subsequent hematogenous
dissemination to the CNS. The infection may spread from contiguous struc-
tures like paranasal sinuses, orbit, mastoid, or skull bone and by direct inocu-
lation from surgery or trauma.
The size of the conidia or yeast, the virulence factors, and angioinvasive-
ness of the fungus are important in the pathogenesis. The interplay between
host defenses and the strategy of the pathogen to evade immune attack,
acquire nutrients, degrade extracellular matrix, and disseminate are not yet
completely understood.
The immune status, portal of entry, type of the fungus, and virulence
of the organism determine the pathology which in turn manifests as the
clinical syndrome. The clinical syndromes include meningitis, intracranial
space-occupying lesion, stroke-like manifestation, or spinal syndrome. The
pathology includes abscess, granuloma, meningitis, infarct with or without
vii
viii Preface II
8 Aspergillosis�������������������������������������������������������������������������������������� 91
Sundaram Challa
9 Candidiasis��������������������������������������������������������������������������������������� 107
M. Altay Atalay
10 Mucormycosis���������������������������������������������������������������������������������� 121
A. Serda Kantarcioglu
11 Histoplasmosis and Coccidioidomycosis���������������������������������������� 155
María del Rocío Reyes-Montes, Maria Lucia Taylor, Esperanza
Duarte-Escalante, and María Guadalupe Frías-De-León
ix
x Contents
12 Cryptococcosis���������������������������������������������������������������������������������� 167
Anita Mahadevan and Shankar Krishna Susarla
13 Blastomycosis and Phaeohyphomycosis���������������������������������������� 187
María Guadalupe Frías-De-León, Erick Martínez-Herrera,
María del Rocío Reyes-Montes, and
Gustavo Acosta-Altamirano
14 Cladophialophora bantiana�������������������������������������������������������������� 195
Hurriyet Deniz Ozgun, Darren L. Jacobs,
and Steven A. Toms
15 Cladosporium spp., Fusarium spp., Bipolaris spp.,
Schizophyllum commune, and Scedosporium apiospermum���������� 205
A. Serda Kantarcioglu
CNS Central nervous system The description and the scientific study of fungal
CSF Cerebrospinal fluid infections remains an interesting and attractive
USA United States of America topic since the antiquity. The father of the docu-
mented medicine, Ηellenic Hippocrates of Kos,
was the first physician who observed (Ancient
Greek word παρατήρισις) and described the can-
1.1 Introduction didiasis phenomenon with white patches into the
oral cavity in a weak and infirm patient. His main
Scientific research documented in the particular contribution initiated the human society’s devel-
field of fungal infections should be explored opment of medicine through a well-documented
through the perspectives of continuously chang- and delicate blending of the art of healing and
ing biological, medical, and public profiles. In scientific observations and studies (Fig. 1.1)
this chapter, a brief review of ancient to modern (Syrmos et al. 2010; Giannouli and Syrmos 2011;
approaches to manage the central nervous system Syrmos 2011).
(CNS) infections caused by fungal pathogens
will be given according to the historical refer-
ences in the following subheadings of the history 1.3 Mid Modern Period
of medicine: “Ancient Greece,” “Mid Modern
Period,” and “Contemporary Period.” Pier Antonio Micheli (1679–1737 AD) (Fig. 1.2),
a catholic priest from Pisa, Central Italy, in his
observations and studies, described Aspergillus
(Schaechter 2011). Initially, his aim was to study
the nine different fungal species that resembled
aspergillum (Sumbali and Johri 2005). This was
very often used, perforated, to sprinkle the holy
N. C. Syrmos (*) · V. Giannouli water during Christian ceremonies (Schaechter
Medical School, Aristotle University of Thessaloniki, 2000). Antonio also became a well-known aca-
Thessaloniki, Macedonia, Greece demic botanist, a ranking professor, and also a
M. Turgut curator of the Orto Botanico di Firenze, in Central
Department of Neurosurgery, Aydın Adnan Menderes Italy (Giardino dei Semplici). He made various
University School of Medicine, Aydın, Turkey
Candida albicans (Antoniades et al. 2008; with mice transmission (Huntington Jr. 1986). He
Genkal et al. 2011). They also described Candida performed clinical studies of the disease in a docu-
meningitis for the first time (Gavito-Higuera mented series of cases of coccidioidomycosis in a
et al. 2016; Pappas et al. 2016; Correia and patient who died because of disseminated coccidi-
Campos 2003). oidomycosis, the first documented case of the dis-
ease in the history (Hirschmann 2007). After
decades of studies and many efforts, the research-
1.4 Contemporary Period ers made the clear definition and manifestation of
coccidioidomycosis in scientific publications.
Ophuls made the first human report of a coccidioi- Further description of the disease was reported by
dal brain lesion during his studies around 1905 Evans in 1909. The first case with both coccidioidal
(Huntington 1976, 1985). The next huge step in meningitis and hydrocephalus was described by
understanding the disease was made by William Ryfkogel. He managed also to perform an accurate
Ophuls (Patrick Ophuls) (1871–1933 AD) description of coccidioidal meningitis (Veterans
(Fig. 1.7), an academic pathologist in 1921, in Affairs Armed Forces, 1955–1958) (Correia and
Stanford Medical School, USA. Ophuls managed Campos 2003).
to clearly identify the fungal nature of this organism Adolfo Lutz (1855–1940 AD), a Brazilian
physician and pioneer tropical medical doctor,
was the first to describe paracoccidioidomycosis
in 1908 (Fig. 1.8). He performed zoological
studies, epidemiological investigations, and
infectious diseases research (Correia and
Campos 2003).
Morris was the first to report coccidioidomy- 1924, Morris reported a unique case of coccidioi-
cosis as the only site of dissemination outside of domycosis outside the pulmonary function as a
the pulmonary area in 1924 (Morris 1924). result of dissemination (Smit et al. 2003). Then, a
Abbott and Cutler made review studies regarding total of 14 cases of typical CSF findings were
14 cases in 1936 with description of the typical reviewed by Abbott and Cutler in 1936 (Abbott
CSF findings (Correia and Campos 2003). and Cutler 1936). The association of meningeal
Subsequent pathological reports were very involvements with coccidioidal infections of the
important in order to clarify the association CNS was documented in an accurate pathologi-
between meningeal involvement and CNS coc- cal report.
cidioidal infection (Spellberg et al. 2005). In general, histiocytes were studied with
Edmond Isidore Etienne Nocard (1850– Histoplasma capsulatum. On the other hand, rhi-
1903 AD) performed studies in Provins (Seine- nocerebral zygomycosis was accurately described
et-Marne, France) (Fig. 1.9) (Haas 2000). He was and presented in a series of three cases by
the first scientist who managed to describe the Gregory Binford in Maryland, USA, in 1943
acid-fast aerobic cattle actinomycetes (Mathijsen (Chiller 2016). In 1952, he published a case
2003). Trevisan called them by the name report of brain abscess attributed to Cladosporium
Nocardia farcinica in 1889 (Pospischil 2002). An (Chiller 2016).
essential step was performed in 1891 by Eppinger Moreover, we have to mention that before the
with the first documented report of metastatic use of the antifungal drugs, such cases with com-
human cerebral nocardiosis from the lung bination of fungal infections of the CNS and later
(Pospischil 2002). improvement following surgical removal of cere-
Histoplasmosis was described by Darling bral abscess and evacuation of granulomatous
around 1906: the report was about a documented lesion were rarely published in the current litera-
disseminated granulomatous infection in a patient ture. In 1903, antifungal chemotherapy started
(Beolens et al. 2011). About 18 years later in successfully using potassium iodide in cases of
cutaneous or subcutaneous sporotrichosis
(Chiller 2016). The next decades involved with
the introduction of mucosal and systemic myco-
ses: in 1953, nystatin, the first useful polyene
drug, and in 1956, amphotericin B, the second
useful polyene drug. Amphotericin B remains till
today the best option about these infection types
(Moen et al. 2013). Although these infections
have been recognized for over a century, until the
use of amphotericin B, fungal infections of the
CNS remained a pathological situation with dif-
ficult effective treatment.
During the next few years, in 1964, flucyto-
sine (5-fluorocytosine); in 1970, azole drugs; in
1978, miconazole; in 1981, ketoconazole; in
1990, fluconazole; in 1992, itraconazole; and in
2000–2010, other drugs against the fungal infec-
tions of the CNS were developed (Chiller 2016;
Moen et al. 2013). In order to avoid the toxicity
of amphotericin B, the following were intro-
duced: liposomal amphotericin B with/without
Fig. 1.9 A photograph of Edmond Isidore Etienne
lipids, triazoles like voriconazole and posacon-
Nocard (1850–1903 AD) azole, and echinocandins like anidulafungin,
8 N. C. Syrmos et al.
caspofungin, and micafungin (Chiller 2016). Gavito-Higuera J, Mullins CB, Ramos-Duran L, Olivas
The use of these combinations in situations such Chacon C, Hakim N, Enrique Palacios E. Fungal
infections of the central nervous system: a pictorial
as cases of patients with severe invasive mycoses review. J Clin Imaging Sci. 2016;6:24.
provided good results and in this way improved Genkal SI, Bondarenko NA, Schur LA. Diatoms in the
the outcome and ameliorated the health-related lakes of the Southern and Northern Eastern Siberia.
quality of life of these patients (Moen et al. Rybinsk: Rybinsky Dom Pechati; 2011. p. 71.
Geronimus D. Piero di Cosimo: visions beautiful and
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Finally, Fragoyannis, van Wyk, and de Beer Gholami-Shabani M, Zamani S, Moosa H, Ghahfarokhi
reported of North American blastomycosis in M, Jamzivar F, Mehdi Razzaghi-Abyaneh M. Recent
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Epidemiology of Central Nervous
System Fungal Infections 2
Sanjeet Singh Dadwal
(Baumgardner et al. 1992; Dworkin et al. 2005). 2017), and the risk correlates with the status of the
The sinonasal fungal infection when invasive in cell-mediated immunity.
nature can extend beyond the extracranial com- The CNS IFI can manifest as meningitis,
partment into the brain (Kourkoumpetis et al. which is the inflammation of the meninges. The
2012; McCarthy et al. 2014) and may manifest as symptoms are often protean and can vary from
meningitis or space-occupying lesions/abscess. acute onset to subacute and chronic in nature.
Certain epidemiologic exposures such as drown- Patients often present with headaches that are
ing have led to infection of the CNS with subacute to chronic and may have visual changes,
Scedosporium apiospermum (Kantarcioglu et al. cranial nerve abnormalities, and other symptoms
2008) and Aspergillus spp. (Kowacs et al. 2004). of elevated intracranial pressure. Nuchal rigidity
Diabetic ketoacidosis, steroid use, and iron over- may or may not exist. At times, patients may
load are known risk factors for mucormycosis present with the symptoms/signs of cerebritis
(Spellberg et al. 2005). Furthermore, iatrogenic suggesting parenchymal involvement. The
fungal infections have occurred in the setting of inflammatory component can result in endarteri-
outbreaks such as with Exserohilum rostratum tis that may result in stroke manifesting with
due to contaminated compounded methylpred- focal neurologic defects. In patients with immu-
nisolone for spinal injections in 2013 (Chiller nocompromised state, symptoms and signs may
et al. 2013), Exophiala infection from contami- be minimal or atypical that often result in delayed
nated injectable steroids (From the Centers for diagnosis. Intracranial abscess-/mass-like lesions
Disease Control and Prevention 2003), and are mostly encountered with invasive mold infec-
Aspergillus meningitis after spinal anesthesia in tions. Mold infections of the CNS are mostly
pregnant women (Gunaratne et al. 2007). encountered in the immunocompromised patients
Perhaps, the most significant group that contrib- and typically present with space- occupying
utes to the burden of IFI is the immunocompro- parenchymal lesions/abscesses and less likely to
mised patient. The high-risk group include, be associated with meningitis.
neutropenia in patients undergoing cytotoxic che- This chapter will provide an outline of the epi-
motherapy for hematologic malignancy, those who demiology for CNS infections related to common
have undergone allogeneic HCT (Kontoyiannis fungal pathogens (true and opportunistic), and
et al. 2010), especially those with graft-versus-host the description will be centric to the specific
disease (GVHD) requiring immunosuppressive organism.
therapy, SOT (Pappas et al. 2010; Singh 2003;
Singh and Paterson 2005), use of biologic agents
such as TNF-α (Warris et al. 2001), use of Bruton 2.3 Yeast Infections of the CNS
tyrosine kinase inhibitor, ibrutinib (Bercusson et al.
2018; Peri et al. 2018), congenital immunodefi- 2.3.1 Cryptococcus neoformans
ciency such as chronic granulomatous disease
(CGD) (Alsultan et al. 2006; Dotis et al. 2007; This pathogen belongs to basidiomycetous fungi
Henriet et al. 2013), and caspase recruitment and is the leading cause of CNS fungal infection
domain-containing protein 9 deficiency (CARD9) manifesting as meningitis and may be accompa-
(Gavino et al. 2014; Lanternier et al. 2015; Rieber nied by brain abscesses “cryptococcomas.” Its
et al. 2016). Within the SOT recipients, the type of distribution is worldwide and is ubiquitously
organ transplant has an impact on the risk of IFI found in soil, bird droppings, and animals.
(Munoz et al. 2016). ICU patients are also at high Humans can be colonized with it as well. There
risk for IFI: mainly candida and aspergillus are eight genotypes, with the most common
(Denning 2004; Pittet et al. 1994). Patients with being C. neoformans var. neoformans in the
human immunodeficiency virus/acquired immuno- USA and Europe. C. neoformans var. grubii is
deficiency syndrome (HIV/AIDS) are at high risk most common in the rest of the world. The most
for cryptococcal infection (Rajasingham et al. significant burden from this pathogen is in the
2 Epidemiology of Central Nervous System Fungal Infections 13
HIV/AIDS patient population, and the risk of soil, on inanimate objects, and on hospital sur-
developing this infection is directly correlated faces and colonize the respiratory/GI tract of nor-
with the cell-mediated immunity that increases mal and immunocompromised hosts. The risk
with the declining CD4 count (most cases occur factors for candidemia include critically ill neo-
when CD4 is <100 cells/μl) (Rajasingham et al. nates associated with prematurity, low APGAR
2017). With the availability of HAART, the score, shock, intubation and congenital malfor-
incidence of cryptococcal CNS infection has
mations, neutropenic host, HCT recipients with
decreased in the USA, although it is still a major mucositis or graft-versus-host disease (GVHD) of
cause for morbidity and mortality in the develop- the gut, SOT, use of broad-spectrum antibiotics,
ing world (Rajasingham et al. 2017). After HIV/ central venous catheter, intravenous drug use,
AIDS, SOT recipients have the highest rate of total parenteral nutrition, gastrointestinal surgery,
CNS infection (Pappas et al. 2010), but it is rare diabetes, sepsis, pancreatitis, ICU stay, and dialy-
in recipients of HCT (with more cases observed sis (Blumberg et al. 2001). C. albicans is the most
in autologous than allogeneic) (Kontoyiannis frequent cause of infection in the USA followed
et al. 2010). In the SOT, Cryptococcus contrib- by C. glabrata (Matsumoto et al. 2014), and there
uted to 8% of all IFI in a large prospective study is variability in the distribution of species based
(Pappas et al. 2010), and the CNS is the most on geography and patient population (Pfaller et al.
common extrapulmonary site of infection. 2012); C. glabrata is more frequently isolated
Increased risk for CNS involvement was corre- from SOT and elderly patients. Additionally, there
lated with abnormal mental status, late-onset has been emergence of drug-resistant Candida
disease, and high serum cryptococcal antigen spp. such as triazole and echinocandin resistance
titer (Osawa et al. 2010). in C. glabrata (Pfaller et al. 2010; Pfaller et al.
C. gattii is an emerging pathogen worldwide 2011). Candida auris, a multidrug-resistant can-
that was previously described primarily in trop- dida species, has gained notoriety in recent years
ical and subtropical areas such as Australia, as a cause of outbreaks associated with high mor-
New Guinea, Hawaii, Southern California, bidity and mortality (Sarma and Upadhyay 2017).
Central Africa, and Southeast Asia (Maziarz Candida used to be the most common cause of
and Perfect 2016). Originally described with fungal meningitis, but that has been replaced by
eucalyptus trees, the recent outbreaks noted C. neoformans. Candida meningitis/brain
association with trees such as oaks and firs. abscesses occur in the context of disseminated
There has been an evolving epidemic in the candida infection in premature infants and neo-
Vancouver Island and Pacific Northwest, USA, nates (Faix 1984; Fernandez et al. 2000) and
and cases have been described in multiple states patients with AIDS (Casado et al. 1997), neutro-
across the USA (Harris et al. 2011). This patho- penia from chemotherapy (Flynn et al. 1993),
gen primarily affects the healthy individuals CGD (Cohen et al. 1981), and SCID (Smego Jr
although there is an association with anti-GM- et al. 1984). Direct inoculation may occur with
CSF antibodies and C. gattii infection (Rosen traumatic injuries (Brenier-Pinchart et al. 1999),
et al. 2013; Saijo et al. 2014). The disease pro- CNS ventricular shunts (Baradkar et al. 2009;
cess is frequently severe with meningitis and Shapiro et al. 1989) and polymer wafers used
cryptococcomas in the immunocompetent in local chemotherapy are additional risk factors
patients (Chen et al. 2014). (Glick et al. 2010).
Candida species are an important cause of health- The disease caused by Coccidioides is com-
care-associated infection presenting as dissemi- monly referred to as coccidioidomycosis or
nated infection (candidemia). They are found in “valley fever,” named after the common
14 S. S. Dadwal
prevalent in the Ohio and Mississippi river val- disseminate in the immunocompromised
leys. Outside of the USA, it has been reported patients (Barros et al. 2011). CNS involvement
from Mexico, South American countries, parts of has been reported in patients with underlying
Asia, and Southeast Asia. Exposure to soil rich in immunodeficiency (Gullberg et al. 1987;
bird or bat guano is a risk factor for the acquisi- Hardman et al. 2005) and mostly manifests as
tion of Histoplasma (Wheat et al. 2016). The meningitis.
activities that are mostly reported to be signifi-
cant exposures include farming, cave explora-
tion, remodeling of old buildings, clearing 2.4 Mold Infections of CNS
brushes, or cutting trees at sites that had sup-
ported blackbird roosting. The highest numbers 2.4.1 Aspergillus
were seen in the context of HIV/AIDS epidemic
(Assi et al. 2007; Kaur and Myers 1983) and, The increase in the number of at-risk patients
subsequently, exposure in the immunocompro- undergoing transplantation, chemotherapy for
mised patients with T cell dysfunction, exposure hematologic malignancies, and use of novel
to TNF-α blockers, SOT, and HCT (Wheat et al. immunosuppressive medications has led to a
2016). It is encountered more commonly in the spurt in invasive aspergillosis. Aspergillus spp.
SOT group, while a lower incidence is observed have a ubiquitous distribution in the nature and
in HCT recipients (Kauffman et al. 2014). are commonly found in soil, decaying vegetation,
Dissemination to the CNS is infrequent and is and food material. The primary route of acquisi-
mostly observed in the immunocompromised tion is inhalational, although infection related to
patient with development of meningitis or focal skin patch dressing and trauma has been observed.
lesions in 5–10% of the cases (Chen et al. 2014). Risk factors for invasive aspergillosis include
A recent retrospective study reviewing 77 cases neutropenia in patients undergoing induction che-
noted male predominance with most frequent motherapy for hematologic malignancy and HCT
underlying diagnosis of HIV/AIDS in 44% fol- especially in the context of GVHD that requires
lowed by transplantation in 13%, and 14% had treatment with steroids or agents such as inflix-
other immunocompromising conditions (Wheat imab, and ibrutinib (bruton tyrosine kinase inhibi-
et al. 2018). Morbidity and mortality are high in tor) and in SOT (Bercusson et al. 2018;
patients with CNS involvement. Kourkoumpetis et al. 2012; McCarthy et al. 2014;
Pappas et al. 2010; Peri et al. 2018; Singh and
Paterson 2005). Inherited conditions such as CGD
2.3.6 Sporothrix schenckii and CARD9 deficiency are also associated with
increased risk of Aspergillus infection (Alsultan
S. schenckii is a dimorphic fungus that is most et al. 2006; Dotis et al. 2007; Henriet et al. 2013;
commonly found in the tropical and subtropical Rieber et al. 2016). Patients with diabetes, recent
areas. It has been reported mostly from Japan, CNS surgery, lumbar puncture, paranasal sinus-
India, Mexico, Brazil, Uruguay, and Peru. In the itis, chronic steroid use, intravenous drug use,
USA, outbreaks related to pine seedlings and pulmonary tuberculosis, and alcoholic liver dis-
manipulation of the moss have been reported ease are also at risk.
from the Mississippi Valley (Barros et al. 2011). Aspergillus is now the most common cause of
The activities associated with risk for acquisi- IFI in the allogeneic HCT patients having sur-
tion of Sporothrix are agriculture, floriculture, passed Candida as reported in a large prospective
wood exploration, mining, and exposure to cats database (Kontoyiannis et al. 2010). In SOT, the
that are infected with this fungus (veterinarians, highest incidence is noted in lung, lung-heart
owners, and caretakers of cats) (Barros et al. transplant (about 6%), and liver and kidney trans-
2004; Vilela et al. 2007). The most common site plants (Pappas et al. 2010). CNS involvement
of infection is the skin although it can be occurred in 15.4% of cases in the context of dis-
acquired via inhalation and has the propensity to seminated disease from a large study in Europe
16 S. S. Dadwal
(Gavalda et al. 2005). A. fumigatus, A. terreus, Although Cladophialophora infections are
and A. flavus are the most common species asso- reported worldwide, the majority are in areas that
ciated with CNS disease. CNS aspergillosis can have a warm and humid climate (Kantarcioglu
manifest as meningitis, infarction, or a brain et al. 2017). A systematic review of C. bantiana
abscess with the latter two presentations being cases reported that the majority of cases are from
more common. India, the USA, Brazil, Canada, France, Spain,
South Africa, and Italy, with sporadic cases from
various other countries. The majority of patients
2.4.2 Non-Aspergillus Mold were immunocompetent (58.3%) and 97% had
Infections brain abscess. Regardless of the immune status,
mortality was high at 65% (Kantarcioglu et al.
2.4.2.1 Mucormycosis 2017).
Mucormycosis is an infection caused by fungi Exophiala dermatitidis notoriously causes
from the Mucorales order (Mendoza et al. 2014), brain abscesses and is mostly reported from the
with Rhizopus spp. the most common offending Asian countries. CARD9 deficiency has been
agent. The organism is found in the decaying identified as a risk factor (Lanternier et al. 2015).
organic matter such as vegetables, seeds, fruits, Other molds such as Lomentospora prolificans,
manure, and compost. It releases spores that Alternaria spp., Exserohilum rostratum,
when airborne can be inhaled. Scopulariopsis spp., Curvularia spp., Bipolaris
The incidence of this devastating illness has spp., Chaetomium, and Ochroconis gallopava are
been increasing over the last decade, in the more often encountered in immunocompromised
HCT and SOT, patients with hematologic hosts (Kontoyiannis et al. 2010; Pappas et al.
malignancy (HM) undergoing cytotoxic che- 2010). In a review of 72 cases of phaeohyphomy-
motherapy, uncontrolled diabetes mellitus with cosis (Revankar et al. 2002), the majority of
acidosis, burns, and trauma (Roden et al. 2005; patients (76%) had underlying immunodefi-
Walsh et al. 2012). Voriconazole and echino- ciency, and CNS involvement was identified in
candin prophylaxis has been associated with 22/72 (30.5%). Only three of the patients with
increased risk of mucormycosis, while tacroli- CNS infection did not have an underlying immu-
mus is protective (Singh et al. 2009). nologic deficit (two caused by Curvularia spp.
The spread to CNS is primarily via the and one by Wangiella dermatitidis). A case series
hematogenous route, although direct extension of 12 SOT patients with Ochroconis gallopava
from the sinuses to the intracranial compart- infection described high mortality rate in those
ment is well known. In a large retrospective with CNS involvement that reached 80%
study of 929 patients, CNS involvement was (Shoham et al. 2008).
described in one-third of the patients, and of From an iatrogenic standpoint, a large out-
that 69% were related to sinonasal source break of fungal meningitis due to Exserohilum
(Roden et al. 2005). Injection drug users mani- rostratum in the USA resulted in patients who
fest predominantly with cerebral involve- had received contaminated compounded methyl-
ment—abscesses or infarcts (Fong et al. 1990; prednisolone used for spinal/epidural injections
Stave et al. 1989). (Chiller et al. 2013).
2.4.2.2 Phaeohyphomycoses
(Dematiaceous Fungi) 2.5 Miscellaneous Fungi
This is a diverse group of pigmented fungi that
are emerging as a cause of CNS fungal infec- Scedosporium apiospermum is ubiquitously found
tions. Many of the fungi in this group are neuro- in the environment, especially polluted environ-
tropic, such as Cladophialophora bantiana, ment of high human activity, agricultural soil, and
Exophiala dermatitidis, and Rhinodadeiella polluted water (Ramirez-Garcia et al. 2018).
mackenziei (Chakrabarti 2007). Scedosporium and Lomentospora accounted for
2 Epidemiology of Central Nervous System Fungal Infections 17
the majority of non-Aspergillus mold infections in Bariola JR, Perry P, Pappas PG, Proia L, Shealey W,
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Cleveland K, Esguerra E, Johnson J, Wright P, Douglas
Morphological Classification
of Fungal Infections (Yeasts, Mold, 3
Dimorphic)
Lakshmi Vemu Gorthi
3.2.2 Y
east Colonies (Roberts 3.2.5 Fission Yeasts (Bennett 1992;
and Goodman 2009; Lehmann 2009)
Bennett 1992)
A second form of cell division in the yeasts is by
Yeast colonies on solid media resemble bacterial fission. The parental yeast cell elongates and sep-
colonies, soft, dry, opaque, 1–3 mm in size and arates from the progeny by a septum, resulting in
cream coloured. However, colony morphology is several shapes (e.g. Talaromyces (Penicillium)
not a definitive differentiating feature among the marneffei).
yeasts (Lehmann 2009).
These fungi predominantly are one-cell forms The filamentous fungi are popularly known as
with round or oval bodies which reproduce by an molds. They form true mycelium and reproduce
asexual process called budding in which the cell by formation of different types of reproductive
3 Morphological Classification of Fungal Infections (Yeasts, Mold, Dimorphic) 25
which is the usual type of reproduction seen in ing irregularly. Zygomycetes rarely have septa,
the laboratory cultures while the sexual state is but if present, the septa are complete and at the
rarely manifested. The only exception is site of formation of a zygospore.
Pseudoallescheria boydii, an ascomycete, which
is seen in its sexual state in the clinical specimen. Mucoromycotina
Its asexual state, Scedosporium apiospermum The characteristics of the asexual spore-bear-
may also be seen (Chander 2018). ing structures are distinct and help in differen-
tiating the several genera. The important
features that have to be examined include
3.6.3 C
lassification of Fungi into height and branching of sporangiophore,
Phyla Based on Sexual arrangement of sporangiospores and sporan-
Reproduction (Rippon 1988; giophores, size of sporangiospore, sporangium
Brandt et al. 2011) shape and size, presence or absence of colu-
mella (a sterile column of hyphae within a
The large numbers of morphological specifica- spore-bearing structure, extending as a sup-
tions associated with sexual sporulation in fungi is porting stalk), shape and size of columella and
an important classification feature. And based on apophyses and presence or absence of rhizoids
these features, medically important fungi are clas- and their location (nodal or internodal) (a root-
sified into three major phyla: the Glomeromycota, like structure, a filamentous branch-like exten-
Ascomycota and Basidiomycota. sion for feeding rather than reproduction)
(Brandt et al. 2011).
–– Glomeromycota—fertilized cell becomes a
The distinct morphological features of the
zygote.
main genera of subphylum Mucoromycotina are:
–– Ascomycota—sexual spores are contained in
an ascus or sac.
–– Mucor: Rhizoids are absent and the sporan-
–– Basidiomycota—sexual spores are produced
giophores tend to branch and often bear large
at the end of the basidium.
round sporangia.
–– Rhizopus: Rhizoids are large and originate
3.6.3.1 Phylum Glomeromycota
immediately on the stolon adjacent to sporan-
(Formerly Zygomycota) (Rippon
giophore (nodal). The latter are usually
1988; Brandt et al. 2011)
unbranched, long and terminate in a columella
Further classified as the subphylum Mucormyco-
and a dark round sporangium with oval colour-
tina which accommodates the order Mucorales,
less to brown spores.
which consists of the medically important genera,
–– Absidia: Rhizoids arise on the stolon between
Absidia, Mucor, Rhizomucor and Rhizopus. The
two sporangiophores (internodal). Rhizomucor:
subphylum Entomophthoromycotina includes the
Is a variant of Mucor, but has poorly developed
genera Basidiobolus and Conidiobolus which are
rhizoids and can grow at 60 °C (Chander 2018;
agents of subcutaneous infections.
Rippon 1988).
Macroscopic Morphology/Colony
Characteristics on Culture Entomophthoromycota (Formerly Subphylum
Grow rapidly on agar in grey-white brown cot- Entomophthoromycotina)
tony or woolly colonies without distinct margins. Species in the Entomophthoromycota generally
share several characteristics. Their vegetative
Microscopic Morphology cells are coenocytic; sporulation occurs by pro-
This group of lower fungi is characterized by an duction of forcibly discharged dispersive or
aseptate thallus. They typically develop coeno- infective conidia; and their zygospores (which
cytic hyphae that are sparsely septate, broad also function as resting spores) are homothallic
(10–15 μm), colourless and ribbon like, branch- (Rippon 1988; Brandt et al. 2011).
28 L. V. Gorthi
3.7.3 C
onidiogenous Cell (Rippon Arthrospore is a conidium formed from restruc-
1988; Brandt et al. 2011) turing the walls of a septate hypha, so that it
breaks into separate segments (hyphal fragmen-
Conidiogenous cells directly produce conidia. tation), e.g. Geotrichum (Brandt et al. 2011).
They may be determinant (no growth of the conid-
iophore after the formation of conidia) or sympo-
dial (a mode of growth which results in the 3.9 ruiting Structures (Brandt
F
development of conidia on a geniculate or zig-zag et al. 2011)
rachis). Conidiogenous cell may be non-specialized
or specialized into a phialide or an annellide. These are due to certain modifications of the
hyphal structures.
3.7.3.1 Phialide (Brandt et al. 2011)
Phialide is a typically a flask-shaped cell that –– Synnemata—A group of erect conidiophores that
develops an open end from which conidia are are cemented together producing conidia at the
produced in basipetal succession. Phialides are a apex and/or along the sides of upper portion.
characteristic of Penicillium and Aspergillus spp. –– Sporodochium—A cushion-shaped stroma
covered with conidiophores.
3.7.3.2 Annellide (Brandt et al. 2011) –– Pycnidium—It is globose to flask-shaped
Annellide is a specialized conidiogenous cell fruiting body with an inner lining of conidiog-
producing conidia in basipetal succession by a enous cell containing conidia. It is large up to
series of short percurrent proliferations (annella- several millimeters in diameter and may have
tions). The tip of an annellide increases in length a hard wall surrounded by peridial hyphae.
and becomes narrower as each subsequent conid- Some strains of Trichophyton mentagrophytes
ium is formed. It is a complex phialide with a col- produce pycnidia, especially when grown on
lar developing for each conidium produced soil—hair agar (Chander 2018; Rippon 1988).
(Rippon 1988).
Other types of conidiogenesis include blasto-
conidium, aleuroconidium and poroconidium 3.10 Significance of Fungal
(Chander 2018). Morphology (Webster
and Weber 2007; Köhler
et al. 2015)
3.8 Conidium (Chander 2018;
Rippon 1988) Mycologists are interested in the structure of the
reproductive bodies and the manner in which
Conidium is an asexually formed spore; a rela- they are produced as these features constitute the
tively thin-walled spore that is terminal on the most important basis for classification and tax-
conidiophore and deciduous at maturity. onomy of the fungi.
30 L. V. Gorthi
als. Healthy individuals with intact immune sys- 2005; Aristizabal et al. 2002). The incidence rates
tem can defend against fungal diseases (Kohler of blastomycosis are 12 times more among Asians
et al. 2014; Taylor et al. 2001). Despite the rise in than in other races (Roy et al. 2013).
fungal diseases, treatment options are limited due Some fungi are known to cause endemic sys-
to availability of fewer drugs and emergence of temic disease that can lead to CNS manifesta-
drug resistance. The virulence traits of most com- tions. Histoplasmosis in Central America and
mon invasive fungal infections are the ability to Midwest United States (Kauffman 2007),
grow at human body temperature, adaptation to Paracoccidioidomycosis in South America
host conditions, phenotypic switching, morpho- (Colombo et al. 2011), Cryptococcosis in
logical diversity, secreted factors, biofilms, and Vancouver Island, along the pacific coast and
cell wall (Polvi et al. 2015). Targeted therapeutic Florida (Byrnes et al. 2009; Kunadharaju et al.
approach is based on the understanding about the 2013), Coccidiomycosis in Southwestern United
pathogenesis of fungal infections (Juvvadi et al. States and Central America (Flaherman et al.
2017). The pathogenetic mechanisms involved 2007), Blastomycosis in Midwestern United
include predisposing/epidemiological factors, States (Khuu et al. 2014) and infections due to
immunoregulation, genetic factors in fungi, mor- Penicillium marneffei from Southeast Asia (Lee
phological changes facilitating entry and adher- et al. 2012) are some of the endemic systemic dis-
ence, and factors for invasion and dissemination. eases occurring across the world.
Hobbies like spelunking, uncontrolled diabe-
tes also predispose to fungal infections (Santelli
4.2 Pathogenesis et al. 2006). Consequent to greenhouse effect, the
rise in temperature has helped fungi to selectively
The word “fungi” derived from the Latin fungus, adapt. This has resulted in broadening the spec-
meaning mushroom, are eukaryotic saprotrophic trum of fungi that can survive at host body tem-
organisms with membrane-bound nuclei that perature (Garcia-Solache and Casadevall 2010). It
derive nutrition from decomposition of organic has been reported most of the genes that enable
matter. Fungal infection is acquired through fungi to grow at higher temperature also contrib-
inhalation of fungal spores from environmental ute toward virulence (Bhabhra et al. 2004).
soil (Shih and Koeller 2015). Fungi are mainly Parenteral drug abuse (Breneman and Colford Jr
classified into molds, yeast, and intermediate 1992), immune-suppressing infections like HIV
forms. Fungi can be true pathogen or opportunis- (Pfaller and Diekema 2010), patients on immuno-
tic (Sharma 2010). Central nervous system suppressive medications like corticosteroids, bio-
(CNS) is relatively resistant to fungal infection logic response modifiers due to hematopoietic
owing to high blood supply and blood-brain bar- stem cell transplants, hematologic malignancies
rier (Davis 1999). Fungal diseases of the CNS (Mendoza et al. 2015) are some of the factors pre-
are usually opportunistic infections resulting disposing to fungal infections. The horizon of pre-
from hematogenous dissemination in susceptible disposing factors for fungal infection has been
hosts (Shih and Koeller 2015). ever expanding with advanced healthcare
facilities.
dendritic cells (DCs), natural killer (NK) cells, dritic cells and CD4 T-lymphocytes. Thus, adap-
innate-like lymphocytes, and epithelial cells tive immune system involves stimulation of T cells
(ECs) (Drummond and Brown 2011). by antigen-presenting cells. Antigen- presenting
The innate immune response is required for cell can produce T helper (Th)1, Th2, T regulatory
recognition of fungal pathogen-associated cell, or Th17 responses. Th1 responses generate
molecular patterns (PAMPs). Pattern recogni- IFN-gamma, which can control many dissemi-
tion receptors (PRRs) like Toll-like receptors nated fungal infections. Whereas Th2 cell response
(TLRs), complement components, C-type lec- produces IL-4 and IL-5 favoring persistence of the
tin receptors (CLRs), and mannose receptors fungal infections (van de Veerdonk and Netea
(MR) present on phagocytes and dendritic cells 2010; Hole and Wormley Jr 2012), regulatory T
are used for recognition of conserved pathogen- cells (Treg cells) inhibit Th1 and Th2 activity
associated molecular patterns. C-type lectin while promoting Th17 responses. Treg cells scav-
receptors (CLRs) are an important large class enge IL-2 through their high-affinity IL-2 receptor
of PRRs. Under C-type lectin receptors, those and increase Th17 cells (Pandiyan et al. 2011;
with antifungal activity include dectin-1, dec- Whibley et al. 2014).
tin-2, and Mincle. Upon entry of fungi into the There are various immunoregulatory signal-
host, these receptors bind to the fungi and bring ing pathways and checkpoints that influence the
about cellular immune response like phagocy- outcome of fungal infection (Verma et al. 2015;
tosis, production of proinflammatory cytokines, Roussey et al. 2016) (Fig. 4.1). Interleukin-10
and induction of respiratory burst through intra- (IL-10) signaling, the programmed cell death
cellular signaling cascade (Hardison and Brown protein-1 (PD- 1) signaling pathway, and the
2012). The common signaling pathway used by cytotoxic T lymphocyte-associated protein 4
CLRs includes kinase Syk and signaling adap- (CTLA-4) signaling pathway are the three impor-
tor CARD 9 (Drummond et al. 2011). The pro- tant pathways that modulate adaptive immune
inflammatory cytokines are produced as a responses (Rutz and Ouyang 2016; Freeman
specific response to fungal stimuli like et al. 2000; Lin et al. 1998). The consequence of
GM-CSF, IL-1β, IL-6, and TNF-α. These have interaction between the immune system and
been found to be defective in CARD 9 defi- fungi can be either protective tolerance or
ciency due to mutation (Drewniak et al. 2013). immune reconstitution inflammatory syndrome
Following fungal infection, host immune (IRIS) (Romani and Puccetti 2006; Shelburne
response through different TLR changes accord- 3rd et al. 2002).
ing to the route of infection, genus and species of The IL-10 signaling pathway plays a major
fungi, and also their morphotype, viz., yeast or role in the development of fungal infections.
hyphal forms (Bellocchio et al. 2004). IL-10 is produced by DCs and regulatory T cells
TLR-dependent cellular responses lead to the (Treg cells). The blockade of the IL-10 signaling
production of type I interferons (IFNs) and pro- pathway is protective against fungal infections
inflammatory cytokines TNF-α and IL-12 and because of enhanced Th1 and Th17 responses
the promotion of adaptive immunity by activa- and increased activation of effector macrophages
tion of T cells (Juvvadi et al. 2017). The respira- (Murdock et al. 2014). The programmed cell
tory burst and degranulation activities of death protein-1 pathway consists of the receptor
neutrophils which are specific antifungal activ- PD-1 and its ligands PD-L1. PD-1 is expressed
ity of neutrophils are influenced by TLR. The on activated T cells and antigen-presenting cells.
specificity and quantity of toxic products It inhibits T-cell proliferation and effector activ-
released by neutrophils determine the fungicidal ity (Freeman et al. 2000). CTLA-4 competes
activity resulting in inflammatory cytotoxicity with CD28 reducing costimulatory signaling via
(Bellocchio et al. 2004). CD80 and CD86 binding to CD28 and also
Effective host defense against fungal infections actively inhibits T-cell activity upon binding
are dependent on local interactions between den- these ligands (Roussey et al. 2016) (Fig. 4.1).
34 C. S. Banushree and N. S. Madhusudhan
Fungi
PAMP Pathways mediating
PRRs Adaptive Immune
APC response
IL-10 - IL-10 signaling
- PD1 signaling
Innate Response Adaptive Response - CTLA4 signaling
Inhibition
Gamma- IFN IL-4, IL-5 IL-17
Stimulation
Antibodies confer protection against fungal may be necessary to bridge the immunological
infections by multiple mechanisms that include communication between cells in the CNS and
direct neutralization of fungi and their antigens, cells in the peripheral tissues (Li et al. 2015;
inhibition of growth of fungi, modification of gene Prinz and Priller 2014).
expression and signaling, lipid metabolism, caus-
ing iron starvation, inhibition of polysaccharide
release, and biofilm formation. Antibodies promote 4.5 Evolution of Fungi
opsonization of fungi and their phagocytosis, com-
plement activation, and antibody-dependent cell 4.5.1 Role of Genes
toxicity (Elluru et al. 2014). in Pathogenesis: Lessons
In CNS, immunological reactions comprise of Learnt from Comparative
both an innate response and an adaptive response. Genomic Studies (CGA)
An innate response is initiated by microglia, and
adaptive response depends upon the presentation The novel genes are acquired at the telomeric
of antigen by antigen-presenting cells such as proximal regions of the pathogenic fungi which
macrophage and dendritic cells present in the are nonexistent in their nonpathogenic relative
leptomeningeal and perivascular region which species (Moran et al. 2011). This phenomenon
has the potential to elicit T-cell response is particularly well noticed in Aspergillus spp.
(Engelhardt et al. 2016). Microglia and perivas- Coordinated epigenetic regulation of expression
cular macrophages release chemokines that regu- of genes for virulence is facilitated by cluster-
late the neuroinflammatory response by increased ing. In a mouse infection model, about 30% of
recruiting of dendritic cells, neutrophils, and clustered genes were induced during initiation
lymphocytes from peripheral tissue. The interac- of invasive aspergillosis (McDonagh et al.
tion of microglia and perivascular macrophages 2008). Though horizontal gene transfer is seen
4 Pathogenesis of Fungal Infections 35
Telomeric Expansion
Fig. 4.2 Schematic diagram depicting genetic evolution of human fungal pathogens
in some fungi, their role in virulence of human • Rarely horizontal transfer—proline racemase
pathogenic fungi is not clear (Moran et al. in Candida parapsilosis. The genetic basis for
2011). Coccidioides spp. which are regarded as evolution is summarized in Fig. 4.2.
pathogenic fungi have lost genes required to
survive on plants as in Aspergillus. This sup-
ports that loss of gene during evolution has 4.5.3 Secretory Factors
helped to create habitat specificity (Sharpton
et al. 2009). Not only acquisition of genes but Enzymes produced by fungi, viz., protease, phos-
functional expression of these is crucial in viru- pholipase, and elastase, cause tissue damage and
lence of the fungi. This has been studied in impairment of host defenses (Romani 2004).
Candida spp. by transcript profiling and chro- Fungi produce enzymes like catalase, substrates
matin immunoprecipitation-microarray (ChIP- like mannitol, and melanin that act as scavengers
chip) analysis of the transcriptional networks of oxidative killing, thereby countering the
(Tuch et al. 2008). effects of reactive oxygen intermediates (ROI).
Thus, innate immune response against fungi is
hampered (Hamilton and Holdon 1999).
4.5.2 G
enetic Basis for Evolution
of Pathogenic Fungi (Moran
et al. 2011) 4.6 Mechanism of Pathogenesis
• Gene duplication and expansion—genes for Cutaneous and mucosal physical barriers resist
metalloproteases in Coccidioides, Saps, and fungi infection. However, fungi often develop
ALS gene in Candida. both virulence mechanisms and morphologic
• Gene loss and pseudo-genetization—HYR1 in changes that facilitate their multiplication within
Candida dubliniensis, galactose metabolism the host. The development and severity of disease
in C. glabrata. by fungal organisms depends upon the size of the
• Telomeric expansion—TLO genes in Candida inoculum, magnitude of tissue destruction, the
albicans, EPA genes in Candida glabrata, ability of the fungi to multiply in tissues, as well
secondary metabolite clusters in Aspergillus. as the immunologic status of the host (Kobayashi
36 C. S. Banushree and N. S. Madhusudhan
Table 4.1 Prevalence of common systemic fungal infec- et al. 2003). Coccidioidomycosis is caused by
tions involving CNS Coccidioides immitis which converts from a
Systemic fungal mold form in the environment to a unicellular
infection Prevalence of CNS involvement spherule containing sporangia in infected tissue
Candidiasis 50% (Pendelbury et al. 1989)
(de Pauw 2011). Blastomycosis is caused by
Aspergillosis 14–40% (Jantunen et al. 2003)
Blastomyces dermatitidis by inhalation of
Blastomycosis 5–40% (Friedman et al. 2000)
Histoplasmosis 10–20% (Wheat et al. 1990)
microconidia from soil. Blastomyces adhesin-1
present only on yeast form mediate binding of
yeast cells to human macrophages through their
1996). The prevalence of common systemic fun- CD14 and CR3 (CD11b/CD18) receptors (Long
gal infections involving CNS is represented in et al. 2003; Newman et al. 1995).
Table 4.1. The mechanism includes entry and Paracoccidioidomycosis is caused by thermally
adherence, invasion, colonization and dissemina- dimorphic fungi, Paracoccidioides brasiliensis.
tion in the host tissue, and damage to the host Inhalation of microconidia causes a primary
tissue by evasion of immune system (Khan et al. lung infection and is followed by the morpho-
2010). logical transition into the pathogenic yeast form.
Glycoprotein 43 of cell wall helps in binding the
macrophage (Tronchin et al. 2008; Lupi et al.
4.6.1 Portal of Entry and Adherence 2005). Candida species are normal commensals
of humans and are frequently isolated from the
Fungal species are widely found in soil, plant skin, gastrointestinal tract, and urine. The most
debris, and other organic substrates and are common species that cause invasive infections
eukaryotic species present on earth, although are C. albicans, C. glabrata, C. parapsilosis, C.
only a limited number of species are human tropicalis, and C. krusei (Garcia-Vidal et al.
pathogens (Dadiee and Hashemizadeh 2014). 2013). Candida albicans is the predominant
The incidence of invasive fungal infections is cause of invasive fungal infections from yeasts.
much lower than superficial fungal infections, Candida albicans as a commensal organism
but the high mortality rate associated with inva- exists in a unicellular yeast-like morphology,
sive fungal infections is of great concern (Brown but when it invades tissues, it becomes filamen-
et al. 2012). Invasive fungal infections can be tous (de Pauw 2011). Candida albicans ability
due to dimorphic(endemic) mycoses that are for mucosal adherence and biofilm formation is
caused by true pathogenic fungi or the an important virulence factor. Aspergillus spe-
opportunistic mold and yeast infections that
cies are found in soil, air, food, and common
are s aprophytes which only infect an decaying organic material. Among 200 recog-
immunocompromised host. Histoplasmosis,
nized species, A. fumigatus is the most common
coccidioidomycosis, blastomycosis, and para-
cause of invasive fungal infection, followed by
coccidioidomycosis cause dimorphic mycoses. A. flavus, A. terreus, A. niger, and A. nidulans.
The most common opportunistic fungi are The conidia produced by aspergillus disperse
Candida and Aspergillus (de Pauw 2011). In into the air, and when inhaled by an immuno-
dimorphic mycoses, conversion to pathogenic suppressed patient, they germinate and become
yeast form occurs after inhalation of microco- hyphae causing the invasive aspergillosis
nidia and small mycelial fragments by the host. (Garcia-Vidal et al. 2013).
Histoplasmosis is caused by Histoplasma cap- In CNS, fungal infections may develop via
sulatum, a dimorphic intracellular fungal patho- hematogenous dissemination from a distant focus
gen. The cell surface heat shock protein (hsp)60 such as lung, through direct implantation after
mediates binding and internalization of yeasts trauma or secondary to the local extension from
and conidia by macrophages, via the CD18 fam- sino-nasal, orbital, or spinal infections (Mohan
ily integrin receptor (Tronchin et al. 2008; Long et al. 2012). Fungi enter from the bloodstream
4 Pathogenesis of Fungal Infections 37
and possess surface components that allow them 2004). The destruction of host tissues by
to traverse the capillary tight junctions and cause Candida in the local environment may be facili-
the infection (Sundaram et al. 2011). tated by the release of hydrolytic enzymes such
One of the key factors contributing to patho- as secreted aspartyl proteinases (Saps), phos-
genesis of these organisms is morphogenetic pholipases, lipases, and hemolysins (Garcia-
variation like formation of yeasts, hyphae, and Vidal et al. 2013). Saps aids in nutrient uptake,
spherules that facilitate their multiplication tissue invasion, adherence, and dissemination
within the host at higher temperature. Candida (Khan et al. 2010).
albicans switches between the growth forms Glucuronoxylomannan is the major constituent
like round yeast, elongated pseudohyphae and of the capsule produced by Cryptococcus neofor-
filamentous hyphae in the host. Yeast cells of mans. Glucuronoxylomannan causes immunosup-
many Candida species form filamentous pseu- pressive and immunodysregulatory effects on the
dohyphae and hyphae in tissues, whereas host by partially activating TLR-dependent signal
Cryptococcus neoformans yeasts become coated transduction pathways (Romani 2004).
with a capsule. Coccidioides immitis develops Titan cells play a central role in C. neofor-
swollen, septated spherules in the host, and mans pathogenicity. Titan cells can be of 100 μM
other fungi like Histoplasma capsulatum, and are formed from C. neoformans cell of
Blastomyces dermatitidis, and Penicillium 5–10 μM through various signaling pathways.
marneffei form filamentous mycelia in the envi- They will have a thicker cell wall, denser capsule,
ronment but convert to yeast morphology upon and tetraploid or octaploid DNA content which
contact with the human host (Rappleye and prevents phagocytosis (Zaragoza et al. 2010;
Goldman 2006). Zaragoza and Nielsen 2013). Melanization of C.
Temperature plays a key role in the morpho- neoformans occurs during infection, and overex-
genetic transition of C. albicans from yeast to pression of melanin results in decreased recogni-
filamentous growth. The heat shock transcrip- tion by the host and modulation of host cell
tion factor Hsf1 and the molecular chaperones immune responses (Nosanchuk et al. 1999).
Hsp70 and Hsp90 regulate the heat shock Urease expression in C. neoformans is linked
response in Candida albicans (Leach and with increased invasion across the blood-brain
Cowen 2013). C. albicans expresses adhesin barrier, and phospholipases A, B, C, and D help
agglutinin-like sequence (AIs) proteins. Among in tissue invasion and adherence (Khan et al.
at least eight AIs, Als1p, Als3p, and Als5p play 2010; Olszewski et al. 2004).
a major role in adherence of C. albicans to the H. capsulatum antigenic cell surface β glucans
host cells. Als1p and Als3p help in binding to are hidden under a layer of α-(1,3)-glucan to
endothelial and epithelial cells, whereas Als5p avoid interaction with host phagocytes. After
binds to extracellular matrix (ECM) proteins. internalization through hsp60, interactions with
The hyphal wall protein 1 (Hwp1p) adhesin CR3 do not result in a strong immune response
present at the germ tube surface mediates tight allowing H. capsulatum to grow and survive
binding to buccal epithelial cells and is required within macrophages (Rappleye et al. 2007).
for biofilm formation (Tronchin et al. 2008; Catalase present in H. capsulatum prevents from
Loza et al. 2004). oxidative killing (Khan et al. 2010).
C. albicans hyphae are hydrophobic and Coccidioides immitis develops swollen, sep-
adhere to various biomaterials, such as cathe- tated spherules in the host which is required for
ters, prostheses, or medical implants, and form dissemination; hyphal phase can tolerate
biofilms which contribute to tissue colonization. pH 2–12. Elastase enzyme secreted causes
Biofilms are complex three-dimensional destruction of lung interstitium and blood ves-
surface-associated communities of yeast and sels, and estrogen-binding protein accelerates
hyphal cells within an extracellular matrix spherule maturation and endospore release
(Hawser and Douglas 1994; Kojic and Darouiche (Kobayashi 1996; Khan et al. 2010).
38 C. S. Banushree and N. S. Madhusudhan
Blastomyces adhesin-1 in Blastomyces derma- Ywp1 that weaken adherence and promote dis-
titidis acts as an adhesin and immunomodulator. semination. This bidirectional morphotype con-
α-1,3-Glucan in cell wall helps in adhesion and version augments the ability of C. albicans to
masking of cell surface receptors from being invade and disseminate. C. albicans invade epi-
recognized by immune cells (Khan et al. 2010; thelial cells by induced endocytosis and active
Newman et al. 1995). penetration (Lin et al. 2015).
Mucormycosis is a serious life-threatening C. neoformans can cross brain endothelial
infection most commonly caused by Rhizopus cells by proteolytic degradation of tight junc-
oryzae that occurs in patients who are immuno- tions, induced endocytosis, and macrophage
compromised because of diabetic ketoacidosis, transport. Degradation of intercellular tight
neutropenia, organ transplantation, and/or junction is facilitated by urease produced by C.
increased serum levels of available iron (Ibrahim neoformans. Binding of C. neoformans hyal-
et al. 2012). Fungi obtain iron from the host by uronic acid to CD44 present on brain endothe-
using high-affinity iron permeases or lial cell activates the protein kinase C (PKC)-α
low-molecular-weight iron chelators (sidero- signaling pathway resulting in rearrangement of
phores) as iron is required for cell growth and endothelial cell actin microfilaments and the
development of fungi (Stearman et al. 1996). formation of pseudopods that engulf the organ-
Moreover, glucose-regulated protein (GRP78) ism and draw it into the endothelial cell. C. neo-
acts as a receptor that mediates binding and pen- formans can invade the brain via a Trojan horse
etration of endothelial cells. The other virulence mechanism, whereby the organism is phagocy-
factors are aspartic proteinases and active ketone tosed by a monocyte, which then diapedeses
reductase system which accelerates the growth in across the blood-brain barrier into the brain,
the acidic and glucose-rich environment seen in carrying the fungus with it (Sheppard and Filler
ketoacidotic states (Farley and Sullivan 1998; 2015).
Anand et al. 1992). In A. fumigatus, exposure of surface β-1,3-
glucans of germinating conidia induces the acti-
vation of phospholipase D via a dectin-
4.6.2 Invasion and Dissemination 1-dependent mechanism which helps in endocy-
tosis by epithelial cells (Han et al. 2011). Hyphae
The invasion of nonphagocytic host cells by fungi will penetrate more deeply into pulmonary tis-
plays an important role in the pathogenesis of sues and invade pulmonary vascular endothelial
fungal infections. The invasion of natural cellular cells from their abluminal side. Later, traverse the
carriers such as vascular endothelium or pulmo- cell to gain access to the vascular compartment.
nary epithelium prevents phagocytosis by neutro- From vascular compartment, hyphal fragments
phils and macrophages. The invaded host cell can disseminate to distal sites by invading vascu-
may serve as a source of nutrients like carbon and lar endothelial cells from the luminal side before
nitrogen for the fungus to sustain biosynthetic penetrating further into other deep tissues such as
processes. Fungi have liking for certain carbon the brain (Kamai et al. 2009; Filler and Sheppard
and nitrogen sources that are rapidly metabolized 2006). Angiotropism is an important virulence
and therefore provide quick energy for growth factor, in particular among Zygomycetes leading
and niche colonization (Sheppard and Filler to cavernous sinus thrombosis, ischemic infarc-
2015; Ries et al. 2018). tion, and rupture of mycotic aneurysms (Rangel-
The yeast form of C. albicans is seen in com- Guerra et al. 1996). Dissemination to various
mensal state while filamentous form in invasive organs occurs in some fungal infections due to
disease. The hyphal form can escape from phago- bloodstream infection. Infection of bloodstream
cytic cells. Sustained polarized hyphal growth may occur through breaks in mucosal barrier as
prevents the entrapment of C. albicans intracel- in systemic candidiasis (Eggimann et al. 2003;
lularly. Yeast cells also produce proteins like Pappas et al. 2009).
4 Pathogenesis of Fungal Infections 39
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Predisposing Factors
5
Shruti Gupta and Sanjay Behari
time of trauma might also result in CNS disease 5.5.2 Filamentous Fungi
(Sethi et al. 2012).
The large filamentous fungi with septate or asep-
tate hyphae, like Aspergillus and Zygomycetes,
5.4 athogenesis of CNS Fungal
P can obstruct large- and intermediate-sized arter-
Mycoses ies. They result in the development of necrotizing
angiitis, thrombosis, and large-sized infarcts. The
A myriad of factors influence the ability of the haemorrhagic infarcts later convert to septic
fungus to infect the CNS. These include host fac- infarcts with abscesses and cerebritis. Skull base
tors as well as the virility of the fungi. The host involvement and invasive infections can occur
susceptibility depends upon the immune system from the paranasal sinuses. Invasive infection, in
and its response to fungal inoculation. If a host its most severe forms, may spread to the orbit
fails to eliminate the fungi at a primary site, for causing visual loss, to the palate causing necro-
example, the lungs, hematogenous dissemination sis, as well as to the brain leading to a high mor-
to the brain occurs. Once the brain comes in con- tality. In addition to this, aspergillosis can result
tact with the fungus, the phagocytic cells such as in the formation of an intracranial space occupy-
microglia as well as astrocytes are activated. ing lesion and, often, can lead to the occurrence
Both cellular and humoral immune responses are of chronic infection in the immune-competent
activated, and cytokine release occurs (Sethi host (Chakrabarti 2007; Sethi et al. 2012; Bozorgi
et al. 2012). et al. 2016).
The virility of the fungus depends on the mode
and the size of inoculum, the ability of fungi to
adapt to the body temperature of the host, the 5.5.3 Dimorphic Fungi
hypoxic state at the cellular level and its ability to
overcome the host immune response. These fungi are commonly associated with self-
limited pulmonary illness or mild systemic illness
when their manifestations occur in the mildest
5.5 Pathobiology of Fungal forms. Dissemination to the CNS constitutes
Lesions nearly one-third to half of the extra-pulmonary
dissemination. Common manifestations include
The pathobiology of the fungal manifestations chronic basilar meningitis with or without hydro-
depends on the morphology of the fungus cephalus and the development of vasculitic
(Chakrabarti 2007; Sethi et al. 2012; Murthy infarcts (Drake and Adam 2009).
2007; Low and Rotstein 2011).
parenchyma, intracranial vasculature, cerebro- involve the humoral immune system which
spinal fluid as well as the calvarium or the skull, involves B-lymphocytes and antibody production
most commonly the paranasal sinuses and the or the cellular immune system which involves
skull base (Scully et al. 2008; Murthy 2007; T-lymphocytes, or both (Low and Rotstein 2011).
Naik et al. 2015). Consequently, the patient Phagocyte and complement function may also be
may present with a clinical syndrome related to impaired. Examples of defective humoral immu-
any part of the cranio-spinal axis as fungal CNS nity include common variable immunodeficiency
infection may result in meningitis, encephalitis, (CVID) and transient hypogammaglobulinemia
abscess formation, intracranial fungal granu- of the infant; defective cellular immunity may
loma, vasculitis and subsequent infarcts (Scully occur in chronic mucocutaneous candidiasis and
et al. 2008; Murthy 2007; Naik et al. 2015; DiGeorge syndrome, whereas both cellular and
Shankar et al. 2007). Certain fungi have spe- humoral deficiencies occur in severe combined
cific involvement patterns, such as the rhinocer- immunodeficiency (SCID). Apart from these,
ebral involvement by the Mucor and Rhizopus chronic granulomatous disease and cyclic neutro-
and the skull base involvement by Aspergillus. penia also favour the invasion by opportunistic
Overall, the most common symptoms are head- infections owing to phagocyte dysfunction.
ache, nausea and vomiting, fever, focal neuro- Prophylaxis with both antifungal medications
logical deficits, seizures and cranial nerve and antibiotics is recommended in these immu-
palsies (Chakrabarti 2007; Sethi et al. 2012; nodeficiency states.
Bozorgi et al. 2016).
CD4+ count <200 per μL of blood. Areas state helps in proliferation of the fungus, and the
endemic for HIV/AIDS show a high preva- acidotic environment causes failure of phagocy-
lence of cryptococcal meningitis, and approxi- tosis, thus protecting the fungus from the host
mately 5–10% patients with HIV have this immune responses (Sethi et al. 2012; Bozorgi
meningitis as an AIDS-defining illness et al. 2016). This is especially true for the fila-
(Shankar et al. 2007). Cryptococcal fungemia mentous fungi belonging to the order Mucorales
can follow inhalation of fungal spores, and viz. Rhizopus and Mucor.
Cryptococcus neoformans can then cross the
blood-brain barrier by transcytosis. It leads to 5.7.2.5 Immunosuppressive
leptomeningeal and brain inflammation with Medication and Solid Organ
CD8+ lymphocytes and macrophage- Transplant
predominant infiltrates. The most commonly affected patient population
belongs to the group that has undergone a small
5.7.2.2 Intravenous (IV) Drug Abuse bowel transplantation; and the risk of fungal
Fungal infections may account for up to 5–50% infection is relatively lower in those patients who
of serious infections in IV drug abusers (Leen have undergone a renal or liver transplantation
and Brettle 1991). The most common fungi iso- (Low and Rotstein 2011). Both medical and sur-
lated are Candida spp. and Aspergillus spp. gical factors play an important role.
Fungal infections in intravenous drug abusers are Immunosuppressive medications used in these
chiefly due to contaminated, unsterile needles patients including various chemotherapeutic
and injections. Multiple injections and sharing of agents, high-dose steroids as well as anti-
injecting needles also predispose this group of lymphocyte globulins directly inhibit cell-
patients to a high risk of HIV infection, which is mediated immunity and place the afflicted
an independent risk factor for mycotic infections individuals at an increased risk of local, systemic
(Low and Rotstein 2011; Leen and Brettle 1991). and CNS mycoses. Extrinsic factors, such as
radiation, further aggravate this inhibition of host
5.7.2.3 Chronic Renal Failure immune responses. Surgical factors include pro-
It is stated that end-stage renal disease results in longed ischemia time, renal failure requiring
an impairment of both humoral and cellular multiple haemodialysis, fulminant hepatitis,
immunity owing to the uremic milieu, and the re- transplantation, older age at transplanta-
degree of impairment is directly related to the tion and graft-versus-host disease (Low and
duration of renal failure (Gandhi et al. 2005). Rotstein 2011).
Defects in humoral immunity lead to an impaired
T-helper cell function and antibody production; 5.7.2.6 Haematological Malignancies
defects in the cellular immunity cause defective and Haematopoietic Stem Cell
lymphocytic response to antigen exposure and Transplant (HSCT)
impaired phagocytosis. The use of chronic Patients with haematological malignancies like
indwelling catheters, multiple antibiotic use, cor- acute leukaemia are at a risk of fungal infections,
ticosteroid treatment, repeated dialysis, the use of especially associated with their invasive and
iron chelating agents, endotracheal or nasogastric acutely fulminant forms. The risk of infection in
tube, hyperalimentation and metabolic acidosis these patients depends on the type of leukaemia,
are conditions that further increase the chances of duration of leukaemia, degree of neutropenia and
mycoses in these patients (Gandhi et al. 2005). the type of chemotherapeutic agents used for
treatment. Specific at-risk populations include
5.7.2.4 Diabetes Mellitus those with de novo leukaemia; those in post-
Diabetes mellitus, more commonly the diabetic remission status; those afflicted with the hairy
ketoacidotic state, is a fertile ground for the mul- cell, acute myelogenous or acute lymphocytic
tiplication of fungi. The carbohydrate-rich ketotic leukaemic variants; those with relapsed leukae-
5 Predisposing Factors 47
matosis are at a higher risk of infections with (Scully et al. 2008; Sethi et al. 2012; Low and
fungi like Aspergillus, Zygomycetes, Candida, Rotstein 2011). Both the duration and the
Cryptococcus, Histoplasma and Paracoccidioides severity of neutropenia influence the extent and
(Alexander et al. 2006). outcome of CNS mycotic infections.
There are two mechanisms that have been pro- Neutropenia due to aplastic anaemia and bone
posed for fungal infections in iron overload marrow failure may be responsible for the
states. It has been observed that a virtually iron- highest level of risk. Filamentous fungi, like
free environment is needed for the proper func- Aspergillus spp., commonly affect neutropenic
tioning of the immune responses, and hence, the patients.
presence of iron impairs the host immune
response. The second mechanism proposed is
that over the years, fungi have developed sophis- 5.7.3 Environmental Exposure
ticated mechanisms to utilize iron, which act as to Fungal Spores
cofactors in several enzymatic reactions and
hence favour the growth of fungi in an iron-rich Fungi are ubiquitous in distribution and are abun-
environment. dant in the soil and in the decaying waste.
Environmental exposure to fungal spores dis-
5.7.2.11 Malnutrition persed in the air as well as encountered in the
Nutritional deficiency is an important cause of farm and animal waste, like bat or pigeon drop-
secondary immunodeficiency state. When the pings, has been implicated in CNS fungal infec-
body weight falls to 80% of the recommended tions. Specific fungi like Cryptococcus have also
level for the age and height, the immune system been associated with eucalyptus tree plantations
is impaired, and when it falls to 70% of the rec- and pigeon droppings; Histoplasma infections
ommended level, the immune system impairment have been associated with bat droppings in caves
is severe. The deficiency of one or more essential (Sethi et al. 2012).
micronutrients, such as calcium, zinc or sele-
nium, also impairs the immune response of the
host and hence predisposes to a multitude of 5.8 Clinical Outcome
infections including fungal infections.
Mortality rates as high as up to 80% have been
5.7.2.12 Trauma reported in CNS mycoses (Bozorgi et al. 2016).
Trauma in both the civilian (motor vehicle acci- The mortality rate in immunocompromised
dents, agriculture-related and natural calamities) patients is higher than in the immunocompetent
and the military (explosive devices) setting has counterparts (Scully et al. 2008; Sethi et al.
been associated with fungal infections. Trauma 2012; Low and Rotstein 2011). Apart from the
leads to direct inoculation with fungi especially immunocompromised state, the severity of neu-
from the soil. Elasticised bandages of wounds tropenia, the age at presentation, the neurologic
have also been implicated in infection with fungi, status at presentation and the duration of hospi-
e.g., Zygomycetes. tal stay are independent risk factors associated
with morbidity and mortality. With the advent of
5.7.2.13 Neutropenia novel antifungal drugs, such as liposomal
Neutrophils are rapidly destructive for fungal amphotericin B and voriconazole, and with
hyphae and constitute an important defence extensive surgical debridement that is now pos-
mechanism against fungal infections. A reduc- sible with advances in surgical techniques,
tion in the neutrophil count is termed as neu- the mortality rates have been reduced to as
tropenia. Neutropenia is defined as an absolute low as 10–15% in the recent times (Bozorgi
neutrophil count of <1500 per μL of blood et al. 2016).
5 Predisposing Factors 49
An Indian study including 130 histopathologi- form at 25 °C (filamentous in nature) and trans-
cally confirmed CNS fungal infection cases form into yeast (spherules) at normal human body
(Sundaram et al. 2006) reported that the most fre- temperature (37 °C). Encapsulated yeast, C. neo-
quent pattern was granulomatous inflammation in formans, preserves its morphology in normal
the majority of patients (74 cases), followed by human tissues and in the environment, similar to
angioinvasion with infarcts and abscesses in 31 some septate and nonseptate mycetes.
cases and angioinvasion with infarcts in 9 cases. In general, fungal infections are becoming
Macroscopically, mycotic aneurysm with rupture more frequent because of expansion of at-risk
and subarachnoid hemorrhage were seen in two populations (transplanted patients, those receiv-
patients, chronic meningitis in two, abscess in ing immunosuppressive and chemotherapeutic
eight (single in three, multiple in five), and enceph- agents, HIV-infected patients, premature infants,
alitis with vasculitis and infarcts in four patients. the elderly, and patients undergoing major sur-
Fungal infections of the CNS could be caused by gery) (Table 6.1) and also because availability of
a large group of organisms; in addition, they are not treatment schemas permit longer survival of these
including in a mandatory report disease by the pub- patients (Naggie and Perfect 2009). Certain vari-
lic health authorities, and therefore, robust epide- ations in the geographical distribution of endemic
miological information on their global burden is not fungal infections can be attributed to climate
available (Schwartz et al. 2018). However, accord-
ing to reports from reference centers (Schwartz Table 6.1 Fungal pathogens of the CNS associated with
et al. 2018; Naggie and Perfect 2009), and the specific conditions
review in the present book, the most important and Fungi
frequent entities include candidiasis, aspergillosis, Disease-induced immunosuppression
cryptococcosis, mucormycosis, histoplasmosis, HIV infection Cryptococcus
coccidioidomycosis, and blastomycosis. neoformans
Fungi are ubiquitous environmental organ- Diabetes and iron overload Mucorales
Hematological malignancies Aspergillus spp., and
isms that may be unicellular (yeast), filamentous
(e.g., acute leukemia) other molds
(molds), or show a dimorphic morphology. More Neutropenia (e.g., aplastic Aspergillus spp.
than one million known mycotic species exist in anemia)
nature, and around 200 species are known to be Prematurity Candida spp.
pathogenic for the human being. However, only Drug-induced immunosuppression
about 20 fungal species produce invasive sys- Corticosteroids Aspergillus spp., and
temic infections, including CNS invasion other molds
Biological drugs (TNFα Molds, dimorphic fungi,
(Guarner and Brandt 2011). Cryptococcus spp.
inhibitors)
According to its morphology, fungi can be Immunomodulatory agents Aspergillus spp.,
classified as follows: pseudomycetes/yeasts (e.g., ibrutinib) Cryptococcus spp.
(Candida spp., Cryptococcus spp., Histoplasma Hematopoietic stem cell Aspergillus spp.,
spp., Blastomyces spp., Coccidioides spp., transplantation non-Aspergillus molds
Paracoccidioides spp., and Sporotrichum spp.), Solid organ transplantation Candida spp.,
Aspergillus spp.,
septate mycetes (Aspergillus spp., Penicillium non-Aspergillus molds
spp., Cephalosporium spp., Cladosporium spp., Medical interventions
Diplorhinotrichum spp., Hormodendrum spp., Neurosurgery, spinal Aspergillus spp., other
and Paecilomyces spp.), and nonseptate mycetes anesthesia, injection molds, Candida spp.
(Mucor spp., Rhizopus spp., Absidia spp., Intravascular or intracranial Candida spp.
Basidiobolus spp., Cunninghamella spp., and devices
Other
Mortierella spp.).
Intravenous drug use Candida spp.
Dimorphic fungi such as Histoplasma spp.,
Adapted from: Lancet Neurol. 2018;17(4):362–72.
Coccidioides spp., Blastomyces spp., Sporotrichum https://doi.org/10.1016/S1474-4422(18)30030-9
spp., and Paracoccidioides spp. display a mycelial (Schwartz et al. 2018)
6 Histopathology 53
changes, an extension of human habitats, ease of dis are thick-walled, broad-based budding yeasts;
travel, and shifting populations. Therefore, dur- P. brasiliensis is big yeasts with small yeasts
ing the last decades, a shift in the epidemiology attached given a so-called pilot-wheel configura-
of human mycoses has occurred (Guarner and tion; and Coccidioides species manifest them-
Brandt 2011). selves as large spherules that contain endospores
Before the twenty-first century, bloodstream (De Pauw et al. 2008; McCarthy et al. 2014).
infections were mostly caused by Candida spp., Histopathological examination represents a
and the most frequent invasive pulmonary rapid and cost-effective approach of providing a
infections included primarily aspergillosis and presumptive or definitive diagnosis of an invasive
endemic mycoses. Nowadays, fungi previously fungal infection. Nevertheless, microbiologists,
not considered pathogenic, including mucora- pathologists, and clinicians need to be aware of the
ceous genera (formerly called zygomycetes), limitations of histological diagnosis, the pitfalls of
and many hyaline and dematiaceous molds are morphological diagnosis, and the additional tests
frequently seen affecting immunocompromised that can be performed (Guarner and Brandt 2011).
patients. Therefore, diagnosis of infection ver- In this chapter, we review the most representa-
sus colonization with these fungi is a common tive microscopic findings and interpretation pitfalls
issue that has important treatment implications regarding the most frequently encountered fungi in
(Guarner and Brandt 2011). Moreover, advances CNS infections. We also present the complemen-
in diagnostic imaging and specific patient sup- tary or alternative tests that can be performed in the
port have permitted the possibility of collecting biopsy specimen and other samples.
biopsy specimens for histological examination
from sites previously not reachable; neverthe-
less, these advantages come with challenges 6.2 isease Caused by
D
related to the limited amount of tissue obtained Aspergillus spp.
and the architectural distortion produced by the
procedure (Gavito-Higuera et al. 2016). The genus Aspergillus is represented by molds
In histological terms, evidence of damaging (Fig. 6.1a, c (Courtesy of D. Palacios M.D.), e
tissue is a sufficient diagnostic proof of a CNS (Courtesy of Z. DL Rosa R.M.)) that reproduce
infection, even in patients with negative results in asexually producing conidia (Fig. 6.1b, d
cultures and other tests. However, this demonstra- (Courtesy of Z. DL Rosa R.M.)) (Bennett
tion requires a brain biopsy, which is one of the 2009). These fungi are ubiquitous and have
most important limitations (De Pauw et al. 2008). been used for centuries for industrial proposes.
Frequently, fungi when present in tissue are not This genus is able to survive in a variety of hab-
easily visible using routine stains; thus, Gomori itats from leaf litter to human tissues. The most
methenamine silver or periodic acid-Schiff (PAS) frequent species associated with human disease
staining can be used to improve their visibility is A. fumigatus, and other species, such as A.
and allow a better morphological identification niger, cause disease in immunosuppressed indi-
(Schwartz et al. 2018; De Pauw et al. 2008). Some viduals. Aspergillus fungi produce three partic-
microscopic fungal characteristics can allow its ular clinical entities: allergic bronchopulmonary
identification to a genus level. For example, aspergillosis, chronic pulmonary aspergillosis/
Aspergillus species can be differentiated from aspergilloma, and invasive or systemic asper-
Mucorales by their septate, dichotomous branch- gillosis (Riscili and Wood 2009).
ing hyphae, whereas Mucorales have an irregular Invasive pulmonary aspergillosis generally
shape of mostly uniseptate or pauciseptate occurs in critically immunosuppressed patients,
hyphae. Some organisms show specific morpho- including patients with prolonged neutropenia,
logical characteristics, which can help in their organ transplant recipients, patients with AIDS,
identification. For example, H. capsulatum are premature newborns, and patients with chronic
small intracellular, budding yeasts; B. dermatiti- granulomatous disease (Sherif and Segal 2010).
54 L. F. Jurado and R. d. P. López-Panqueva
a b
c d
Fig. 6.1 Aspergillus spp. (a) Illustration, septate acute- hyphae, Gomory, 40×. (d) Vesicle with conidia from cul-
angle (45°)-branching hyphae. (b) Illustration, vesicle ture. (e) Appearance in culture. All illustrations are origi-
with conidia. (c) Septate acute-angle (45°)-branching nal by Jurado LF
6 Histopathology 55
Apparition of neurological signs of stroke or sei- 83% on cases with septated, acute- angle-
zures usually indicates that the fungus has branching hyphae and the presence of Aspergillus
reached the CNS. spp. in culture; Fusarium spp., Trichophyton
Aspergillus spp. are one of the most frequent spp., and others were recovered in culture from
fungi isolated from neurological infections, most the discordant cases. Among the Aspergillus spe-
infections of the CNS are due to A. fumigatus, cies isolated, A. fumigatus, A. flavus, A. niger, A.
which gets into the CNS by hematogenous spread nidulans, and A. terreus were identified.
from the primary site of infection (lungs) or from Cases of mixed infection, involving
contiguous anatomical sites, such as the paranasal Aspergillus spp. and Candida or mucoraceous
sinuses (McCarthy et al. 2014). Focal lesions or a genera, have been described, posing an important
brain abscess are the predominant findings dilemma. To be able to identify mixed infections,
(Barrera-Herrera et al. 2015), while cerebral infarc- it is crucial to use alternative diagnostic testing
tion caused by septic embolism, vascular thrombo- (Hofman et al. 2010). In cases of invasive pulmo-
sis, or mycotic aneurysms are less frequent nary aspergillosis, cultures are positive in only
(McCarthy et al. 2014), and meningitis (without 50% of bronchoalveolar lavage (BAL) fluid spec-
parenchymal involvement) is rarely reported imens, and organisms recovered from BAL fluid
(Antinori et al. 2013). Microscopically, Aspergillus samples could reflect colonization rather than the
spp. are thin (3–12 μm), septate, acute-angle (45°), actual infection. In cases of invasive disease,
or dichotomous branching hyphae (Fig. 6.1a, c), isolation of Aspergillus spp. in blood cultures is
and when present in cavitary lesions, vesicles with approximately 5% (Sherif and Segal 2010).
conidia can be also identified (Fig. 6.1b, d). Regarding the complementary test that can be
In a retrospective study performed by performed when a case of aspergillosis is suspected,
Sundaram et al. (2006), 56% of the cases were galactomannan and (1 → 3)-β-d-glucan, which are
caused by Aspergillus spp., 25% of them had a components of the fungi cell wall, can be measured
positive culture, and the majority of patients were in body liquids using commercially available kits
immunocompetent. The most common source of (Sherif and Segal 2010). However, this analysis
spreading was continuity from sinuses, orbit or presents false-positive results in approximately
ear, followed by the hematogenous route; how- 50% of individuals taking antibiotics (piperacillin,
ever, in eight cases, no source of infection was amoxicillin) and all patients receiving substances
identified. Histologically, granulomas and dense that contain products of A. niger fermentation (plas-
fibrosis were the most frequent features. Eight malyte). This test also presents cross-reaction with
cases presented isolated intracerebral granulo- other fungi, such as Penicillium spp. and
mas; fibrosis was less marked in these lesions. Histoplasma spp. (Guarner and Brandt 2011). A
The granulomas observed differed from tubercu- study (Chong et al. 2016) that evaluated galacto-
lous granulomas by the prominence of multinu- mannan testing in CSF from 17 patients with CNS
cleated giant cells, an abundance of neutrophils Aspergillus infection and 27 controls reported a sen-
and plasma cells with lymphocytes and few epi- sitivity of 88% and a specificity of 96%, which indi-
theloid cells and marked fibrosis. Microscopic cates a high diagnostic performance.
identification of the fungus was performed on The (1 → 3)-β-d-glucan is present in a broad
GMS and PAS, and the culture positivity was range of fungi (Alexander et al. 2010). There are
25%. The most common isolated organisms were commercially available assays for testing circu-
A. flavus, A. fumigatus, A. niger, and A. terreus. lating (1 → 3)-β-d-glucan; it has been detected in
Lee et al. (2010) reported a series of 393 patients with systemic fungal infections (invasive
patients with evidence of fungal infection on his- aspergillosis, candidemia, and Pneumocystis
tologic examination (a total of 231 (58.8%) were pneumonia) (Persat et al. 2008). The evidence on
rhinocerebral infections); they analyzed the cul- CNS infection is scarce; recently a study showed
ture-histology concordance of filamentous fungi that its measurement in CSF represents a good
in 122 specimens; they showed concordance in approach for diagnosing and therapeutic
56 L. F. Jurado and R. d. P. López-Panqueva
a b
Fig. 6.2 Candida spp. (a) Illustration, budding yeasts inflammatory reaction H&E, 100×. (c) Budding yeasts
and thin branching, pseudohyphae or filaments showing from culture. (d) Appearance in culture. All illustrations
periodic constrictions. (b) Pseudohyphae over tissular are original by Jurado LF
6 Histopathology 57
During the last decades, Candida species have philic inflammation with few lymphocytes and
evolved from infrequent to relevant and common macrophages, fibrin, and coagulative necrosis
human pathogens causing a wide spectrum of (van de Veerdonk et al. 2010); sparse giant
clinical syndromes. Candida albicans usually cells and granulomas can also be found. Due
colonizes the oropharynx and vagina, and viable to the bloodstream nature of the infection,
organisms can be cultured from these surfaces mycotic aneurysms or thrombophlebitis can
(Southern et al. 2008). When there are microbial develop. In candidemia cases, necrotizing vas-
imbalances caused by antibiotic use, hormonal culitis has been described; interestingly,
deregulations, and immunosuppression (HIV organisms are not observed in affected ves-
infection, diabetes), superficial infections in the sels, supporting the idea that Candida soluble
gastrointestinal or genitourinary tract can occur fractions are responsible for this pathogenic
(Concia et al. 2009). Invasive candidiasis occurs pattern (Sargent et al. 2010). In neutropenic
frequently as a healthcare-associated infection, patients, the necrosis is frequently accompa-
and patients at risk include those under broad- nied by hemorrhage, and few lymphocytes and
spectrum antibiotic treatment, immunosuppres- macrophages can be seen (Schuetz and Walsh
sant drugs, those with vascular access devices, 2015).
cancer diagnosis, and neutropenia (Darouiche Candida spp. are yeasts that produce pseudo-
2009). hyphae (Fig. 6.2a, b), thus requiring differentia-
CNS infections caused by Candida species tion from other yeasts and molds that produce
usually arise from hematogenous spread, and hyphae in tissues, such as Aspergillus spp. and
most of these are caused by Candida albicans Trichosporon spp. Elongated Candida pseudo-
(Kullberg and Arendrup 2015). Epidemiological hyphae can appear to be branching but can be
data on the Candida species distribution in CNS differentiated because pseudohyphae are thin
infections is not available; in the published series, and do not have septations (Fig. 6.2b). Another
Candida spp. usually appears in the second or pitfall is the germinating blastospores that
third place of frequency (Brumble et al. 2017; appear to be branching but can be distinguished
Sundaram et al. 2006). Meningitis is the most due to the absence of a constriction between the
common clinical form of CNS involvement due base of the blastospore and the germ tube
to Candida spp., but chronic meningitis, brain (Guarner and Brandt 2011; Schuetz and Walsh
abscess formation, ventriculitis, mycotic aneu- 2015).
rysms, and vasculitis have also been reported When the invasive disease is suspected, blood
(Zimmermann et al. 2016; Merwick et al. 2015; cultures are the most important tool for diagno-
Fennelly et al. 2013). sis; after all, blood culture positivity is around
Candida organisms can form mats of budding 50–70% of tested cases. Additionally, the peptide
yeasts and thin branching pseudohyphae, also nucleic acid fluorescent in situ hybridization
called filaments, that may show periodic con- assay (PNA FISH) can be used to identify the
strictions (Fig. 6.2a). The organisms can be seen most common Candida species in smears made
with H&E (Fig. 6.2b), GMS, and PAS stains, but from positive blood culture bottles (Posch et al.
C. glabrata does not produce filaments. During 2017).
histopathologic examination, it is very important Another diagnostic option for invasive infec-
to identify invasion of tissues and vessels, con- tion is the multiplex-tandem PCR, which can be
sidering that isolation from the skin, lungs, and performed in whole blood, serum, or plasma and
the gastrointestinal or genitourinary tract may be has yielded better and faster results compared
indicative of colonization (Southern et al. 2008; with blood culturing; nevertheless, this method-
Darouiche 2009). ology is still for research use only and needs to be
The usual tissue reaction, both in superfi- validated for diagnostic purposes (Lau et al.
cial and invasive disease, consists of neutro- 2010).
58 L. F. Jurado and R. d. P. López-Panqueva
a b
Fig. 6.3 Mucorales. (a) Illustration, usual appearance of Mucor spp. (b) Illustration, usual appearance of Rhizopus spp.
(c) Illustration, hyphae with some septations (pauciseptate) and a 90° angle branching. (d) Rhizopus spp. from culture
6 Histopathology 59
d e
Fig. 6.3 (continued)
Entomophthorales. The former was originally Mucorales are ubiquitous in the environment
identified as affecting insects and causing muco- and are usually found in soil and decomposing
cutaneous disease in immunocompetent human organic matter (Ribes et al. 2000). Their spores
hosts, while the Mucorales cause a spectrum of are easily airborne, frequently causing contamina-
predominantly angioinvasive disease in immuno- tion in the laboratory; considering this, isolation
suppressed patients (Fig. 6.3f (Courtesy of of these molds must be correlated with the clinical
A. Agudelo M.D.)). Among the Mucorales, history, in order to define their clinical signifi-
Rhizopus is the most frequent genus that causes cance. Inhaled spores can produce respiratory dis-
human disease; conversely, Mucor spp. produce ease in immunosuppressed individuals, and the
disease in less than 20% of cases (Naggie and spores can also invade the skin and subcutaneous
Perfect 2009). tissue by traumatic inoculation, contaminated
60 L. F. Jurado and R. d. P. López-Panqueva
needles, and even insect bites; less commonly, not be appreciated in the specimen. In these
spores can be swallowed and cause gastrointesti- cases, the pathologist must describe the degener-
nal disease (Prabhu and Patel 2004). ate hyphal elements observed in the specimen.
When the immune response is not able to con- This identifies the tissue where the fungus is
trol the initial infection, the spores germinate and found, ruling out the common possibility of cul-
invade the surrounding tissue. Initially, there is an ture contamination.
edematous reaction, and by the time the hyphae In immunosuppressed patients, the hyphal ele-
invade blood vessels, (Fig. 6.3f) the tissue ments are usually found immersed in abundant
becomes necrotic and acquires a characteristic necrosis, hemorrhage, and blood vessel thrombo-
black color. By contrast, immunocompetent indi- sis (Ben-Ami et al. 2009). Another important
viduals develop an intense inflammatory response diagnostic feature is the identification of fungal
and can present with a mass in the skin, respira- elements invading the blood vessel wall or inside
tory sinuses, or the gastrointestinal tract (Roilides the lumen (Fig. 6.3f). Neutrophilic inflammation
et al. 2014). could also be identified surrounding the lesion.
In general terms, mucormycosis presents itself It is important to be careful in differentiating
in three main clinical ways, which are rhinocere- Mucorales from other fungi that produce nonpig-
bral (Fig. 6.3g (Courtesy of A. Rueda M.D.)), mented hyphae in tissue (Aspergillus spp.), other
pulmonary, and cutaneous. Mortality due to dis- hyaline septate molds (Fusarium and
seminated disease is extremely high, and it is Scedosporium), and Candida spp. (Ribes et al.
influenced by the predisposing factors and the 2000). An important morphological pitfall is the
clinical presentation (Roden et al. 2005). Early presence of many septations and acute-angle
identification of the initial infection site is imper- branching, which suggest Aspergillus spp. or
ative to start a proper surgical and antifungal another hyaline septate mold, while the identifi-
treatment. Additionally, for diagnosis, tissue cation of yeasts and pseudohyphae formation
specimens should be both cultured and histopath- should suggest Candida spp. When using GMS,
ologically analyzed. poorly stained hyphae are observed, which
Identification of these molds in tissues is very should suggest mucormycosis. Therefore, spe-
important since it allows distinguishing the cifically identifying Mucorales in tissues or
presence of the fungi as a pathogen from a cul- detecting dual infections by Mucorales genera
ture contaminant and also is indispensable to and other fungi, immunohistochemistry, in situ
define the presence of blood vessel affectation hybridization, or PCR can be very useful
(Fig. 6.3f). Mucorales genera are characterized (Hofman et al. 2010).
by nonpigmented, wide (5–20 μm), thin-walled, Like in most mycotic infections, culture is
ribbon-like hyphae with some septations (pauci- indispensable for organism-specific diagnosis.
septate) and a 90° angle branching (Ribes et al. Furthermore, during sample-processing, is
2000) (Fig. 6.3c, e). The hyphae may vary in important to handle the specimens carefully,
width, appear folded or crinkled, and be sparse or since aggressive grinding of the tissue may ren-
fragmented. In lesions exposed to air and in cul- der the fragile fungal elements nonviable (Ribes
ture media, thick-walled spherical structures can et al. 2000). Mucorales genera are fast-growing
form at the ends of the hyphae (Fig. 6.3a, b, d). fungi, but unfortunately, the yield of cultures is
Routine H&E stains may show only the cell wall low. Also, although serologic tests have been
without structure inside (Fig. 6.3e, f); in cyto- attempted, they are not recommended (Guarner
logic specimens, the hyphae can be identified and Brandt 2011).
using Papanicolaou and calcofluor white stains; The retrospective Indian study performed by
and GMS and PAS can also help highlight the Sundaram et al. (2006) included a total of 130
fungal wall (Naggie and Perfect 2009). CNS fungal infection cases during a 17-year
In some situations, the hyphae may look period; in the study, 30% of the cases (39
degenerated, and many of the characteristics may patients) were mucormycosis, and of these
6 Histopathology 61
patients, 37 developed the rhinocerebral form. alveolar macrophages and change to the yeast
In three patients, the disease was limited to the phase (Fig. 6.4b, c). The organisms are able to
CNS, and their clinical presentation was stroke- survive inside macrophages for weeks, and the
like and diffuse encephalopathy. The most fre- migration of macrophages to the lymph nodes
quent histologic pattern was hemorrhagic facilitates its dissemination. The disseminated
infarction with angioinvasion and neutrophilic form can occur both after initial infection
infiltrates. And the identified fungal elements and as reactivation of latent infection in
were hyaline hyphae, pale and nonseptate with immunocompromised individuals (HIV-AIDS,
irregular or right-angle branching (Fig. 6.3c, e). hematologic cancer patients, solid organ trans-
Cultures were performed in 15 (38%) patients planted individuals, corticosteroids, and tumor
and were positive in 33% (5 cases), and Rhizopus necrosis factor antagonists users) (Hage et al.
oryzae was the most frequently isolated 2010).
organism. Patients with high exposure loads or those
who are immunosuppressed are at high risk of
developing acute pneumonia or ARDS (Wheat
6.5 Disease Caused by et al. 2016; Bueno-Fischer et al. 2009). The
Histoplasma spp. migration of macrophages to mediastinal lymph
nodes can make patients present with mediastini-
Histoplasma capsulatum is a cosmopolitan fun- tis, and as macrophages travel through the body,
gus that can be found in old buildings, caves, and the fungi spread to other organs, such as the skin,
soil rich in bird and bat droppings; nevertheless, gastrointestinal tract, liver, spleen, and bone mar-
there are areas of high concentration where the row; further, although infrequent, central nervous
disease is endemic; these areas include the Ohio system compromise can occur (Wheat et al.
and Mississippi River valleys in the United States, 2016).
some countries in Central and South America, Histoplasma capsulatum var. capsulatum in
southern Europe, areas in Africa, and southeast- tissue is an oval 2–4 μm yeast and may show
ern Asia (Wheat et al. 2016; Colombo et al. 2011). narrow-based buds (Wheat et al. 2016)
In most areas of the world, human histoplasmosis (Fig. 6.4b, c). With H&E staining, the basophilic
is due to H. capsulatum var. capsulatum; never- yeast cytoplasm looks separated from the sur-
theless, in western and central regions of sub- rounding tissue by a clear zone corresponding to
Saharan Africa, the African clade of Histoplasma the cell wall, and using GMS and PAS stains, it is
capsulatum, formerly named H. capsulatum var. possible to highlight the cell wall. Due to the
duboisii, can be found as the etiological agent of initial immune response, the yeasts are
the disease. Frequently, histoplasmosis occurs in phagocytized by histiocytes, so they appear to be
outbreaks related with old buildings renovation/ clustered; therefore, some authors suggest this as
demolition, or when tourists visit caves, but most an important diagnostic clue (Fig. 6.4b, c).
of these cases are sporadic (Loulergue et al. 2007). Further, this yeast aggregation inside macro-
As with most fungi that cause systemic dis- phages and occasionally neutrophils is the usual
ease, the infection spreads by airborne route. In presentation of Histoplasma in fluids stained
other words, the human being gets infected of with Papanicolaou or blood smears stained with
histoplasmosis by inhaling the microconidia, and Giemsa (Gupta et al. 2009).
the immediate clinical response depends on the There is scarce information regarding the his-
amount of fungus inhaled and the immune tologic presentation of acute pulmonary histo-
response; individuals may show no symptoms, plasmosis; some authors have described nodular
may have acute or chronic pulmonary disease, or areas of parenchymal and vascular necrosis asso-
may have a disseminated presentation (Bueno- ciated with lymphohistiocytic vasculitis
Fischer et al. 2009; Kauffman 2009). After the (Mukhopadhyay and Katzenstein 2010). This
conidia are inhaled, they are phagocytized by histopathologic pattern simulates the lymphoma-
62 L. F. Jurado and R. d. P. López-Panqueva
a b
Fig. 6.4 Histoplasma spp. (a) Illustration, saprophytic hyphae over necrotic tissue, H&E, 40×. (e) Fungal ele-
form. (b) Illustration, parasitic form, yeast inside macro- ments invading a blood vessel, H&E, 40×. (f) Macroscopic
phages. (c) Multiple yeast infecting cells, Gomory, 40×. appearance of encephalic Mucormicosis, frontal lobes
All illustrations are original by Jurado LF. (d) affected by hemorrhagic necrosis causing cavitary lesions.
Nonpigmented, thin-walled, ribbon-like pauciseptate All illustrations are original by Jurado LF
toid granulomatosis but scattered small granulo- distorts the organ architecture and produces
mas and the presence of yeasts must suggest the necrotic areas. Considering that the morphology
diagnosis of histoplasmosis. of H. capsulatum is not specific, it is important to
The chronic pulmonary cases that radiograph- always perform clinical-epidemiologic correla-
ically appear as coin lesions show a granuloma- tion (Kauffman 2008).
tous inflammation with the necrotic and calcified Many fungi can be morphologically confused
material, and it is common to find yeasts within with H. capsulatum var. capsulatum when
the necrotic-calcified material (Wheat et al. observed in tissue sections (Bueno-Fischer et al.
2016). When immunosuppressed patients 2009); for example, in the case of capsule-
develop disseminated disease, it is usual to deficient cryptococci, size variation and weakly
observe sheets of macrophages filled with yeasts positive mucicarmine-stained yeasts may help to
(Fig. 6.4b, c). The abundance of macrophages differentiate cryptococcosis from histoplasmosis.
6 Histopathology 63
Another case could be the small variant of B. der- being reported as negative (Wheat et al. 2016;
matitidis, where it is useful to look for the pres- Kauffman 2008). On the other hand, there are
ence of broad-based budding and larger forms, immune-based methods, where testing for anti-
which can help in making the diagnosis of B. der- bodies can be performed using complement
matitidis infection. The endospores of fixation or immunodiffusion; however, in immu-
Coccidioides spp. can also be a challenging case, nodeficient patients, production of antibodies
where looking for rest of a ruptured spherule or may not even occur (Kauffman 2009). In addi-
an intact spherule is paramount for its differentia- tion, false-positive serology results can occur in
tion. Another important example involves individuals with lymphoma, tuberculosis, and
Pneumocystis jirovecii, as when distinguishing other fungal infections, especially in cases of
this organism, it is important to know that it lacks blastomycosis (Wheat et al. 2016).
budding and has an intracystic focus. For the Through enzyme immunoassay, it is possible
cases of Candida glabrata, this organism may to detect certain antigens in urine and serum;
show more size variability than histoplasmosis, after all, the antigen is concentrated in the urine,
and the inflammation is mostly neutrophilic. making Histoplasma antigen detection more reli-
Finally, in Penicillium marneffei infection, it is able (Wheat et al. 2016). In the same way to anti-
important to keep in mind that this organism body testing, there are false-positive results with
forms a transverse septum rather than a budding antigen testing; particularly, the cross-reactivity
pattern. with blastomycosis is problematic because histo-
In addition to the mentioned fungi, some par- plasmosis and blastomycosis have overlapping
asites can also appear morphologically similar to endemicity and histopathologically can have a
Histoplasma spp., agents of leishmaniasis, toxo- striking resemblance. Furthermore, in patients
plasmosis, and Chagas’ disease, which also with localized disease (nondisseminated), the
show intracellular organisms (Gupta et al. 2009). antigen burden is lower, and thus sensitivity is
One of the most relevant histopathologic differ- lower. With those limitations, combining the
ences between these protozoans and Histoplasma results of detection of antigen in urine and serum
is that H&E stains the entire organism, and none may increase the sensitivity in patients with
of them show the halo produced by the fungal localized histoplasmosis (Swartzentruber et al.
cell wall. Kinetoplasts (a distinct hematoxylin- 2009).
stained bar located to the side of the nucleus that
represents a mass of mitochondrial DNA) should
be observed in the cases of Leishmania or 6.6 isease Caused by
D
Trypanosoma infections. Finally, it is important Coccidioides spp.
to remember when toxoplasmosis and Chagas’
disease are suspected, that the infected cells are Coccidioidomycosis is a disease of the Western
somatic (cardiomyocytes or neurons) rather than hemisphere caused by a dimorphic soil-dwelling
macrophages. In summary, to perform a fungus of the genus Coccidioides. It was first rec-
definitive diagnosis of histoplasmosis from tis- ognized as a clinical condition in Argentina in
sue sections is challenging, and if cultures were 1882, and, soon after, another case in the San
not requested, alternative testing must be Joaquin Valley in California (USA) was reported
demanded. (Rixford and Gilchrist 1896).
The culture of blood samples can help in There are two primary species that cause the
diagnosing disseminated disease, but since
disease. Coccidioides immitis is endemic in some
Histoplasma spp. are an intracellular organism, parts of the United States, particularly in the
lysis-centrifugation methods must be used to lib- California desertic areas (Adam et al. 2009);
erate the yeasts from infected cells. Additionally, additionally, Coccidioides posadasii is present in
this fungus has a long generation period, so cul- desertic regions of the United States (Arizona,
tures must be incubated for 4–6 weeks before Utah, New Mexico, and West Texas), northwest
64 L. F. Jurado and R. d. P. López-Panqueva
Mexico, and desertic zones in Argentina, When susceptible individuals inhale the
Paraguay, and areas of Central America (Colombo arthroconidia (Fig. 6.5a), this fungus reaches the
et al. 2011; Ampel 2009). Nevertheless, differ- alveoli and transforms into multinucleated spher-
ences in morphology or clinical presentation ical structures that contain hundreds of endo-
have been found between the entities produced spores (Fig. 6.5b, c (Courtesy of D. Palacios
by each species. An interesting correlation M.D.)) (Parish and Blair 2008). It is estimated
between the incidence of the disease and specific that approximately 60% of infected people have
environmental factors is commonly reported; for no symptoms, while the remainder may present a
example, coccidioidomycosis incidence increases clinical condition that simulates an acute
when there are rainy summers followed by dry community-acquired pneumonia, also known as
winters, after earthquakes, or when humans “valley fever” (Adam et al. 2009; Ampel 2009).
establish themselves on the previously recog- The chest X-rays may show lobar opacities and
nized endemic areas. Thus, when any of these hilar adenopathy, and in some patients, cutaneous
situations take place, Coccidioides arthrospores manifestations (erythematous rashes) are devel-
are released in higher concentrations compared oped as reflections of the immune response to the
with the usual baseline (Parish and Blair 2008). acute infection (Gavito-Higuera et al. 2016).
a b
Fig. 6.5 Coccidioides spp. (a) Illustration, saprophytic with multiple endospores inside. H&E, 40×. All illustra-
form, showing arthroconidia development. (b) Illustration, tions are original by Jurado LF
spherules with multiple endospores inside. (c) Spherules
6 Histopathology 65
Most of acute infections resolve with no com- tant hallmark of coccidioidomycosis (Li et al.
plications; however, in a minority of patients, the 2005). Eosinophil infiltrates can also be abundant,
infection may progress to a chronic condition, which produces eosinophilic major basic protein,
developing either a cavity or a nodule (Wheat creating the Splendore-Höeppli phenomenon (an
et al. 2016). It is recognized that specific risk intense cover of eosinophilic material around the
groups (African Americans, Asians, pregnant fungal elements) (Read et al. 2005).
women, patients with diabetes, patients receiving The microorganism to consider for differential
corticosteroids) are more likely to develop dis- diagnosis, is Rhinosporidium seeberi, a parasite
seminated disease (Adam et al. 2009). The most that causes polyps in the upper respiratory tract,
common sites of extrapulmonary involvement are produces big sporangia (some can be seen with
the skin, lymph nodes, bones, and joints; never- the naked eye) with multiple internal endospores.
theless, the most feared is the extension to the This parasite has very similar morphology to
CNS (Kauffman 2008). In those cases, the most Coccidioides; however, its sporangia and endo-
frequent presentation is meningitis. Therefore, the spores are bigger than the fungal spherules, and
usual imaging findings are meningeal enhance- its internal sporangial wall stains with mucicar-
ment and hydrocephalus, but focal brain lesions, mine (Malo et al. 2014).
infarcts, or areas of cerebritis or cerebellitis can Considering that one important characteristic of
also be observed (Lammering et al. 2013). Coccidioides is the presence of spherules, it is
The most characteristic morphological feature important to remember that endospores outside
of Coccidioides is the presence of spherules of spherules or immature spherules without endo-
diverse sizes (10–100 μm) with multiple endo- spores can be confused with yeasts such as
spores (2–5 μm); those can be identified with Blastomyces, Histoplasma, Candida, or
H&E staining (Fig. 6.5c) (Saubolle 2007). Pneumocystis (Saubolle 2007). It also needs to be
Sometimes, the walls of the spherules are rup- remembered that in immunosuppressed patients,
tured, and the endospores can appear spilled over more than one organism may coexist; thus, in areas
the surrounding tissue. It is usual that active of endemicity, Pneumocystis and Coccidioides
lesions contain multiple organisms, while lesions could be found in the same specimen.
in resolution show a lower number of fungal Detection of antibodies can be a helpful diag-
structures (Guarner and Brandt 2011). nostic tool. Nowadays, IgM and IgG are gener-
Using GMS, it is possible to highlight spher- ally measured using EIA or immunodiffusion;
ule and endospore walls. In contrast, PAS stain however, it is also possible to use tube precipita-
affinity varies with age of the structures; there- tion to measure IgM and complement fixation for
fore, immature endospores and spherules stain IgG antibodies. False-negative results have been
strongly, while mature structures appear less reported in up to 38% of patients with hematog-
stained (Saubolle 2007). Occasionally, in the enous infection and 46% of fatal cases (Adam
cavitary lung or cutaneous lesions, mycelia can et al. 2009). Detection of antigens in the urine
be observed (Saubolle 2007). The sensitivity of using EIA has shown positive results in 71% of
histopathology for Coccidioides identification is patients with coccidioidomycosis but has a cross-
84% and 75% for cytology (Adam et al. 2009). reaction in 10% of patients with other endemic
The predominant inflammatory response to mycoses (Durkin et al. 2008).
endospores is neutrophilic, whereas the reaction
to spherules is mostly granulomatous. Thus, early
in the infection process, the histologic pattern 6.7 isease Caused by
D
tends to look mixed (pyogranulomatous) because Cryptococcus spp.
the concentration of both fungal structures is high.
In addition, lymphocytic clusters of B and T cells Human cryptococcosis is a systemic mycosis
next to well-constituted granulomas with necrosis caused by some species of the Cryptococcus
have been described and appear to be an impor- genus. Up to 40 species have been described, but
66 L. F. Jurado and R. d. P. López-Panqueva
few are recognized as a human pathogen (May and brain than C. neoformans. The occurrence of
et al. 2016). The most relevant are C. neoformans the disseminated disease depends on the host
and C. gattii, but two other species, C. albidus immune status; in immunocompetent patients the
and C. laurentii, have been reported in rare cases, most frequent presentation is pulmonary, while
producing disease in humans (Johnson et al. immunosuppressed patients commonly develop
1998; Kordossis et al. 1998). CNS disease (Williamson et al. 2017).
These organisms are found in soil and are Cryptococcus spp. is a spherical to oval
related with pigeon droppings. Infection involves encapsulated yeast, measuring approximately
most frequently the lungs and the CNS and less 5–10 μm in diameter in clinical specimens with
frequently can compromise the skin and the skel- a narrow-based budding and a capsule ranging
etal system. Because its incidence is high in in size from 1 to >30 μm (Fig. 6.6a, d). In speci-
immunocompromised patients, especially indi- mens isolated from the outside environment,
viduals with AIDS and organ transplant recipi- yeast tends to be smaller and with a thinner cap-
ents, cryptococcosis is considered an sule (Neilson et al. 1997). The thick, polysac-
opportunistic disease (May et al. 2016). charide capsule gives the yeasts a characteristic
Cryptococcal meningoencephalitis is a fre- appearance of having a clear space around them
quent and life-threatening complication in (Fig. 6.6b, c (Courtesy of M. Tuñon M.D.), d).
patients with HIV infection (Williamson et al. When analyzing CSF, India ink is the ideal
2017) and is the most common fungal infection negative stain to highlight the capsule (Fig. 6.6d).
of the CNS worldwide (223,100 estimated cases Due to the capsule, the buds appear separate
in 2014) (Rajasingham et al. 2017) causing a sub- from the mother cell. The capsule can be stained
stantial disease burden in countries with poor with Alcian blue and Mayer’s or Southgate’s
access to medical care and high numbers of peo- mucicarmine. As it happens with all other yeasts,
ple living with HIV. the wall stains with GMS and PAS; moreover,
C. neoformans is the causative agent for the for cryptococci, Fontana-Masson also results in
majority of infections in immunocompromised a stain due to the presence of melanin
individuals, while C. gattii causes disease in (Williamson et al. 2017).
immunocompetent hosts (Huston and Mody The histopathological reaction varies from
2009). Additionally, C. neoformans var. grubii well-formed granulomas, where the yeasts are
(serotype A) and C. neoformans var. neoformans found inside histiocytes and giant cells, to mini-
(serotype D) have a worldwide distribution and mal inflammatory response with abundant extra-
are primarily associated with pigeon guano. HIV cellular organisms that distorts the tissue
infection is the most important risk factor for architecture (Gazzoni et al. 2009). When the
cryptococcal disease; however, other conditions granulomatous pattern is present, the granulomas
associated with cryptococcosis include chronic show a spectrum from abundant necrosis to fibro-
lung, liver, and renal disease, autoimmune dis- sis (Fig. 6.6b, c). In some cases, the fibrosis is
eases, immunosuppressant use, and cancer (Gala- intense, with abundant stocky-spindle fibroblast
nis and MacDougall 2010). cells in a storiform pattern accompanied by a
Irrespective of the species, inhaling cryptococ- background of lymphocytes and plasma cells,
cal yeasts or basidiospores infects humans; there- giving the appearance of an inflammatory pseu-
fore, the lung is always the first infected organ dotumor (Sing and Ramdial 2007). Some authors
(Huston and Mody 2009). In contrast to other have suggested that the histological reaction is
pathogenic fungi, few exposed individuals remain related to the immune status of the patient and the
asymptomatic; most develop pneumonia, crypto- presence or absence of capsule (Rajasingham
coccomas, or pleural effusion. From the lungs, et al. 2017).
cryptococci can disseminate to the CNS, resulting In some cases, the yeast produces a thinner
in meningitis or cryptococcomas. Further, C. gattii polysaccharide capsule; thus, these organisms
more frequently causes solid lesions in the lungs may look similar to other yeasts, such as Candida
6 Histopathology 67
a b
c
d
Fig. 6.6 Cryptococcus spp. (a) Illustration, narrow-based thick polysaccharide capsule gives the yeasts a character-
budding yeast. (b, c) Granulomatous reaction, multiple istic appearance of having a clear space around them.
yeasts inside multinucleated giant cells, abundant lym- India ink, 40×. All illustrations are original by Jurado LF
phocytic infiltrate, H&E, 40×. (d) Yeast in CSF smear. Its
spp. or Histoplasma. In this situation, staining in serum and CSF may not be useful in patients
these specimens with Fontana-Masson may show with poorly encapsulated cryptococci because
evidence of melanin, which is a hallmark of cryp- most of the serologic tests detect capsular anti-
tococci. The use of cryptococcal antigen analysis gens (Gazzoni et al. 2009).
68 L. F. Jurado and R. d. P. López-Panqueva
Cryptococcal antigen testing using latex Great Lakes and the Saint Lawrence River, which
agglutination or EIA can be performed in serum include multiple states in the United States
and CSF, showing a sensitivity and specificity of (southeastern, south central, and upper
above 90%; nevertheless, false-negative results Midwestern states); it has also been found in sev-
can occur due to a low fungal burden or a prozone eral Canadian provinces (McKinnell and Pappas
phenomenon. Alternatively, false-positive results 2009). Outbreaks of blastomycosis after a source
have been reported in infected individuals with exposure have been reported, but most cases
Trichosporon spp. or Klebsiella pneumoniae, in occur sporadically in the endemic areas.
those with a positive rheumatoid factor, or when Occasionally, cases are reported in areas where
the reagent was incubated with the specimen the disease is not endemic, such as Colorado,
beyond the recommended time (Williamson et al. Texas, Kansas, and Nebraska in the United States,
2017). Cultures, using canavanine-glycine- and from other countries around the world
bromothymol blue medium, that turn blue in the (Shukla et al. 2009).
presence of C. gattii are helpful to identify the Patients infected with B. dermatitidis can
infective species and are indispensable for mea- develop pneumonia, extrapulmonary disease, or
suring antifungal susceptibility when indicated. both. Lung involvement often imitates an acute
bacterial pneumonia, lung cancer, or tuberculo-
sis. Cutaneous lesions, which can be present as
6.8 Disease Caused by verrucous or ulcerative lesions, are the most com-
Blastomyces dermatitidis mon extrapulmonary manifestation, followed by
bone, prostate, and CNS disease.
Gilchrist described blastomycosis in Baltimore In the yeast phase, the organism appears as
(USA) during the 1890s as a skin infection spherical, budding, thick-walled yeast with a
caused by what he thought was a protozoan daughter cell forming a single bud with a broad
organism (Gilchrist 1894); thus, the disease was base. Further, size varies from 5 to 15 μm, and
named as Gilchrist’s disease. Initially, because most are spherical to oval and have a double cell
skin manifestations of blastomycosis were very wall appearance, which consists of the interior
striking, it was considered a dermatologic and exterior components of its thick cell surface
condition (Gilchrist and Stokes 1898). The con- (Fig. 6.7c). The yeast may be found inside or out-
cept of airborne transmission was not recognized side of macrophages in the pyogranulomatous
until pathologic descriptions allowed the patho- tissue response (McKinnell and Pappas 2009).
physiologic mechanisms to be elucidated (Sarosi After inhalation of the conidia, a variety of
and Davies 1979). There are infrequent cases of responses can occur in the lungs, from asymp-
direct cutaneous inoculation in laboratory work- tomatic disease to acute and chronic pneumo-
ers and veterinarians, but virtually all cases of nia, and fatal acute respiratory distress
blastomycosis are considered to originate primar- syndrome (McKinnell and Pappas 2009; Patel
ily from lung disease (Sarosi and Davies 1979). et al. 2010). In up to 40% of cases, the disease
The etiological agent is the dimorphic fungus has become systemic at the time of diagnosis,
Blastomyces dermatitidis. The organism exists in affecting the skin, soft tissue, bone, genitouri-
nature in the mold or mycelial phase and converts nary tract, or the CNS (McKinnell and Pappas
to the yeast phase in tissues at body temperature 2009). Blastomycosis in immunocompromised
(Fig. 6.7a, b). The mold is the infective form, pro- hosts appears to be more severe and frequently
ducing conidia that can be aerosolized and subse- fatal. In patients with CNS involvement, diabe-
quently inhaled. In culture, B. dermatitidis grows tes mellitus is an important risk factor (Bariola
from 25 to 28 °C as a mold and at 37 °C as a yeast et al. 2010).
(Nemecek et al. 2006). B. dermatitidis in tissue appears as yeasts that
This fungus has been isolated from soil in the measure 8–15 μm in diameter, have a thick
Mississippi and Ohio River valleys, around the refractile cell wall, and usually show a single,
6 Histopathology 69
a b
Fig. 6.7 Blastomyces dermatitidis. (a) Yeast form, with pop” morphology. (c) Thick-walled yeast with broad-
broad-based budding. (b) Mycelial or saprophytic form, based budding, showing its “footprint” morphology. All
forming single conidium showing its characteristic “lolli- illustrations are original by Jurado LF
broad-based bud (Fig. 6.7a, c). The yeasts can be degrees of neutrophilic infiltrate; therefore, as in
identified in many samples, such as sputum, the cases of coccidioidomycosis, it has been
bronchoalveolar lavage, fine-needle aspirates, described as pyogranulomatous inflammation
CSF, and biopsy specimens. When the H&E stain (Patel et al. 2010).
is used, the thick refractile cell wall gives the Few studies have compared the identification
appearance of a space between the fungal cell of broad-based budding yeasts in histopathologic
contents and the surrounding tissue. The multiple or cytologic specimens with culture or other
nuclei of the yeast stain with hematoxylin. diagnostic methods that confirm the diagnosis of
Sometimes, this fungus can show small yeast blastomycosis. Patel et al. (2010), in a
forms, called microforms. Additionally, B. der- retrospective study of 53 cases, reported that
matitidis can be identified with routinely used Coccidioides immitis, Candida albicans, and
preparations such as Papanicolaou and KOH. The Aspergillus were recovered from 10% of the
contour of the yeast is best appreciated with sil- pathologic specimens, demonstrating broad-
ver stains such as PAS or GMS (Patel et al. 2010; based budding yeasts in the direct histopatho-
Lemos et al. 2000). logic examination. A previous study by Lemos
The histological reaction produced by the et al. (2000) of patients with blastomycosis
yeasts is mainly granulomatous with diverse showed that in a high percentage of the cultures,
70 L. F. Jurado and R. d. P. López-Panqueva
Candida was isolated. These results suggest that and accurate diagnosis is always mandatory. The
not all broad-based budding yeasts in the 8–15 μm broad spectrum in clinical and pathogenic pre-
diameter range are Blastomyces. sentation makes its diagnosis a big challenge.
Considering that histopathologic and cyto- When a tissue or liquid specimen is available,
logic studies can provide results before the cul- the pathological analysis is a very useful tool for
ture, there is pressure to use these findings to rapid and accurate diagnosis. Usually, pathologi-
guide treatment, particularly because B. derma- cal studies can provide results before the culture
titidis can take up to 3 weeks to grow or may not or other analyses are available. Nevertheless, it
grow. The sensitivity of culture varies depending is important to remember the limitations of this
on the specimen that was obtained and may range approach because many of the fungal structures
from 62% to 100% (Lemos et al. 2000; among different species are very similar.
Martynowicz and Prakash 2002). The diagnostic Therefore, it could be really easy to misdiagnose
yield of histopathology will depend on the exper- a specific fungal infection, which would have a
tise of the pathologist (McKinnell and Pappas relevant impact on therapy. Thus, it is always
2009). Because of the possibility of false-positive important to consider the clinical information, as
results, pathologists must describe the yeast and well as complementary analysis, in order to
budding pattern observed in the tissue and also obtain a definitive ethological diagnosis.
should add a commentary in the report about the
yeasts that can have similar morphology. In addi-
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Molecular Genetics and Genomics
of Fungal Infections 7
Shivaprakash M. Rudramurthy
and Arunaloke Chakrabarti
demia may lead to CNS infection. CNS fungal reported for early diagnosis and monitoring ther-
infections have also been reported after intrave- apy by those tests, none of those tests, except
nous drug abuse. Other than haematogenous Cryptococcus antigen, has received United States
route, direct inoculation of CNS may occur by Food and Drug Adminstration (US FDA)
any fungus as a result of surgery, trauma and approval (Kami et al. 1999a; Buitrago et al. 2011;
contaminated medical supplies. Direct extension Binnicker et al. 2011; Vucicevic et al. 2010;
from adjacent structure like paranasal sinuses, Klingspor and Jalal 2006; Elsayed et al. 2001;
mastoid and orbit to CNS is also possible by fun- Ralph and Hussain 1996). Researchers have also
gus (McCarthy and Walsh 2017). attempted the molecular genomic technology in
CNS fungal infection may manifest as menin- diagnosis of fungal diseases, but the studies are
gitis, meningoencephalitis, hydrocephalus, few and far between. In the present chapter, we
space-occupying lesion, acute cerebrovascular have reviewed the existing knowledge on molec-
events and spinal infections (Preuner and Lion ular genomic tests for diagnosis and monitoring
2009). The outcome of CNS fungal infections of CNS fungal diseases.
depends on early diagnosis and prompt manage-
ment. But, the early diagnosis faces stiff chal-
lenges due to general considerations of rarity of 7.2 DNA-Based Molecular
CNS fungal infection, non-specific clinical Diagnosis of CNS Fungal
symptoms and signs, difficulty in collection of Infections
samples for brain and absence of any standard-
ized molecular tests. However, CNS fungal The currently available techniques such as con-
infection has drawn a lot of international atten- ventional and biomarker tests have many limita-
tion after Exserohilum rostratum meningitis out- tions and often fail to provide a rational basis for
break in the United States (Gade et al. 2013), institution of appropriate antifungal therapy.
though similar post-tsunami Aspergillus menin- There is a need for the use of molecular tech-
gitis outbreak in Sri Lanka due to contaminated niques, which are rapid and sensitive and may
medical supplies occurred much earlier detect the causative agent accurately that would
(Gunaratne et al. 2007). help appropriate and prompt antifungal therapy.
Imaging including computerized tomography Molecular techniques are applied either for
(CT) and magnetic resonance imaging (MRI) detection of nucleic acid in clinical samples or
helps in suspecting CNS fungal infection. Any for the accurate identification of fungi isolated
patient at risk of CNS fungal infection or docu- from the clinical specimen. Detection of nucleic
mented fungal infection of lung or sinuses should acid in clinical sample faces the challenge of
be screened with neuroimaging. To confirm the extraction of nucleic acid, inadequate amount
diagnosis, invasive samples from CNS are nucleic acid in sample and contamination during
required. Cerebrospinal fluid (CSF) sample helps test procedure. However, DNA-based molecular
in the diagnosis of meningitis or meningoenceph- assays for the identification of majority of the
alitis by direct demonstration of fungus on fungi isolated from clinical specimen and for out-
microscopy and/or culture, whereas biopsy sam- break investigation are well established.
ple is required for space-occupying lesion. The
collection of biopsy sample may be a challenge
in majority of the cases. In this difficult scenario, 7.2.1 Molecular Test Formats
non-culture-based diagnostic tests of CSF may
help in early detection of CNS mycosis. Among Polymerase chain reaction (PCR) is the com-
the non-culture-based diagnosis, molecular bio- monly used test format for the molecular diagno-
markers and antigen detection in CSF have been sis of fungal infections. Either standard PCR or
developed as pan-fungal marker or pathogen- different modifications of the PCR including
specific tests. Though promising results are nested PCR, real-time PCR, multiplex PCR and
7 Molecular Genetics and Genomics of Fungal Infections 77
PCR-ELISA (enzyme-linked immunosorbent cies level (Atkins and Clark 2004), and cross-
assay) are used to amplify the fungal nucleic acid reactivity with phylogenetically related fungi
from the specimen and simultaneous detection. may be a problem for identification (Bialek et al.
The amplified products are identified by either 2002). The diagnostic performance of ITS pan-
DNA sequencing or use of DNA microarray fungal PCR has shown high concordance (>80%)
chips or analysis by restricted fragment length with routine conventional methods including
polymorphism or use of specific hybridization microscopy and isolation. The sensitivity and
probes captured to microbeads (Luminex tech- specificity were 57% and 97% even in cases with
nology) (Preuner and Lion 2009; Landlinger fungal infections having negative microscopy
et al. 2009). Of all the PCR amplification formats (Lass-Flörl et al. 2013). The other less frequently
available, real-time platforms are generally pre- used targets include mitochondrial gene, beta
ferred due to (a) less chance of contamination in tubulin gene, calmodulin gene, etc.
a closed system and without the requirement of
post-PCR processing; (b) simultaneous quantifi-
cation of the fungal load in the clinical sample; 7.2.3 DNA Extraction
(c) simultaneous detection of the species either
by use of specific probes or by melt curve analy- Extraction of the DNA from clinical sample may
sis of amplified product; and (d) possible multi- not be easy as the number of fungal cells present
plex assay to detect multiple pathogen in single in the CSF is low in patients with space-
reaction (Khot and Fredricks 2009). occupying lesions. Hence, the extraction proce-
dure should be of high quality without much
damage to the DNA. Bialek et al. (2002) demon-
7.2.2 Targets for PCR Amplifications strated that a simple freeze and boil procedure
may be sufficient to free the Cryptococcal DNA
The PCR-based assays allow the detection of from the brain tissue homogenates. Similarly,
either the broad range of fungus or specific spe- the DNA extracted from frozen neural tissue of
cies or genus. For the detection of presence of patients with amyotrophic lateral sclerosis
unknown fungi in the clinical specimen, pan- revealed multiple fungal genera including
fungal primers that enable amplification of broad Candida, Malassezia, Fusarium, Botrytis,
range of fungus are used. Many pan-fungal Trichoderma and Cryptococcus, which were
primers have been designed to amplify different responsible for the severity of the disease
regions of ribosomal genes such as internal (Alonso et al. 2017). Different commercial kits
transcribed spacer (ITS) regions 1 and 2, 18s are also being used for efficient DNA extraction
region and 28s/26s region (Fig. 7.1).The multi- from clinical specimen (DNeasy Blood & Tissue
copy of ribosomal genes also improves sensitiv- Kit, TaKaRa MiniBEST Universal Genomic
ity of detection. The ITS region is specifically DNA Extraction). The DNA from pure culture
used, as this region has both the high conserved can be extracted using the physical methods such
and variable areas. White et al. (1990) first time as boiling, grinding in liquid nitrogen, bead beat-
described the universal primers for the amplifica- ing and use of cell-degrading enzymes or deter-
tion of the ITS region, which is now widely used. gents such as sodium dodecyl sulphate followed
Occasionally, this region may not be sufficient to by purification by phenol/chloroform isoamyl
discriminate the fungi to the species and subspe- alcohol precipitation.
Fig. 7.1 Schematic diagram of ribosomal gene used as targets for molecular diagnosis of fungal infection or identifica-
tion of fungi
78 S. M. Rudramurthy and A. Chakrabarti
syndromic-based for the diagnosis of select men- microbiologic diagnosis, mere isolation from
ingitis and encephalitis pathogens including non-sterile clinical specimens may be suspicious
Cryptococcus (Liesman et al. 2018). In addition due to ubiquity of this pathogen (Simoneau et al.
to the common bacterial and viral pathogens, it 2005). Biomarker testing including galactoman-
also has the target for C. neoformans/C. gattii. nan and glucans has good specificity but variable
Liesman et al. (2018) evaluated FilmArray ME sensitivity (Barnes 2008; Leeflang et al. 2008).
panel on large number CSF samples (n = 291) The test results do not identify the species of
and demonstrated an overall 52% (26/50) agree- Aspergillus implicated with the disease, which
ment for Cryptococcus neoformans/Cryptococcus may be important due to variability of antifungal
gattii detection when compared with susceptibility among the species. Thus, molecu-
Cryptococcus antigen by LFA. However, when lar methods are introduced, keeping in mind
compared directly to the results of routine fungal those inherent shortcomings in diagnosis. The
smear or culture, overall agreement improved to rapidity of results, high sensitivity and ability to
92.3% (12/13). In another study performed in discriminate the species by molecular tests are
human immunodeficiency virus (HIV)-infected useful for diagnosis and initiation of targeted
patients, the multiplex panel could detect antifungal therapy. However, only few studies
Cryptococcus culture-positive cases with 100% compared PCR diagnosis to culture and serol-
sensitivity and specificity and differentiate ogy. Valero et al. (2016) developed a real-time
between relapse and paradoxical immune recon- PCR assay using fluorescent-labelled sequence-
stitution inflammatory syndrome (Rhein et al. specific probes. They validated their assays in 60
2016). However, culture-positive cases may be clinical samples, including five CSF samples,
missed occasionally by this panel (Chew et al. and recorded 100% sensitivity. Overall sensitiv-
2018). Therefore, this test should be used as an ity was 83.3% and sequencing could be avoided
adjunct to other tests, such as culture, in 67.7% of the samples. The test could identify
Cryptococcus antigen detection test and/or the pathogen within 24 h of sample collection
pathogen-specific PCR assay (Hanson 2016; (Valero et al. 2016). Nested PCR testing was
Demogines et al. 2015). used in 113 CSF samples from 55 immuno-
compromised patients with proven/probable,
possible or no CNS aspergillosis. CNS aspergil-
7.4 olecular Diagnosis of CNS
M losis could be diagnosed with 100% sensitivity
Aspergillosis and 93% specificity in all the proven/probable
cases. PCR was positive in 4 out of 22 possible
CNS aspergillosis can present as meningitis, cases and 2 negative CNS aspergillosis cases.
encephalitis, cerebral abscesses or strokes. Hummel et al. (2006) used PCR-based diagnosis
Cerebral abscesses are common presentation. on the 35 CSF samples from 6 patients with
The disease may occur by direct extension from proven/probable and possible CNS aspergillosis;
sinuses and ear or haematological dissemination. each patient had at least 1 positive sample by
The major risk factors for CNS aspergillosis are PCR. One of the earliest studies using molecular
prolonged neutropenia, corticosteroid therapy, techniques for CNS aspergillosis diagnosis was
haematopoietic and solid-organ transplant recip- from Japan. PCR was performed on CSF of five
ients and haematological malignancies on che- patients with pulmonary aspergillosis and focal
motherapy (Shamim et al. 2010). CNS neurological signs. PCR was positive in all five
aspergillosis is difficult to diagnose as the symp- patients, but CSF cultures were negative in those
toms overlap with infections due to other disease patients, whereas serology was positive in four
aetiologies, leading to high mortality. Early patients (80%) (Kami et al. 1999a). Other stud-
definitive diagnosis is crucial to administer ies carried out with one or two patients have
appropriate antifungal therapy. Though culture- been included in Table 7.1 (Verweij et al. 1999;
based assays are considered the mainstay of Kami et al. 1999b; Moling et al. 2002; Komatsu
Table 7.1 Review of genomic-based diagnosis of central nervous system fungal infections
80
Positive culture/
positive microscopy/
Reference, No of samples/patients histology/staining/ Sensitivity/
Major fungal classes country (controls) Specimen smear or serology Positive PCR specificity Comments
Dimorphic fungi
Histoplasmosis Buitrago 1 CSF NA 1/1 – Possible case of
et al. (2011), histoplasmosis, real-time-
Spain PCR was used
Coccidioidomycosis Binnicker 2 CSF Culture and serology 2/2 – Real-time PCR
et al. (2011), negative
USA
Vucicevic 5 CSF 1 CSF culture 0/5 – 1 case of proven
et al. (2010), positive for coccidioidomycosis, 1
USA Coccidioides probable, 3 unconfirmed
Mitchell 82 pleural and spinal fluids CSF – – – Real-time PCR using BD
(2015), USA Max
Cryptococcosis Saha (2009), 46 diseased, 30 controls CSF Culture positive 46/46 100%/100% Urine and serum were also
India 44/46, India ink tested by PCR, with 100%
43/46, LAT 46/46, sensitivity and specificity
EIA 46/46
Paschoal 56 samples from acute CSF Culture positive 52/56 92.9%/100% Proven and probable cases
et al. (2004), cryptococcal meningitis, 16 43/56, India ink of cryptococcal meningitis
Brazil samples from bacterial/ positive 48/56
viral meningitis
Iyer (2002), 17 CSF Culture positive 13/17 – 18S rRNA was targeted
India 13/17
Rappelli et al. 21 samples from acute CSF Culture and India ink 21/21 – Nested PCR
(1998), Italy cryptococcal meningitis, 19 positive 21/21
samples from bacterial/
viral meningitis
Candidiasis Klingspor 24 CSF 1/25 4/24 – 18S rRNA was targeted
and Jalal
(2006),
Sweden
Elsayed et al. 4 CSF Culture positive 0/4, 2/4 – Ribosomal DNA used as
(2001), serology positive 2/4 target
Canada
S. M. Rudramurthy and A. Chakrabarti
Ralph and 7 samples from 1 patient CSF Culture positive 1/7 4/6, 1 sample not – –
Hussain tested
(1996),
Canada
Non-Aspergillus moulds
Mucormycosis Bialek et al. 2 brain biopsy samples Brain Histopathology 2/2 – Nested PCR of 18S rRNA
(2005), biopsy positive 2/2
Germany tissue
Exserohilum Gade et al. 359 all case samples CSF Cultures positive for All case sample: 23%/38% 2 samples were not tested.
rostratum (2015), USA 729 all case specimens E. rostratum (2), 2 83 (23%) by All the culture-positive
(461 case patients and 171 for E. nigrum, 1 for conventional PCR cases were also detected by
PCR positive for E. C. cladosporioides and 138 (38%) by both conventional and
rostratum) real-time PCR real-time PCR
All case
specimens: 91
(13%) by
conventional PCR
and 174 (24%) by
real-time PCR
Aspergillosis Valero et al. 5 CSF samples (60 clinical CSF Microbiologic 5/5 100% Pan-fungal qPCR against
7 Molecular Genetics and Genomics of Fungal Infections
Positive culture/
positive microscopy/
Reference, No of samples/patients histology/staining/ Sensitivity/
Major fungal classes country (controls) Specimen smear or serology Positive PCR specificity Comments
Hummel 35 samples from 6 CSF Microbiologic Multiple samples NA Nested PCR 18S rRNA
et al. (2006), suspected patients of CNS criteria positive in 4 tested per patient;
Germany aspergillosis cases (proven/ each patient had
probable CNS IA) one positive CSF
2 cases of possible sample
IA
Bialek et al. 2 brain biopsy samples Brain Histopathology 2/2 – Nested PCR of 18S rRNA
(2005), biopsy positive 2/2
Germany tissue
Kami et al. 5 CSF Culture positive 0/5, 5/5 100%/100% –
(1999a, b), serology positive 4/5
Japan
Verweij et al. 26 samples from 1 patient CSF Culture positive 1/7 4/26 – Genus-specific hot start
(1999), PCR
Netherlands
Komatsu 1 CSF Culture and serology 1/1 – 18S rRNA
et al. (2004), negative
Japan
Moling et al. 2 CSF Culture positive 2/2, 1/2 – 18S rRNA
(2002), Italy serology 1/2
S. M. Rudramurthy and A. Chakrabarti
7 Molecular Genetics and Genomics of Fungal Infections 83
et al. 2004). One study used nested PCR on par- diagnosed by Exserohilum-specific PCR who
affin wax-embedded brain biopsy samples were negative by broad-range PCR. Of CSF sam-
(Bialek et al. 2005). They could detect Aspergillus ples from 345 patients, 82 (24%) were positive
in the brain biopsy samples, which correlated for E. rostratum, and one CSF sample had
with histopathology findings (Bialek et al. 2005). Cladosporium DNA. The researchers later devel-
Due to the promising results in patients with CNS oped a validated TaqMan real-time PCR for the
aspergillosis, PCR testing could be used espe- detection of E. rostratum in the body fluids
cially in patients who are not subjected to inva- (majority of samples were CSF) and identified 57
sive procedures (Reinwald et al. 2013). additional cases; 19%, 29% and 47% cases were
positive by culture, conventional PCR and real-
time PCR, respectively. They suggested that
7.5 Non-Cryptococcal and Non- beta-d-glucan assay was more appropriate than
Aspergillus CNS Infections the real-time PCR for monitoring the response to
therapy, though cautioned about the possibility of
Candida species may rarely cause meningoen- contamination in both the test formats. Therefore,
cephalitis in patients with haematological malig- the results of the tests should be interpreted with
nancy, post-neurosurgery and premature neonates clinical finding and epidemiological data (Gade
(Pappas et al. 2004). The majority of the reported et al. 2015). A highly sensitive species-specific
cases of Candida meningoencephalitis have been molecular beacon real-time PCR assay that has
diagnosed by culture methods. Badiee et al. used the potential to detect and quantify E. rostratum
the TaqMan probes designed from 18s region of in clinical sample has also been developed (Zhao
rDNA, to detect the species-specific Candida in et al. 2013).
the CSF and serum of patients with suspected PCR assay for the diagnosis of coccidioidomy-
non-Cryptococcal fungal meningitis. Of 38 cosis was attempted by Vucicevic et al. They
patients evaluated, they diagnosed 4 cases of included 153 respiratory and 5 CSF samples
Candidal meningitis, 3 due to C. albicans and 1 (Vucicevic et al. 2010). The ITS2 LightCycler
C. glabrata (Badiee and Alborzi 2011). real-time PCR was positive in five respiratory
The large outbreak of fungal meningitis asso- samples, but the CSF samples were negative in all
ciated with the epidural injections of contami- cases. Later using the same technique, two cases
nated methylprednisolone acetate solution in the of meningeal coccidioidomycosis were diagnosed
United States (Centers for Disease Control and successfully, and the PCR results were positive
Prevention (CDC) 2012) has helped in our under- 3 days before the serology and 3 weeks before the
standing on the utility of the molecular tests for culture results (Binnicker et al. 2011). PCR test
the diagnosis of the fungal meningitis. Though A. for meningeal coccidioidomycosis requires fur-
fumigatus was isolated from the index case, the ther evaluation. In case of histoplasmosis, though
vast majority of the laboratories confirmed the molecular PCR-based techniques are available for
infections due to Exserohilum rostratum. For the the detection and identification of Histoplasma
development of the molecular diagnostic test to from culture and animal model, application of
investigate this outbreak, two sets of primers, (1) molecular tests directly on clinical samples is
broad spectrum primers (ITS3 and ITS4) that very few and less successful (López et al. 2017).
could amplify 350 bp fragment of any fungus and
(2) Exserohilum specific primer that amplifies
250 bp variable region of ITS2, were used (Gade 7.6 Molecular Identification
et al. 2013). The amplified products were identi- of the Fungal Isolates
fied by DNA sequencing. A total of 115/413
(28%) cases were positive for fungal DNA by Identification of the fungi to the species level is
PCR and sequencing. Broad-range PCR was pos- important for the initiation of appropriate anti-
itive in 96 cases; additionally 25 more cases were fungals. Conventional mode of identification of
84 S. M. Rudramurthy and A. Chakrabarti
the fungal culture usually relies on morphologi- technique, they could differentiate all major
cal and physiological characters. It requires in- molecular types of C. neoformans and C. gattii.
depth knowledge on the minute details of each This technique is promising for the detection and
species. In clinical laboratory this method of differentiation of major Cryptococcus species
identification is usually difficult and takes time directly from the clinical specimen. The semi-
due to lack of expertise and slow-growing nature nested PCR used in this technique increased the
of many species. Accurate identification may be a detection limit to ~1 pg. HCRA had been devel-
problem even for experts in many cases due to oped earlier as well that targeted ITS regions of
morphological similarities of closely related spe- rDNA. The technique could only differentiate the
cies. Molecular techniques have been used suc- known molecular types (Kaocharoen et al. 2008).
cessfully for identification of the fungi. Currently Recently, real-time PCR assay, targeting inter-
DNA sequencing is considered as the gold stan- genic spacer-1 region of rDNA locus, has been
dard for the identification of the fungi. Different developed, which could differentiate the cultures
conserved and variable genes have been used, of C. neoformans sensu lato and C. gattii sensu
ITS region of the rDNA being the commonest. lato (Tavares et al. 2016). Though the detection
ITS2 region is commonly used as it contains both limit of this technique is not better than the
conserved and variable regions. The International above-mentioned probe-based techniques, this
Society for Human and Animal Mycology method is affordable and has less chance of con-
(ISHAM) has created the barcode database for tamination, as it eliminates post-amplification
identification of majority of the pathogenic fungi processing.
using ITS2 region. Identification of Aspergillus to the species
Differentiation between C. neoformans and C. level is essential as few newly described species
gattii is essential for epidemiology and manage- A. lentulus, A. calidoustus, A. terreus and A. ver-
ment, as they differ in their distribution, ecology, sicolor have elevated MICs to several antifungal
clinical presentation, antifungal susceptibility drugs (Balajee et al. 2005). The ITS sequences
and therapeutic outcomes (Trilles et al. 2012). of Aspergillus do not have sufficient variation
Identification and differentiation of these two and fail to differentiate various Aspergillus spe-
species by biochemical tests take time. Molecular cies complexes. Other protein-coding loci, such
technique has been developed to differentiate the as for beta-tubulin (BenA), calmodulin (CaM)
two species from culture, but the technique was and RNA polymerase 2 (RPB2), are used to dif-
not successful for differentiating directly on clin- ferentiate Aspergillus species. Using the oligo-
ical specimen. With the aim to develop the sensi- nucleotide probe and PCR-enzyme immunoassay,
tive technique for the detection and differentiation de Aguirre et al. (2004) successfully identified
of these two species directly from the clinical seven medically important Aspergillus species
specimens, Trilles et al. (2012) developed hyper- (A. flavus, A. fumigatus, A. nidulans, A. niger, A.
branched rolling circle amplification (HRCA) terreus, A. ustus and A. versicolor). ISHAM
technique to differentiate all seven major molec- working group recommends the use of ITS
ular types of C. neoformans (Trilles et al. 2014). sequence for the identification up to Aspergillus
Phospholipase B gene (PLB) was amplified by species complex and BenA and CaM for identi-
semi-nested PCR. They designed the species- fication of species within species complex. For
specific padlock probes that produce minimum Candida species ITS region and D1/D2 region of
secondary structure at the 5′-end probe binding large subunit of rDNA (26s) are targeted. Robust
arm. For effective padlock probe binding, the techniques such as high-resolution melting curve
annealing temperature was adjusted close to or analysis of ITS 1 and 2 region may also be
above the ligation temperature. These probes applied to speciate Candida (Mandviwala et al.
were ligated to the amplified product, and HCRA 2010). Nonamplified nucleic acid probes target-
reactions were performed in real-time PCR ing the 26S rRNA region have been evaluated for
machine to amplify the probe signals. Using this the identification of fungi, employing peptide
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Part II
Fungal Pathogens: Pathogenesis,
Pathology and Diagnosis
Aspergillosis
8
Sundaram Challa
orbit, base of skull, parasellar area, and cribri- by BBB, blood-cerebrospinal fluid barrier, and
form plate into anterior, middle, and posterior intrinsic immune system (Dotis and Roilides 2007;
fossae (Shankar et al. 2007; Chimelli and Mahler- Nau et al. 2014). The pathogenesis and pathology
Araujo 1997). depend on the immune status of the host, route of
entry, pathogen species, and its virulence (Fig. 8.1).
Normal host even when exposed to conidia does Microglia and perivascular, meningeal, and cho-
not develop disease due to adequate host defenses. roid plexus macrophages along with complement
In the lung or sinus, when the conidia begin to constitute the innate immune system in CNS
swell, the macrophages recognize the pattern- (Nau et al. 2014). Microglia is the major immune
associated molecular patterns (PAMPs) on the surveillance cell of CNS and is similar to myeloid
surface of the conidia, phagocytose them, recruit derived peripheral macrophages in its functions.
neutrophils, and elaborate chemokines and cyto- The cerebrospinal fluid (CSF) also contains a
kines to kill and eliminate the germinating trafficking population of mononuclear cells,
conidia. However, immunosuppressed individu- comprising T cells, B cells, monocytes, and den-
als, especially those with qualitative or quantita- dritic cells (Ransohoff and Engelhardt 2012).
tive defects in neutrophils, cannot prevent The microglia and the other immune cells con-
germination of conidia into hyphae. The hyphae tinuously sample the brain microenvironment
enter the host epithelial cells, grow within them and subarachnoid space to identify any pathogen-
by preventing apoptosis, then invade blood related antigens (Dando et al. 2014). Microglia
vessels from abluminal surface, produce throm- expresses germ line-encoded pattern recognition
bosis, cause endothelial damage, and disseminate receptors (PRRs), on the surface or in the cyto-
to extrapulmonary sites including CNS (Filler plasm, which are crucial in the recognition of
and Sheppard 2006; Hohl and Feldmesser 2007; pathogens. These include Toll-like receptor-2/4
Dagenais and Keller 2009). (TLRs), Dectin-1 and Dectin-2, complement
Immunocompetent hosts exposed to constant receptor-3 (CR-3), CD45, CD86, and others.
exposure to high inocula of conidia, aided by hot Complement aids in the recognition and killing
and humid climate, also cannot prevent germina-
tion of conidia. The proteases secreted by the
Portal of
fungus degrade the extracellular matrix and pro- entry
vide nutrients as well as facilitate invasion Immune Size of
(Hedayati et al. 2007; Krishnan et al. 2009; status conidia
Raksha et al. 2017).
Neuro Antigenic
tropism character
of the pathogen as well as bridging functions to phagocytosis, oxidative burst, and release of
other parts of the immune network (Ricklin et al. damaging compounds by binding to corre-
2010). Neuronal and glial cells express TLRs as sponding receptors on immune cells. Aspergillus
well as complement receptors (CR); however, also drives complement activation by interac-
production of complement in response to injury tion with the pattern recognition molecule pen-
is controlled by fine-tuned regulatory mecha- traxin-3 (PTX-3). When A. fumigatus is
nisms to protect the brain from injury (Veerhuis opsonized with PTX-3, the complement cas-
et al. 2011). cade can be activated either by interaction
between PTX-3 and C1q via the classical path-
way or by interaction between PTX-3 and fico-
8.1.7 H
ost Defenses to Aspergillus lin-2 via the lectin pathway (Ma et al. 2009;
Infection in CNS Moalli et al. 2010).
The host tries to inhibit the spread of fungus
The Aspergillus spp. hyphae on reaching the by forming abscess and sealing it off from adja-
cerebral microvasculature interact with endothe- cent brain parenchyma (Rambach et al. 2008;
lial cells, microglia, and astrocytes. The microg- Speth and Rambach 2012). The cytokine IL-6
lial PRRs, TLR-4, CR-3, and Dectin-1, recognize sequesters iron intracellularly, which is an impor-
the PAMPs, the β-glucan, and the mannose, on tant nutrient for Aspergillus, and restricts its
Aspergillus hyphae (Mogensen 2009; Hadas growth (Erta et al. 2012).
et al. 2010). This recognition and interaction
trigger both humoral- and cell-mediated immune
responses (Dotis and Roilides 2007). 8.1.8 Strategy of Pathogen
The microglia and other resident immune
cells express major histocompatibility complex The virulence factors of Aspergillus help to evade
Class I and Class II molecules. Microglia act as immune attack and facilitate invasion (Rambach
antigen-presenting cells and stimulate prolifera- et al. 2005). The fungi hide from recognition,
tion of T cells. Upon activation, the effector sig- mask the receptors, acquire or produce comple-
nal transduction pathways are initiated leading to ment regulatory molecules from the host, and
NF-kB activation and production of cytokines secrete proteases to degrade complement factors
and chemokines such as interferon-γ, TNF-α, (Rambach et al. 2008; Speth et al. 2008). A.
interleukin-1β, IL-6, and IL-12. These enhance fumigatus secretes alkaline protease which
phagocytosis and kill the hyphae by the produc- cleaves and destroys complement in the CSF and
tion of free radicals, nitric oxide, and superoxide reduces the capacity to opsonize the hyphae
anion. The chemokines recruit dendritic cells, (Rambach et al. 2010; Balenga et al. 2015).
neutrophils, CD4+ and CD8+ T cells, B lympho- Fungal hyphae limit the surface deposition of C3
cytes, and macrophages from periphery and and thus interfere with complement-dependent
along with activated resident cells potentiate phagocytosis. The level of complement deposi-
inflammation to kill the hyphae (Dotis and tion on different Aspergillus species correlates
Roilides 2007). inversely with their pathogenicity: highly viru-
On recognition of Aspergillus hyphae by lent species like A. fumigatus and A. flavus bind
PRRs, complement cascade gets activated and less C3 on their surface than nonpathogenic spe-
leads to the cleavage of C3 and formation of cies like A. glaucus or A. nidulans (Henwick
membrane attack complex. Astrocytes, oligo- et al. 1993).
dendroglia, neurons, and invading macrophages The melanin pigment blocks the C3 binding
show increase in complement synthesis, site and prevents phagocytosis and protects fun-
whereas microglia show only minor amounts. gus against reactive oxygen species (ROS) to
Opsonization of conidia and hyphae with com- enable its survival in the host (Tsai et al. 1998;
plement fragments like C3b and iC3b mediates van de Veerdonk et al. 2017).
8 Aspergillosis 95
During its growth, A. fumigatus produces sev- cytokine/chemokine profile illustrates that CNS
eral enzymes and toxic substances. Mycotoxins, fungal infection is the result of an ineffective
which include soluble galactosaminogalactan immune response, possibly due to an insufficient
(GAG), gliotoxin, and fumagillin, inhibit phago- antifungal effector function of endogenous glial
cytosis, reduce ROS production by neutrophils, cells resulting from competing pro- and anti-
and inhibit T cell responses (Dagenais and Keller inflammatory cytokines (Dotis and Roilides 2007;
2009; Tomee and Kauffman 2000). GAG induces Licinio and Wong 1997). Specific T cell subsets
neutrophil apoptosis and inhibits the IL-1- which are activated as part of host response to A.
mediated inflammation and induces Th-17 T cells fumigatus include both protective and disease-
that protect the fungus (van de Veerdonk et al. promoting T cells (van de Veerdonk et al. 2017).
2017). Fumagillin inhibits the function of neutro- The antifungal effect of complement depends
phils. Gliotoxin can damage and kill microglial on its concentration. CNS is a vulnerable organ
cells, astrocytes, and neurons via apoptosis and as the complement levels are low in CSF and
decreases macrophage function that is required complement activation might be insufficient for
for optimal phagocytosis and killing of the patho- an effective antifungal defense (Speth and
gens (Tomee and Kauffman 2000; Fallon et al. Rambach 2012).
2010; Schlam et al. 2016). Secretion of gliotoxin The formation of abscess to contain the spread
makes the fungal conidia less susceptible to hides the fungus from the attack of the comple-
opsonization, increasing the propensity of the ment and allows its growth (Rambach et al. 2008;
fungus to invade the CNS through endothelial Speth and Rambach 2012). The strategy of the
cell endocytosis (Rambach et al. 2008; Tomee pathogen versus the host defense is given in
and Kauffman 2000). A. fumigatus proteases Fig. 8.2.
damage and degrade the host tissue, facilitating
the acquisition of essential nutrients, such as iron
and zinc, which are required for its metabolism 8.1.10 Pathology
(van de Veerdonk et al. 2017). A. fumigatus has a
remarkable adaptability to hypoxic conditions Damage from Aspergillus spp. can result from
and changes its transcriptional profile and metab- fungal growth and tissue invasion or from inflam-
olism to survive and invade in the host (van de matory response (Hohl and Feldmesser 2007). The
Veerdonk et al. 2017). A. flavus produces afla- ineffective clearing of the organism and inability
toxin, and the strains that produce higher amounts to down regulate the inflammatory response also
of aflatoxin are pathogenic and exhibit increased contribute to the pathology. Neuropathologic fea-
host cell cytotoxicity (Qureshi et al. 2014). tures include hemorrhagic infarcts and/or necrosis,
vascular thrombosis, meningitis, granuloma, and
formation of solitary or multiple abscesses
8.1.9 Limitation of Host Defense (Kleinschmidt-DeMasters 2002; Sundaram et al.
2006; Shankar et al. 2007; Walsh et al. 1985b;
The protective action of microglia against infec- Hope et al. 2005; Ruhnke et al. 2007) (Fig. 8.3).
tions critically depends on the cross talk with cir- The infarcts and abscess are usually due to A.
culating granulocytes and monocytes, before fumigatus and are a result of hematogenous dis-
circulating leukocytes enter the brain and semination in immunosuppressed hosts, whereas
CSF. Neutropenia, a common risk factor for dis- granulomas are usually due to A. flavus resulting
seminated aspergillosis, probably affects microg- from contiguous spread from sinus, orbit, or ear in
lial function by impairing this cross-talk (Nau immunocompetent hosts. Spinal cord involvement
et al. 2014). Though microglia has a protective is rare. It is reported as direct extension from lung
role, microglial activation and cytokine release or thoracic vertebrae and presents as epidural mass
disrupt BBB and increase the risk of CNS inva- causing cord compression (Seres et al. 1972; Sheth
sion (Streit 2002; Olson and Miller 2004). The et al. 1985).
96 S. Challa
Fig. 8.2 Central
nervous system Host Pathogen
aspergillosis: strategy of
pathogen against host
defense. Abbreviations:
PRR pattern recognition Express PRRs on microglia to
Hide recognition, mask receptors
receptor, PAMP recognize PAMPs
pathogen-associated
molecular pattern, ROS Stimulate T cell proliferation, Produces GAG, gliotoxin, fumagilin to
reactive oxygen species, elaborate cytokines, chemokines, prevent phagocytosis, induce neutrophil
GAG phagocytosis, produce ROS, kill apoptosis, impair macrophage function,
galactosaminogalactan, hyphae kill microglia, neurons, astrocytes
ECM extracellular
matrix
Activates complement, Produces reglatory proteins, proteases
opsonization, phagocytosis, to degrade complement, limits surface
produce ROS, kill hyphae deposition
a b c
d e f
Fig. 8.3 (a) Coronal section of brain showing infarct (d) Slender septate dichotomously branching hyphae
(arrow). (b) Photomicrograph showing granulomas with (Gomori’s methenamine silver ×40). (e) Slender septate
predominance of multinucleated giant cells and extensive hyphae (periodic acid-Schiff ×40). (f) Angioinvasion with
fibrosis (hematoxylin and eosin ×10); inset showing nega- destruction of elastic tissue (GMS ×10); inset showing
tive staining septate hyphae within giant cell (H&E ×40). septate hyphae of Aspergillus spp. in the vessel wall
(c) Photomicrograph showing abscess with necrotic mate- (GMS ×40)
rial and numerous septate hyphae in the wall (H&E ×4).
8 Aspergillosis 97
8.1.11 Hemorrhagic Infarcts with sinus lesion, dural based with basal meningitis,
or completely intraparenchymal (Sundaram et al.
These involve anterior and middle cerebral artery 2006; Shankar et al. 2007; Chimelli and Mahler-
territories and are usually acute and necrotizing. Araujo 1997; Sundaram and Murthy 2011). They
These are due to direct invasion of vessel wall or are characterized by epitheloid cells, predominance
secondary to septic embolus. In view of the angioin- of multinucleated giant cells, and infiltrates of neu-
vasive nature of Aspergillus, the infarcts are usually trophils, lymphocytes, plasma cells, and eosino-
hemorrhagic with areas of necrosis, which may phils. The stroma is very dense and fibrous.
convert into septic infarcts (Chimelli and Mahler- Intraparenchymal granulomas with no obvious
Araujo 1997). Arteritis with bland infarcts due to source of infection in the sinus, ear, or lung are
Aspergillus spp. is reported (Uppin et al. 2007). reported (Sharma et al. 1997; Sundaram and Murthy
Histology shows vascular invasion with thrombosis 2011; Challa et al. 2007). They need to be differen-
and necrosis. The inflammatory infiltrate is usually tiated from tuberculomas in countries endemic for
sparse (Sundaram et al. 2006; Shankar et al. 2007; tuberculosis, by the location, extensive fibrosis,
Chimelli and Mahler-Araujo 1997). minimal necrosis with prominence of foreign body
giant cells, and nature of infiltrate along with appro-
priate fungal stains (Sundaram et al. 2006;
8.1.12 Mycotic Aneurysm Sundaram and Murthy 2011; Challa et al. 2007).
8.1.13 Abscess
8.1.16 Diagnosis
These may be solitary or multiple and are located
at grey-white matter junction. They may be well Due to high morbidity and mortality of CNS asper-
circumscribed or poorly delineated from adjacent gillosis, early and accurate diagnosis is essential
brain parenchyma depending on the immune sta- for institution of appropriate treatment. The anti-
tus of the host. The abscess contains central fungal susceptibility varies among different spp. of
necrotic material, surrounded by hemorrhagic Aspergillus. Hence it is important to diagnose
necrosis and neutrophilic infiltrate with inter- Aspergillus and differentiate from other filamen-
spersed fungal hyphae (Shankar et al. 2007). tous fungi with similar morphology and also get
species identification for specific treatment.
The diagnostic tests include direct microscopy,
8.1.14 Granuloma culture, serology, and molecular tests. These tests
are applied according to the clinical scenario and
These are usually located in frontal or temporal availability. Surgical excision and submission of
regions. These may be extracerebral in continuity tissue for histopathology and culture are done
when there are mass lesions extending from sinus/
98 S. Challa
orbit/ear or intracranial space-occupying lesions special stains like PAS and GMS delineate the
(ICSOLs). In immunocompromised hosts with morphology of fungal hyphae much better. In the
CNS involvement, biopsy from a peripheral site tissue, the Aspergillus spp. are seen as slender,
like lung may be obtained (Guarner and Brandt septate, hyaline, nonpigmented, and dichoto-
2011). Serological and molecular tests are useful, mously branching hyphae. Fungal hyphae may be
in disseminated aspergillosis, subject to availabil- seen as negative staining structures within the
ity. The advantages and limitations of various giant cells and in the stroma. PAS and GMS are
diagnostic tests are given in Table 8.1. complementary to each other with a concordance
rate of 84% (Hope et al. 2005; Heaton et al.
2016). Frozen sections and squash smears are
8.1.17 Direct Microscopy less sensitive (60%) but highly specific (98%)
for the detection of fungal filaments; these,
These include (1) wet mounts with or without though useful in the rapid diagnosis, should not
potassium hydroxide, (2) fluorescent techniques be used as a standalone method (Heaton et al.
applied to fresh fluid or tissue, and (3) frozen/ 2016; Sundaram 2003). In suspected cases of
formalin-fixed paraffin-embedded (FFPE) sec- CNS aspergillosis, a combination of H&E, PAS,
tions stained with hematoxylin and eosin (H&E), GMS, and Masson-Fontana stains helps in mak-
periodic acid-Schiff (PAS), and Gomori’s methe- ing a presumptive diagnosis (Hope et al. 2005;
namine silver (GMS) (Hope et al. 2005; Guarner Guarner and Brandt 2011).
and Brandt 2011; Barton 2013). The advantages
include high sensitivity and rapid turnaround
time, but the major disadvantage is its inability to 8.1.20 Limitations of Histopathology
differentiate Aspergillus from other filamentous
fungi and species identification, compromising Morphology cannot provide the fungal genus
the diagnostic accuracy (Hope et al. 2005; and species; speciation needs culturing and bio-
Guarner and Brandt 2011; Barton 2013). chemical characterization (Shankar et al. 2007;
Guarner and Brandt 2011). The septate hyaline
dichotomously branching morphology alone
8.1.18 Wet Mounts cannot differentiate Aspergillus spp. from other
hyaline molds like Fusarium spp. and
The wet mounts of the specimen delineate the Scedosporium spp. (Guarner and Brandt 2011).
hyphal morphology, and addition of 10% potas- In addition, there may be difficulty in distin-
sium hydroxide causes partial digestion of the guishing Aspergillus spp. from Mucorales,
proteinaceous material for better visualization. which are pauci/aseptate, and dematiaceous
The fluorescent dye, Calcofluor-white, selec- fungi with less melanin in the cell walls, by
tively binds to the polysaccharides in the fungal using histopathology alone (Guarner and Brandt
cell wall, but the stain is not specific to Aspergillus 2011; Tarrand et al. 2003; Sangoi et al. 2009;
spp. (Chander et al. 1993). Lee et al. 2010; Sundaram et al. 2014; Challa
et al. 2014). The concordance between morpho-
logic diagnosis on histopathology and culture
8.1.19 Histopathology varies from 79% to 88% (Tarrand et al. 2003;
Sangoi et al. 2009; Lee et al. 2010; Sundaram
Histopathology is very useful in the diagnosis, et al. 2014; Challa et al. 2014).Misdiagnosis and
especially tissue obtained from sinus or ICSOL; misclassification can occur in about 20% cases
however, it may not be possible to obtain tissue (Sundaram 2003; Tarrand et al. 2003; Sangoi
from lesions located in deep white matter of brain et al. 2009; Lee et al. 2010; Sundaram et al.
or vascular lesions. Routine histology with H&E 2014). Tissue fungal infection burden of less
stain helps in assessing tissue reaction, whereas than 103 CFU/gm of tissue usually proves to be
8 Aspergillosis 99
Table 8.1 Diagnostic tests for central nervous system aspergillosis: advantages and limitations
Test Sample Advantages Limitations Comment
Culture Tissue Species identification Yield less than 50%; Yield on tissue better
Blood/BAL/CSF Allows antifungal long turnaround (79%); blood/BAL/
susceptibility/ time; may fail to CSF usually negative
resistance grow or sporulate
Histopathology Tissue from sinus/ Rapid, cost-effective Cannot identify Antifungal
orbit/ICSOL presumptive diagnosis genera/species; susceptibility differs
Core biopsy from of invasive fungal cannot differentiate among species/
lung/skin in disease; differentiates from other hyaline genera;
disseminated from contamination septate fungi like misclassification up
disease Fusarium and to 20%; use of IHC/
Scedosporium; in situ hybridization/
sometimes, difficult PCR on FFPE tissue
to differentiate from increases diagnostic
Mucor/Candida/ accuracy; however,
dematiaceous fungi; not yet validated
architectural
distortion; yield is
79%
Galactomannan Blood/BAL Useful in disseminated False-positive Higher positive result
disease in certain results in patients than imaging and
subpopulations like in (1) receiving direct microscopy;
HSCT/hematologic antibiotics like low sensitivity in
malignancy; rapid; piperacillin, non-neutropenic
early marker of amoxicillin, or patients
invasive disease; ticarcillin; (2)
sensitivity 40–100%; receiving substances
specificity 56–100% that contain products
of fermentation of A.
niger; (3) with
infections with other
fungi including
Penicillium,
Paecilomyces,
Alternaria, and
Histoplasma spp.;
cutoff value
controversial
(1,3)-β-d Glucan Serum Rapid Not specific for Marker of invasive
Aspergillus; disease in certain
false-positive results subpopulations like
in patients receiving in HSCT/
piperacillin/ hematologic
tazobactam malignancy
PCR Blood/BAL/FFPE/ Early marker of Yield on FFPE Technique not yet
other fluids invasive disease; lower than in blood recommended for
sensitivity 84%; routine use;
specificity 86% combination of GM
with validated PCR
improves diagnosis
of definite disease
Others Fungal antibodies, Not useful for
metabolites diagnosis
Abbreviations: BAL brochoalveolar lavage, CSF cerebrospinal fluid, ICSOL intracranial space-occupying lesion, HSCT
hematopoietic stem cell transplant, FFPE formalin-fixed paraffin-embedded, PCR polymerase chain reaction, GM
galactomannan
100 S. Challa
histologically negative (Kahn et al. 1986). Tissue 8.1.23 Limitations of Culture Studies
reaction depends on the immune status of the
host and may be minimal in severely immuno- The turnaround time is long and may take a few
suppressed host. days. The yield of organisms may be low; organ-
isms may fail to grow or fail to sporulate, and
sometimes the growth characteristics are atypical
8.1.21 Ancillary Techniques (Tarrand et al. 2003). Hence culture studies may
to Histology be insensitive and nondiagnostic (Hope et al.
2005; Guarner and Brandt 2011).
These include immunohistochemistry (IHC), immu-
nofluorescence, in situ hybridization, and polymerase
chain reaction (PCR). Immunohistochemical 8.1.24 Molecular Tests
reagents that detect Aspergillus spp. in tissue are
commercially available; however, the widespread In disseminated aspergillosis or IA involving
presence of common antigens in fungi has resulted CNS, when tissue cannot be obtained, molecular
in cross-reactivity with other hyaline septated molds, tests are useful. Blood cultures are usually nega-
Mucorales, and some yeasts (Phillips and Weiner tive. The molecular tests include (1,3)-β-d-glucan
1987; Reed et al. 1993; Schuetz and Cohen 2009). In (BG) test, galactomannan (GM) antigenemia test,
culture-proven aspergillosis, IHC positivity on FFPE and PCR. Most of the tests are standardized and
tissues was reported to be 88–100% (Challa et al. validated in serum or BAL fluid for A. fumigatus
2015; Jung et al. 2015). IHC was found to be par- involving the lung. Galactomannan and validated
ticularly useful to differentiate Aspergillus spp. from PCR applied to blood can be used as screening
Mucor spp. when culture studies are not available tools to identify patients at high risk of develop-
(Challa et al. 2015; Jung et al. 2015). ing IA (Jones and McLintock 2003). Detection of
The yield of fungal DNA from FFPE is low. antibodies and metabolites of Aspergillus spp.
These tests still need validation for routine diag- was not found to be useful for diagnosis of IA
nostic use (Patterson et al. 2016). (Hope et al. 2005).
Aspergillus spp. have the ability to grow at 37 °C (1,3)-β-d-Glucan is a cell wall component of
and hence can be distinguished from other patho- most fungi including Aspergillus spp. Serum
genic fungi. It grows readily in blood agar, choc- assays are recommended for diagnosing IA in
olate agar, and brain-heart infusion broth but certain high-risk populations like hematologic
better with Sabouraud dextrose agar, which is a malignancies and HSCT, but they are not specific
fungal-specific medium (Hope et al. 2005). The for Aspergillus (Patterson et al. 2016). False-
recovery is improved by inhibiting growth of positive reactions are known to occur in some
bacteria and environmental molds by the addition patients who are receiving piperacillin/tazobac-
of chloramphenicol/gentamycin and cyclohexi- tam (Metan et al. 2010).
dine, respectively (Sutton 2003). The speciation
is done by conidial morphology. In addition to
diagnosis, culture enables susceptibility testing 8.1.26 Galactomannan (GM) Test
to antifungals as well as determines resistance.
Certain species of Aspergillus like A. terreus and GM is a polysaccharide antigen present on the
A. nidulans are resistant to amphotericin, and cell wall of Aspergillus, and it is released into the
hence the culture studies help in therapeutic blood during invasive infections. It can be
options (Walsh et al. 2003). detected in serum or brochoalveolar lavage
8 Aspergillosis 101
(BAL) fluid, CSF, or pleural fluid days before Histopathology/cytology and culture are
clinical symptoms appear and hence an effective strongly recommended for the diagnosis of IA till
method for early diagnosis of IA (Arvanitis et al. molecular methods are available for routine use
2014; Feng 2015). It is a fairly sensitive and spe- (Patterson et al. 2016). Despite the limitations,
cific test, less time consuming and yields higher histopathology and culture remain gold standard
positive results compared to imaging and direct for the diagnosis of CNS aspergillosis where tis-
microscopy. The sensitivity and specificity range sue can be obtained. Molecular tests are useful in
from 40% to 100% and 56% to 100%, respec- disseminated disease.
tively (Feng 2015). The positive cutoff value is
controversial (Zhou et al. 2017). It gives a false-
positive result in approximately 50% of patients 8.2 Conclusion
receiving antibiotics like piperacillin, amoxicil-
lin, or ticarcillin, 100% of patients receiving sub-
CNS aspergillosis occurs in both immunosup-
stances that contain products of A. niger
pressed and immunocompetent hosts with geo-
fermentation (plasmalyte), and various percent-
graphical variations. Ineffective defense
ages of patients with infections with other fungi,
mechanisms and strategy of pathogen to evade
like Penicillium, Paecilomyces, Alternaria, and
immune attack result in disease. The pathology
Histoplasma (Hachem et al. 2009). In non-
and clinical manifestations depend on the immune
neutropenic patients, the value of the detection of
status, risk factors, route of spread, and the species
circulating GM is limited because of low sensi-
involved. Early diagnosis is important for institu-
tivity (Erjavec et al. 2009).
tion of appropriate therapy as CNS aspergillosis is
associated with high morbidity and mortality.
Histopathology and culture remain gold standard
8.1.27 Polymerase Chain Reaction where tissue can be obtained and molecular meth-
(PCR) ods are important tools in disseminated disease.
PCR amplifies target of rDNA and subsequent Acknowledgment The author, who was former Dean and
sequencing. Using real-time quantitative PCR Senior Professor of Pathology at Nizam’s Institute of
allows fast and accurate diagnosis of IA and Medical Sciences, Hyderabad, gratefully acknowledges
all the colleagues, students, and technical staff for their
identification of species. PCR can be applied to contribution.
blood, BAL, or FFPE on the extracted DNA (Feng
2015). Aspergillus DNA can be detected very
early in patients with IA, much before onset of References
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Candidiasis
9
M. Altay Atalay
AIDS Acquired immunodeficiency Candidiasis can be seen at all ages worldwide and
syndrome is manifested by the acute and/or chronic infec-
CMC Chronic mucocutaneous tions of the skin, mucosa, internal organs, and
candidiasis systems, which are caused by a genus of yeast
CNS Central nervous system or called Candida species. Candida species are
Coagulase-negative staphylococci found in the mouth and the gastrointestinal tract
CSF Cerebrospinal fluid (GIT) in approximately 30–50% of healthy indi-
CT Computed tomography viduals. They are also found in the genital system
CVC Central venous catheter flora in 20–30% of the females. In addition, they
DM Diabetes mellitus live in the soil and on a wide variety of plants and
ESCMID European Society of Clinical foods. Their isolation from the soil is regarded as
Microbiology and Infectious a sign of fecal contamination. Although there are
Diseases more than 200 species of Candida, there are fre-
GIT Gastrointestinal tract quently isolated pathogens from human beings.
HIV Human immunodeficiency virus Among them are C. albicans predominantly fol-
IC Invasive candidiasis lowed by C. glabrata, C. tropicalis, C. parapsilo-
ICU Intensive care unit sis, C. krusei, C. guilliermondii, C. lusitaniae, C.
IDSA Infectious Diseases Society of dubliniensis, C. pelliculosa, C. kefyr, C. lipolyt-
America ica, C. famata, C. inconspicua, C. rugosa, and C.
IL Interleukin norvegensis. Recently, another species of
MRI Magnetic resonance imaging Candida, Candida auris, has been introduced,
NAC Non-albicans Candida presenting with multidrug resistance and a high
NPV Negative predictive value mortality rate as a dangerous health threat in some
PPV Positive predictive value countries. Another recent development in the lit-
TPN Total parenteral nutrition erature is renaming and the taxonomic rearrange-
ment of Candida species. These rearrangements
also include moving of a number of species to
other genera. Some of these can be referred here
as follows: Candida krusei → Pichia kudriavze-
M. A. Atalay (*) vii, Candida norvegensis → Pichia norvegensis,
Department of Medical Microbiology, Erciyes Candida guilliermondii → Meyerozyma guillier-
University School of Medicine, Kayseri, Turkey
mondii, Candida lusitaniae → Clavispora lusita- all hematogenous infections, where Candida spe-
niae, Candida kefyr → Kluyveromyces marxianus, cies were the causative microorganisms, the use
and Candida pelliculosa → Wickerhamomyces of broad-spectrum antibiotics was identified,
anomalus. Furthermore, a group of following suppressing the normal bacterial flora and elimi-
species of Candida including C. glabrata, C. nating the effects of natural antagonists, which
parapsilosis, and C. haemulonii are today prevent the fungal colonization in the mucosa
regarded as species complexes (Correia et al. (Das et al. 2011). In addition, in a cohort study in
2006; Mersequel et al. 2015; Pfaller et al. 2005; which intraabdominal major surgical procedures
Tavanti et al. 2005). were associated with Candida infections, it was
reported that 50% of 107 patients with candi-
demia had recently undergone a surgical proce-
9.2 Epidemiology dure (Block et al. 2009). Neutropenic patients
suffering from acquired immunodeficiency syn-
Despite the fact that numerous species of Candida drome (AIDS) and oncologic diseases are almost
are isolated from animals at primitive levels and always reported to have oropharyngeal and
from the environment, the candidiasis infections esophageal candidiasis.
in the human beings are usually of endogenous Recent epidemiologic studies have demon-
origin. In the human body, Candida species can strated that the frequency of candidiasis ranged
be isolated from the GIT, oropharynx, vagina, from 1.2 to 25 in 100,000 patients (Arendrup
and the skin. The leading species among these 2010). Candida species are the fourth most com-
isolates is C. albicans. The variabilities in the mon fungal agents associated with the hematog-
rates of identified C. albicans isolates are influ- enous infections in the hospitals in the United
enced by personal characteristics of the individ- States. More than 50% of the invasive Candida
uals including their eating habits and ages. A infections are due to non-albicans Candida
study has reported that the rates of identified C. (NAC) species (Pfalller et al. 2014). It has been
albicans isolates obtained from the oral cavity observed that NAC species has received medical
vary in a range from 1.9% to 41.4%. These rates attention with increased rates of isolation approx-
have been found to be in a range of 0–55% and imately over the past 30 years. One of the under-
2.2–68% for the GIT and vagina, respectively, in lying causes of this fact is explained by the
the same study (Odds 2010). While C. tropicalis evidence that the widespread use of azole deriva-
species are isolated mostly from the oropharynx, tives, including fluconazole, leads to the exces-
C. glabrata species are isolated from the vagina sive proliferation of the species such as
and GIT. Moreover, it has been demonstrated that C. glabrata and C. krusei in the perianal region
the colonization of the Candida species in the and other areas of the body due to their relatively
hospital inpatients is more common compared to lower susceptibility to these antifungal agents. A
healthy individuals. Candida species associated prospective surveillance program including
nosocomial infections may emerge with the patients with invasive candidiasis determined a
involvement of the hospital personnel and via spurt in 2496 NAC infections between years
contaminated materials. The impact of these fac- 2004 and 2008. The species identified in these
tors on the emergence of nosocomial infections infection spurts are listed as follows according to
has been determined by molecular diagnostic their frequencies: C. glabrata complex (46.4%),
methods. C. parapsilosis complex (24.7%), C. tropicalis
The prevalence of Candida infections has (13.9%), C. krusei (Pichia kudriavzevii; 5.5%),
been reported to be on the rise in the last 20 years. C. lusitaniae (Clavispora lusitaniae; 1.6%), C.
The two major underlying causes of this fact are dubliniensis (1.5%), and Candida guilliermondii
the pathological/iatrogenic immunosuppression (Meyerozyma guilliermondii; 0.4%). The same
and the increase in the use of broad-spectrum study has identified two or more Candida species
antibiotics. Another study reported that in 92% of in 4.4% of candidiasis cases. Although variations
9 Candidiasis 109
are observed in different studies, C. albicans is of the neutrophils followed by the monocytes and
the most frequent agent isolated from candidiasis eosinophils, which can terminate Candida spe-
cases (Nieto et al. 2015). cies by oxidative and non-oxidative pathways. In
particular, the risk proneness of the neutropenic
patients for the development of candidiasis
9.3 Pathogenicity stresses the importance of neutrophils for the
host defense systems against Candida species.
An important feature of host defense against can-
didiasis is the intact skin barrier. Otherwise, an
entry gate for these pathogenic microorganisms 9.4 Candida Infections
like C. albicans is created when the integrity of
this barrier against the outer world is disrupted Candida species cause numerous infections in
resulting from the insults due to medical devices the range from the mucosal colonization to mul-
or surgical procedures. The adherence capacities tiple organ involvements.
of the Candida species to epithelial cells (espe-
cially of C. albicans) or to the synthetic poly-
meric structures like intravascular or urethral 9.4.1 Superficial Candida Infections
catheters (C. tropicalis) have been associated
with their virulence. Although these fungi present These infections usually originate from the own
with their abilities to secrete proteases and microbial flora of the individuals although they
lipases, facilitating their invasion, the clinical may be acquired contagiously on some occasions.
significance of these enzymes has not been clari-
fied, yet (Matthews 1994). 9.4.1.1 Oral Candidiasis
The intensity of colonization by the associated Oral candidiasis may present with various differ-
agent plays an important role in the development ent clinical forms:
of candidiasis. Candidiasis typically affects the
inpatients who have stayed in the hospital for –– Acute pseudomembranous oral candidiasis: It is
long-term periods. Fifty-one percent of the most commonly encountered in breastfed
hematogenous Candida infections are associated infants and in the old-age individuals. Otherwise,
with the patient acceptance to the intensive care these infections may occur in human immuno-
unit (ICU) (Das et al. 2011). Another factor con- deficiency virus (HIV) (+) patients with a CD4
tributing to the pathogenesis is the presence of (+) cell count of <200/mm3, in cancer patients,
Candida species, which may cause biofilm for- and in patients who use steroid inhalers. There
mations in central venous catheters, contact are studies demonstrating the correlation
lenses, intrauterine devices, and medical devices between the frequencies of asymptomatic oral
like pacemakers. The use of toremifene, a broad- Candida carriage and HIV viremia in HIV-
spectrum antibiofilm compound, has been shown infected individuals. Furthermore, compared
to reduce biofilm formation by Candida species. to CD4 cell counts, the high plasma levels of
This is an important development for patients HIV-1 RNA identified during the early stages
with recurrent infections associated with Candida of HIV infections have been reported to be a
and with other biofilm-forming bacteria, espe- better preliminary indicator for the develop-
cially due to the presence of catheters (Donlan ment of oral candidiasis (Oberoi 2010). In
and Costerton 2002; Glockner 2011). patients infected with HIV, oral candidiasis
The clinical outcome of the infection is deter- has been associated with the long-term or
mined primarily by the state of host defense con- intermittent use of fluconazole or other azole
ditions. After the invasion of the dermis or the derivatives and with other patient factors
circulating blood by the pathogen, the primary including being over the age of 35, being
defense systems are involved, consisting mainly addicted to intravenous substance use, and
110 M. A. Atalay
being black-raced. The most frequently iso- tropicalis, C. krusei, and C. stellatoidea, which
lated agent in oral candidiasis cases is C. albi- have been reported as the other causative agents.
cans, followed by C. glabrata complex, C. A number of studies have identified risk factors
parapsilosis complex, C. tropicalis, C. krusei for developing C. albicans esophagitis; however,
(Pichia kudriavzevii), and C. dubliniensis. In the evidence for non- C. albicans species as the
the oropharyngeal candidiasis cases in the causative agents is limited (Kakati et al. 2015).
patients suffering from cancer, the most fre-
quently isolated agents are particularly the flu- 9.4.1.3 Candida Vulvovaginitis
conazole and itraconazole resistant clades of and Balanitis
the C. glabrata complex. It has been reported Seventy-five percent of females suffer from at
that prophylaxis with fluconazole reduces the least one candidal vulvovaginitis episode in their
oropharyngeal C. albicans carriage rates from life spans. Recurrences and persistent symptoms
46% to rates between 0% and 10% in leuke- may occur in some cases due to treatment fail-
mia patients who underwent bone marrow ures. C. albicans is the causative agent in 80% of
transplantation, whereas, it leads to the the cases. It is followed by C. glabrata complex
increased colonization rates of C. glabrata in 5% of the cases. In recent years, C. africana
complex and C. krusei (Pichia kudriavzevii) species have been reported as another causative
mainly in the perianal region and in other parts agent of vaginitis in the literature. Diabetes mel-
of the body (Garcia-Cuesta et al. 2014). litus (DM), pregnancy, and the use of broad-
–– The clinical picture of the disease manifests spectrum antibiotics are the most common
with pseudomembranes appearing as white significant risk factors. Itching, erythema, and
patches in the hard palate and gingiva and burning sensation in the vagina and vulva and
occasionally in the throat. The patches consist dysuria and dyspareunia are the common find-
of desquamated epithelial cells, leukocytes, ings in patients suffering from this clinical condi-
yeasts, and bacteria. Removal of these mem- tion. A thick and whey-like vaginal discharge
branous structures results in bleeding from may occur.
the underlying tissues. Oral candidiasis is Penile candidiasis (balanitis) is most com-
usually self-limiting except in patients suffer- monly associated with DM in men. Vulvovaginitis
ing from AIDS. may occur in the sexual partners of females
–– Chronic atrophic candidiasis: This clinical suffering from this type of infection. Patients
form of oral candidiasis is also known as with long-term catheter implementations may
“denture stomatitis” in patients using dentures also develop chronic infections. Itching, ery-
and having intraoral lesions. The most com- thema, and vesiculopustular lesions are evident
mon manifestation is “angular cheilitis” iden- in the glans penis (Achkar and Fries 2010).
tified by crusting in the corners of the lips.
–– Chronic hyperplastic candidiasis: It is also 9.4.1.4 Cutaneous Candida Infections
known as candidal leukoplakia as well, and
malignancies may develop from 15% to 20% –– Intertrigo is a common disease in obese
of the lesions. females. Vesicles and pustules develop in
occluded body areas prone to maceration such
9.4.1.2 Candida Esophagitis as the inguinal areas, areas under the breasts
This disease, which can also be classified under and axillae.
deep candidiasis, may be associated with oropha- –– The candidal infection of the skin between the
ryngeal candidiasis or may emerge as an inde- toes presents with painful lesions due to the
pendent entity. It is most common in patients erythematous lesions and cracks in the skin.
suffering from AIDS. C. albicans is the most This clinical condition is often seen in indi-
common underlying cause of fungal esophagitis viduals who are frequently in contact with
followed by other Candida species including C. water.
9 Candidiasis 111
9.4.2.2 Acute Disseminated isolation rates from the blood are relatively lower
Candidiasis (Rivoisy et al. 2018).
It is a fulminant infection usually with an anti-
bacterial resistant fever. It can be seen in patients 9.4.2.6 Myocarditis
with and without neutropenia. The most common Myocarditis occurs usually as a symptom of a
complications are meningitis, brain abscesses, hematogenously disseminated infection or as a
renal abscesses, myocarditis, endocarditis, endo- complication of endocarditis. Approximately
phthalmitis, and cutaneous abscesses. 50% of the patients with Candida myocarditis die
of disseminated infection. Making the diagnosis
9.4.2.3 Chronic Disseminated can be a challenging process. Cardiac abscesses
Candidiasis identified in patients with widespread candidiasis
It develops during the neutropenic period in or candidemia are treated as the complications
patients with leukemia. There may not be any secondary to the existing infection. The isolation
evidence of organ involvement in the presence of of the fungus from the lesions allows making the
persistent fever. The neutrophil count may return precise diagnosis (Einarsdóttir et al. 2002).
to normal levels; however, fever and weight loss
persist. Hepatomegaly and splenomegaly can be 9.4.2.7 Thrombophlebitis
seen. Alkaline phosphatase levels are usually Thrombophlebitis is associated with the presence
very high. Multiple lesions are identified in com- of intravascular devices. Partial or complete
puted tomography. Cultivation positivity of the blockages are formed in greater vessels. Besides
biopsy samples is approximately 30%. Blood the clinical examination, venography and mag-
cultures are negative. netic resonance imaging (MRI) are other useful
tests. Fungi can be cultivated from the venous
9.4.2.4 Gastrointestinal Candidiasis blood (Block et al. 2009).
It is a rare clinical condition with ulcer forma-
tion in the mucosa. It is most commonly seen in 9.4.2.8 Osteomyelitis
cancer patients at advanced stages and in Osteomyelitis develops usually as a result of the
patients suffering from AIDS. It is reported that, hematogenous dissemination. Sometimes it may
in newborns, diarrhea cases may be encountered develop in association with aspiration or corti-
associated with C. albicans, which can be sone injections, and more rarely it may develop
treated with oral nystatin treatment (Parra- after a trauma (Fig. 9.1a, b). It is more common
Herran et al. 2010). in cancer patients and in low birth weight infants.
Blood cultures are usually negative. The confir-
9.4.2.5 Endocarditis mation of diagnosis requires examination of the
Two percent of all endocarditis cases develop due biopsy samples and cultivation (Fig. 9.1c) (Kohli
to Candida species. Those infections are on the and Hadley 2005).
rise in patients with prosthetic heart valves and in
patients with intravenous substance dependency. 9.4.2.9 Arthritis
Specific diagnostic findings include large pieces Arthritis develops as a result of direct inoculation of
of vegetation and embolization of the greater ves- the microorganism following hematogenous spread,
sels. Blood cultures and cultivations from the dissemination from an infected bone, or trauma.
cloth samples obtained from the embolism cases Usually larger joints are involved such as the shoul-
are the pathognomonic evidence. However, filter- der and knee with the development of many non-
ing out of the yeast in the capillary bed following specific symptoms. Cultivation of the Candida
their entry to the circulation prevents them to species from the synovial fluid is important in mak-
enter the venous circulation. Eventually, their ing the diagnosis (Kohli and Hadley 2005).
9 Candidiasis 113
specific clinical and radiological evidence is diseases, and congenital malformations are other
identified. The identification of the presence of risk factors. The microbiological diagnosis of
yeasts in the sputum or in the sample fluids invasive candidiasis in ICU patients can be estab-
obtained from bronchoscopic procedures is not lished only during the late phases of infection
pathognomonic as Candida species can be iso- despite the advanced technology. Retrospective
lated in 3–85% of the sputum cultures due to studies associated the late diagnosis and timing
oropharyngeal contaminations. The material of antifungal treatment with the increased mor-
obtained from the open pulmonary biopsies or tality rates. Therefore, prediction models have
from the percutaneous aspirations of the pulmo- been developed to identify the high-risk patients
nary lesions are the appropriate samples for prone to invasive candidiasis. In order to assess
examination (Kontoyiannis et al. 2002). the process after the colonization in patients
undergoing surgical interventions, Pittet et al.
(1994) proposed the term “Candida colonization
9.5 he Risk Factors
T index” in 1994. This index is the ratio of the num-
for Candidemia and Invasive ber of different body regions colonized with the
Candidiasis in the ICU same strain of Candida species (culture positive)
Patients to the sum of the number of the body regions
where samples for cultivation are collected. The
Candidemias account for 50–70% of the invasive threshold value is 0.5 meaning that the index will
candida infections. Candida species rank the be increased if the ratio is above. Empirical treat-
fourth after coagulase-negative staphylococci ments given to the patients with higher ratios
(CNS), Staphylococcus aureus, and enterococci above the threshold value achieved significant
in leading to 8–10% of nosocomial infections in reductions in the incidence of invasive candidia-
the United States (25–50% of the cases are ICU sis. However, the model has some disadvantages
patients), and they are the major causes of mor- including the very low levels of positive predic-
bidity and mortality (Pappas 2006). Mortality tive value (PPV) and the increases in the labora-
due to candidemia is reported as 30%; however, it tory workload. Nevertheless, the colonization
may reach rates of 50–70% in ICU patients. The index is important for the assessment of the early
most important risk factors for invasive candidia- colonization dynamics in the ICU patients prone
sis (IC) include prolonged periods of stay in the to invasive candidiasis. In addition, the higher
ICU, antibiotic use, hemodialysis, central venous negative predictive values (NPVs) indicate that
catheters (CVCs), underlying major diseases, the model will be useful in the prediction of the
total parenteral nutrition (TPN), GIT perforations low-risk patients. Another concept in diagnosis is
or operations, acute pancreatitis (invasive candi- the “Candida score.” For the first time in 2006,
diasis occurs as a complication in 25% of cases), Leon et al. (2006) scored the risk assessments
steroid or immunosuppressant use, mechanical and suggested that colonization, major surgical
ventilation, multiple blood transfusions, Candida procedures before being accepted in the ICU,
species colonization in different areas of the evidence of severe sepsis, and TPN were the
body, and diabetes. The issue is bigger in the independent risk factors. The authors reported
pediatric ICU patients, ranking the second in the that they obtained a score by multiplying several
septicemia cases in the United States. It has been factors with either 1 or 0 when these factors were
reported that the most commonly associated risk present or absent respectively. They calculated
factors with candidemia in this patient group the specificity and the sensitivity of estimating
include prolonged TPN, CVC, and topical anti- the possibility of the present disseminated infec-
fungal use. In newborns, low birth weight tion when the score was >2.5. There are a number
(<1250 g), premature birth, low APGAR scores, of studies on the colonization index and the
Candida species colonization in different body Candida scores. The most important outcome is
regions or in catheters, H2 receptor blockers, GIT the higher NPV values in the assessment of those
116 M. A. Atalay
parameters, meaning that they do not allow mak- Alternatively, the first-line treatment with
ing a diagnosis; however, they allow the exclu- echinocandins in the hematogenous infections
sion of possible clinical entities in the differential caused by C. parapsilosis has been shown to have
diagnosis. no negative effects on the treatment outcomes.
The ESCMID recommendations for fluconazole
differ from the IDSA recommendations, and the
9.6 Treatment 2009 IDSA guideline is being updated currently.
In addition, both ESCMID and IDSA recom-
Fungal Infection Study Groups of both the mend the use of echinocandins in neutropenic
Infectious Diseases Society of America (IDSA) patients (Fernández-Ruiz et al. 2014).
and European Society of Clinical Microbiology It is recommended as a standard modality that
and Infectious Diseases (ESCMID) have pub- the treatment is continued for 14 days after termi-
lished practice guidelines for the treatment of nation of candidemia. If the patient is in a stable
invasive candidiasis in various patient groups clinical condition and able to receive orally
(Cornely et al. 2012; Cuenca-Estrella et al. administered medications, the treatment is
2012; Hope et al. 2012; Lortholary et al. 2012). switched to fluconazole following a 10-day intra-
The recent European guidelines recommend a venous therapy in the lower line of treatment
7–10-day treatment with fluconazole 100 mg/ (Ullmann et al. 2012). Removal of any perma-
day as the first-line treatment for the treatment nent intravenous catheters is recommended defi-
of oropharyngeal candidiasis of both adult and nitely. If removal of the catheter is not possible,
pediatric HIV-infected patients. Potential alter- treatment with liposomal amphotericin B or echi-
natives to fluconazole are listed in the guideline nocandins may be started (Hope et al. 2012;
as follows: miconazole 10 or 50 mg/day for Lortholary et al. 2012; Pappas et al. 2009).
7–14 days, itraconazole oral solution 100 or For the treatment of CNS candidiasis, the rec-
200 mg/day for 14 days, and/or posaconazole ommended dose of lipid formulations of ampho-
200 mg to be administered on the first day of the tericin B with or without flucytosine is 3–5 mg/
treatment continued with 100 mg/day kg. Flucytosine dose should be maintained at a
(Lortholary et al. 2012). However, topical use level of 40–60 μg/ml. As a former line of treat-
of antifungal medications (e.g., amphotericin B ment, a high-dose fluconazole treatment of 400–
lozenges or nystatin) has not been recom- 800 mg/day can be administered. The use of
mended due to their poor tolerability (bitter echinocandins in central nervous infections is
taste, side effects associated with GIS, the limited due to their poor permeability through the
requirement for the administration of frequent blood-brain barrier although improvement with
doses) and lower efficacy. Echinocandins caspofungin has been reported in a patient with
should not be preferred in the treatment of tri- Candida meningitis resistant to amphotericin B
azole-susceptible Candida species as they are deoxycholate and fluconazole (Cornely et al.
more costly compared to fluconazole and only 2012; Sugar and Liu 2000).
the parenteral forms of this medication are The appropriate treatment options to prevent
available. The ESCMID guideline recommends the development of invasive candidiasis in
echinocandins (caspofungin, anidulafungin, high-risk neonates include fluconazole, nystatin,
and micafungin) as the first-line medications or alternatively lactoferrin ± Lactobacillus. The
for the treatment of non-neutropenic adult can- complication of invasive candidiasis in the new-
didemia patients (Hope et al. 2012). Compared born period is the development of the dissemi-
to echinocandins, the effect sizes of liposomal nated disease most likely including central
amphotericin B and voriconazole are similar; nervous system infections. Amphotericin B
however, they are recommended less frequently. deoxycholate, liposomal amphotericin B, ampho-
On the other hand, fluconazole is recommended tericin B lipid complex, fluconazole, miconazole,
with marginal strength (Lortholary et al. 2012; and caspofungin can be used in the treatment
Ullmann et al. 2012). (Hope et al. 2012).
9 Candidiasis 117
Although the efficacy of fluconazole has been reported that formic acid, a low-cost substance,
established, primary antifungal prophylaxis is can be used for treatment at low doses, although
not recommended in Europe to prevent the devel- it is toxic at higher doses (Lastauskienė et al.
opment of oropharyngeal and esophageal candi- 2014).
diasis in HIV patients. The disadvantages of the
primary prophylaxis include the possible drug
interactions between the highly effective antiret- 9.6.1 Antifungal Resistance
roviral medications and azoles, development of
resistance to fluconazole and/or other azoles, the The mechanism of antifungal resistance is either
presence of effective treatments for oropharyn- primary or secondary. The development of resis-
geal candidiasis, cost, and potential toxic effects tance may be associated with single individual
of triazoles. Thus, the best prophylaxis of oro- factors although it may be acquired as well due to
pharyngeal and esophageal candidiasis is the use the ability of the causative fungi to inhibit the
of appropriate antiretroviral therapies (Lortholary antifungal mechanism of action of a specific drug
et al. 2012). and/or a class of drugs or due to the low concen-
Laser treatment has been used in the treatment trations of the drug (Cowen et al. 2014). Candida
of several diseases in dermatology in recent species are able to develop resistance against
years. It has been reported that Nd-YAG laser antifungal agents by (1) decreasing the intracel-
therapy can be used as an alternative for patients lular concentrations of medications, (2) decreas-
disqualified to be treated with antifungal treat- ing the concentrations and structures of targeted
ments (Naouri and Mazer 2013). Due to the antifungal proteins, or (3) changing the sterol
capacity of Candida species to develop resistance composition in the cell membrane (Perlin et al.
to various treatment agents and due to some of 2015; Sanglard and Odds 2002). Recently, it has
the established toxic effects of the antifungal been suggested that CARD9 is a central regulator
agents, attempts have been made in recent years of neutrophil-dependent antifungal immunity in
to develop alternative medications. It is estab- the CNS (Fig. 9.2) (Drummond and Lionakis
lished that formic acid, as well as acetic acid, 2018). 5-Flucytosine resistance occurs either by
leads to apoptosis in Candida species. It has been reducing the uptake of the drug into the cell or via
enzymatic changes involving the conversion of Alexander BD, Johnson MD, Pfeiffer CD, Jiménez-
5-flucytosine to 5-fluorouracil or of 5-fluorouracil Ortigosa C, Catania J, Booker R, Castanheira M,
Messer SA, Perlin DS, Pfaller MA. Increasing echino-
to the 5-fluorouridine monophosphate. The candin resistance in Candida glabrata: clinical failure
examination of the azole resistance has revealed correlates with presence of FKS mutations and ele-
that various mechanisms may lead to the devel- vated minimum inhibitory concentrations. Clin Infect
opment of resistance by Candida species the Dis. 2013;56:1724–32.
Arendrup MC. Epidemiology of invasive candidiasis.
against azole derivative drugs. The most common Curr Opin Crit Care. 2010;16:445–52.
mechanisms are the excessive expression of the Bassetti M, Taramasso L, Nicco E, Molinari MP, Mussap
MDR or CDR encoded drug efflux pumps and M, Viscoli C. Epidemiology, species distribution,
the development of a point mutation in the antifungal susceptibility and outcome of nosocomial
candidemia in a tertiary care hospital in Italy. PLoS
gene encoding the target enzyme (ERG11). One. 2011;6:e24198.
Echinocandin resistance is always acquired Block AA, Thursky KA, Worth LJ, Slavin MA.
during treatment in susceptible Candida species. Thrombolytic therapy for management of complicated
The mechanism of developing resistance is alter- catheter-related Candida albicans thrombophlebitis.
Intern Med J. 2009;39:61–3.
ations in the amino acids in the HS1 and HS2 Cornely OA, Bassetti M, Calandra T, Garbino J, Kullberg
regions of the FKS subunit of glucan synthase, BJ, Lortholary O, Meersseman W, Akova M,
leading to a reduced drug sensitivity to this Arendrup MC, Arikan-Akdagli S, Bille J, Castagnola
enzyme. Fungal adaptation to the effects of drugs E, Cuenca- Estrella M, Donnelly JP, Groll AH,
Herbrecht R, Hope WW, Jensen HE, Lass-Flörl C,
develops due to the increased numbers of resis- Petrikkos G, Richardson MD, Roilides E, Verweij PE,
tant FKS types in response to increased cellular Viscoli C, Ullmann AJ, ESCMID Fungal Infection
stress. Primary resistance against the azole deriv- Study Group. ESCMID* guideline for the diagnosis
atives and/or echinocandins is most frequent with and management of Candida diseases 2012: non-
neutropenic adult patients. Clin Microbiol Infect.
C. glabrata complex among Candida species 2012;18:S19–37.
(Alexander et al. 2013). Correia A, Sampaio P, James S, Pais C. Candida bracaren-
sis, sp. nov., a novel anamorphic yeast species pheno-
typically similar to Candida glabrata. Int J Syst Evol
Microbiol. 2006;56:313–7.
9.7 Conclusion Cowen LE, Sanglard D, Howard SJ, Rogers PD, Perlin
DS. Mechanisms of antifungal drug resistance. Cold
Candidiasis of CNS is uncommon and is one of Spring Harb Perspect Med. 2014;5:a019752.
Cuenca-Estrella M, Verweij PE, Arendrup MC, Arikan-
the most serious consequences of invasive candi- Akdagli S, Bille J, Donnelly JP, Jensen HE, Lass-Flörl
diasis. The risk factors include prematurity, C, Richardson MD, Akova M, Bassetti M, Calandra
immunosuppression, breach in mucocutaneous T, Castagnola E, Cornely OA, Garbino J, Groll AH,
barrier as in patients on parenteral nutrition, Herbrecht R, Hope WW, Kullberg BJ, Lortholary O,
Meersseman W, Petrikkos G, Roilides E, Viscoli C,
patients with indwelling catheters or those receiv- Ullmann AJ, ESCMID Fungal Infection Study Group.
ing broad spectrum antibiotics or with prolonged ESCMID* guideline for the diagnosis and manage-
ICU stay. Diagnosis of CNS candidiasis by ima- ment of Candida diseases 2012: diagnostic proce-
geology is difficult and differential diagnosis dures. Clin Microbiol Infect. 2012;18:S9–18.
Das I, Nightingale P, Patel M, Jumaa P. Epidemiology,
includes bacterial and tuberculous infections. clinical characteristics, and outcome of candidemia:
High index of clinical suspicion is required for experience in a tertiary referral center in the UK. Int J
prompt diagnosis and treatment. Infect Dis. 2011;15:e759–63.
Donlan RM, Costerton JW. Biofilms: survival mecha-
nisms of clinically relevant microorganisms. Clin
Microbiol Rev. 2002;15:167–93.
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Mucormycosis
10
A. Serda Kantarcioglu
a b
c d
Fig. 10.1 Microscopical morphology of (a) Mucor sp., primary culture media, (d) Coenocytic hyphae and spo-
(b) Rhyzopus sp. (Lactophenol cotton blue stained, ×100), rangium (Lactophenol cotton blue stained, ×100)
(c) Rhizopus arrhyzus on cooked sheep’s blood agar as
philic with a minimum growth temperature about air and the later are less prone to aerosolization.
20 °C and maximum extending up to Spore size and hydrophilic/hydrophobic charac-
60 °C. Others can grow at temperatures below ter impacts the dispersal of fungi.
0 °C. Most of the pathogenic Mucorales are het- Several members of Mucorales including
erothallic, and in their sexual development, Rhizopus oryzae (R. arrhizus), the most common
hyphae of the two different but compatible mat- environmental member of the Mucoraceae fam-
ing types, [(−) and (+)], sense each other and ily and the most prevalent agent of mucormyco-
undergo fusion to form zygospores, which later sis, may, under particular in vitro or in vivo
germinate to form a sporangium at the apex cul- conditions, form a variety of cellular forms,
minating in sexual meiospores. However, in most branching mycelium, budding yeasts and
species zygospore production is rarer due to the spherule-like chlamydoconidia (Kantarcioğlu
conditions necessary for their formation. The et al. 2006). Some species of order Mucorales are
asexual sporangiospores are produced in massive able to grow under anaerobic conditions, while
quantities, and they are thought to be able to most of them require aerobic conditions. During
serve as the major source of dissemination and the transition of hyphae to yeast and vice versa of
infection, whereas sexual zygospores are consid- dimorphic Mucorales, there occurs a series of
ered to be dormant (Mendoza et al. 2015; alterations in metabolism. Some of these altera-
Muszewska et al. 2014). Mucorales produce wet tions seem to be related only to a change in habi-
and dry sporangiospores, the former disperse by tat, aerobic to anaerobic or the reverse (José
124 A. S. Kantarcioglu
1985). Furthermore, while the majority of the In vitro studies revealed the potential for bio-
Mucorales only grow at high water activities, film formation for R. oryzae, L. corymbifera and
some of them are able to grow in salt concentra- R. pusillus, but not for A. elegans (Singh et al.
tions of at least 15%. 2011). The biofilm matrix is formed with glucos-
Several Mucorales genomes have been amine as the dominant dry component. Rhizopus
sequenced. Based on the sole gene count and oryzae is claimed to possess extraordinary
genome size comparisons, Rhizopus oryzae (R. genome plasticity owing to a whole genome
arrhizus) is an exception regarding the number of duplication, which resulted in an elevated num-
genes in the whole collection of currently ber of genes, including those involved in host-
sequenced Mucorales, with the average gene num- pathogen interactions (Muszewska et al. 2014;
ber of 11.000 genes. R. oryzae possess extraordi- Lewis et al. 2012; Ma et al. 2009).
nary genome plasticity owing to a whole genome
duplication, which resulted in an elevated number
of genes, including those involved in host-patho- 10.5 Ecology and Seasonality
gen interactions (Muszewska et al. 2014).
The knowledge of pathogenic Mucorales ecology
is often anecdotal and conflicting. The major mode
10.4 Cell Wall and Biofilm of transmission might generally be inhalation of
Formation aerosolized sporangiospores. However, Mucorales
very rarely found on nasal mucus, suggesting that
Mucorales exhibit a special structure of cell wall. spores in the mucus of airway mucosa are cleared
They differ in cell wall composition having less by mucociliary transport or that there is a low level
glucan and more chitin and without galactoman- of airborne contamination (Richardson 2009).
nan compared with Ascomycota. Mucorales cell However, in susceptible hosts, infection may
wall mainly composed of chitin and chitosan, the occur.
deacetylated homopolymer of chitin, as structural Mucorales are thermotolerant, fast-growing
polysaccharides. Chitin is built of β-1,4 bonded moulds and ubiquitous in nature and widely
N-acetyl glucosamine. The cell wall of R. oryzae found on organic substrates including spoiled
and other Mucorales contains a high percentage of food and bread, over mature or decaying fruits
chitin and chitosan, which are synthesized by chi- and vegetable matter, crop debris and soil. The
tin synthases (23 genes) and chitin deacetylases fungus grows and acquires nutrients from dead
(34 genes), respectively. Fungal hyphae grow at and decaying matter. Some species are parasites
the tip. Therefore, specialized vesicles, the chito- of plants causing soft rot, of insects, and small
somes, bring precursors of chitin and its synthesiz- animals, while others form symbiotic relation-
ing enzyme, chitin synthetase, to the outside of the ships with plants. The fungus is also found on air,
membrane by exocytosis. The enzyme on the dust, compost piles and animal particularly herbi-
membrane catalyses glycosidic bond formations vores’ faeces and manure; however, human
from the nucleotide sugar substrate, uridine pathogenic Ascomycota are broadly encountered
diphospho-N-acetyl-d-glucosamine. The nascent in air samples, whereas Mucorales are less abun-
polysaccharide chain is then cleaved by the dant in both indoor and outdoor air samples in
enzyme chitin deacetylase. The enzyme catalyses different geographic areas, in Poland (Ejdys
the hydrolytic cleavage of the N-acetamido group 2001; Gniadek and Macura 2007), Lebanon
in chitin. After this the chitosan polymer chain (El-Herte et al. 2012), Turkey (Çolakoǧlu 2003;
forms microfibrils. These fibres are embedded in Sen and Asan 2009; Asan et al. 2010) and even
an amorphous matrix consisting of proteins, glu- India (Sharma et al. 2001; Thirumala and Nathu
cans (which putatively cross-link the chitosan 2013; Pavan and Manjunath 2014) where the dis-
fibres), mannoproteins, lipids and other com- ease incidence is relatively higher (Chakrabarti
pounds (Muszewska et al. 2014; Li et al. 2011). and Singh 2014). Mucorales are soilborne fungi
10 Mucormycosis 125
and survive in the soil as spores. Unusual genera Mucorales in nature. Moisture is critical and
including Apophysomyces and Saksenaea are there should be enough moisture (75–85%) for
also soil saprophytes occurring mainly in tropical spores to germinate and mycelia to grow; there-
and subtropical climates (Gomes et al. 2011). fore, seasonal variation in atmospheric condi-
The isolation of Mucorales from soil is probably tions may affect the abundance of spores present
influenced by soil characteristics. Mucorales are in nature. Rainy seasons might permit high spore
found soil rich in composite vegetation, decaying concentration in soil due to the washing of
vegetables and fruits (Richardson 2009). Spore Mucorales spores from decaying plant material
populations increase in the soil and severely and precipitation, and winds might disperse them
decline during winter. Fruits that have fallen on around in dust. Spores usually do not germinate
the soil surface are infected through contact with at a higher relative humidity. The optimum tem-
infested soil. Throughout the fall and winter, perature for sporulation, mycelial growth and
spores are dislodged from the decaying fruit into spore germination is between 23 and 28 °C, while
the soil. Spores are also dispersed by mowing, rotting and tuber decay develop between 15 and
which scatters pieces of infected fruit. The fun- 23 °C; however, there are numerous reports of
gus survives best in cool, dry soils. Mucor spp. causing decay of vegetables in cold
Fruit become most successful to infection storage. The high spore density of Mucorales
1 month before harvest. Fruits that are overma- depending on climatic conditions in soil and the
ture are more susceptible to infection. Rhizopus outdoor or indoor environment close to human
oryzae, Mucor racemosus, M. circinelloides, M. habitat may probably make easy to exposure and
piriformis and several other species of Mucorales invasion of susceptible individuals.
are plant pathogens that causes a soft rot on sweet Apophysomyces species complex, a recently
potatoes, tomatoes, apples, pears and other fruits emerged pathogen causing ROCM predomi-
and roots. All these fungi are saprophytic so they nantly in healthy individuals, was found signifi-
initially produce pectic enzymes that break down cantly associated with low nitrogen content of the
vegetable tissues. Once a spore lands on a soil in India (Prakash et al. 2016).
wounded tissue, it germinates and starts growing Mucorales are found occasionally in water-
on the surface, producing a thick mycelium damaged buildings, as demonstrated by air sam-
which at the same time produces cell-degrading pling, and analysis of settled dust by quantitative
enzymes (pectinases, amylases) that denature the polymerase chain reaction (PCR). Moreover,
tissues in advance, thus leading to infection. The inhalation of sporangiospores in dust has been
disease infection usually occurs in wounded linked to outbreaks of RCM or pulmonary mucor-
areas after cracking of fruits during sale after har- mycosis due to excavation, construction or con-
vest, transports, market shelf and storage when taminated air-conditioning filters. Whereas most
moisture conditions are favourable. Infected fruit Mucorales infections are community- acquired,
may completely decay after about 2 months in nosocomial acquisition due to percutaneous
cold storage. Decaying fruit become very juicy routes of exposure is very important (Richardson
and within this juice are abundant spores of the 2009).
fungus. Rhizopus stolonifer is more commonly
known as black bread mould. Mucorales uses the
soil and decaying fruits, and plant materials as a 10.6 Etiologic Agents
reservoir, and fungal spores can be inhaled from
disturbed soil, decaying plants, raw vegetables or Most cases of human infection are caused by
overmature fruits to form human mucormycosis. members of Mucoraceae family, predominantly
Although mucormycosis is being reported by Rhizopus species. Rhizopus, Mucor and
globally, cases are relatively common in tropical Lichtheimia species (formerly Absidia) cause
and subtropical countries, where the climatic 70–80% of all mucormycosis cases, with
conditions favour the survival and growth of Lichtheimia species as the second and third most
126 A. S. Kantarcioglu
abundant agent in Europe and the USA, respec- R. oryzae (R. arrhizus), was the most frequent
tively (Gomes et al. 2011; Roden et al. 2005; fungus isolated (Sundaram et al. 2005; Song
Skiada et al. 2011; Schwartze and Jacobsen 2014; et al. 2017), and case fatality rates were high.
Lanternier et al. 2012). Other common causative CNS infection by the Mucoraceae was first
agents include species of Apophysomyces, described in 1885, in a man with multiple brain
Rhizomucor, Cunninghamella and Saksenaea. abscesses who died with widely disseminated
Rhizopus oryzae (Rhizopus arrhizus) is the most infection (Paltauf 1885). In 1943, the description
common environmental member of the genus. of three cases established the classic triad that
Approximately 60% of all the culture-proven characterizes most cases of mucormycosis: dia-
cases of human mucormycosis and nearly 90% of betes mellitus with ketoacidosis, naso-orbital
the rhinocerebral cases are caused by R. oryzae necrotizing infection and meningoencephalitis
(Richardson 2009; Murthy and Sundaram 2014). (Gregory et al. 1943; Sepkowitz and Armstrong
The fungus has been recovered as a saprobe from 1997). Shortly, thereafter, this was recognized as
the nasal cavities and paranasal sinuses of healthy the most prevalent form of mucormycosis.
individuals (Kantarcioğlu et al. 2006) it becomes The emergence of mucormycosis is being
pathogenic under some particular conditions. reported globally, with a rise in the number of
Mucorales colonize a high number of patients but cases in patients with uncontrolled or poorly con-
do not necessarily cause invasion (Bouza et al. trolled diabetes. In recent years, mucormycosis
2006). Apophysomyces elegans, a saprophytic appears to be increasing worldwide, but its exact
soil fungus, has recently emerged as a pathogen prevalence and incidence remain unclear because
species that, unlike the other members of of the difficulty to diagnose and neglect to report
Mucorales, has been reported to cause rhino- mainly in developing countries. A Working Group
orbitocerebral infections in immunocompetent on zygomycosis was formed by the European
individuals (Chakrabarti and Singh 2014; Liang Confederation of Medical Mycology (ECMM) in
et al. 2006; Singh et al. 2017). In English litera- 2004 and collected cases of proven and probable
ture, the majority of patients with A. elegans zygomycosis in 13 European countries to analyse
ROCM had no predisposing conditions, except a the clinical characteristics, microbiology, treat-
few cases occurred following facial or head ment practices and outcome of zygomycosis
trauma (Liang et al. 2006; Singh et al. 2017; through a voluntary case registry, because no
Wolkow et al. 2017). Although few in number, in large prospective studies had been undertaken
English literature, four of the five reported cases until then (Skiada et al. 2011; Petrikkos et al.
of ROCM due to Saksenaea were in previously 2014). Recent population-based studies and/or a
healthy individuals (Baradkar et al. 2008; few multicentre retrospective studies of proven
Shatriah et al. 2012; Kaufman et al. 1988; Taj- and probable cases of mucormycosis (Chakrabarti
Aldeen et al. 2012). and Singh 2014; Roden et al. 2005; Kennedy et al.
2016; Kontoyiannis et al. 2016; KÖMÜR et al.
2016; Bitar et al. 2009) suggested that mucormy-
10.7 Epidemiology cosis has emerged as a prevalent infection after
candidiasis and aspergillosis primarily in patients
Case series on mucormycosis have been reported with underlying risk factors. In most studies, hae-
from medical centres in India; reported cases matologic malignancy was reported as the most
were also reviewed in retrospective studies common underlying disease as well as uncon-
(Chakrabarti and Singh 2014; Roden et al. 2005; trolled diabetes mellitus in some other studies.
Petrikkos et al. 2014; Kennedy et al. 2016; While the majority of patients had at least one
Kontoyiannis et al. 2016; Rueping et al. 2009); underlying condition, the others had no predis-
central nervous system (CNS) or rhinocerebral posing factors.
was the most common site of Mucorales infec- The fungus may affect one of several organ
tion (14.6–69%): Rhizopus species, particularly systems, most commonly the paranasal sinuses
10 Mucormycosis 127
and the brain. Sinus involvement consisting of clinical mucormycosis suggested ROCM may be
rhinocerebral, sinus and sino-orbital infections closely related to high environmental exposure.
constituted the majority of infections followed by Zygomycete research in the scientific commu-
orbital and cerebral involvement. Rhinocerebral nity is presently stimulated and coordinated on
disease is the most common form of mucormyco- an international basis by the ECMM-ISHAM
sis and occurs in the setting of diabetic ketoaci- (International Society for Human and Animal
dosis. ROCM is uncommon in patients with Mycology) Working Group Zygomycoses, coor-
acquired immunodeficiency syndrome (AIDS). In dinated by George Petrikkos. The Working Group
patients with haematologic diseases, the infec- was founded in 2004 under ECMM. After orga-
tion most frequently occurs in neutropenic phase nizing the first International Forum on
(Bouza et al. 2006). One of the most common Zygomycosis in 2008 at Cape Sounion, Greece, a
underlying conditions in paediatric population supplement of Clinical Microbiology and
was profound dehydration (Talmi et al. 2002; Infection was published. A second Forum was
Sheikh and Amr 2011). held in 2010 at Porto Heli, Greece. A website was
In the literature, seasonal variations in the made in the same year (www.zygomyco.net)
incidence of mucormycosis with respect to tem- with a database where cases of mucormycosis
perature, rainfall and humidity have also been can be submitted online. An overview of zygo-
noted (Nithyanandam et al. 2003). Environmental mycosis in Europe was published with 230 cases
factors, such as tropical and subtropical humid accrued by the registry of the Working Group
climate and high temperature typical for India, (Skiada et al. 2011). The Working Group joined
provide an optimum set-up for survival of these ISHAM in 2009. Several members of the group
fungi (Chakrabarti and Singh 2014). Davies et al. took part in a meeting in Chicago, organized by
(2017) reported an increased incidence of rhino- Thomas Walsh with the support of the Hank
orbital-cerebral mucormycosis after Colorado Schueler foundation, and a special issue of
flooding in the USA. The authors hypothesized Clinical Infectious Diseases was published
that the combination of immunocompromised (Walsh et al. 2012). Subsequently a Special
status and environmental exposure resulted in Interest Group (SIG) meeting was organized by
increased incidence (Davies et al. 2017). There Kerstin Voigt and Sybren de Hoog in conjunction
are several reports suggesting that the incidence with the International Mycological Congress on
of mucormycosis increases in summer and early the Biology of Fungi (IMC9) held in Edinburgh,
autumn (El-Herte et al. 2012; Talmi et al. 2002; Scotland, August 2010. A follow-up meeting
Shpitzer et al. 2005; Al-Ajam et al. 2006). The concentrating on zygomycete biodiversity took
authors reported their experience of 19 cases in place in Utrecht, the Netherlands, March 2011.
Israel, that all except two cases presented between This meeting enabled fruitful discussions, inspir-
August and December, and hypothesized that ing an exchange of ideas and providing an
ROCM may have seasonal incidence peaking in updated view on this group of fungi. The out-
the fall and early winter (Talmi et al. 2002; come of this meeting formed the basis of the
Sheikh and Amr 2011). Then, the authors retro- present special issue of Persoonia.
spectively analysed their own data during a
25-year period, trying to define the seasonal
occurrence of RCM and a peak observed in the 10.8 Pathogenesis
month of September; however, no association
was noted between meteorologic conditions and The pathogenesis of the infection is still poorly
the incidence of RCM (Shpitzer et al. 2005). understood as well as the role of specific viru-
El-Herte et al.’s (2012) review on the seasonal lence determinants and the interaction with the
variation of the air concentration of spores of host immune system. Mucorales are able to pro-
Mucorales and other fungi and the variation of duce various proteins and metabolic products
128 A. S. Kantarcioglu
toxic to humans, but the pathogenic role of these laxis against Aspergillus fumigatus, which
potential virulence factors is still unknown. reduces the frequency of Aspergillus infections
(Voigt et al. 2013; Binder et al. 2014). However,
in the literature, there were very few patients with
10.8.1 Host Factors in Mucormycosis ethmoidal sinus infection with orbital extension
or brain involvement among VRZ-receiving
10.8.1.1 Predisposing Factors, patients, and all of them were also neutropenic
Underlying Conditions (Marty et al. 2004; Vigouroux et al. 2005).
Mucorales may cause CNS infections both in
previously healthy individuals and having one or 10.8.1.2 Previously Healthy Patients
more underlying diseases and/or predisposing Patients with no identifiable risk factors were
factors. Uncontrolled diabetes mellitus was the also reported. Unlike RCM caused by the more
frequent underlying disease reported in cases common genera of Mucoraceae (Rhizopus,
caused by the more common genera of Mucor and Lichtheimia), RCM due to A. elegans
Mucoraceae (Rhizopus, Mucor and Lichtheimia). appears to occur in immunocompetent patients.
The other main risk factors are ketoacidosis (dia- In addition, several cases occurred following
betic or other), renal failure, iatrogenic immuno- facial or head trauma (Liang et al. 2006). The
suppression, use of corticosteroids or majority of the reported patients with RCM due
deferoxamine, defects in innate immunity, par- to Saksenaeceae (though few) had no predispos-
ticularly of phagocytic effector cell functions, ing conditions (Wolkow et al. 2017; Sundaram
neutropenia due to cancer treatment, haemato- et al. 2005).
poietic and solid organ transplantation, injectable From the first report of CNS infection by
drug use, disruption of mucocutaneous barriers Mucorales up to April 2018, in the literature, 65
by catheters and other devices and direct implant cases have been described in previously healthy
during neurosurgical procedures (Mendoza et al. patients (Sharma et al. 2001; Liang et al. 2006;
2015; Skiada et al. 2011; Liang et al. 2006; Wolkow et al. 2017; Baradkar et al. 2008;
Kennedy et al. 2016; Rueping et al. 2009; Shatriah et al. 2012; Adelman and Aronson 1969;
Petrikkos et al. 2003). Bichile et al. 1985; Blodi et al. 1969; Chmel and
Diabetic ketoacidosis and deferoxamine- Grieco 1973; Hameroff et al. 1970; Masucci et al.
treated patients are uniquely predisposed to 1982; Pierce et al. 1982; Wetli et al. 1984; Woods
mucormycosis. Haemodyalisis patients receiving and Hanna 1986; Kasantikul et al. 1987;
the bacterial siderophore deferoxamine for treat- Mackenzie et al. 1988; Miller et al. 1988; Oliveri
ing iron overload are uniquely predisposed to et al. 1988; Stave et al. 1989; Fong et al. 1990;
highly lethal and frequently disseminated mucor- Riefler III et al. 1991; Bhattacharyya et al. 1992;
mycosis (Arizono et al. 1989). Deferoxamine Gollard et al. 1994; Hopkins et al. 1994; Siddiqi
prevents iron overload toxicity via efficiently and Freedman 1994; Hussain et al. 1995; Radner
chelating iron from the host; however, it is known et al. 1995; Rangel-Guerra et al. 1996; Fairley
that Rhizopus possess cell surface receptors to et al. 2000; Garcia-Covarrubias et al. 2001;
ferric-rich form of deferoxamine and ferriox- Chakrabarti et al. 2003; Khor et al. 2003; Rao
amine (Baldin and Ibrahim 2017). et al. 2006; Schütz et al. 2006; Verma et al. 2006;
Another predisposing factor of mucormycosis Elinav et al. 2009; Air et al. 2010; Tsung et al.
is considered the use of voriconazole (VRZ) in 2010; El et al. 2011; Parsi et al. 2013; Sarrami
high-risk patients, either for prophylaxis or treat- et al. 2013; Angali et al. 2014; Reddy and Raju
ment of other fungal infections. Voriconazole has 2015; Ginsberg et al. 1987; Gaing et al. 1992;
been shown to be inactive against Mucorales Terk et al. 1992; Bhadani et al. 2007; Watson
in vitro. During recent years Mucorales have et al. 1985; Pillai et al. 2016). Only three of the
emerged as agents of disease in hospitalized previously healthy patients were posttraumatic
patients. Presumably this is partly due to prophy- cases, one of them had a penetrating head injury
10 Mucormycosis 129
(Mackenzie et al. 1988), one had a history of factors that enable the organism to cause disease.
closed-head trauma (Garcia-Covarrubias et al. Although increasing frequency of causative
2001) and another one fell from a tractor and then agents of disease, little is known about the patho-
fell into a water-filled ditch (Radner et al. 1995). genic potential of most mucoralean fungi. In gen-
Twenty-nine of the 65 previously healthy patients eral, adaptation, survival and replication with the
were i.v. drug abusers. Further three intravenous host environment are likely to contribute to the
(i.v) drug abusers were human immunodeficiency virulence potential of fungi.
virus (HIV)-positive patients (Cuadrado et al.
1988; Escobar and Del Brutto 1990), and one 10.8.2.1 Spore Sizes
more i.v. drug abuser had cervix cancer (Scully The small conidial size of fungi (e.g. Aspergillus
et al. 2012). One of the previously healthy fumigatus) is often regarded as a putative viru-
patients had a history of surgery for arteriove- lence factor because the size allows conidia to
nous malformation (Rangel-Guerra et al. 1996), enter the host via respiration. Spore size of the
and one had cataract surgery and dental abscess Mucorales is variable, depending on the species
(Wolkow et al. 2017). An unusual case was 3–11 μm, but in general bigger than those of A.
described in an immunocompetent host who had fumigatus, for example (Binder et al. 2014).
a previous history of primary cutaneous mucor- However, the spores can be inhaled and cause
mycosis, but no cutaneous portal of entry had disease in the human lungs and sinuses.
been identified (El et al. 2011). In an immuno-
competent child with Absidia brain abscess, phy- 10.8.2.2 Stress and Temperature
sicians had postulated that the previous aggressive Tolerance
use of antibiotics coupled with the breakdown of The clinically relevant species of Mucorales are
the gastrointestinal mucosa during severe diar- thermotolerant and have the ability to grow at
rhoea might have led to mucormycosis (Tsung 37 °C, some even at higher temperatures
et al. 2010). (Table 10.1). However, no clinical correlation
between growth speed at host temperature and
differences in virulence potential was detected in
10.8.2 Virulence and Pathogenic a recent study (Schwartze et al. 2012).
Potential of Mucorales The fungi require robust stress response to
survive in human host. Wide variation in stress
In addition to host factors that predispose patients responses was noted in mucoralean fungi to both
to mucormycosis, Mucorales possess virulence osmotic and oxidative stresses and acidic pH.
Table 10.1 Maximum growth temperature of the common etiologic agents of invasive mucormycosis belonging to
Mucorales (De Hoog et al. 2000)
Maximum growth
Family Genus Species temperature (°C)
Mucoraceae Rhizopus Oryzae >37
Microsporus >37
Azygosporus >37
Schipperae >37
Mucor Circinelloides >37
Indicus >37
Rhizomucor Pusillus >37
Lichtheimia (Absidia) Corymbifera >37
Apophysomyces Elegans >37
Cuninghamellaceae Cunninghamella Bertholletiae >37
Saksenaeceae Saksenaea Vasiformus >37
Syncephalastraseae Syncephalastrum Racemosum >37
130 A. S. Kantarcioglu
Rhizopus arrhizus (R. oryzae), the commonest 2007). Blocking the function of CotH proteins
cause of human infection, showed high stress tol- either biochemically by using anti-CotH antibod-
erance in comparison with other species of ies or genetically by attenuating CotH expression
Mucorales (Singh et al. 2016). The utilization of reduces the ability of Rhizopus delemar to invade
macronutrients, e.g. carbon sources, in the host and injure endothelial cells in vitro and reduces
affects growth in vivo and might thus contribute disease severity in mice (Gebremariam et al.
to virulence. Nutrients within the host are most 2014). The most commonly isolated Mucorales
likely available as complex molecules, e.g. pro- from patients (Rhizopus, Mucor and Lichtheimia)
teins, rather than free amino acids. contain three to seven copies of CotH, while
those that are only occasionally the cause of the
10.8.2.3 Surface Proteins disease such as Apophysomyces, Cunninghamella,
Tissue invasion and destruction are important for Saksenaea and Syncephalastrum) only contain
mucormycosis. Primarily, interaction with blood one to two copies (Chibucos et al. 2016).
vessels seems to play a crucial role in the patho-
genesis of mucormycosis. R. arrhizus was shown 10.8.2.4 F actors Modulating GRP78-
to be able to invade endothelial cells with their CotH Interactions
subsequent damage as surveyed by in vitro assays The unique predisposition of diabetic ketoacido-
(Schwartze et al. 2012; Ibrahim et al. 2005). It sis patients and deferoxamine-treated patients to
was also shown that invasion depends on specific mucormycosis points to the importance of hyper-
recognition of the endothelial receptor glucose- glycaemia, iron and acidifying ketone bodies in
regulated protein 78 (GRP78) which is a heat- the virulence of Mucorales. Diabetic patients suf-
shock protein involved in stress-related responses fer from an elevated concentration of glucose.
(Wang et al. 2009). This recognition causes host Hyperglycaemia can induce excessive glycosyl-
cellular death by induction of the endothelial ation of proteins such as transferrin and ferritin,
cell-mediated fungus endocytosis (Baldin and diminishing their iron affinity (Ribes et al. 2000).
Ibrahim 2017), and blocking of the interaction by On the other hand, in the presence of an acidotic
anti-GRP78 antibodies protects diabetic ketoaci- condition due to accumulation of ketone bodies
dosis mice from mucormycosis (Liu et al. 2010) (e.g. β-hydroxy butyrate [BHB]), the low pH in
and results in strongly decreased mortality in the blood vessels strongly impairs the ability of
mice (Schwartze et al. 2012; Liu et al. 2010). It transferring into chelate iron (Artis et al. 1982).
was shown that when GRP78 is blocked other Glucose, iron and BHB enhance the growth of
factors involved in Rhizopus interacting with fungus (Fu et al. 2004; Gebremariam et al.
endothelial cells such as the platelet-derived 2016) and also induce the expression of GRP78
growth factor (PDGF) pathway are activated and CotH, resulting in augmented fungal inva-
(Chibucos et al. 2016). sion and subsequent injury of the endothelium
The fungal ligand that binds to GRP78 during in vitro (Liu et al. 2010; Gebremariam et al.
invasion of the endothelium belongs to the spore- 2016). The role of GRP78-CotH interactions is
coating (CotH) protein family. CotH proteins are currently unknown in the neutropenic host and
universally present in Mucorales and absent from the other patient populations susceptible to
any other organisms which the genome has been mucormycosis.
sequenced. In other pathogenic fungi, invasion of
host cells is mediated by other cell surface pro- 10.8.2.5 Toxin-Like Substances
teins, such as agglutinin-like sequence (Als) pro- It was shown that nonviable Rhizopus, killed by
teins that have been reported to act as invasins for heat or chemicals, was able to cause a comparable
C. albicans and bind to different host receptors amount of damage to endothelial cells as viable
(cadherins), while Aspergillus fumigatus invasins cells (Ibrahim et al. 2005). These results suggest
thaumatin-like proteins (CalA) binds to integrins the contribution of toxin-like substances in
(Wächtler et al. 2012; Liu et al. 2016; Phan et al. mucormycosis pathogenesis. Thus, it was specu-
10 Mucormycosis 131
lated that the presence of toxin-like secondary nal body parts and form a chronic infection. The
metabolites produced from Mucorales, which interplay between pathogens and hosts varies
mediate the interaction between the pathogen and among fungal agents and the host condition. The
the host (Baldin and Ibrahim 2017; Ibrahim et al. innate immune system and its complex interplay
2005). with the adaptive immune system are important
in the pathogenesis of chronic inflammatory dis-
10.8.2.6 Iron Uptake (Acquisition) eases such as chronic rhinosinusitis due to
Iron is an essential element for cell growth and Mucorales (Muszewska et al. 2014; Ooi et al.
development, contributing to many vial processes 2008). However, most of the mechanisms are
of the cell. Pathogenic fungi can use multiple known from in vitro studies or animal data and
processes for obtaining iron from the host, and should be considered with caution as the inflam-
the level of available, unbound iron in serum matory reaction may be different in humans.
plays a critical factor in predisposing patients
with diabetic ketoacidosis to mucormycosis 10.8.3.1 Innate and Adaptive
(Artis et al. 1982; Howard 1999; Boelaert et al. Immunity to Mucorales
1993). In mammalian hosts, iron is bound to host
carrier proteins, such as transferrin, ferritin and The First Line of Defense
lactoferrin, and this sequestration avoids toxic Aerosolized Mucorales sporangiospores may be
effect of free iron (Artis et al. 1982; Howard inhaled and enter to the nose, deposited in the
1999). This strategy of limiting iron availability nasal turbinates and paranasal sinuses in immune-
is also a major universal host defense mechanism competent hosts. Among diabetic and other
against microbes and against Mucorales in par- patients having predisposing conditions, the
ticular, because R. oryzae grows poorly in normal inhaled sporangiospores germinate to form
serum unless exogenous iron is added (Artis et al. hyphae that invade tissues causing locally
1982; Boelaert et al. 1993). Patients with diabetic destructive sinus, orbital and rhinocerebral infec-
ketoacidosis have elevated levels of free iron in tions (Spellberg et al. 2005). The first line of
their serum. Patients receiving dialysis who are defense is the physical and chemical barriers of
treated with the iron chelator deferoxamine are nasal mucosa. Nasal mucociliary clearance
also susceptible to mucormycosis (Ibrahim et al. serves as the primary mechanism by which the
2012). Fungi can obtain iron from the host by airway epithelium removes pathogens from the
using high-affinity iron permeases or low- airway lumen. Antimicrobial-rich mucus gel is
molecular-weight iron chelators (siderophores). composed by mucin proteins. Mucin produced by
Rhizopus is known to secrete rhizoferrin, a sid- the mucus cells serves as sticky binding sites that
erophore that supplies Rhizopus iron. Another trap inhaled pathogens. Physical obstruction of
mechanism by which fungi can obtain iron from sinus ostia or mucostasis can cause hypoxic con-
the host is through use of heme. The Rhizopus ditions within the cellular environment. However,
genome project revealed two homologs of the some species of the order Mucorales, such as
heme oxygenase that may obtain iron from host Mucor spp. and Rhizopus spp., are able to grow
haemoglobin and might explain the angioinva- under anaerobic conditions. These near-anaerobic
sive nature of R. oryzae (Ibrahim et al. 2012). conditions may permit the survival of fermenta-
tive Mucorales species and significantly contrib-
ute to the pathogenesis of sinonasal mucormycosis
10.8.3 Host-Pathogen Interactions, (Hariri and Cohen 2016).
Host Defense Against Sinonasal epithelial cells also generate and
Mucorales secrete antimicrobial compounds to directly
counteract pathogens. These compounds have
Human-infecting Mucorales are usually thermo- various antibacterial, antifungal and antiviral
tolerant, which makes them able to invade inter- effects and include proteins such as defensins,
132 A. S. Kantarcioglu
cathelicidins and reactive oxygen and nitrogen tects the underlying sinonasal tissue from inhaled
species, e.g. nitric oxide (Abbas et al. 2014a, b). pathogens. Cell junctions are the intracellular
It is important for the immune system to be able connections responsible for cellular adhesion,
to recognize the presence of these organisms. which make such a physical barrier possible.
Host sinonasal epithelium plays an important Mucorales may harm the integrity of the sinona-
role in initially recognizing the presence of sal epithelial cell barrier by producing proteases
microbes and responding by increasing produc- (Ma et al. 2009) that can cleave tight junction
tion of antimicrobial peptides and cytokines, with proteins. The first line of defense against
recruitment of phagocytes and lymphocytes of Mucorales is the upper sinonasal epithelial cells
the adaptive immune system, to eliminate the that are encountered at the initial site of infection.
infection (Ooi et al. 2008). Intrinsically faulty or having dysfunction or dam-
To mount an appropriate immune response, age in epithelial cells that extends to the base-
there must be mechanisms by which the cells of ment membrane exposes extracellular matrix
the sinonasal epithelium can detect and react in a proteins. R. oryzae resting spores have been
measured manner to potentially harmful threats shown to adhere to the basement membrane pro-
in the airway. Pathogen recognition receptors teins laminin and type IV collagen (Bouchara
(PRRs) recognize pathogen-associated molecular et al. 1996). Following adhesion to the basement
patterns (Ooi et al. 2008). Proper detection of membrane proteins, Mucorales spores germinate
these pathogens is significant for sinonasal epi- and invade host cells (Ghuman and Voelz 2017).
thelial cells to be able to prepare a defensive
response. PRRs have been implicated as sensors Innate Immune Response
able to detect the presence of the pathogens and Sporangiospores may colonize the mucus.
certain compounds that they secrete. Activation Immune dysfunction, whether overt or subtle, is
of these receptors also triggers innate immune the key factor predisposing to fungal invasion of
responses to prevent or counteract infection, sinonasal tissues. Presumably, fungi are unable to
including mucociliary clearance and the produc- penetrate the epithelial layer when the immune
tion and secretion of antimicrobial compounds system is functioning normally (Hontelez et al.
(e.g. defensins) (Hariri and Cohen 2016). 2012). Following the successful crossing of
Defensins are an antimicrobial peptide family physical barriers, Mucorales encounter cells of
distributed in epithelial cells and phagocytes. innate immune system, including macrophages,
Members of both defensin subfamilies, α- and neutrophils, microglial and dendritic cells (DC),
β-defensins, are expressed in sinonasal epithelial and lead to variable host responses. The main line
cells. They are capable of affecting membrane of innate host response to filamentous fungi con-
permeabilization in both bacteria and fungi. sists of circulating polymorphonuclear neutro-
Cathelicidins are another major class of antimi- phils (PMNs), mononuclear cells (MNCs) and
crobial peptides. Cathelicidin LL-37 is produced macrophages. The function of these cells is both
in the human nasal mucosa. However, the effects to damage the invading organisms and to regulate
of these two antimicrobial peptides on Mucorales innate immune response through secretion of
should be investigated. Reactive oxygen and cytokines and chemokines.
nitrogen species generated by the sinonasal epi- Phagocytes are capable of damaging fungal
thelium can play an important role in upper air- spores and hyphae through oxygen-dependent
way innate immunity. Nitric oxide (NO) in the and oxygen-independent mechanisms. The
upper airway originates predominantly from the oxygen- dependent mechanisms consist of a
paranasal sinuses, particularly the maxillary series of reactions starting with the production of
sinuses, and provides protection against superoxide anion (Ooi et al. 2008; Delves and
pathogens. Roitt 2000), which is dismutated into hydrogen
Sinonasal mucosal epithelial cells adhere to peroxide. Myeloperoxidase then catalyses the
one another to form a physical barrier that pro- conversion of hydrogen peroxide and halides to
10 Mucormycosis 133
generate hypohalides, such as hypochlorite and 1982; Waldorf 1989). M. circinelloides belongs
chloramines, which exert potent antifungal activ- to the order Mucorales and is a dimorphic fungus
ities (Babior 2000; Hampton et al. 1998). Cationic that grows as a budding yeast anaerobically and
peptides (defensins and cathelicidins) are part of as a filamentous fungus aerobically. The larger
the oxygen-independent pathway of phagocytic sporangiospores germinate inside and lyse
cells (De Lucca and Walsh 1999; Ramanathan macrophages, whereas the smaller sporangio-
et al. 2002; Yang et al. 2002). A variety of cyto- spores do not (Orlowski 1991; Lübbehüsen et al.
kines, chemokines, and growth factors play an 2003).
important role in the host response against fila-
mentous fungi (Romani 2004). Most of these Polymorphonuclear Leucocytes
in vitro studies have been performed with If germination of sporangiospores evades or
immune cells obtained from healthy volunteers, escapes this first line of defense, functional neu-
because their primary objective was to elucidate trophils are required to damage hyphae and pre-
the basic properties of normal host response vent their invasion of surrounding tissue
against fungal pathogens (Roilides et al. 2012). (Diamond and Clark 1982). Neutrophils are the
most abundant type of leucocytes found in the
Macrophages blood and are rapidly recruited to the site of
Innate immune system acts to prevent pathogenic pathogenic infection. These innate immune cells
spread, resists the establishment of infection and are able to phagocyte and destroy pathogens via
triggers the adaptive immune response. Tissue cationic peptides and oxidative burst, in a non-
macrophages and neutrophils will serve as func- specific manner in healthy immune conditions
tional effector cells. Macrophages ingest and (Abbas et al. 2014a, b). Neutrophils exhibit fun-
then kill sporangiospores by non-oxydative gicidal activity mediated by the production of
mechanisms, thereby preventing their germina- cationic peptide activity. It was found that R.
tion to hyphae (Roilides et al. 2012; Levitz et al. oryzae spore developmental stage influences the
1986). Mucoralean hyphae have often a bigger efficacy of neutrophil-killing activity (Ibrahim
diameter than those of Ascomycota. Hyphae et al. 2012).
diameter and size determine the efficacy of Gil-Lamaignere et al. (2005) compared the
phagocytosis by macrophages. Mainly the litera- antifungal function of human pectin lyase (PNL)
ture on Mucorales-macrophage interactions is against hyphae of R. oryzae, R. microspores and
limited and associated with murine or human Absidia corymbifera and found that both PNL-
pulmonary macrophages. However, based on oxidative burst in response to hyphae and PNL-
their anatomical location, functional specializa- induced hyphal damage were significantly lower
tion and expression of surface markers, macro- in response to Rhizopus species than in response
phages are characterized by a high degree of to A. corymbifera. Interferon-ϒ (IFN ϒ) and
heterogenicity (Gordon and Taylor 2005; Gordon granulocyte-macrophage colony-stimulating fac-
et al. 2014; Gordon and Plűddemann 2013). tor (GM-CSF) augmented this activity in a time-
None of the available experiments were associ- dependent manner. IFN-ϒ significantly reduced
ated with sinus-resident macrophages and/or interleukin-8 (IL-8) release in response to all spe-
parenchymal macrophages (Ghuman and Voelz cies tested. Treatment of PNLs with the combina-
2017). tion of cytokines enhanced the release of tumour
Although macrophages are unable to kill fun- necrosis factor-α (TNF-α) in response to R.
gal spores, macrophages, monocytes, and human microspores and A. corymbifera but not in
neutrophils can damage and kill fungal hyphae response to R. oryzae hyphae. The authors sug-
without prior phagocytic uptake by means of oxi- gested intergenus differences in host response
dative stress and cationic peptides (Mendoza to Mucorales. Rhizopus hyphae had been shown
et al. 2015; Levitz et al. 1986; Chinn and Diamond to induce the expression of tlr2 mRNA in
1982; Diamond and Clark 1982; Diamond et al. human PNLs, which indicates that Toll-like
134 A. S. Kantarcioglu
of the Hsp70 chaperone family, and proposed IFN-γ. These cytokines have been shown to stimu-
that it was a required host receptor that mediates late proliferation and differentiation of myeloid
and damage of human endothelial cells by R. ory- progenitor cells to neutrophils (G-CSF, GM-CSF)
zae and invasion and damage of endothelial cells or monocytes and eosinophils (GM-CSF), to
was in a receptor-dependent manner. It was upregulate chemotaxis, phagocytosis and respira-
shown that Mucorales internalization was host tory burst of phagocytic cells (neutrophils, mono-
iron dependent (Liu et al. 2010). R. oryzae unger- cytes, macrophages) (G-CSF, GM-CSF, IFN-γ)
minated spores, but not germinated spores or and to regulate/enhance protective T-helper type 1
hyphae, attach to the matrix proteins laminin and (Th1) responses (IFN-γ) (Lohmeyer 1997; Roilides
type IV collagen in vitro (Bouchara et al. 1996). et al. 1998). Most of the data on the role of these
cytokines in modifying host response against the
T-Cell Response to Mucorales Mucorales originate from in vitro and in vivo stud-
In general, Th1-type cell-mediated immunity is ies (Gil-Lamaignere et al. 2005; Liles et al. 1997;
required for clearance of a fungal infection, while Pursell et al. 2003; Saoulidis et al. 2011), clinical
Th2 immunity usually results in susceptibility to evidence on their efficacy as adjunctive treatment
systemic infection or allergic responses. Th1 in patients with mucormycosis remains limited
cells produce predominantly cytokines such as (Roilides et al. 2014).
IFN-ϒ and IL-10 and promote protective cell- Interestingly, fungal morphology also affects
mediated immunity to fungi and phagocyte acti- the chemotactic potential of R. oryzae on neutro-
vation. In contrast, TH2 cells produce phils. Although inactive spores do not induce
predominantly cytokines such as interleukins 3 neutrophil migration, active spores and hyphae
and 4 (IL-3 and IL-4) and tend to promote anti- present a potent chemotactic signal (Waldorf and
body production (Ghuman and Voelz 2017; Diamond 1985). After encountering R. oryzae
Blanco and Garcia 2008). hyphae, human polymorphonuclear neutrophils
Mucorales-specific T cells can be found both activate the expression of Toll-like receptor 2 and
in patients and in healthy individuals. Potenza NF-kB pathway-related genes (Chamilos et al.
et al. (2011) investigated Mucorales-specific T 2008). Additionally, dectin-1-dependent activa-
cells in patients with invasive mucormycosis and tion of interleukin 23 production by human den-
found that Mucorales-specific T cells belonging dritic cells induces pro-inflammatory Th17
to both CD4+ and CD8+ subsets were present dur- responses (Chamilos et al. 2010). In contrast,
ing infection. The authors also investigated macrophages from diabetic or corticosteroid-
Mucorales-specific cytokine profiles and found treated mice fail to inhibit spore germination
that IL-4, IFN-ϒ, IL-10 and IL-17 to be most (Waldorf et al. 1984a, b). Neutrophils from dia-
abundantly produced. Additionally, IFN-ϒ- betic patients with hyperglycaemia and diabetic
producing T cells were demonstrated to induce ketoacidosis retain the ability to damage fungal
Mucorales hyphal damage. hyphae but show reduced responses to R. oryzae
Th17 cells produce IL-17 and implicate in chemotactic factors (Chinn and Diamond 1982).
mucosal immunity against fungi. In vitro stimu- This might interrupt downstream signalling and
lation of T cells by R. oryzae showed the genera- the activation of appropriate downstream cyto-
tion of Th17 cells. Th17 cell production of IL-17 kine responses.
has an impact on neutrophil activity by acting as
a chemoattractant and inducing the production of
antifungal defensins by neutrophils (Chamilos 10.9 Portal of Entry and Routes
et al. 2010). of Infection
Cytokines studied so far include the haematopoi-
etic growth factors, granulocyte colony-stimulating The major mode of transmission might generally
factor (G-CSF) and granulocyte-macrophage be inhalation of aerosolized sporangiospores.
colony-stimulating factor (GM-CSF), as well as Based on radiologic and/or histologic observa-
10 Mucormycosis 137
tions, several routes of CNS invasion have been ment in the absence of sinus involvement had
documented. Mainly, CNS involvement may also been documented mainly in intravenous
occur from haematogenous spread or direct inva- drug abusers (Sundaram et al. 2005; Spellberg
sion, or direct inoculation. et al. 2005). With instance of recycling of used
Mucorales species fungus has a propensity syringes and catheters for i.v. administration,
for growing along the walls of blood vessels. haematogenous inoculation of Mucorales spores
Mucorales are vasotropic and vasoinvasion and can lead to primary CNS lesions, without overt
neurotropism were considered the common predisposing factors.
pathologic features of invasive mucormycosis. Infection with Apophysomyces in previously
Inhaled sporangiospores can be spread to the healthy humans typically develops after trau-
brain by the haematogenous route invading the matic implantation of the spores or inhalation
intracranial vasculature. Cells of Mucorales into the sinuses (Wolkow et al. 2017).
may possess appropriate virulence factors or
specific surface receptors to adhere the endo-
thelial cell surface proteins, to cross the blood- 10.10 Signs and Symptoms
brain-CSF barriers and escape the action of
host mechanisms (Spellberg et al. 2005) and Initial symptoms of RCM are nonspecific, involv-
may extend to CNS via lumens and walls of ing sinus pain, headaches, nasal congestion, altered
vessels. mental status, fever, soft tissue swelling and eye
Apart from angioinvasion in RCM, direct syndrome, lacrimation, irritation or periorbital
spread through cribriform plate of the ethmoid anaesthesia. Unilateral vision disturbance and fur-
bone into the anterior cranial fossa can occur ther changes involving ptosis, proptosis or loss of
(Melsom and Khangure 2000; Hosseini and extraocular muscle function are signs of the pro-
Borghei 2005), and it was suggested that this rep- gressing infection towards the retro-orbital region
resents perineural spread. Mucorales can spread or the CNS. Necrotic black lesions on the hard pal-
up the nerve roots into the CNS (Parsi et al. 2013; ate, necrotic turbinates and septum perforation
McLean et al. 1996; Orguc et al. 2005; Margo should be carefully inspected. Extension to the
et al. 2007). Histologically proven perineural eyes is possible, leading to blurred vision or even
extension of disease from cavernous sinus to complete loss of vision. From the eyes the disease
pons along the trigeminal nerve with no apparent can progress towards the central nervous system
meningeal or intraparenchymal brain involve- resulting in altered consciousness, cranial neuropa-
ment was reported (McLean et al. 1996). thies or cerebral abscesses (Muszewska et al. 2014;
Perineural invasion had been considered unusual Sheikh and Amr 2011; Binder et al. 2014; Spellberg
but contrast-enhanced MRI studies have docu- et al. 2005; Teixeira et al. 2013). The most common
mented perineural invasion via the trigeminal presenting signs and symptoms of ROCM caused
nerve (Hosseini and Borghei 2005; Mohindra by A. elegans are similar to the classic features of
et al. 2007; Ghuman et al. 2015). High percent- those caused by other Mucorales species (Liang
age of perineural invasion in patients with inva- et al. 2006).
sive mucormycosis was reported and RCM progresses rapidly if not treated and
hypothesized that perineural invasion was another extends to neighbouring tissues, causing throm-
possible mechanism involving extension of the bosis of the sphenopalatine vessels in the ptery-
fungi into CNS reported perineural invasion in gopalatine fossa and/or anterior ethmoid vessels
90% of biopsies that contained peripheral nerves in the orbit, nasal ulceration and further necro-
(Cornely et al. 2014; Frater et al. 2001; Sravani sis associated with painful black-coloured
et al. 2014). eschar on the palate or mucosa of the nasal cav-
Direct inoculation through penetrating head ity, septum or osseous walls of the maxillary
injury or during therapeutic or narcotic parenteral sinus, uni- or bilaterally (Hosseini and Borghei
administration was also described. Brain involve- 2005).
138 A. S. Kantarcioglu
10.11 Clinical Presentations Most of the patients with RCM also developed
acute ocular symptoms (Hosseini and Borghei
Rhino-orbito-cerebral disease defines an infec- 2005; Sponsler et al. 1992; Onerci et al. 1991;
tion that originates in the paranasal sinuses with Weprin et al. 1998; Fisher et al. 1991; Langford
gradual and fast extension to the bony structures, et al. 1997; Aköz et al. 1999; Luna et al. 1996) as
eyes, facial soft tissues and the brain in a few a result of invasion of the pterygopalatine fossa
days, following inhalation of spores. The angio- and inferior orbital fissure. Invasion of the optic
invasive nature of these fungi causes cerebral nerve and/or central thrombosis of the retinal
infections and brain abscesses; occasionally it artery results in visual loss. Fundoscopic exami-
may break out into the subarachnoid space nation reveals retinal atrophy secondary to isch-
(Gottfredsson and Perfect 2000). Chronic presen- aemia. Paralysis of the ocular muscles may occur
tation of rhinocerebral mucormycosis was also due to infiltration of the retrobulbar and extraocu-
described with indolent and slowly progressive lar muscles. This paralysis is usually complete,
course, often occurring over weeks to months though at times it may be selective (Brown and
(Sheikh and Amr 2011) (Fig. 10.2). Lau 2001). Both the involvement and lack of
Brain involvement in RCM without sinus involvement of the lamina papyracea (orbital
invasion had been reported in 37 i.v. drug abusers lamina) which is a smooth bone plate forming the
of 64 previously healthy patients. Meningitis due lateral surface of the labyrinth of the ethmoid
to Mucorales spp. in two (Sohail et al. 2001; bone have been reported (Raj et al. 1998; Soto-
Kasliwal et al. 2009) and Absidia corymbifera in Aguilar et al. 1997; deShazo et al. 1997). In such
one patient (Mackenzie et al. 1988) have been patients with ocular signs and no black necrosis
reported. of the nasal mucosa, CT scan of the brain may be
normal, causing a delay in diagnosis. In these
cases, biopsy and frozen sections from the nasal
mucosa and/or sinuses can help achieve the diag-
nosis (Baldin and Ibrahim 2017; Langford et al.
1997; Ibrahim and Kontoyiannis 2013).
10.12 Radiology
a b
c d
Fig. 10.3 (a) CT 1: Axial computer tomography scan of inflammation of the intraconal and extraconal fat and
the sinuses showing mucosal thickening and opacifica- extraoculer muscles extending to zygomatic region and
tion in the maxillary sinuses and ethmoid air cells with invading ipsilateral cavernous sinüse; (d) MR T1C:
bony erosions of the medial wall of the maxillary sinuse Axial contrast enhanced fat suppressed T1 weighted MR
suggesting invasive fungal sinusitis; (b) CT 2: Axial scan showing soft tissue thickening and enhancement in
computer tomography scan showing proptosis and defor- the preseptal region of the right globe extending the
mation of the right globe and soft tissue thickening of zygomatic region, proptosis and deformation of the right
the right preseptal region. Also opacification of the eth- globe. (Courtesy of Civan Islak, Prof, MD, University of
moid air cells; (c): MR T2: Axial T2 weighted MR scan Istanbul-Cerrahpasa, Cerrahpasa Medical Faculty Dept
showing mucosal thickening of the ethmoid air cells, of Radiology)
There were several reported radiologic clues to teristic changes, including sinus opacification,
diagnosis. Plain sinus radiographs seldom reveal inflammatory changes in the paranasal sinuses,
an air-fluid level as seen in acute bacterial sinus- erosion of bone and obliteration of deep facial
itis; however, bony erosion might be found. Both planes. Frontal lobe involvement may show little
CT scan and MRI of the brain may show charac- or no ring enhancement (Meyers et al. 1979).
140 A. S. Kantarcioglu
a b
c d
Fig. 10.4 (a) Axial computer tomography scan showing sfenoid sinüse; (d) Axial T2 weighted MR scan showing
opacification and mucosal thickening of the ethmoid cel- fluid collection, mucosal thickening in right sfenoid
lular airs extending to right sided extraconal fat and right sinuse and ethmoid cellular airs suggesting sinusitis
cavernous sinuse; (b) Coronal nonenhanced T1 weighted extending to extraconal fat and ipsilateral cavernous sinus.
MR scan showing fluid collection, mucosal thickening in (Courtesy of Civan Islak, Prof, MD, University of
right sfenoid sinuse suggesting sinusitis extending to ipsi- Istanbul-Cerrahpasa, Cerrahpasa Medical Faculty Dept of
lateral cavernous sinus; (c) Coronal contrast enhanced T1 Radiology)
weighted MR scan showing collection of fluid in the right
Extension of the infection into the cavernous four case reports (Ghuman et al. 2015; Al-Shafai
sinus, with cavernous sinus thrombosis and inter- and Mikulis 2006; Mathur et al. 2007; Alsuhaibani
nal carotid artery narrowing, was well demon- et al. 2012).
strated on pre- and postgadolinium MRI scans. Linear enhancement on MRI beginning at the
Infarctions in the anterior choroidal artery distri- orbital apex might be correlated with fungal
bution suggested intracranial invasion of the tracking of the trigeminal and lacrimal nerves.
infection, and watershed distribution of infarc- Mucormycosis can spread considerable dis-
tions attested to the tenuousness of flow through a tances from its primary focus of infection along
narrowed cavernous portion of carotid artery peripheral nerves, a phenomenon that can be
(Yousem et al. 1989). MRI demonstrated isch- identified clinically with contrast-enhanced MRI
aemic optic nerve involvement due to ROCM in (Margo et al. 2007).
10 Mucormycosis 141
a b
Fig. 10.5 (a-c) Fungal morphology in imprinted tissue preparations: broad coenocytic hyphae (Giemsa stained, ×100)
Mucorales. BHI broth may be used routinely by of mucormycosis and may be of value for out-
some ophthalmologists for corneal scrapings. break investigation. Identification to the genus
Mucorales will grow rapidly, often filling the and species level is strongly supported for a bet-
entire Petri dish within a few days (Fig. 10.1c, ter epidemiological knowledge of the disease.
d). In any case, negative cultures do not rule out Recovery of Mucorales from cultures of clini-
the infection. A culture result from a non-sterile cal specimens allows not only for diagnosis but
body site is not in itself diagnostic of infection also for the identification of the causative organ-
because these fungi are common in the environ- ism to species level. Direct microscopy is not
ment (Lass-Flörl 2009; Torres-Narbona et al. useful for species identification. Classical identi-
2007a, 2008). fication is based primarily on sporangial mor-
phology. This includes the arrangement and
number of sporangiospores, the shape, colour,
10.14.4 Serology presence or absence of columellae and apophyses
as well as the arrangement of the sporangio-
There are no standardized assays available for the phores and the presence or absence of rhizoids.
detection of Mucorales-specific antigens, and Species can be differentiated by elements such as
serological tests for mucormycosis cannot be rec- rhizoids, stolons and columella which are usually
ommended without further clinical evaluation seen on lactophenol cotton blue-stained slides
and are not available for routine use at this time. under microscope. Lactophenol cotton blue can
1,3-β-d-Glucan is a common component of be used to achieve better visualization (Fig. 10.1d)
the cell wall of a wide variety of fungi but not of (Ribes et al. 2000). Growth temperature studies
the Mucorales. They are galactomannan nega- (25, 37, 45 °C) can be helpful in identifying cul-
tive. Therefore, serum tests based on these fea- ture characteristics. Sporulation may be stimu-
tures are not recommended for the diagnosis of lated by the use of nutrient-deficient media, such
CNS mucormycosis. as cornmeal-glucose-sucrose-yeast extract agar
or Czapek Dox agar.
Carbon assimilation is moderately supported,
10.14.5 Preoperative Cytology and molecular identification is strongly sup-
ported in comparison with morphology. The best
Nonspecific signs of meningitis might be found technique for molecular identification is ITS
in cerebrospinal fluid, although the culture yield sequencing. There are currently limited data for
was extremely low (Jones et al. 1981). MALDI-TOF as an identification method.
Preoperative cytology is an effective technique to Molecular techniques are more reliable than phe-
establish a diagnosis of mucormycosis and obvi- notypic identification of Mucorales in culture to
ates the need for a preoperative biopsy. Frozen the species level. Sequencing of ITS is currently
section is a specific and sensitive method to make the best molecular technique for species identifi-
a quick initial diagnosis of RCM (Sheikh and cation. Carbon assimilation profiles using the
Amr 2011; Lackner et al. 2014; Hofman et al. commercialized kits ID32C and API 50 CH
2003). (bioMerieux, Marcy l’Etoile, France) allowed
precise and accurate identification of Mucorales
to the species level (Schwarz et al. 2007).
10.14.6 Genus and Species Alternative techniques such as matrix-assisted
Identification laser desorption/ ionization time-of-flight
(MALDI-TOF) mass spectrometry are promising
There is no strong evidence that identification to but not yet validated for all species (Cornely et al.
the genus/species level may be important to guide 2014). Although MALDI-TOF identification of
treatment. Identification to the species level is of Mucorales seems promising, more data are
interest for a better epidemiological knowledge needed to validate this technique and commer-
144 A. S. Kantarcioglu
cially available databases should be validated before (Gomez-Lopez et al. 2001; Ortín et al.
(Dolatabadi et al. 2015). 2004; Honda 1998; Zeilender et al. 1990).
Terbinafine was active against all species tested,
except for R. oryzae, M. circinelloides and R.
10.15 Antifungal Susceptibility variabilis. Azole drugs showed various levels of
activity. Itraconazole showed activity against
10.15.1 In Vitro Susceptibility only R. pusillus and Lichtheimia corymbifera.
Testing Similar results have been found in other studies
(Dannaoui et al. 2003; Rogers 2008). Itraconazole
European Committee on Antimicrobial has also shown good activity in animal models of
Susceptibility Testing (EUCAST) and CLSI infection with L. corymbifera (Dannaoui et al.
(M38-A2) (CLSI 2008; EUCAST 2017) refer- 2002; Mosquera et al. 2001). Therefore, itracon-
ence microdilution methods are used as standard azole could be useful for some cases of mucor-
assays for antifungal susceptibility testing of mycosis when susceptible strains are involved.
Mucorales. Using methods other than the refer- Voriconazole has no in vitro activity against these
ence assays such as Etest (Caramalho et al. 2015; fungi. In addition, it has been shown that patients
Torres-Narbona et al. 2007b; Khan et al. 2009) or with leukaemia and bone marrow transplant
XTT assay (Antachopoulos et al. 2006; Spreghini recipients on VRZ prophylaxis can develop
et al. 2010) remains investigational. With these breakthrough infections caused by Mucorales
fungi it is often difficult to achieve accurate and species (Kontoyiannis et al. 2005). Ravuconazole
consistent endpoints. As the interpretive minimal showed some activity against M. corymbifer, R.
inhibitory concentrations (MIC) breakpoints pusillus, R. oryzae, R. microsporus and
have not yet been defined for zygomycetes/ Actinomucor elegans, although isolates resistant
Mucorales and the correlations between clinical to this drug were found in most of the species.
response and MIC values for a given strain are Posaconazole was the azole drug which showed
uncertain, the use of antifungal susceptibility the best in vitro activity (Alastruey-Izquierdo
testing in mucormycosis for routine clinical deci- et al. 2009).
sions is not recommended at this time (Cornely In a retrospective analysis of 16 patients
et al. 2014). Except for posaconazole, moderate infected with A. elegans, an AmB MIC of <1 lg/
(<80%) correlation of Etest and Sensititre mL correlated with recovery. Of those infected
YeastOne with the CLSI M38-A2 method was with strains with an AmB MIC of ≥1 lg/mL, 43%
noted in antifungal susceptibility testing of failed to respond (Chakrabarti et al. 2010).
Mucorales (Cornely et al. 2014; CLSI 2008). Animal studies for determination of in vitro-in
Rapid (within 6–8 h) susceptibility testing can be vivo correlation are also limited. In murine mod-
achieved with the XTT assay (Antachopoulos els of infections due to Rhizopus microsporus
et al. 2006). Currently, there are no validated (Rodríguez et al. 2009) and R. oryzae (Rodríguez
MIC breakpoints for any of the drugs against fun- et al. 2008, 2009), posaconazole was shown to be
gal genera in Mucorales, and so determination of more effective in infections due to strains with an
susceptibility categories (S, I and R) is not pos- MIC of 0.25 lg/mL compared with those with an
sible. A correlation between the generated MIC MIC of 2 lg/mL. On the other hand, a low mini-
and clinical outcome was addressed in only a few mum fungicidal concentration, i.e. 0.5 lg/mL of
studies. posaconazole, was associated with response in
AmB, which is the antifungal of choice for mice infected with R. oryzae. High posaconazole
this mycosis, was the most active agent against minimum fungicidal concentration values, i.e.
all isolates, with the exception of those belonging >16 lg/mL, correlated with clinical failure in a
to the genera Cunninghamella and similar murine model (Spreghini et al. 2010).
Apophysomyces. High drug MICs for Antifungal susceptibility testing of the strains
Cunninghamella species have been reported in the order Mucorales has been performed
10 Mucormycosis 145
mostly for epidemiological purposes. The data However, it was not superior to AmB (0.8 mg/kg/
presented in these studies provide significant day) alone (Rodríguez et al. 2008). In vitro com-
clues for the expected susceptibility profiles and bination studies have also been performed to
are useful to evaluate genus-, species- and strain- explore the interaction of antifungal agents
based variations in susceptibility. Fluconazole, against members of the order Mucorales.
voriconazole, echinocandins and flucytosine lack
meaningful in vitro activity against Mucorales.
In general, AmB and posaconazole are the most 10.16 Treatment and Outcome
active drugs in vitro. The comparative activities
of AmB and posaconazole may vary depending CNS mucormycosis is perhaps one of the most
on the genus and species of the infecting strain. aggressive fungal infections of humans, and the
AmB, posaconazole and isavuconazole are cur- early cases were uniformly fatal. The first survi-
rently the most active agents against Mucorales; vor, reported by Harris in 1955, was a 14-year-
however, their activity remains suboptimal, and old girl with diabetes who was treated successfully
new therapeutic strategies are needed. by rigid control of her diabetes and administra-
Combination therapy could be a promising tion of systemic iodides (Harris 1955).
approach (Dannaoui 2017; Alastruey-Izquierdo AmB was the drug of choice for primary treat-
et al. 2009; Rodríguez et al. 2009; Sun et al. ment of mucormycosis. Antifungal therapy was
2002; Almyroudis et al. 2007; Arikan et al. 2008; considered as essential part of a combined thera-
Vitale et al. 2011). peutic approach also involving surgical debride-
Species-specific differences in azole and terbi- ment of all devitalized tissue and reversal of
nafine susceptibilities are noted particularly for underlying predisposing conditions. AmB deoxy-
Rhizopus and Mucor (Dannaoui et al. 2003; cholate had been used as standard treatment when
Rodríguez et al. 2009; Vitale et al. 2011). Strain- no alternative was available (Walsh et al. 2012;
based variations have also been described, as for Jacobs et al. 2002). The efficacy of AmB has been
posaconazole susceptibility of R. oryzae strains reproducibly demonstrated in both laboratory (in
(Rodríguez et al. 2009). vitro and in vivo) investigations and in clinical
Despite the lack of preference for its use in studies (Antachopoulos et al. 2006; Spreghini
treatment of mucormycosis, itraconazole MICs et al. 2010; Dannaoui et al. 2003; Chakrabarti
are relatively low for a number of strains, includ- et al. 2010; Rodríguez et al. 2009; Sun et al. 2002;
ing those of Rhizomucor (Alastruey-Izquierdo Arikan et al. 2008; Rodriguez-Tudela et al. 2003).
et al. 2009; Vitale et al. 2011) and Lichtheimia Although interpretive breakpoints for determina-
(Dannaoui et al. 2003; Alastruey-Izquierdo et al. tion of in vitro susceptibility to AmB have not
2009; Sun et al. 2002; Vitale et al. 2011). Efficacy been determined, apparent in vitro resistance,
of combination therapy was addressed in murine with elevated MICs, may be observed in clinical
models of mucormycosis. Improved survival was isolates, especially among Cunninghamella spe-
observed with the combination of AmB lipid cies (Alastruey-Izquierdo et al. 2009; Vitale et al.
complex and caspofungin (CAS) compared with 2011; Pastor et al. 2010). These in vitro properties
monotherapy and untreated controls in diabetic are consistent with the poor prognosis of mucor-
ketoacidotic mice infected with a more virulent mycosis caused by Cunninghamella bertholle-
brain isolate of R. oryzae. However, improved tiae, where among 34 reported cases, overall
organ clearance was not achieved with combina- mortality was 76% (Orguc et al. 2005).
tion therapy (Spellberg et al. 2005). In a murine Liposomal AmB (LAmB) may be superior to
model of disseminated mucormycosis caused by AmB lipid complex (ABLC) for CNS infection
R. oryzae, posaconazole was combined with as evidenced by higher CNS concentrations and
AmB at low dose (0.3 mg/kg/day) prolonged sur- more rapid clearance of fungus in animal mod-
vival, and reduced tissue burden was observed els (Groll et al. 2000). The optimal dosage of
compared with monotherapy and controls. lipid formulations is unknown. Based on in vivo
146 A. S. Kantarcioglu
and in vitro data, the initial dosage may be Isavuconazole (ISA) was reported having a
5.0 mg/kg/day. Higher dosages of LAmB (e.g. significant effect in the CNS as demonstrated in
7.5–10 mg/kg/day) may allow for greater CNS animal models (Falci and Pasqualotto 2013).
penetration and may be safely achieved in Peixoto et al. (2014) reported that after failing
immunocompromised patients (Walsh et al. PSZ and being intolerant to AmB, a patient with
2001) and may be appropriate for CNS mucor- disseminated mucormycosis including CNS
mycosis (Walsh et al. 2012; Jacobs et al. 2002). infection was treated successfully with ISA for
Despite this aggressive medical approach, sur- over 6 months. The authors proposed that ISA
gical resection of infected CNS tissue is usually may become an option to treat patients with
necessary. Reversal of immunosuppression, mucormycosis, especially those who cannot tol-
recovery from neutropenia and control of dia- erate AmB therapy.
betes mellitus are also important cornerstones Hyperbaric oxygen (HBO) is sometimes
of therapy of cerebral mucormycosis. Local used as adjunctive therapy in management of
irrigation and intracavitary/interstitial and mucormycosis (Kantarcioğlu et al. 2006). In
intrathecal administration of AmB have been vitro studies had previously demonstrated that
attempted in cases unresponsive to conven- high pressures (10 atm absolute, ATA) of 100%
tional therapy. Intrathecal AmB is not indicated oxygen were reported as fungicidal for some
in treatment of cerebral mucormycosis due to Mucorales, including Rhizopus spp. in vitro
complications that outweigh any putative ben- (Gamaletsou et al. 2012). However, HBO mode
efits (Jacobs et al. 2002). of therapy was not recommended for routine
Although combination treatment with an AmB primary treatment of mucormycosis (Jacobs
formulation and CAS has been described as suc- et al. 2002).
cessful in a limited number of predominantly dia- ESCMID and ECMM Joint Clinical Guidelines
betic patients with ROCM (Reed et al. 2008), low focus on the diagnosis and management of mucor-
penetration of CAS into CSF following intrave- mycosis. Imaging is strongly recommended to
nous administration of standard doses was determine the extent of disease. For adults and
reported in humans (Strenger et al. 2017) con- children, surgical debridement was recommended
firming published data from animal studies. In whenever possible in addition to immediate first-
models of neutropenic and ketoacidotic mice, the line antifungal treatment with liposomal or ABLC
combination of LAmB and posaconazole (PSZ) with a minimum dose of 5 mg/kg/day. AmB
also did not improve survival rates or reduce fun- deoxycholate was recommended better avoided
gal tissue burden (Ibrahim et al. 2009). because of its toxicity and severe adverse effects.
PSZ was the first drug in the azole class to For salvage treatment posaconazole prophylaxis
show a broad spectrum of activity against the 200 mg four times daily was strongly recom-
Mucorales species (Alastruey-Izquierdo et al. mended in this guide. Reversal of predisposing
2009) and had proven to be useful in combination conditions was underlined in patients with ongo-
with AmB to treat RCM or patients for whom ing neutropenia, controlling hyperglycaemia and
treatment with AmB alone has failed (Rueping ketoacidosis in diabetic patients and limiting glu-
et al. 2009; Mullane et al. 2007; Perkhofer et al. cocorticosteroids to the minimum dose required.
2008) hypothesized that inflammatory The authors recommended against using defera-
disturbance of the blood-brain barrier may facili- sirox in haematological patients outside clinical
tate the penetration of PSZ into the CSF and the trials and marginally support a recommendation
cerebral abscess fluid. Based on their clinical for deferasirox in diabetic patients. HBO is sup-
data, the authors proposed that PSZ may be an ported with marginal strength only. Furthermore,
option in the treatment of cerebral fungal infec- continuing treatment until complete response
tions, as has been formerly suggested by the demonstrated on imaging and permanent reversal
promising results from a clinical trial by of predisposing factors was strongly recom-
Pitisuttithum et al. (2005). mended (Cornely et al. 2014).
10 Mucormycosis 147
Blodi FC, Hannah FT, Wadsworth JA. Lethal orbitocer- Cornely O, et al. ESCMID and ECMM joint clinical guide-
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Histoplasmosis
and Coccidioidomycosis 11
María del Rocío Reyes-Montes, Maria Lucia Taylor,
Esperanza Duarte-Escalante,
and María Guadalupe Frías-De-León
long enough to allow the fungus growth or in Blastomyces dermatitidis, Candida glabrata,
body fluids and tissue samples using different Pneumocystis jirovecii, Coccidioides spp.,
stains and direct microscopy. The adequate stain Talaromyces (formerly Penicillium marneffei),
to identify H. capsulatum in clinical samples is Leishmania spp., Toxoplasma gondii, and
calcofluor white. The culture sensitivity for the Trypanosoma cruzi. For the correct identifica-
detection of the fungus depends on the clinical tion, specific histochemical stains such as Gomori
manifestation (pulmonary versus disseminated), methenamine silver (GMS) and periodic acid-
host immunity, and the burden of disease. Patients Schiff (PAS) should be used, which are the most
with disseminated histoplasmosis have a higher useful to visualize H. capsulatum in tissue prepa-
rate of positive cultures (74%) than patients with rations (Fig. 11.1) (Knox and Hage 2010). The
acute pulmonary histoplasmosis (42%) (Hage microscopic examinations, as well as the histopa-
et al. 2015). In patients with HIV/AIDS, the cul- thology, require a good level of experience, in
tures of samples from airways can be positive in order to identify correctly H. capsulatum and dis-
up to 90%, while blood cultures may be positive card similarities with other microorganisms.
in up to 50% (Kauffman 2007). In the histopatho- The serological test used in the diagnosis of
logical examination of H. capsulatum, consider- histoplasmosis is the double immunodiffusion
ation must be given to microorganisms with (ID) which detects the presence of serum anti-
similar morphology, such as Cryptococcus spp., bodies that precipitate in the agar gel after
a b
c d
Fig. 11.1 (a) Numerous yeast cells. Hematoxylin and characteristic macroconidia (echinulate conidia) of
eosin stain; (b) numerous yeast cells PAS stain positive; Histoplasma capsulatum. (From Chang and Meaux
(c) numerous yeast cell of Histoplasma GMS; and (d) (2015), with permission)
11 Histoplasmosis and Coccidioidomycosis 159
bonding with antigens H and M of H. capsula- was designed from random amplified polymor-
tum. The band M is detected in the majority of phic DNA (RAPD) patterns of fungal isolates
patients with acute histoplasmosis (80%) but from different countries in the Americas. This
persists for long periods of time; the band H is marker has been tested through PCR simplex in
rarely seen (20%), but when present, it confirms clinical and environmental samples with good
the acute infection. The ID is an inexpensive specificity (Frías De León et al. 2012). Buitrago
technique but has variable sensitivity and speci- et al. (2013) conducted a multicenter study to
ficity, with predictive values of 86–100% accord- validate the Hcp100, and 1281–1283(220)
ing to the antigen used, and has low sensitivity in markers, in comparison with the amplification
immunocompromised patients who produce low by real-time quantitative PCR (qPCR) of the
levels of immunoglobulins. The complement fix- rDNA ITS1. The authors showed that the
ation (CF) test is more sensitive than the ID tech- marker detected by qPCR was the most sensi-
nique (90% versus 80%, respectively). Through tive, followed by Hcp100 and 1281–1283(220).
CF, a titre 1:8 results positive, indicating prior Therefore, molecular methods are useful for the
exposure to H. capsulatum, while a titre ≥1:32 is histoplasmosis diagnosis, although these meth-
suggestive of active infection (Wheat et al. 1990). ods have not been approved yet for rou-
The ELISA technique is widely used in patients tine clinical use by the Food and Drug
with acute pulmonary histoplasmosis or dissemi- Administration (FDA).
nated histoplasmosis (Kasuga et al. 2003). This
test is used in the detection of antigens in urine
samples and/or serum. The sensitivity and speci- 11.1.6 Treatment Options
ficity of the urinary antigen detection (UAg) and
testing of serum antigens (SAg) vary depending The treatment of CNS histoplasmosis should be
on the type and extent of the infection. The detec- aggressive and prolonged. Daily treatment with
tion of the UAg is more sensitive than the SAg in liposomal amphotericin B 5.0 mg/kg, until add-
patients with disseminated histoplasmosis ing up to a total of 175 mg/kg administered for
(Wheat and Kauffman 2003), so Libert et al. 4–6 weeks, is recommended. Subsequently, treat-
(2018) support the detection of UAg to discard ment should be switched to high doses of itracon-
suspected histoplasmosis. azole (200 mg two or three times a day) or
Nowadays, molecular techniques have had fluconazole (600–800 mg per day) for at least
greater acceptance because of their sensitivity, 1 year, until the detection of antigens becomes
specificity, and speed. In the last two decades, negative in cerebrospinal fluid (CSF). In
several molecular assays using different molecu- patients with severe immunodeficiencies, such as
lar markers for the detection of H. capsulatum in AIDS, maintenance therapy is needed for life
clinical samples through PCR have been devel- (Wheat et al. 2007).
oped (Babady et al. 2011; Bialek et al. 2002;
Bracca et al. 2003; Dantas et al. 2013; Frías De
León et al. 2012; Gago et al. 2014; Guedes et al. 11.2 Coccidioidomycosis
2003; Haynes et al. 1995; Martagon-Villamil
et al. 2003; Tang et al. 2006; Ueda et al. 2003). 11.2.1 Introduction
Due to its high sensitivity and specificity, the
Hcp100 marker is used in the diagnosis of histo- Coccidioidomycosis is caused by the fungi
plasmosis (Bialek et al. 2002). This molecular Coccidioides immitis and C. posadasii (Fisher
marker was validated on clinical samples by et al. 2002). The infection is acquired by suscep-
Muñoz et al. (2010), showing sensitivity and tible hosts through the inhalation of its arthroco-
specificity of 100% and 95.2%, respectively. nidium present in the soil of endemic areas,
Another marker described for the detection of H. which include arid and semiarid regions of the
capsulatum is named 1281–1283(220), which Southwestern USA, Northern Mexico, Central
160 M. del Rocío Reyes-Montes et al.
America, and some regions of South America et al. 2013). However, recent studies suggest that
(Hector and Laniado-Laborín 2005). the geographic range of Coccidioides spp.
Although 60% of the infections are subclini- extends toward the north, particularly in the state
cal and self-limited, approximately 1% of these of Washington, where several cases were reported
result in infections with extrapulmonary dissemi- in 2010 and 2011, without the patients reporting
nation, especially in immunocompromised recent travels to endemic areas (Marsden-Haug
patients, in which the dissemination to the CNS et al. 2013, 2014).
is associated with high morbidity and mortality In the rest of the American continent, the spe-
(Adam et al. 2009). In addition, neurological cies associated with coccidioidomycosis is C.
manifestations caused by Coccidioides spp. in posadasii. However, the actual incidence is
the CNS are variables, from meningitis to menin- unknown because it is not mandatory to report
goencephalitis and meningomyelitis (Adam et al. this disease; furthermore, it has been poorly stud-
2009). CNS coccidioidomycosis should be con- ied. In Mexico, the coccidioidomycosis data is
sidered in the differential diagnosis of patients restricted to the publication of clinical cases and
with neurological symptoms, especially if they retrospective studies in different hospital centers
are immunosuppressed and live in regions where (Baptista Rosas and Riquelme 2007; Laniado-
the disease is endemic. Laborin 2007; Mondragón-González et al. 2005;
Muñoz-Hernández et al. 2004, 2008); however, it
is suggested that the current situation of the dis-
11.2.2 Epidemiology ease may have followed a development like that
of the USA (Nguyen et al. 2013). Therefore,
The geographical distribution of Coccidioides is Hector et al. (2011) propose that the coccidioido-
restricted to the American continent in endemic mycosis is an emerging disease in Mexico due to
regions that include the USA (Arizona, California, the increase in the rate of infection recorded in
New Mexico, Nevada, Utah, Washington, and recent years.
Texas), Mexico, and some areas in Guatemala, In Central and South America, the epidemio-
Honduras, Venezuela, Brazil, Argentina, and logical situation is no different, as there are few
Paraguay (Hector and Laniado-Laborín 2005). In studies on this disease. In Brazil, Cordeiro et al.
the USA, the annual incidence of coccidioidomy- (2009), through a serological survey with 229
cosis is variable; however, it has increased in volunteers in the northeastern part of the country,
recent years with a rate that goes from 5.3 per found a 7.42% of positive samples and showed a
100.000 inhabitants in 1998 to a rate of 42.6 in direct relationship with endemic areas. In
2011 (Centers for Disease Control and Prevention Argentina, Canteros et al. (2010) performed a ret-
(CDC) 2013). The rate of positive skin prick tests rospective study of the coccidioidomycosis cases
for Coccidioides is found in ranges of 50–70% in from 1892 to 2009, finding an increase in the
Kern County, including the City of Bakersfield number of cases in this period, so they proposed
and neighboring counties of Tulare and Kings. that the disease is emerging in the country.
On the other hand, unlike C. immitis, C. posada- In endemic regions of the USA, the most
sii has a wider region of endemicity which affected people are the construction and farm
includes the central and southern Arizona in west workers, military personnel, archaeologists,
Texas and southern New Mexico. The region excavators, inmates, and officers in correctional
with a higher concentration of C. posadasii is facilities. Also, it is known that in these endemic
Arizona, where most of the skin prick tests and regions, the epidemics have occurred after dust
positive cases of coccidioidomycosis occur in the storms, earthquakes, and excavations where the
Maricopa County (including the city of Phoenix), dispersion of arthroconidia is facilitated
Pima (including the City of Tucson), and Pinal (Stockamp and Thompson III 2016).
Counties. It is also present, sporadically, in sites In Mexico, the majority of coccidioidomycosis
in the southern part of Utah and Nevada (Brown cases are acquired in the place of residence
11 Histoplasmosis and Coccidioidomycosis 161
(endemic areas), although some cases are related without eliminating them. There is evidence that
to migration to the USA, where the disease is the phagosome-lysosome fusion does not occur,
acquired (Baptista Rosas and Riquelme 2007). thus preventing the arthroconidia to come into
While in Brazil, an association between the infec- contact with the lytic enzymes of the PMN (Frey
tion caused by Coccidioides spp. and armadillo and Drutz 1986). Hence, the arthroconidia grow
(Dasypus novemcinctus) hunters in endemic areas and transform into immature spherules, which
has been demonstrated (Brillhante et al. 2012). induce phagocytosis and the production of ROS,
The main risk factor for acquiring the infec- TLR-2, and Dectin-1, as well as the signals medi-
tion and developing the disease is exposure to ated by MyD88 and intracellular adapters, which
dust by occupational or recreational causes in lead to the activation of the transcription factor
endemic areas. It has also been described that the NF-kB that produces proinflammatory cytokines
disease is more common in men, as well as in such as TNF and macrophage inflammatory pro-
African Americans and Filipinos, and in patients tein 2 (MIP-2) and interleukin 6 (IL-6), which are
with immunocompromised caused by different essential effectors of the cellular response of the
reasons such as HIV, transplant recipients, and Th1 and Th17. The dectin-1 also mediates the
renal failure, among others (Laniado- Laborin production of cytokines Th1 and Th17, IL-23,
2007). IL-17A, IL-22, IL-12, and IFN. In addition, the
A recent work concludes that the major risk spherules increase the expression of the RNAm
factors for the development of disseminated coc- and other factors for evasion in the host. In the
cidioidomycosis are exogenous immunosuppres- following days (two or three), the spherules
sion (caused by steroids biological products), undergo a nuclear division and mature into large
pregnancy, race/ethnicity, and discrete genetic septate cells (30–80 μm) that contain endospores.
defects, such as defects in the interleukin-12/ These spherules are too large to be phagocyted;
IFN-γ pathway and the signal transducer and acti- besides, they produce an alkaline extracellular
vator of transcription 3 (STAT3) (Odio et al. 2017). matrix (ECM) that prevents contact with PMN. In
the fourth or fifth day, the spherules break to
release mature endospores. Host response is to
11.2.3 Pathogenesis and Pathology attract PMNs and macrophages, which phagocyte
the endospores easily. However, phagocytosis can
The initial encounter between Coccidioides spp. present some drawbacks, as the endospores can
and the host begins when the arthroconidium of remain in large bunches joined by fibrillary struc-
the fungus is inhaled by a susceptible host, who tures coming from the outside wall of the spherule
visits or lives in endemic areas, and they reach the and protected by the ECM. From this moment on
lungs and alveoli. Epithelial cells of the lung are continues the parasitic cycle of the fungus in the
equipped with pattern recognition receptors host (Smith et al. 2013). Furthermore, dissemina-
(PRRs), such as Toll-like receptors (TLRs) and tion of the fungus can occur through the blood-
dectin-1, which are capable of inducing immedi- stream or lymphatic stream toward any anatomical
ate effector responses besides influencing the site of the host, including the CNS (Johnson and
regulation of alveolar macrophages. In the lungs, Einstein 2006), frequently causing meningitis, a
the innate immune cells recognize components of clinical picture of great relevance due to the high
the fungus through multiple receivers, inducing rates of morbidity and mortality.
phagocytosis and the production of reactive oxy-
gen species (ROS). In a matter of hours,
Coccidioides induces an influx of activated poly- 11.2.4 Clinical Features
morphonuclear (PMN) cells, which, at the same
time, can increase the formation of spherules. The Meningitis caused by Coccidioides spp. is the
PMN responds in a similar manner to macro- most important disseminated form, due to the
phages, that is, surrounding the arthroconidia high risk that represents for the host. Meningitis
162 M. del Rocío Reyes-Montes et al.
occurs in almost one-third of the immunocom- results of images. In particular, the symptoms of
promised patients, in most cases, in weeks or CNS invasion by Coccidioides spp. are non-
months after the primary infection. The most specific, so the diagnosis is complicated. On the
common symptom is headache. The patient can other hand, biopsies of the CNS are considered
have altered mental states, with or without fever, too risky in severely ill patients, especially in
personality changes, nausea, and vomiting. In populations of patients with hematological low
addition, walking abnormalities and neurologic platelet counts or neutropenia. However, the
deficits may occur in some cases (Johnson and biopsy material stained with hematoxylin-eosin
Einstein 2006; Sharma 2010). On the other hand, (HE), GMS, or PAS is useful for the diagnosis
there may be some complications of meningitis, (Góralska et al. 2018).
which include hydrocephalus, myocardial infarc- Coccidioidal meningitis is usually diagnosed
tion, vasculitis, and abscesses. The vasculitis is of by the detection of anti-Coccidioides antibodies
great importance since it is characterized by cere- in the CSF, in an 80% of cases, through sero-
bral infarction due to inflammatory changes in logical tests as ID, CF, or enzyme immunoassay
the walls of small- and medium-sized brain arter- (EIA) (Kassis et al. 2015; Stockamp and
ies and veins that lead to vascular insufficiency Thompson III 2016). In addition, the analysis of
and neurologic deficits that threaten the patient’s CSF usually shows an elevated white blood cell
life (Tager et al. 2017). In addition to meningitis count with a mixed or lymphocytic pleocytosis
and vasculitis, brain inflammation can occur as a and a high level of protein (occasionally quanti-
result of the fungus dissemination along the walls fiable in grams per deciliter instead of milli-
of vessels or perivascular spaces (Shih and grams per deciliter) and a low blood glucose
Koeller 2015). It has also been described that it is level.
common for coccidioidomycosis to extend into A recent study showed that the quantitative
the spinal cord as microscopic foci of the disease, test of Coccidioides antigen detection in CSF,
although it is rare that discrete lesions develop in performed in microtiter plates coated with anti-
the CNS (Bañuelos et al. 1996). Coccidioides antibodies, is effective in the diag-
On the other hand, the pattern of neuroimag- nosis of coccidioidal meningitis, as it showed a
ing of coccidioidomycosis is similar to that of 93% sensitivity and a 100% specificity (Kassis
meningitis caused by tuberculosis, which shows et al. 2015).
a thick exude and an abnormal increase in basal Although the imaging studies are useful to
cisterns and the subarachnoid space which can evaluate the complications associated with men-
further evolve into hydrocephalus or vasculitis ingitis, the initial characteristics of the disease
(Shih and Koeller 2015). It has also been can be difficult to distinguish from other causes,
described the presence of subarachnoid hemor- especially with tuberculosis and even autoim-
rhage as a result of granulomatous inflammation mune diseases, without detailed evidence
of large vessels (Shih and Koeller 2015). (Stockamp and Thompson III 2016).
According to the criteria of the European In the case of CNS coccidioidomycosis, flucon-
Organization for Research and Treatment of azole has become the most commonly used pri-
Cancer/Invasive Fungal Infections Cooperative mary therapy at a dose of 400 mg/day, although
Group and the National Institute of Allergy and higher doses have been suggested which can
Infectious Diseases Mycoses Study Group range from initial doses of 400 mg/day or 800–
(EORTC/MSG), the diagnosis of invasive fungal 1200 mg every 24 h, as they can decrease the rate
infections is made based on a combined interpre- of therapeutic failure. Also, the itraconazole has
tation of risk factors, clinical symptoms, and been used as primary therapy, usually in doses of
11 Histoplasmosis and Coccidioidomycosis 163
200 mg every 12 h, with fatty foods and an acidic capsulatum DNA in human tissue. J Clin Microbiol.
drink to increase absorption. However, the intra- 2002;40:1644–7.
Bongomin F, Gago S, Oladel RO, Denning DW. Global
thecal amphotericin B deoxycholate is the origi- and multi-national prevalence of fungal diseases-
nal gold standard. It can be administered via estimate precision. J Fungi (Basel). 2017;3(4):57.
direct lumbar or cisternal injection, and it is cur- Bracca A, Tosello ME, Girardini JE, Amigot SL, Gomez
rently used as rescue therapy upon failure of the C, Serra E. Molecular detection of Histoplasma cap-
sulatum var. capsulatum in human clinical samples. J
azoles. The therapy often begins with low doses Clin Microbiol. 2003;41:1753–5.
that increase gradually unless the patient experi- Brillhante RS, Moreira Filho RE, Rocha MF, Castelo-
ence symptoms or signs of toxicity (Galgiani Branco Dde S, Fechine MA, Lima RA, Picanço YV,
et al. 2016). Cordeiro Rde A, Camargo ZP, Queiroz JA, Araujo
RW, Mesquita JR, Sidrim JJ. Coccidioidomycosis in
armadillo hunters from the state of Ceará, Brazil. Mem
Inst Oswaldo Cruz. 2012;107(6):813–5.
11.3 Conclusion Brown J, Benedict K, Park BJ, Thompson GR
III. Coccidioidomycosis: epidemiology. Clin
Epidemiol. 2013;5:185–97.
Meningitis caused by Histoplasma spp. and Buitrago MJ, Cuenca-Estrella M. Current epidemiology
Coccidioides spp. has increased in the last and laboratory diagnosis of endemic mycoses in Spain.
decades. These mycoses have a high morbidity Enferm Infecc Microbiol Clin. 2012;30(7):407–13.
and mortality mainly due to a delay in the diag- Buitrago MJ, Canteros CE, Frías De León G, González
Á, Marques-Evangelista De Oliveira M, Muñoz CO,
nosis and treatment failure. The success of the Ramirez JA, Toranzo AI, Zancope-Oliveira R, Cuenca-
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ing a high index of clinical suspicion as well as Histoplasma capsulatum DNA through a multicenter
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Canteros CE, Toranzo A, Ibarra-Camou B, David V,
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N, Capece P, Gorostiaga J, Chacón YA, Tonelli R,
Boscaro G, Abiega C, Mendieta S, Fernández C,
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Cryptococcosis
12
Anita Mahadevan and Shankar Krishna Susarla
AIDS Acquired immunodeficiency syndrome A large number of fungi can cause meningitis in
ART Antiretroviral therapy humans, which include yeasts (such as
BBB Blood-brain barrier Cryptococcus and Candida), filamentous fungi
CM Cryptococcal meningitis (such as Aspergillus and Zygomycetes), dematia-
CNS Central nervous system ceous molds (Phaeohyphomycetes), and dimor-
CSF Cerebrospinal fluid phic fungi (such as Histoplasma spp., Coccidioides,
DC Dendritic cell Paracoccidioides, and Blastomyces). They are
EFA Early fungicidal activity uniformly fatal unless detected early, with institu-
HAART Highly active antiretroviral therapy tion of prompt and effective treatment.
GalXM Galactoxylomannan Cryptococcus is considered an opportunistic
GXM Glucuronoxylomannan pathogen as it causes disease in the immunocom-
HIV Human immunodeficiency virus promised host. This so-called “sugar-coated” yeast
IL Interleukin is a unique model of eukaryotic virulence which
INOS Inducible nitric oxide synthase has helped in understanding fungal pathogenesis,
IRD Immune restoration disease developing robust diagnostic tests, and standard-
IRIS Immune reconstitution inflammatory izing treatment modalities.
syndrome
MP Mannoprotein
MRI Magnetic resonance imaging 12.2 Epidemiology
TNF Tumor necrosis factor
There have been three waves of epidemics in this
infection. The first peak in incidence was in the
mid-1980s with the advent of acquired immuno-
deficiency syndrome (AIDS). Cryptococcal men-
ingitis (CM) was reported in 8% of patients with
human immunodeficiency virus (HIV)/AIDS in
the USA and as much as 40% of these patients in
A. Mahadevan (*) · S. K. Susarla other parts of the world (Powderly 2000). A
Department of Neuropathology, National Institute recent epidemiologic analysis projected that
of Mental Health and Neurosciences (NIMHANS), there are around one million cases of c ryptococcal
Bangalore, India
meningitis in AIDS patients each year that are Genomic analyses established that serotypes A
responsible for over 600,000 annual deaths (Park and D are distinct strains, called C. neoformans
et al. 2009). var. grubii and C. neoformans var. neoformans,
In India, CM is reported as the most common respectively. C. neoformans var. grubii (serotype
opportunistic infection of the CNS in patients A) is the most common causative agent of the cen-
with HIV/AIDS (Wadia et al. 2001; Satishchandra tral nervous system (CNS) disease in HIV/AIDS
et al. 2000). At our institute in south India, cryp- with high mortality and morbidity. The different
tococcal meningitis at autopsy was seen in 31.3% strains have varying epidemiological features. In
of cases (Shankar et al. 2005). Its overall inci- low-income countries, most cases of cryptococco-
dence declined with the availability of highly sis are caused by serotype A (C. neoformans var.
active antiretroviral therapy (HAART), though grubii), whereas, in the West, it is restricted to
cryptococcosis is responsible for 15–44% of infections in HIV-positive patients only. In con-
deaths in HIV/AIDS patients in sub-Saharan trast, serotype D (C. neoformans var. neoformans)
Africa and remains one of the most common is a predominant strain found in Western Europe
AIDS-defining illnesses in India, Brazil, and and is uncommon in the rest of the world. In our
Thailand. institute in south India, Cryptococcus neoformans
A second outbreak started with the increase in was the common isolate in HIV-associated crypto-
successful solid organ transplantation and the coccosis. The majority of isolates from HIV-
expanding armamentarium of immunosuppres- positive patients from India are serotype A var.
sive drugs for treatment of cancers and connec- grubii consistent with the serotype prevalent
tive tissue diseases such as anti-tumor necrosis worldwide (Banerjee et al. 2004; Kwon-Chung
factor (TNF), anti-CD54 monoclonal, antibodies, and Bennett 1984) and a few reports of var. gattii
etc. (Pyrgos et al. 2013). C. neoformans accounted (Chakrabarti et al. 1997; Banerjee et al. 2001).
for 10% of fungal infections in transplant recipi- C. gattii (serotypes B and C) causes 70–80%
ents (Singh and Forrest 2009; Hosseini- of cryptococcal infections among immunocom-
Moghaddam and Husain 2010). petent hosts restricted to tropical and subtropical
A third outbreak of infection with C. gattii areas. However, a recent outbreak (1999–2003),
occurred in Vancouver and Pacific Northwest involving patients with apparently normal
islands (1999–2003) that has been attributed to immune system, was reported in some areas of
the emergence of hypervirulent C. gattii strains Canada and Northwest USA. The incidence of
due to climatic changes (Billmyre et al. 2014). cryptococcal infection does not significantly dif-
fer in relation to age, race, or occupation (Kidd
et al. 2004).
12.3 Mycology
In our institute, a study conducted by the Paradoxically, treatment efforts to reverse the
department of Neuromicrobiology revealed that immunodeficiency, e.g., through antiretroviral
the predominant source of cryptococci in the therapy, worsen symptoms through an exuberant
environment in our geographical location was inflammatory response: the immune reconstitu-
pigeon excreta (33%) followed by soil contami- tion inflammatory syndrome (IRIS).
nated with pigeon/bird droppings (13%), while There are three basic steps for fungi to suc-
soil surrounding eucalyptus trees, the tree bark cessfully infect the brain—establish local infec-
and leaves of eucalyptus tree was less common tion, disseminate into the bloodstream, and cross
(Nagarathna et al., personal communication). the blood-brain barrier to reach the CNS.
Predilection for these ecological niches is
attributed to the requirement for certain com-
pounds in the environment for its replication. The 12.5.1 Step 1: Local Infection
human fungal pathogen Cryptococcus can com-
plete its sexual cycle during a pathogenic associ- 12.5.1.1 Host Factors
ation with plants, as the inositol found in plants Basidiospores of cryptococci that are the result
stimulates sexual reproduction in Cryptococcus of sexual reproduction are the infectious forms
species accounting for the predilection for growth of the yeast that reach the pulmonary alveoli fol-
in eucalyptus trees (Xue et al. 2007). Similarly lowing inhalation. Containment of the fungus
sexual reproduction of C. neoformans occurs in within the lung requires cell-mediated, innate
media enriched with pigeon guano, accounting immunity, and antibody responses (Eisenman
for its ecological niche in pigeon droppings et al. 2007). The first line of defense is phagocy-
(Nielsen et al. 2007). tosis mediated by macrophages and complement
The recent outbreak of C. gattii infection in (Feldmesser et al. 2001). Other factors include
immunocompetent individuals in Vancouver CD4+ and CD8+ T cells and cytokines including
Island, Canada, and the Pacific Northwest suggests TNF-α, interferon (IFN)-γ, and interleukin (IL)-
evolution of host range, geographic location, and 18 (Huffnagle and Lipscomb 1998). The first
virulence of this pathogen and its emergence as a critical step for successful control of C. neofor-
travel-associated pathogen (Byrnes et al. 2009). mans infection in the lung is polarization of
CD4+ T cells toward T-helper 1 (Th1) phenotype
by IFN-γ production and monocyte-derived den-
12.5 Pathogenesis dritic cell (DC) recruitment (Drummond 2017).
Th1 cells stimulate “classic activation” of mac-
C. neoformans is an opportunistic pathogen as rophages (M1) and production of inducible nitric
immunosuppressed individuals are more suscep- oxide synthase (iNOS) for fungal killing (Hole
tible to infection. The fungal structure is respon- and Wormley 2016). Phagocytosis by alveolar
sible for establishing an infection and survival in macrophages is initiated by antibody and com-
the human host, while its virulence factors con- plement mediated (opsonin/non-opsonin depen-
tribute to pathogenicity. However, the outcome of dent) interaction between epitopes on the
the infection is determined by the immune status cryptococcal polysaccharide capsule with the
of the host. The most devastating infections cell surface receptors—such as mannose, dec-
develop in patients with defective cell-mediated tin-1, CD14, and Toll-like receptor 4 (Garcia-
immunity, such as AIDS, solid organ transplanta- Rodas and Zaragoza 2012). Once phagocytosed,
tion, reticuloendothelial malignancy, corticoste- the phagosomes containing cryptococci fuse
roid treatment, long-term immunosuppressive with lysosomes, and CD4+ T cells stimulate kill-
therapy, advanced renal and liver diseases, rheu- ing via IFN-γ and iNOS. CD4+ T-cell depletion
matological diseases, and sarcoidosis, but not in as occurs in AIDS prevents killing of the crypto-
subjects with neutropenia or immunoglobulin cocci within macrophages, allowing it to survive
deficiency (Buchanan and Murphy 1998). and replicate.
170 A. Mahadevan and S. K. Susarla
response (Zaragoza et al. 2009). Capsular forma- recover their immune system following the ini-
tion inhibits antibody (Ab) production, depletes tiation of the HAART. An exaggerated inflam-
complement, inhibits leukocyte migration, and matory response is elicited, producing sudden
induces apoptosis in macrophages and T cells worsening of symptoms and deterioration. IRIS
(Lipovsky et al. 2000; Monari et al. 2006; is common following infection with
Ellerbroek et al. 2002). The striking variations in Mycobacterium tuberculosis and C. neofor-
capsule structure and size result in multiple phe- mans (French 2009).
notypic forms that differ in their recognition by
the host immune system (Zaragoza 2011).
The three morphological and phenotypic vari- 12.5.5 Capsule Structure
ations include changes in capsule structure, size,
and the total size of the cell, by the formation of The major component of the cryptococcal poly-
cryptococcal giant/titan cells or microforms. saccharide capsule is the high molecular weight
These changes facilitate fungal survival, adapta- complex polysaccharide, glucuronoxylomannan
tion, escape from immune destruction and phago- (GXM), constituting 90–95% of the total mass
cytosis, dissemination, and long-term survival. (Cherniak et al. 1980). The remaining 5–10% is
formed by galactoxylomannan (GalXM)
(Cherniak et al. 1982). GXM has a chain of man-
12.5.4 Capsule Size: Phenotypic nose residues, with substitutions of xylose and
Switching glucuronic acid, while GalXM has a chain of
galactose with substitutions of xylose and man-
Variation in capsule size can occur in different nose along with a small proportion of mannopro-
stages of the disease. Following first contact teins (MPs) (Levitz and Specht 2006). The GXM
with the host, there is capsule enlargement is distributed throughout the whole capsule,
(“early” response) to escape phagocytosis and whereas GXMGal and MPs localize close to the
ensure intracellular survival. The capsular rear- cell wall (De Jesus et al. 2010).
rangements prevent recognition by immune The C. neoformans capsule is a double-edged
cells. It also assists in systemic dissemination sword. While on the one hand, it can protect the
and crossing of the BBB. Production of fungus from host defenses, on the other hand, it
micro-cells facilitates both dissemination and promotes effective clearance of the fungus from
brain invasion. In later stages, there is forma- tissues. The capsule protects C. neoformans from
tion of fungal giant/titan cells (a “late” phagocytosis and killing by neutrophils, mono-
response), a key factor that ensures long-term cytes, or macrophages (Kozel and Mastroianni
survival. In immunocompetent hosts, the infec- 1976; Kozel and Gotschlich 1982; Vecchiarelli
tion is controlled, but the fungal cells are not et al. 1994a). The capsule being strong negatively
completely cleared and go into a dormant/latent charged causes electrostatic repulsion of the neg-
state (Dromer et al. 2007). The variation of atively charged host effector cells, reducing cell-
Cryptococcus cell morphology as smooth, cell interactions required for clearance of the
mucoid, or wrinkled form has been described cryptococci (Nosanchuk and Casadevall 1997).
(Guerrero and Fries 2008). The smooth C. gat-
tii cells produced smaller capsules that were 12.5.5.1 Role of Capsule in Promoting
more efficient in crossing the BBB to cause Host Defense:
CNS infection (Jain et al. 2006). The large Immunosuppressive Effect
number of different yeast forms also plays a The capsules of serotype A and D isolates are
role in the immune reconstitution inflammatory chemotactic for neutrophils, and complement is
syndrome (IRIS) or immune restoration disease fixed by cryptococcal capsules by the alternative
(IRD). This occurs in HIV patients who rapidly pathway (Kozel and Pfrommer 1986). C3b and
172 A. Mahadevan and S. K. Susarla
C3bi once deposited on the surface of the crypto- phenoloxidase activity (Shaw and Kapica 1972).
cocci trigger phagocytosis by binding to CR3 Conversion of dihydroxyphenols such as 3,4-dihy-
receptors on leukocytes (Griffin 1981). The cryp- droxyphenylalanine (DOPA) to dopaquinone is
tococcal capsule can mask the C3b and C3bi catalyzed by a phenoloxidase and is a rate-limit-
deposits, thereby preventing complement-ing step. C. neoformans lacks the tyrosinase
mediated killing (Kozel et al. 1984). The capsule enzyme required for endogenous production of
can also prevent opsonization by antibodies to dihydroxyphenol. Hence, to produce melanin, a
GXM, by blocking the Fc portion of the antibody C. neoformans isolate has to find diphenolic com-
(Kozel et al. 1988). The thicker the capsule, the pounds from its growth environment and catalyze
greater is its protective capacity. conversion of these compounds into melanin
Encapsulated isolates effectively impair T-cell intermediates. The brain is rich in catecholamines
response (Collins and Bancroft 1991), especially such as DOPA and is a favorite target for infection
the proliferative responses in T cells due to by C. neoformans. However, the brain is not a pre-
reduced secretion of interleukin-1 (IL-1) ferred niche for growth as the organism cannot
(Vecchiarelli et al. 1994b) and proinflammatory use catecholamines as a sole carbon source
cytokines such as TNF-α, IL-1ß, and IL-6 by (Polacheck et al. 1990) but may serve as a place
monocytes and macrophages (Delfino et al. 1996, for the organisms to survive. Production of mela-
1997). The reduction in TNF-α in infections in nin from brain catecholamines protects the fungus
turn prevents the induction of protective immu- from oxidative damage by scavenging free radi-
nity, with disease progression. cals. But melanized yeast cells are less susceptible
Minimal host tissue responses are observed in to amphotericin B contributing to the difficulty to
patients with disseminated cryptococcosis. This effectively treat infections in immunocompro-
is due to inhibition of leukocyte migration, by the mised hosts (Wang and Casadevall 1994). Melanin
capsular products GXM, GalXM, and MP (Dong also “cloaks” C. neoformans from recognition by
and Murphy 1995). GXM stimulates neutrophils host effector cells to mount a protective T-cell
to shed L-selectin, necessary for initiating neu- response (Huffnagle et al. 1995). Fortunately
trophil migration into tissues (Dong and Murphy though, phenoloxidase activity in C. neoformans
1996). The levels of cryptococcal antigen in is greatly diminished at 37 °C body temperature;
serum and CSF correlate with the severity of thereby, there is no melanin production in vivo
disease, response to therapy, and prognosis (Jacobson and Emery 1991) as demonstrated with
(Diamond and Bennett 1974). Denning et al. sug- Fontana-Masson stains (Kwon-Chung et al.
gested that high cryptococcal antigen concentra- 1981). Laccase, a key enzyme in melanin biosyn-
tions could change the osmolality of the CSF, thesis, plays a role in virulence (Zhu and
affecting outflow and adsorption of CSF via the Williamson 2004).
arachnoid villi and increasing intracranial pres- Production of d-mannitol, a potent scavenger
sure. The increased pressure may be responsible of hydroxyl radicals, may contribute to the sur-
for the severe headaches, visual loss, and early vival of C. neoformans in the host as it protects
death (Denning et al. 1991). The release of man- from heat, osmotic stress, and damage by reac-
nitol by C. neoformans contributes to increased tive oxygen intermediates (Wong et al. 1990).
intracranial pressure and restricting its use in High concentrations of d-mannitol in the CNS
reducing CSF pressure (Staib 1962). may also contribute to brain edema. It is not
A characteristic that differentiates pathogenic known whether different isolates of C. neofor-
isolates from nonpathogenic isolates of mans vary in mannitol production.
Cryptococcus species is the organism’s ability to Similarly, production of superoxide dismutase
form a brown to black pigment on a medium (SOD) by cryptococci, which is scavenger of free
(such as birdseed or caffeic acid agar) containing radicals, is increased at 37 °C which compensates
diphenolic compounds. This melanin-like com- for the decrease in melanin production (Jacobson
pound is produced by C. neoformans isolates with et al. 1994).
12 Cryptococcosis 173
Xu et al. 2014). Occludin, marker of tight junc- tissue of the lung and arteries (Markaryan et al.
tions, rapidly degrades altering integrity of endo- 1994). Steen et al. identified a similar metallo-
thelial tight junction. protease in a rabbit model of cryptococcal men-
The reason for the remarkable neurotropism ingitis (Steen et al. 2003). (Mpr1) in the
for the CNS is not clear. Experimental model of extracellular proteome of C. neoformans
systemic cryptococcosis that closely mimics (CnMpr1) also belonging to the same M36 class
human infection has provided valuable insights of fungalysins (Vu et al. 2014). C. neoformans
into the pathogenesis. Cryptococcus is a faculta- lacking MPR1 (mpr1Δ) failed to cross the BBB
tive intracellular pathogen. It survives within due to its failure to adhere to the brain
macrophages and replicates and escapes the hos- endothelium.
tile host environment (Feldmesser et al. 2000).
Yeast cells escape from phagocytic cells by active
phagosomal extrusion and invasion of other 12.6 Pathology
macrophages.
The capsular polysaccharide, mannitol, the Infection is initiated by inhalation of the sexual
mating type, melanin, phenotypic switching, form of yeast cells (measuring <4 μm dia meter)
phospholipase, prostaglandins, and urease are from the environment of dust containing excreta
important for neurotropism and CNS invasion of pigeons. There is no human-to-human
(Perfect and Casadevall 2002). Fungal cell mor- transmission.
phology and capsule morphology are important The organism is a basidiomycete (Filobasidiella
in crossing the BBB. The giant/titan forms can- neoformans) that exists in two forms: the vegeta-
not be phagocytosed, preventing systemic tive form, as a yeast measuring 2.5–10 μm dia
dissemination as well as BBB invasion. The
meter, and the sexual form (basidiospores), mea-
Cryptococcus cell morphology can be smooth, suring 1.8–3 μm in diameter. The basidiospores,
mucoid, or wrinkled. The smooth C. gattii cells which are the infectious form of C. neoformans,
are more efficient in crossing BBB due to their which is a desiccated form of the yeast, being
smaller capsules (Guerrero and Fries 2008; Jain smaller than 4 μm, can enter the pulmonary alve-
et al. 2006). oli to establish pulmonary infection (Velagapudi
Other cell surface and secreted proteins et al. 2009).
important for CNS invasion include extracellular
phospholipase B (Feldmesser et al. 2000; Cox
et al. 2001; Maruvada et al. 2012), urease 12.7 Pulmonary Infection
(Olszewski et al. 2004), and laccase (Qiu et al.
2012). Ligand-receptor interaction between cap- The inhaled spores lodge with the alveolar spaces
sular hyaluronic acid and CD44 receptor pro- of the lung. Cryptococcus can colonize the respira-
motes adhesion of vascular endothelium in the tory tract. Once in the lungs, the yeast cells become
brain (Jong et al. 2008; Long et al. 2012). A pro- rehydrated and acquire the characteristic polysac-
teomic approach examining the cryptococcal cell charide capsule and convert to encapsulated blasto-
surface proteins identified an assortment of pro- conidia (Fig. 12.1f). The encapsulated yeasts, by
teases and free radical-inducing proteins modu- the antiphagocytic and immunosuppressive proper-
lating CNS invasion (Long et al. 2012; Eigenheer ties of the capsule, avoid phagocyte recognition
et al. 2007). One of the key candidate proteins is and prevent leukocyte migration allowing fungal
a fungalysin metalloprotease belonging to the replication. In the presence of intact T-cell immu-
M36 peptidase class of proteases mediating host- nity, the primary pulmonary infection may be in
fungus interaction that are unique to some fungi. dormant form or mimic influenza-like respiratory
The fungalysin class of metalloprotease in A. infection that resolves spontaneously. An epidemi-
fumigatus breaks down elastin in the connective ological survey found that almost all adults in
12 Cryptococcosis 175
a b
c d
f
e
Fig. 12.1 Systemic involvement in disseminated crypto- shows collections of cryptococci in the renal cortex,
coccosis. (a) Para-aortic lymph nodes enlarged and infil- extending into the medulla along medullary rays (*). (e)
trated by cryptococci (*). Also note small cryptococcal Budding yeast forms of cryptococci highlighted on peri-
nodules on subcapsular surface of the right kidneys odic acid-Schiff stain. (f) Electron micrograph demon-
resembling tubercles. (b) Cryptococcal lymphadenitis strating cryptococcal capsule and the intracellular
involving mesenteric nodes (*). (c) Pulmonary cryptococ- organellar details (d: H&E stain ×Obj.10; e: PAS stain
cal lesions seen as small honeycomb-like aggregates of ×Obj.40; f: Uranyl acetate-lead citrate ×14,000)
cryptococci (*). (d) Photomicrograph from the kidney
176 A. Mahadevan and S. K. Susarla
a b
c d
e f
g h
Fig. 12.2 Cryptococcal meningitis. Thick, glistening, copy as expansion of Virchow-Robin spaces by
gelatinous mucoid exudate seen along the superolateral collections of cryptococci (e, f, *). The cryptococcal cell
convexity of the brain (a, *). The cryptococci are filling wall and budding forms are highlighted by periodic
the arachnoidal granulations along the dural venous acid-Schiff stain (g) and Gomori methenamine silver
sinuses (b, arrows). On microscopy, numerous budding (g), while the mucopolysaccharide-rich capsule of
yeast forms are seen phagocytosed by macrophages and Cryptococcus is highlighted on mucicarmine stains (h).
giant cells (arrows) in subarachnoid space (c). Multiple Note the marked phenotypic variation in sizes and cap-
punched-out pseudocystic parenchymal lesions are seen sule thickness of the yeasts (c, e, f: H&E stain; g: PAS
in caudate (C), putamen (P), and thalamus producing a stain; h: Gomori methenamine silver stain; i: Mucicarmine
soap bubble appearance (d, *). These are seen on micros- stain. Magnification = scale bar)
178 A. Mahadevan and S. K. Susarla
pathology of IRIS is complex but revolves around fungal therapy, with increased CSF fungal load
ART-triggered recovery of the immune response at the end of antifungal therapy. This high resid-
in a previously immunosuppressed tissue envi- ual cryptococcal antigen load probably drives
ronment with high microbial loads, and conse- IRIS when CD4 counts rise due to antiretroviral
quent potential immunopathology reflected by therapy (ART).
florid inflammatory response with predominant Scriven et al. (Scriven et al. 2017) hypothe-
CD8+ T cells. size that the shift in macrophage phenotype is
Cryptococcal IRIS occurs in 10–20% of HIV driven directly by reduction in the HIV viral
patients with median period of 4–9 weeks follow- load and high CD206 expression. CD206 (man-
ing ART initiation and presents with recurrence nose receptor) is a marker of “alternative activa-
of signs and symptoms of meningitis (Jarvis et al. tion” of macrophages, stimulated by T-helper 2
2014). Risk factors for development of IRIS (Th2) CD4+ T cells producing IL-4 and IL-13.
include low CSF cell counts (<5/μl), low levels of Dysregulation of Th2 responses leading to high
proinflammatory cytokines (IFN- γ, TNF- α, levels of activated macrophages in the CSF may
IL-6, and IL-8), and increase in chemokines in be the common pathogenetic pathway for para-
CSF (CCL2/MCP-1, CCL3/MIP-1α) (Boulware doxical and unmasking cryptococcal IRIS.
et al. 2010; Jarvis et al. 2015). Immunomodulation with steroid therapy
increases the risk of a poor outcome as it is found
to slow the clearance of Cryptococcus from CSF
12.9 Prognostic Factors leading to higher mortality and morbidity
(Beardsley et al. 2016). Augmentation with
High Th1 immune response reflected by high IFN-γ has demonstrated benefits in faster fungal
CSF levels of IFN-γ, TNF-α, and IL-6 are associ- clearance when coupled with effective antifungal
ated with low CSF fungal load, faster clearance therapy.
on antifungal therapy, and good survival (Jarvis
et al. 2014; Siddiqui et al. 2005). Flow cytometric
studies have shown that the phenotype of CD4 T 12.10 Diagnosis
cells is more important in determining outcome,
and not just the CD4 counts. Good survival was Diagnostic tests employing detection of crypto-
associated with higher counts of IFN-γ and coccal capsular polysaccharide in serum and
TNF-α secreting CD4+ T cells. CSF by latex agglutination or enzyme-linked
Immune signature in non-survivors was low immunosorbent assay (ELISA) have superseded
Th1 and higher Th2 cytokines—IL-10, IL-6, and the classic India ink test in CSF wherein the
CXCL10 in CSF, with neutrophilia. In addition, capsule is negatively stained by India ink
there was evidence of widespread systemic (Fig. 12.3a). The antigen test has an overall sen-
monocyte deactivation characterized by sitivity and specificity of 93–100% and 93–98%,
decreased HLA-DR expression (Siddiqui et al. respectively. The false-positive rate is less than
2005). This paralleled high plasma concentration 1% and is usually due to technical reasons (e.g.,
of the capsular polysaccharide glucuronoxylo- cross-reactivity with antigens from Trichosporon
mannan (GXM). GXM deactivates monocytes species). These tests can sometimes be positive
and T-cell responses (Retini et al. 1998). prior to viable cryptococcal colonies in cultures.
High intracranial pressure (≥30 cm H2O) was Culture remains the gold standard for confirma-
associated with fewer T cells, a higher fungal tion of diagnosis producing characteristic
burden, and larger size of Cryptococcus. The creamy, smooth, mucoid colonies of
absence of effective Th1 response produces a Cryptococcus on Sabouraud dextrose agar
higher CSF fungal load at baseline due to mono- medium. Caffeic acid agar medium will high-
cyte deactivation, excessive CNS chemokine light black-colored colonies in case of patho-
production, and slower fungal clearance on anti- genic Cryptococcus strains (Fig. 12.3b, c).
180 A. Mahadevan and S. K. Susarla
a b c
Fig. 12.3 Diagnostic tests. (a) India ink preparation in colored colonies in case of pathogenic Cryptococcus
CSF highlights negatively stained cryptococci with thick strains. (Courtesy: Dr. Nagarathna Chandrashekar,
capsules. (b) Characteristic creamy, smooth, mucoid Professor, Department of Neuromicrobiology,
colonies of Cryptococcus on Sabouraud dextrose agar NIMHANS, Bangalore)
medium. (c) Caffeic acid agar medium highlights black-
how these pathogens are controlled within the ner- tral nervous system via transcellular penetration of the
vous system. Unravelling answers to these ques- blood-brain barrier. Infect Immun. 2004;72:4985–95.
Charlier C, Chretien F, Baudrimont M, Mordelet E,
tions will provide new insights into organ-specific Lortholary O, Dromer F. Capsule structure changes
fungal pathogenesis and help develop effective associated with Cryptococcus neoformans crossing of
treatment reducing morbidity and mortality. the blood-brain barrier. Am J Pathol. 2005;166:421–32.
Charlier C, Nielsen K, Daou S, Brigitte M, Chretien F,
Dromer F. Evidence of a role for monocytes in dis-
semination and brain invasion by Cryptococcus neo-
formans. Infect Immun. 2009;77:120–7.
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184 A. Mahadevan and S. K. Susarla
on the cell surface and the reduction of beta(1.3)- of cases, which frequent manifestation are mass
d-glucan that prevents the recognition of the lesions or abscesses (Brick et al. 2013).
yeast by receiver’s dectin-1 in innate immune The manifestations of CNS blastomycosis
cells (Koneti et al. 2008; Richer et al. 2014). The include chronic meningitis, acute meningi-
yeasts develop and multiply in the lower lobes tis, and intracranial abscesses. The variety of
of the lungs or pass toward the interstitial tissue, symptoms include headache, fever, altered men-
generating an inflammatory response with poly- tal status, stiff neck, nausea, vomiting, vision
morphonuclear (PMN) leukocytes, with suppura- changes, seizures, or focal neurologic deficits,
tive changes and granuloma formation. Bronchial such as dysphasia and paresthesia of the extremi-
lesions are common and destroy the mucosa with ties, according to the affected area (Lyons and
dissemination to the lung underlying. After con- Andriole 1986).
version to the phase of yeast, the organisms can In cases of chronic meningitis, the cerebro-
disseminate through hemathogenic way toward spinal fluid (CSF) reveals a predominantly neu-
other body sites, especially the skin, bone/joints, trophilic pleocytosis lymphocytic or high protein
genitourinary system, and CNS (Bradsher and and glucose normal to low (Bariola et al. 2010).
Bariola 2011; Saccente and Woods 2010). T-cell Although meningitis is the most common mani-
activation is necessary for control of this disease festation of CNS blastomycosis, solitary mass
(Wüthrich et al. 2011). It has been speculated that lesions are not an uncommon presentation; some
in some people, the fungal structures can remain patients have presented focal neurologic defi-
viable and reactivate to cause disease extrapul- cits as a result of single lesions of intracranial
monary even after several years (Castillo et al. mass (Chander et al. 2007; Roos et al. 1987).
2016). Meningitis usually presents with headache, men-
ingismus, photophobia, papilledema, decreased
consciousness, and, in advanced cases, seizures
13.1.4 Clinical Features or complications with the increased intracranial
pressure. Fungal meningitis may have a variable
Blastomycosis can be presented as a subclinical severity and can be clinically indistinguishable
disease, a progressive pulmonary or extrapul- from bacterial causes of chronic meningitis.
monary disease, or both (Bradsher et al. 2003; Fungal abscesses generally present with focal
Chapman et al. 1997). Disseminated extrapul- neurologic abnormality, headache, and/or sei-
monary infection can affect nearly all organs, zures, which is the consequence of the local
including the CNS, although infrequently. destruction or compression of the brain tissue.
Infections have also been reported in the eye, It should be noted that fungal and bacterial syn-
endocrine glands, larynx, breast, uterus, and peri- dromes overlap, so a careful review of the risk
toneum (Bradsher and Bariola 2011; Litvinjenko factors of the guest should raise suspicion of
and Lunny 2017). a fungal cause which will lead the assessment
CNS involvement occurs in 5–10% of and appropriate management of patients (Scully
patients with disseminated infection but has et al. 2008).
been observed in up to 33% of the autopsies of
patients with blastomycosis (Bariola et al. 2010;
Litvinjenko and Lunny 2017; Saccente and 13.1.5 Diagnosis
Woods 2010). Patients with acquired immune
deficiency syndrome (AIDS) and blastomyco- Blastomycosis should be considered in the differ-
sis are more likely to manifest affectation of the ential diagnosis of pneumonia lobar or s egmental
CNS (40%) (Castillo et al. 2016). CNS involve- subacute, especially in residents or visitors from
ment also appears to be common in children; it endemic areas. However, even in hyperendemic
has been informed of this complication in 15% counties, diagnosis may be missed or delayed
190 M. G. Frías-De-León et al.
(Baumgardner et al. 2011; Pfaff et al. 2014). been shown to be useful to carry out the diagno-
This disease should be considered especially in sis of CNS blastomycosis (Bariola et al. 2010;
patients with a history of outdoor recreational Ward et al. 1995).
activities and with manifestations of pneumonitis
refractory to initial antibiotic treatment.
The gold standard in the diagnosis of blas- 13.1.6 Treatment Options
tomycosis of the CNS is the cultivation of
CSF or ventricular fluid, since the CSF culture The guidelines of the Infectious Diseases Society
obtained by lumbar puncture is slightly sensi- of America for the treatment of blastomycosis
tive (Bariola et al. 2010). It is important that cul- emphasize that all patients with blastomycosis
tures are maintained for at least 4 weeks before should receive antifungal therapy, regardless of
you discard the presence of the fungus, and the the clinical presentation, due to the high probabil-
colonies can be identified using DNA probes or ity of progression or recurrence of infection if not
DNA amplification by polymerase chain reac- treated (Chapman et al. 2008). The recommended
tion (PCR) (Babady et al. 2011). Another diag- treatment for patients with CNS blastomycosis is
nosis option is the histopathological examination the lipid formulation of amphotericin B, 5 mg/kg
which allows the observation of yeasts of double daily for 4–6 weeks or until the CNS symptoms
wall (Patel et al. 2010). It is also recommended have improved, followed by a reduction therapy
to include a magnetic resonance imaging before with azole antifungals for at least a year (Bradsher
the biopsy for culture and histopathology (Bush et al. 2003). Of the azole antifungals, voriconazole
et al. 2013). The hematoxilin-eosin (HE) and is the ideal because it achieves good concentra-
Gomori methenamine silver (GMS) stains may tions in CSF and has an excellent activity against
increase the sensitivity of detection, while peri- the fungus, while the itraconazole, although it has
odic acid-Schiff (PAS), in situ hybridization, and good activity against B. dermatitidis, levels in CSF
other histochemical stains can help differentiate are low (Bush et al. 2013). The fluconazole has
B. dermatitidis from other fungi (Mukhopadhyay excellent penetration into the CSF and has proven
and Gal 2010). The diagnosis may also be based to be effective at high doses (400–800 mg) in the
on the demonstration of budding yeast of broad treatment of blastomycosis of the CNS in some
base by direct microscopic examination of clini- patients (Brick and Agger 2011; Pappas et al.
cal samples not stained. The serological tests are 1997; Pearson et al. 1992). Posaconazole fails to
not useful. The WI-1 antigen, a protein in the cell reach appropriate levels in the CSF so it should
wall of yeast, has been used to diagnose blasto- not be used. Depending on the response to therapy
mycosis by radioimmunoassay in endemic areas and the immune status of patients with CNS blas-
(Centers for Disease Control and Prevention tomycosis, lifetime therapy with an azole antifun-
n.d.). gal may be needed to prevent relapse (Centers for
The sampling of CSF is indicated in all Disease Control and Prevention n.d.; Chapman
patients with meningitis and may show a pre- et al. 2008). For patients with CNS blastomycosis
dominance of neutrophils in the case of blasto- with lesions of intracranial mass, surgical removal
mycosis. Care is required before taking samples may be curative (Roos et al. 1987).
of the CSF to ensure that increased intracranial
pressure do not place the patient in an undue
risk of lumbar puncture (Davis et al. 2007). The 13.2 Phaeohyphomycosis
meningitis caused by B. dermatitidis is associ-
ated with a CSF pleocytosis. The protein level in 13.2.1 Introduction
CSF is generally high, while the glucose level is
typically normal or reduced. The neurosurgical Phaeohyphomycosis is caused by filamentous,
procedure, excluding the lumbar puncture, has ubiquitous, opportunist fungi which can produce
13 Blastomycosis and Phaeohyphomycosis 191
melanin, the reason they were previously known Nodulisporium species, and Scopulariopsis spe-
as black fungi or dematiaceous, currently called cies (Chakrabarti et al. 2016; Revankar et al.
pheoid fungi (Gottfredsson and Perfect 2000; 2004).
Matsumoto et al. 1994; Revankar et al. 2004;
Rinaldi 1996). These fungi can produce super-
ficial, subcutaneous, or systemic infections, in 13.2.3 Pathogenesis and Pathology
both immunocompetent and immunosuppressed
patients (Rinaldi 1996). Infection of CNS is Little is known about the mechanisms of patho-
rare and shown as brain abscesses or meningitis genicity that these pheoid fungi use to produce
(Gottfredsson and Perfect 2000). infection. It is known that the melanin, a compo-
nent of its cell wall, is one of the main factors of
virulence, and its function is to protect the fungus
13.2.2 Epidemiology of free radicals and the sodium hypochlorite that
phagocytic cells release to the environment with
The largest number of phaeohyphomycosis cases the oxidative stress. Another feature that helps to
has been reported in the USA, affecting people prevent any type of action on the plasma mem-
of any age and ethnicity, without occupational brane is the union of the melanin to hydrolytic
or gender predisposition, although some authors enzymes (Hamilton and Gomez 2002; Jacobson
have reported that it predominates in adult men 2000; Revankar and Sutton 2010). The pheoid
(Brandt and Warnock 2003; Emmens et al. 1996). fungi also have enzymes (peptidases, hyaluroni-
Among the factors that predispose to this myco- dases, proteases, and chitin synthase) that confer
sis are solid organ and bone marrow transplants, them ability to survive and cause infection. In
neutropenia, therapies based on systemic steroids, the case of CNS infections, one of the hypoth-
traumas, intravenous drug abuse, leukemia, the eses that have been proposed is that these fungi
use of catheters, chronic sinusitis, and human penetrate through the airways by inhalation of
immunodeficiency virus (HIV) infection (Ben- conidia, causing mainly a pulmonary asymp-
Ami et al. 2009; Emmens et al. 1996; Gómez and tomatic clinical picture; subsequently the hema-
Cardona-Castro 2016). It has been observed that togenous dissemination occurs from the lungs
the disease also is frequently found in immuno- and by neurotropism spreads to the brain. The
competent persons without obvious risk factors, CNS phaeohyphomycosis can also be acquired
in whom the mortality rate is high (Ben-Ami et al. by traumatic inoculation of the fungus and later
2009). dissemination to the brain (Brandt and Warnock
The fungi more related to CNS phaeohypho- 2003; Dixon et al. 1987). Its incubation period is
mycosis are Cladophialophora bantiana and unknown.
Ramichloridium mackenziei; however, there are
many other fungi that can cause the disease, like
Ochroconis gallopavum, Bipolaris spicifera, 13.2.4 Clinical Features
Fonsecaea pedrosoi, Chaetomium strumarium,
Bipolaris hawaiiensis, Chaetomium atrobrun- The frequent forms in which the CNS phaeohy-
neum, Exophiala castellanii, Wangiella dermatit- phomycosis presents are single brain abscess,
idis, Acrophialophora fusispora, Cladosporium meningitis, encephalitis, myelitis, or arachnoidi-
cladosporioides, Cladophialophora modesta, tis (Litchevski et al. 2014; Ochiai et al. 2012).
Curvularia pallescens, Exophiala jeanselmei, The most common clinical findings in these
Microascus cinereus, Myceliophthora thermoph- patients are headaches, seizures, neurologi-
ila, Rhinocladiella atrovirens, Scedosporium cal deficits, fever, altered mental status, nausea,
prolificans, Scopulariopsis brumptii, vomiting, and meningismus (Litchevski et al.
Cladophialophora species, Exophiala species, 2014; Revankar et al. 2004).
192 M. G. Frías-De-León et al.
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1992;303(5):313–5. management of central nervous system blastomycosis.
Pfaff BL, Agger WA, Volk TJ. Blastomycosis diagnosed Surg Neurol. 1995;43(4):379–88.
in a nonhyperendemic area. WMJ. 2014;113(1):11–8. Wüthrich M, Gern B, Hung CY, Ersland K, Rocco
Randhawa HS, Chowdhary A, Kathuria S, Roy P, Misra N, Pick- Jacobs J, et al. Vaccine-induced protec-
DS, Jain S, et al. Blastomycosis in India: report of tion against 3 systemic mycoses endemic to North
an imported case and current status. Med Mycol. America requires Th17 cells in mice. J Clin Invest.
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Cladophialophora bantiana
14
Hurriyet Deniz Ozgun, Darren L. Jacobs,
and Steven A. Toms
14.2 Discovery of C. bantiana bantiana and predict neurotropic behavior (van
den Ende and De Hoog 1999).
Cladophialophora bantiana was discovered The youngest C. bantiana case noted in the
by Guido Banti in 1911, who was an anatomy literature was a 6-day-old neonate with a skin
professor and a physician at the University of fungal infection (Jayakeerthi et al. 2004). The
Florence at the time. Banti had a female patient youngest C. bantiana abscess in the CNS was
with fatal cerebral phaeohyphomycosis. Later documented in a 6-month-old boy (Jayakeerthi
in her autopsy, Banti spotted numerous dark et al. 2004). C. bantiana presents itself mostly in
brown nodules similar to melanotic sarcoma, the immunocompetent young males, especially
along with phaeoid mycelium and long chained in second and third decades of life (Jayakeerthi
5–10 conidia. After Banti, Pier Andrea Saccardo et al. 2004). In cerebral phaeohyphomycosis
named the samples that he received from Banti as male predominance is 76% and the mean age is
Torula bantiana. 38. The majority of patients (52%) had no iden-
In 1960 Dante Borelli, a medical mycologist, tifiable underlying medical conditions (Satton
named Torula bantiana as Cladosporium trichoi- et al. 2009). A total of 77 (76%) of 101 cases
des, which was a synonym for Cladosporium were in men, and 24 cases (25%) were in women.
bantianum, and observed that it could grow at Predilection for race or geographic location has
42 °C. In 1961 Rafaele Stiglioni proved that not yet been identified (Jayakeerthi et al. 2004).
this fungus belonged to Cladosporium genus by
using the drawings of Torula of dematiaceous
hyphae and photomicrographs of histological 14.3 Clinical Presentation
preparations of Banti. In 1963 another group of of Infection
scientists denied Torula bantiana belonging to
Cladosporium genus. Between 1981 and 1983 Phaeohyphomycosis can cause four types of dis-
variations in size were noticed, which built up on eases, which are superficial, cutaneous and cor-
to the question if they were two different types neal, subcutaneous, and systemic. C. bantiana
of fungi. is observed in systemic phaeohyphomycosis
In 1984, it was discovered that the two fungi (Satton et al. 2009), although it can rarely cause
had same antigens, and in 1986 Borelli claimed cutaneous or subcutaneous infections (Kuan et al.
that they were thermotolerant, which lead to the 2016). Systemic phaeohyphomycosis (dissemi-
conclusion that they were cospecific. In 1992 nated infection) often leads to cerebral disease
again the same question was raised; however in (Perfect et al. 2003), which is rare in animals, and
1995 Sybren G. De Hoog and colleagues ended the transmission to humans from animals has not
the discussion by concluding that Torula ban- been reported yet (Rantala et al. 2015).
tiana and Cladophialophora bantiana were the C. bantiana presents non-specifically with
same organism (De Hoog 1995). chronic headaches in 59% of the cases (Satton
The C. bantiana differ from related fungi et al. 2009). Fewer (54%) present with localizing
in another important aspect important to their signs due to mass effect causing focal neurologi-
growth. Due to the carbohydrate active enzyme cal deficits (Satton et al. 2009), such as weak-
(CAZyme) content in C. bantiana genome, rather ness in the limbs, altered mental status, increased
than cellulose, they decompose hemicellulose tone, numbness, hemiplegia, hemiparesis, cranial
and pectin components, which are abundant in nerve deficits, facial palsy, slurring of speech,
plant cell wall (Kuan et al. 2016). Finally, direct decreased hearing, positive Babinski sign, papill-
gene sequencing and polymerase chain reaction edema, diplopia, motor aphasia, fever, seizures,
(PCR) have been useful in identifying species of and rarely chronic meningitis. These manifesta-
similar fungi. Historically, the introns in the 18S tions can precede diagnosis from weeks to years
rRNA gene subunit of species have been used for (Trinh et al. 2003) based on the disease burden,
random target PCR in phylogenetic identification clinical severity, and impact on the patient’s daily
of species and have also been used to identify C. routine.
198 H. D. Ozgun et al.
a b
Fig. 14.1 MRI of brain in a C. bantiana patient show- diffusion-weighted imaging (b). The lesion demon-
ing a ring and a cystic lesion with a large amount of strated ring enhancement on T1-weigted imaging with
edema on T2-weighted (a) with restricted diffusion on gadolinium (c)
14 Cladophialophora bantiana 199
a b
Fig. 14.2 A case of C. bantiana ventriculitis shows dif- extensive arachnoidal adhesions (b) and fungal plaques on
fuse ependymal enhancement on T1-weighted imaging the ependymal wall (c)
with gadolinium (a). Endoscopic exploration showed
scans demonstrate cerebral vasogenic (Trinh diagnosis should be considered, such as oppor-
et al. 2003) edema, which might cause sul- tunistic infections and lymphomas (Garzoni
cal effacement. C. bantiana will often show et al. 2008).
contrast enhancement and necrotic lesions The abscesses are typically confined to one
with ring enhancement on T1-weighted MRI hemisphere generally and they are more likely to
images with gadolinium (Levin et al. 2004). occur in the frontal lobe of the brain (Ajantha et al.
Larger abscesses may show midline shift in the 2011). Given the likely hematological spread of
brain due to mass effect (Salama et al. 1997). the pathogen to the brain, this localization pref-
The differential diagnosis based on CT and/ erence should not be surprising, although cases
or MRI includes metastases, high-grade glio- of C. bantiana have been observed in choroid
mas (Emmens et al. 1996), and bacterial brain plexus, thalamus, diencephalon, and cerebellum.
abscesses (Garzoni et al. 2008). In immuno- In the rare case in which they localize in pons or
compromised people, wider list of differential brainstem, the consequences may be rapidly fatal
200 H. D. Ozgun et al.
a b
c d
Fig. 14.3 MRI of brain in a C. bantiana patient showing ium with poor branching in cornmeal Tween 80 agar as
a space-occupying lesion in the right parietal lobe and left well as growth at 42 °C in passages, positive urease test
frontal lobe (a, b). Surface colors of all media including result, and cycloheximide resistance suggested C. banti-
Sabouraud dextrose agar medium with cycloheximide ana (d). The isolate was confirmed as C. bantiana based
were olive-gray to black and contained velvety colonies on its DNA sequence analysis (Courtesy of M. A. Atalay,
(c). Lemon-like very long and integrated chains of conid- M.D.)
secondary to respiratory failure (Emmens et al. sive that it is not identified until a post mortem anal-
1996). ysis often revealing extensive necrotizing cerebritis
(Emmens et al. 1996). Since C. bantiana may be
inhaled and since it can affect even immunocom-
14.5 Diagnosis (Biopsy/Surgery/ petent people, it should be treated according to the
Culture) rules of biosafety level 3, which requires a biosafety
cabinet, negative airflow into the laboratory, and
C. bantiana is mostly commonly diagnosed with protective laboratory clothing such as respiratory
a surgical procedure, such as a stereotactic brain protection (Rantala et al. 2015; Badali et al. 2010).
biopsy or cyst aspiration (Garzoni et al. 2008). The staff who handle the specimen must protect
Unfortunately, sometimes the disease is so exten- themselves accordingly (Ajantha et al. 2011).
14 Cladophialophora bantiana 201
During surgical resection or biopsy, the dark In addition to morphology and growth tem-
pigmentation of the tissue is apparent grossly. perature, phylogenetic analysis (Kuan et al.
Microscopically, C. bantiana frozen sections 2016) and molecular identification methods are
should reveal pleomorphic cells, branching more reliable ways to differentiate C. bantiana
hyphae (Ahmad et al. 2017), and multinucleated from other fungi. Identification of C. bantiana
giant cells (Salama et al. 1997; Emmens et al. based on DNA can be done via amplified frag-
1996). Permanent sections of hematoxylin and ment length polymorphism (AFLP). This method
eosin and Gomori silver methenamine should detects genomic restriction fragments by PCR
reveal multinucleated giant cells composed amplification, by selectively amplifying prim-
of fungi (Ahmad et al. 2017), giant cells, and ers without needing prior sequence information
inflammation (Lakshmi et al. 2008) but no focal (Badali et al. 2010). When genetic analysis is
demyelination (Trinh et al. 2003). Lactophenol needed for diagnosis, DNA extraction using malt
cotton blue staining shows darkly pigmented agars cetyltrimethylammonium bromide method
and septate fungal hyphae, 2–3 mm in diameter may be used (Moller et al. 1992). Species can be
(Jayakeerthi et al. 2004) and long, wavy, sparsely confirmed by internal transcribed spacer (ITS)
branching, dry, strongly coherent chains of oval sequencing, which is used for identification based
(Ahmad et al. 2017) conidia (Kuan et al. 2016; on molecular systematics and DNA and has low
Badali et al. 2008). The fungal and bacterial brain variability in C. bantiana (Rantala et al. 2015;
abscesses can be differentiated neuropathologi- van den Ende and De Hoog 1999). 558 base pair
cally via the invasion of the capsule and beyond, intron at position 1768 in the small-subunit rDNA
which occurs in fungal abscesses but not in bac- gene (Badali et al. 2010) is considered strictly
terial ones (Revankar and Sutton 2010). In the specific to C. bantiana (Kantarcıoǧlu and De
case of melanin not being visible with the usual Hoog 2004). For definite diagnosis, results also
stains, Fontana-Masson stain, which is specific should be compared to deposits in GenBank and
to melanin (Ajantha et al. 2011), can be used to database of CBS (Fungal Biodiversity Center) on
highlight the pigment (Satton et al. 2009). black fungi (Rantala et al. 2015).
The fungal media preferred, the temperature
to grow, and the duration of growth should be
considered, to prevent misdiagnosis or failure 14.6 Treatment (Surgery/
to diagnose. C. bantiana can be observed with Antifungals)
microscopic wet mount examination or KOH
wet mount (Kuan et al. 2016; Seyedmousavi Although there is no consensus about a stan-
et al. 2014) and can grow in conventional fun- dard anti-fungal therapy yet, maximal surgical
gal media, such as potato dextrose, oatmeal, resection, wherever possible, seems to improve
and malt agar. Sabouraud agar is not the first survival in C. bantiana infections (Salama et al.
choice because it cannot develop the dark color 1997). Complete surgical resection helps with
of C. bantiana (see Fig. 14.3). However, it can “reducing the pressure effects, reduction of the
grow on cycloheximide-rich media. It is urease fungal load and enhancing the response to the
positive and it is non-proteolytic on casein agar antifungal therapy,” even though the abscesses
(Ajantha et al. 2011). The fungus grows with a might recur (Ajantha et al. 2011).
moderate rate, which takes 7–8 days to see and When complete surgical excision is not pos-
15 days to mature (Ajantha et al. 2011). The high sible, surgical debridement and debulking of
growth temperature of 42 °C is a virulence factor the abscess should be attempted (Perfect et al.
(Ajantha et al. 2011) and a differentiating char- 2003). The physician should be aware that partial
acteristic of saprophytic fungi (Lakshmi et al. decompression might aggravate patient’s status,
2008). The fungi’s thermal stress-responsive pro- by increasing postoperative edema and swelling
teins have contributed to its ability to survive in (Levin et al. 2004). During surgery, it is impera-
the human body (Kuan et al. 2016). tive to avoid ventricular entry, as fungal contami-
202 H. D. Ozgun et al.
nation of the cerebrospinal fluid (CSF) will often ease. Posaconazole may be better tolerated by
lead to fatal ventriculitis (see Fig. 14.2). Repeated the patients since it has low toxicity and less
stereotactic aspiration of the cyst abscess to ster- drug-drug interactions, even though its effect
ilize the lesion leaving the enhancing capsule is more fungi static than fungicidal (Ajantha
intact in addition to long-term oral voriconazole et al. 2011). Both newer azole drugs distribute
and amphotericin B was suggested as a strategy to high volume and can accumulate in tissue,
in treating C. bantiana (Garzoni et al. 2008). in addition to having good oral bioavailability
For the treatment of dematiaceous fungi (Garzoni et al. 2008). The effect of voricon-
including C. bantiana, there are four potential azole is fungicidal and it can infiltrate in the
classes of antifungal medications (Perfect et al. CNS, though it has a high MIC (Ajantha et al.
2003): 1:polyene drugs, such as Amphotericin 2011), which in long-term use showed recovery
B, a systemic drug that show moderate in vitro (Ahmad et al. 2017). Unfortunately, fluconazole
effect; 2:flucytosine, which works better in (Garzoni et al. 2008), ketoconazole, micon-
combination therapies because it develops azole, and uconazole (Ajantha et al. 2011) are
resistance in single use; 3:azole drugs, such as not active against C. bantiana.
fluconazole, which inhibit fungal membrane Combination therapy is more helpful (Perfect
synthesis, which have more efficacy in vitro and et al. 2003) than monotherapy and should be
safe to use in long-term therapies, and triazoles preferred as monotherapy more often resulted
can be used as first line drugs against phaeo- in treatment failure in the literature (Ajantha
hyphomycosis; and 4:echinocandins, such as et al. 2011). The most common current recom-
caspofungin, are newer drugs that inhibit fun- mendations suggest that liposomal amphoteri-
gal beta-glucan synthase in vitro. However, cin B should be combined with high-dose azole
results of trials using this drug are yet to be (Ajantha et al. 2011). Also, in a relatively new
reported (Perfect et al. 2003). Theoretically, case which was published in 2016, post-op oral
C. bantiana may be resistant to echinocandins, voriconazole for 6 months after IV amphotericin
because it doesn’t have much glucan in the cell B and itraconazole administration was clinically
wall (Badali et al. 2010). successful (Kuan et al. 2016).
In clinical scenarios, C. bantiana is resis- In the treatment of C. bantiana, importance
tant to Amphotericin B in vitro, in large part of the time of diagnosis and starting the appro-
because it cannot diffuse well into the CNS priate treatment cannot be overlooked, due to
(Badali et al. 2010). Nevertheless, clinical their effect on mortality rate (Jayakeerthi et al.
responses to Amphotericin B and its com- 2004). If treatment is delayed, CNS infections
bined use with a triazole, possibly flucytosine of C. bantiana can lead to death of the patient
(Garzoni et al. 2008), in addition to surgery, in 1–6 months (Ajantha et al. 2011). The side
were better (Lakshmi et al. 2008; Scheld et al. effects of the specific anti-fungal drugs must be
2004) than results with Amphotericin B alone. considered for each patient. No specific duration
Nephrotoxicity is reduced clinically by using is suggested in literature for continuing the treat-
lipid preparations of Amphotericin B (Revankar ment; however prolonged therapy and the impor-
et al. 2004). tance of radiographic follow-up is clear (Ajantha
When oral flucytosine and azole drugs are et al. 2011). Any residual abscess remaining after
used for a long term, they may cause regres- surgery (Levin et al. 2004) can be seen with CT
sion in the lesions that can be observed in the or MRI (Kuan et al. 2016). Follow-up imaging
CT (Salama et al. 1997). Although itracon- can document progression or resolution of the
azole penetrates poorly in the CSF, it accu- brain abscess or the development of new lesions.
mulates in the brain, which makes it efficient Therefore, imaging every 3–6 months to moni-
(Revankar et al. 2004). New triazoles voricon- tor the infection is required (Garzoni et al. 2008).
azole and posaconazole might work better than At this point, it is unclear how long after the ter-
itraconazole, although there are no random- mination of antifungal agents one should follow
ized studies given the rare nature of this dis- up patients radiographically. Physicians must
14 Cladophialophora bantiana 203
Moller EM, Bahnweg G, Sandermann H, Geiger Salama AD, Rogers T, Lord GM, Lechler RI, Mason
HH. Asimpleand efficient protocol for isolation of PD. Multiple Cladosporium brain abscess in a renal
high molecular weight DNA from filamentous fungi, transplant patient: aggressive management improves
fruit bodies, and infected plant tissues. Nucleic Acids outcome. Transplantation. 1997;63(01):160–2.
Res. 1992;20:6115–6. Satton DA, Rinaldi MG, Sanche SE. Dematiaceous fungi.
Perfect J, Schell W, Cox G. Phaeohyphomycosis. In: In: Anaissie EJ, McGinnis MR, Pfaller MA, edi-
Dismukes W, Pappas P, Sobel J, editors. Clinical tors. Clinical mycology. 2nd ed. Philedelphia, PA:
mycology. New York: Oxford University Press; 2003. Churchill Livingstone/Elsevier; 2009. p. 329–54.
p. 271–82. Scheld MW, Whitley RJ, Marra CM. Infections of the
Rantala M, Attia S, Koukila-Kähkölä P, De Hoog S, central nervous system. 4th ed. Philadelphia, PA:
Anttila M, Katila T. Cladophialophora bantiana Lippincott Williams & Wilkins; 2004. p. 719.
as an emerging pathogen in animals: case report of Seyedmousavi S, Netea MG, Mouton JW, Melchers WJG,
equine endometritis and review of the literature. J Clin Verweij PE, De Hoog GS. Black yeasts and their fila-
Microbiol. 2015;53:3047–53. mentous relatives: principles of pathogenesis and host
Revankar SG, Sutton DA. Melanized fungi in human dis- defense. Clin Microbiol Rev. 2014;27(3):527–42.
ease. Clin Microbiol Rev. 2010;23(4):884–928. Trinh JV, Steinbach WJ, Schell WA, Kurtzberg J, Giles SS,
Revankar SG, Sutton DA, Rinaldi MG. Primary central Perfect JR. Cerebral phaeohyphomycosis in an immu-
nervous system phaeohyphomycosis: a review of 101 nodeficient child treated medically with combination
cases. Clin Infect Dis. 2004;38(02):206–16. antifungal therapy. Med Mycol. 2003;41(04):339–45.
Cladosporium spp., Fusarium spp.,
Bipolaris spp., Schizophyllum 15
commune, and Scedosporium
apiospermum
A. Serda Kantarcioglu
a c
Fig. 15.1 (a): Colonies (on SDA) and (b) microscopy of Cladosporium sphaerospermum, (c) microscopy of
Cladosporium macrocarpum (lactophenol coton blue stain, ×40)
15 Cladosporium spp., Fusarium spp., Bipolaris spp., Schizophyllum commune, and Scedosporium... 207
that of long hyphae, or the combination of these brospinal fluid (CSF) and/or brain tissue or
may be seen (De Hoog et al. 1995). abscess martial. Identification of isolated fungi
was confirmed by molecular techniques in four of
cases (Kantarcioǧlu et al. 2002; Chen et al. 2013;
15.1.4 Ecology and Epidemiology Tauber et al. 2014).
The common primary symptom was head-
Species of Cladosporium are widely distrib- ache; diplopia was reported in one patient with
uted in the air as well as decayed organic mat- abscesses in brain stem (Kantarcioǧlu et al.
ter, and very often they are food contaminants. 2002), fever in two cases (Lalueza et al. 2011;
These fungi are commonly encountered on plant Chen et al. 2013), and acute urinary retention and
and other kinds of debris, frequently coloniz- right flank pain in one patient with acute menin-
ing lesions of plant pathogenic fungi, and are gitis (Chen et al. 2013).
also isolated from air, soil, food, paint, textiles,
and other organic matters as saprobes; they are
also common endophytes (Crous et al. 2007). 15.1.6 Laboratory Diagnosis
The small conidia of Cladosporium species eas-
ily spread in large numbers over long distances Direct microscopy of a potassium hydroxide
and represent the most common fungal compo- (KOH) (10%) preparation of the biopsy or sur-
nents isolated from air (Asl et al. 2017). Central gical specimen shows the presence of phaeoid
nervous system (CNS) infections caused by (dark colored) septate hyphae. Giemsa-stained
Cladosporium species were rarely reported, from preparations of CSF, tissue, or abscess material
Turkey (Küllü et al. 1985; Kantarcioǧlu et al. can show irregularly swollen, septate branched
2002), Spain (Lalueza et al. 2011), Taiwan (Chen hyphae and/or spherical or oblong, thick-walled
et al. 2013), and Germany (Tauber et al. 2014). conidial chains (Fig. 15.2a–d) (Kantarcioǧlu
et al. 2002).
Cladosporium spp grows fast on agars
15.1.5 Clinical Presentations (Sabouraud agar with or without addition of
antibiotics, without actidione, etc.) forming
Five cases of culture-proven CNS infections due darkly pigmented (pigmented molds), olive-
to Cladosporium species have been reported to green to darkly brown colonies of powder-like
date, including a posttraumatic cerebral infec- structure (Fig. 15.1a). The vegetative mycelium
tion in a 30-year-old apparently healthy man due is usually dark-colored (Fig. 15.1b, c). The
to Cladosporium cladosporioides (Kantarcioǧlu colonies are formed after 5 days. The identifi-
et al. 2002), acute meningitis in a 73-year-old wood cation and differentiation of species is possible
worker due to Cladosporium sphaerospermum after cultivation on potato dextrose agar at the
(Chen et al. 2013), brain abscess in a 45-year-old temperature of 25 ° C. The greatest number of
man due to Cladosporium macrocarpum (Lalueza conidia does not multiply at the temperature of
et al. 2011), two mixed fungal encephalitis due to 25 °C (De Hoog et al. 1995).
Cladosporium spp. in a 47-year-old woman who Members of Cladosporium are relatively
was receiving chemotherapy, and in a 48-year- easy to identify to genus and species complex
old man who had cardiac surgery (Tauber et al. based on their typical conidiogenous struc-
2014). Two cases were associated with timber tures. However, morphological identification
puncture (Kantarcioǧlu et al. 2002; Chen et al. of Cladosporium species is difficult given the
2013). Occupational exposure or traumatic inocu- high morphological similarity between closely
lation might be important. C. macrocarpum could related species. It is recommended that pheno-
have reached the brain through blood vessels by typic identifications be confirmed with DNA
directly spreading from the upper gastrointestinal sequencing. Genus-level identification is usu-
tract following celiac plexus neurolysis (Lalueza ally sufficient, and morphological identification
et al. 2011). The fungus was isolated from cere- can be confirmed by ITS and D1/D2 sequence
208 A. S. Kantarcioglu
a b
c d
Fig. 15.2 (a–d) Examples of in vivo fungal elements (stained with Ehrlich–Ziehl–Neelsen); (d) chains of
observed in three subsequent CSF specimens of the swollen cells and septate hyphal element (stained with
patient. (a–c) Irregularly swollen hyphal elements Giemsa)
analysis. Multilocus gene analysis of the ITS, tibility studies (McGinnis and Pasarell 1998;
D1/D2, EF-1α, and actin gene loci is necessary Kantarcioglu and Yucel 2002) were shown in
for accurate species identification (Bensch et al. Tables 15.1 and 15.2.
2012; Asl et al. 2017). This is especially impor- Treatment was done with oral voriconazole
tant for members of the C. cladosporioides com- (VRZ) (400 mg/day) in one patient (Lalueza
plex, which is demonstrated the greatest species et al. 2011) after 30 days he was discharged
diversity, the highest number of species associ- from hospital on oral VRZ (400 mg/day).
ated with clinical samples, and also, the greatest Complete excision of the brain abscess com-
number of undescribed species (Sandoval-Denis bined with antifungal therapy with VRZ or/and
et al. 2015). amphotericin B (AmB) and flucytosine may be
effective in CNS cases.
Table 15.1 In vitro MICs of non-bantiana Cladosporium species mentioned as associated with CNS cases
Species MIC (μg/ml)
Reference AMB 5-FC KTZ FLZ ITZ MCZ TRB
Guarro et al. C. cladosporioides CBS 171.54 C 1 4 >16 32 0.5 1
(1997)a H 4 64 0.5 >64 2 8
C. cladosporioides FMR 5031 C 0.125 0.125 <0.03 16 0.06 0.25
H 0.5 0.125 0.5 32 1 4
C. cladosporioides IFO 4459 C 0.25 0.25 1 0.5 1
H 8 >64 >16 >16 >16
C. sphaerospermum CBS 193.54 C 1 4 4 >64 >16 4
H 2 4 >16 >64 >16 >16
C. sphaerospermum FMR 5030 C 0.5 8 2 >64 0.5 8
H 8 >128 4 >64 1 0.5
SK (2002) caseb C. cladosporioides 8 <0.03 0.25 0.06 <0.03 0.5
Abbreviations: MIC minimal inhibitory concentration, AMB amphotericin B, 5-FC flucytosine, KTZ ketoconazole; FLZ
fluconazol, ITZ itraconazol, MCZ miconazole, TRB terbinafine, CAS caspofungin, MCF micafungin, AND anidulafun-
gin, C with conidial inoculum, H with hyphal inoculum
a
A broth method
b
National Committee for Clinical Laboratory Standards Institute M38-P guidelines
etable tissues. Some are plant pathogens, causing Fusarium pathogens belong to the F. solani com-
root and stem rot, vascular wilt, or fruit rot. They plex and F. oxysporum complex.
can be recovered from the deepest roots in soil, and
they may occasionally cause infection in animals
(Nucci and Anaissie 2007). Several species have 15.2.3 Morphology and Biology
emerged as important opportunistic pathogens in
humans causing hyalohyphomycosis (especially in Macromorphology. Most Fusarium species pro-
burn victims and bone marrow transplant patients) duce woolly to cottony, flat, spreading colonies.
and are documented agents of both superficial and The color of the colony may be white, cream,
systemic infections in humans. Fusarium species tan, salmon, cinnamon, yellow, red, violet, pink,
are frequently found on cereal grains, where it may or purple; and on the reverse, it may be colorless,
cause seedling and head blight and produce myco- tan, red, dark purple, or brown.
toxins and may also cause allergic diseases (sinus- Microscopic morphology. Sporodochia con-
itis) in immunocompetent individuals (Nucci and sist of masses of branched conidiophores. In cul-
Anaissie 2007; Refai et al. 1969). ture they build up and are seen macroscopically
as light-colored-raised bodies. Macroconidia are
borne in sporodochia. They are mostly long, slen-
15.2.2 Taxonomy der, rather pointed at both ends, dorsoventrally
curved, sickle-shaped, and septated, and possess
Fusarium genus is a mold of kingdom Fungi, a basal foot cell. Microconidia may be formed.
phylum Ascomycota, class Sordariomycetes, Typically they are present on the aerial mycelium
order Hypocreales, and family Nectriaceae. of the culture growth, appearing as small, usu-
The genus Fusarium was divided into sections ally one-celled spores, and oval-shaped, tear-drop
(Nelson et al. 1994), but the current classifica- or pear-shaped and sometimes even spherical.
tion scheme replaces the designation “section” Mesoconidia are the fusoid conidia that are lon-
with “complex.” Currently the genus Fusarium ger than microconidia with three to four septae
comprises at least 300 phylogenetically distinct but shorter than macroconidia with lack of foot-
species, 20 species complexes, and 9 mono- shaped and notched basal cell. The production of
typic lineages (Balajee et al. 2009; O’Donnell both fusoid macroconidia (hyaline, multicellular
et al. 2015). Most of the identified opportunistic clusters, macroconidia with foot cells at the base
Table 15.2 MICs and MFCs of non-bantiana Cladosporium species mentioned as associated with CNS cases from small series
210
Species (strain number) MIC (MFC) Results for antifungal agents (μg/ml)
Reference parameter AMB 5-FC VRZ PSZ ITZ TRB CAS AND MCF
McGinnis and C. cladosporioides (3) Range 0.03–2 (0.4) 0.06–1 0.03–0.25
Pasarell (1998)a (0.08) (0.12)
GM
MIC50
MIC90
C. sphaerospermum (5) Range 2–8 (3.48) 0.5–1 0.25–32
(0.87) (0.76)
GM
MIC50
MIC90
SK (2002) Cladosporium sp. (21) Range <0.03–>16 <0.03–16 0.125–128
in vitrob (0.06–>16) (0.5–>16) (1–>128)
GM 0.9 (2.0) 1.4 (4.1) 3.4 (11.9)
MIC50 8 (16) 4 (8) 8–32
MIC90
Sandoval-Denis C. cladosporioides Range 0.06–2 0.06– 0.25–16 <0.03– <0.03–2 <0.03–4 0.125– 0.03–0.5 0.03–
et al. (2015)c complex (57) >32 1 8 0.5
GM 0.73 1.20 1.65 0.40 0.34 0.12 2.78 0.19 0.11
MIC50
MIC90 1 4 4 0.5 0.5 1 8 0.5 0.25
C. cladosporioides (3) Range 0.5–1 1–2 0.5–16 0.25–1 0.25–0.5 0.5–2 1–4 0.125– 0.125
0.25
GM 0.79 1.26 1.59 0.40 0.31 1.00 2.00 0.16 0.13
MIC50
MIC90
Cladosporium sp. (29) Range 0.125–2 0.06– 0.25–8 <0.03– <0.03–2 <0.03–4 0.125– 0.03–0.5 0.03–
>16 1 8 0.5
GM 0.67 1.10 1.73 0.43 0.36 0.09 2.00 0.18 0.10
MIC50
A. S. Kantarcioglu
MIC90 1.00 4.00 4.00 0.50 0.50 1.00 8.00 0.50 0.25
C. sphaerospermum Range 0.125–2 0.06–4 0.5–16 0.06–4 0.25–>16 <0.03–1 0.06–4 <0.03–1 0.06–1
complex (17)
GM 1.13 1.13 1.70 0.64 1.13 0.06 1.27 0.15 0.03
MIC50
MIC90 2 2 8 2 32 0.5 4 0.25 0.125
C. macrocarpum (1) Range 0.5 2 1 0.25 0.5 0.125 1 0.125 0.5
GM
MIC50
MIC90
Abbreviations: MIC minimal inhibitory concentration, MFC minimal fungicidal concentration, AMB amphotericin B, 5-FC flucytosine, KTZ ketoconazole, FLZ fluconazol, ITZ
itraconazol, VRZ voriconazole, PSZ posaconazole, TRB terbinafine, CAS caspofungin, MCF micafungin, AND anidulafungin, ND not determined
In calculating GM values, MICs of ≤0.03 or >16 μg/ml were classed as 0.03, or 32 μg/ml, respectively
50%, MICs (MFCs) at which 50% of isolates are inhibited (killed), respectively
a
National Committee for Clinical Laboratory Standards M27-A guidelines
b
National Committee for Clinical Laboratory Standards M38-P guidelines
c
Clinical Laboratory Standards Institute M38-A2 method
15 Cladosporium spp., Fusarium spp., Bipolaris spp., Schizophyllum commune, and Scedosporium...
211
212 A. S. Kantarcioglu
a b
c d
Fig. 15.3 Colonies of (a) F. oxysporum; (b) F. sporotrichioides; (c) F. solani; (d) reverse of F. soloni (on SDA media)
15 Cladosporium spp., Fusarium spp., Bipolaris spp., Schizophyllum commune, and Scedosporium... 213
Toll-like receptors in the innate immune recognition neutropenia and/or severe T-cell immunode-
of fungi has been recognized (Romani 2004, 2008; ficiency (Table 15.3). Brain abscess was the
Verma et al. 2015), and although little is known main clinical presentation (Cho et al. 1973;
about fusariosis and Toll-like receptors, this system Abramovsky et al. 1974; Antunes et al. 1998;
is likely important in invasive fusariosis as well. Agamanolis et al. 1991; Vincent et al. 2003; Kapp
The importance of T-cell defenses against et al. 2011; Garcia et al. 2015; Peterson et al.
Fusarium is illustrated by the occurrence of dis- 2014; Steinberg et al. 1983; Schwartz et al. 2013).
seminated fusariosis in nonneutropenic hemato- The principal portal of entry for Fusarium
poietic stem cell transplant recipients (HSCT) spp. is the airways, followed by the skin at site
(Nucci et al. 2004). These patients have severe of tissue breakdown and possibly the mucosal
T-cell immunodeficiency caused by multiple membranes (Nucci and Anaissie 2007). Nasal
therapies for their underlying disease and for route was the presumed mode of entry of the fun-
graft-versus-host disease. Further supporting gus into the cerebrum in one patient (Garcia et al.
the importance of T-cell immunity and phago- 2015). Brain abscess and/or meningitis and ven-
cytes is the major impact of corticosteroid ther- triculitis were reported, and concomitant endo-
apy on the outcome of fusariosis, as shown by phthalmitis were reported in one patient (Kapp
the much higher death rate among recipients of et al. 2011).
such therapy than among patients who were not Disseminated disease is the most frequent and
receiving corticosteroids (Nucci et al. 2003). challenging clinical form of fusariosis in immu-
nocompromised patients, accounting for approxi-
mately 70% of fusariosis in this population. Patients
15.2.7 Portal of Entry and Routes at risk for disseminated fusariosis include those with
of Infection acute leukemia and prolonged and profound neutro-
penia and patients undergoing HSCT.
Given the ubiquity of Fusarium species in the
environment, fusariosis may potentially be
acquired in the community, as suggested by the 15.2.10 Histopathology
presence of airborne fusarial conidia in outdoor
air samples. Showering and other water-related Pathologic examination of necrotic purulent
activities appeared to be an efficient mechanism abscess and biopsy materials with slides stained
for the dispersion of airborne fusarial conidia and with hematoxylin and eosin showed inflama-
transmission to the immunocompromised host, as tory cells, branched septate hyphae invading the
shown by the close molecular relatedness between walls of the blood vessels (Peterson et al. 2014;
water and patient isolates (Anaissie et al. 2001; Steinberg et al. 1983), and Gomori methenamine
Boutati and Anaissie 1997; Raad et al. 2002). silver staining showed septated hyphae with acute
angled branching in reported cases (Peterson et al.
2014).
15.2.8 Signs and Symptoms In tissue, the hyphae can be similar to those of
Aspergillus species, with hyaline and septate fila-
Symptoms of CNS fusariosis are nonspecific, ments that typically dichotomize in acute and right
involving severe headache, altered mental sta- angles. However, adventitious sporulation may be
tus, confusion, fever, nausea, vomiting, and neck present in tissue, and the finding of hyphae and
stiffness in meningitis cases. yeast-like structures together is highly suggestive
of fusariosis in the high-risk population. In the
absence of microbial growth, distinguishing fusa-
15.2.9 Clinical Presentations riosis from other hyalohyphomycoses may be dif-
ficult and requires the use of in situ hybridization
Immunocompromised patients at high risk for in paraffin-embedded tissue specimens (Hayden
fusariosis are those with prolonged and profound et al. 2003).
Table 15.3 CNS infections by Fusarium species
Reference Pathogen Age/sex/race/geography Risk factor/underlying disease Clinical syndrome Therapy Outcome
Cho et al. (1973) F. solani 21/2/M/C/USA ALL Disseminated
Abramovsky et al. Fusarium 2/F/B/USA Burned Brain, renal abscess
(1974) sp.
Steinberg et al. (1983) Fusarium 17/F/W/USA Infectious mononucleous Brain abscess AmB Died
sp.
Agamanolis et al. (1991) Fusarium 15/M/USA ALL, contaminated skin Meningoencephalitis MCZ, 5-FC, AmB Died
sp. wound
Antunes et al. (1998) Fusarium / /USA ALL Brain abscess (M) Surgery Survived
sp.
Kapp et al. (2011) Fusarium 69/M/Germany AML Meningitis, AmB Died
sp. endophthalmitis
Vincent et al. (2003) Fusarium 76/M/USA AML, N Disseminated L-AmB, ITZ, VRZ Died
sp.
Schwartz et al. (2013) F. 1/F/Canada AA, HSCT, N Brain abscess, lung, blood L-AmB, VRZ, Died
oxysporum granulocytes
F. solani 15/F/Canada AML, HSCT, N Brain abscess, lung L-AmB, VRZ Died
Peterson et al. (2014) Fusarium 33/F/H/USA none Brain abscess Surgery, VRZ, TRB,
sp. AmB
Garcia et al. (2015) F. solani 50/F/USA DM, ALL, APST, N Brain abscess VRZ, L-AmB, G-CSF
Abbreviations: ALL acute lymphocytic leukemia, AML acute myeloid leukemia, N neutropenia, AA aplastic anemia, HSCT hematopoietic stem cell transplant, DM diabetes mel-
litus, APST autologous peripheral stem cell transplant, B black, C Caucasian, H Hispanic, W white, M multiple, 5-FC flucytosine, AMB amphotericin B deoxycholate, L-AMB
liposomal amphotericin B, KTZ ketoconazole, ITZ itraconazole, MCZ miconazole, VRZ voriconazole, TRB terbinafine, G-CSF granulocyte colony-stimulating factor, w week(s),
15 Cladosporium spp., Fusarium spp., Bipolaris spp., Schizophyllum commune, and Scedosporium...
m month(s)
215
216 A. S. Kantarcioglu
a b
c d
Fig. 15.4 (a) The hyphae of Fusarium in tissue resemble walled chlamydoconidia, (c) macroconidia, (d): acute-
those of Aspergillus spp. (Giemsa-stained imprint slide and right-angled branching hyphae with meso- and
preparation, ×100); (b) mesoconidia and single, smooth- macroconidia (lactophenol cotton blue stained, ×40)
ceptibility of a single strain is difficult. However, success. Based on the data available, ESCMID
at least the species show different tendencies in and ECMM joint guideline recommend VRZ
their susceptibility against various antifungal com- and lipid-based AmB formulations. Lipid-based
pounds (Al-Hatmi et al. 2014). AmB preparations exhibit fewer side effects
Fusarium strains have high levels of intrin- when compared with AmB deoxycholate and
sic antifungal resistance. Recently, diagnostic should be favored. The response rate to a lipid
guidelines recommend AmB and VRZ as the pre- formulation of AmB appeared superior to that
ferred drugs of choice for treatment of deep and of conventional AmB. In addition to antifungal
disseminated infections (Tortorano et al. 2014). treatment, the optimal management of patients
Although AMB and VRZ are sufficient for the with fusariosis includes surgical debridement of
treatment of the majority of Fusarium infections, infected tissues, removal of venous catheters in
some Fusarium species are not susceptible to confirmed catheter-related fusariosis, and rever-
AmB and VRZ or posaconazole (Al-Hatmi et al. sal of the immunocompromised state (Tortorano
2014). However, both drugs may be preferred et al. 2014).
because of good CNS penetration. Fusarium CNS infection in patients with
In vitro antifungal susceptibility profiles of prolonged neutropenia carries a poor prognosis
Fusarium species demonstrate high MICs to (Vincent et al. 2003). The prognosis of fusari-
most antifungal agents. Notably, some species osis in the immunocompromised host is directly
may exhibit different patterns of susceptibility: related to the immune status of the patient, with
F. solani species complex are usually resistant to high death rates in patients with persistent immu-
azoles and exhibit higher AmB MIC values than nodeficiency. In general, patients with localized
other species, whereas F. oxysporum and F. verti- CNS infection are likely to benefit from surgi-
cilloides may be susceptible to VRZ and posacon- cal debridement, while disseminated infection
azole. The echinocandins are not active against requires the use of systemic agents and immu-
Fusarium spp. and lack CNS penetration (Nucci notherapy, when possible (Vincent et al. 2003;
and Anaissie 2007; Al-Hatmi et al. 2014, 2017; Tortorano et al. 2014).
Alastruey-Izquierdo et al. 2008).
Multiresistance to antifungals, observed in
all Fusarium species, is intrinsic; therefore these 15.3 Bipolaris spp.
fungi are notoriously difficult to treat. The execu-
tive board of the European Fungal Infection 15.3.1 Introduction
Study Group (EFISG) of the European Society
of Clinical Microbiology and Infectious Diseases Bipolaris is a large genus of dematiaceous
(ESCMID) and the European Confederation of hyphomycetes with more than 100 species, most
Medical Mycology (ECMM) decided to pro- of them being saprobes in soil and pathogens of
ceed with a pan-European guideline for the plants, while some of the saprobic species are
diagnosis and management of hyalohypho- potentially able to infect humans and animals.
mycosis caused by Fusarium and other non- Hyphomycetes are a form classification of Fungi,
melanized fungi (Tortorano et al. 2014). Due to and dematiaceous or phaeoid (Pappagianis and
the lack of clinical trials and the critical role of Ajello 1994) fungi include a large group of organ-
immune reconstitution in the outcome of fusari- isms darkly pigmented (dark brown, olivaceous,
osis, the optimal treatment strategy for patients or black). In most cases the pigment is mela-
with severe Fusarium infection remains unclear. nin (Dixon and Polak-Wyss 1991). These fungi
Reversal of immunosuppression is recommended are alternately called phaeoid, dematiaceous,
whenever possible. In immunocompromised dark, or black molds. Phaeohyphomycosis, a
patients, VRZ, AmB deoxycholate, lipid-based term introduced by Ajello et al. in 1974, liter-
AmB (L-AmB) formulations, and various com- ally means “infection caused by dark-walled
binations have been reported with varying fungi” (Ajello et al. 1974; Revankar and Sutton
15 Cladosporium spp., Fusarium spp., Bipolaris spp., Schizophyllum commune, and Scedosporium... 219
2010). Most species belonging to the genus closely related. In the past, morphological dif-
Bipolaris are saprobes in nature, found in wood ferentiation of the genera relied upon a combina-
and decomposing plants. The prevalent clinically tion of characters including conidial shape, the
significant saprobes are Bipolaris australiensis, presence or absence of a protruding hilum, the
Bipolaris hawaiiensis, and Bipolaris spicifera. contour of the basal portion of the conidium and
Bipolaris hawaiiensis is particularly associated its hilum, the point at which the germ tube origi-
with CNS infections (Chowdhary et al. 2015; Da nates from the basal cell, and, to a lesser degree,
Cunha et al. 2012). These fungi are able to infect the sequence and location of the first three conid-
both immunocompetent and immunosuppressed ial septa. Drechslera can be differentiated from
patients, mainly in tropical and subtropical areas all other helminthosporoid genera by its ability
(Da Cunha et al. 2012). to develop a germ tube from any of the cells in
A computerized search of the MEDLINE data- the conidia.
base (National Library of Medicine, Bethesda, Molecular phylogenetic analysis based on
MD, USA) was performed for cases reported ITS (internal transcribed spacers and inter-
in the literature, with (by cross- referencing) vening 5.8S nrDNA) and GPDH (partial
the terms “Helminthosporium,” “Drechslera,” glyceraldehyde-3-phosphate dehydrogenase)
“Bipolaris,” “Bipolaris hawaiiensis,” “Bipolaris genes (Berbee 2001) showed Drechslera and
spicifera,” “cerebral,” “brain abscess,” “menin- Bipolaris to be two distinct genera. Bipolaris
gitis,” “central nervous system infection,” “rhi- and Curvularia share many morphological simi-
nocerebral infection,” “rhino-orbito-cerebral larities, and both genera have sexual morphs in
infection,” “allergic fungal sinusitis,” “dissemi- Cochliobolus. According to molecular analyses
nated,” “immunocompetent,” and “otherwise of ITS and GPDH sequence data, some Bipolaris
healthy.” These keywords were used alone and/ species clustered with Curvularia. Manamgoda
or in combination with an “and” statement. et al. (Manamgoda et al. 2012) have found that
Additional cases were obtained by scanning the glyceraldehyde-3-phosphate dehydrogenase
references cited in the original articles. Original (GPDH) gene is the best single marker for spe-
full texts of all the relevant articles were obtained cies of Bipolaris. Generic boundaries between
via MEDLINE, TUBITAK-ULAKBIM (Turkish Bipolaris and Curvularia are revised and pre-
Academic Network and Information Center), and sented in an updated combined ITS and GPDH
other international libraries and were used for the phylogenetic tree. The genus Bipolaris belongs
analysis. to Ascomycota, Dothideomycetes, Pleosporales,
and Pleosporaceae. Its sexual morph, the genus
Cochliobolus, is not common in nature, but it is
15.3.2 Taxonomy occasionally produced under laboratory condi-
tions (Manamgoda et al. 2014).
Traditionally, darkly pigmented fungi have been
collectively indicated under umbrella terms, such
as “dematiaceous” or “phaeoid” fungi, refer- 15.3.3 Morphology
ring to the presence of brown hyphae or yeast
cells. Today, the leading principle of fungal clas- Macroscopic morphology: Rapidly growing dark
sification is molecular phylogeny. Species in colonies on SDA are hairy and expanding, effuse,
Bipolaris were initially described in the genus grey to blackish brown, woolly, suede-like to
Helminthosporium; after several taxonomic floccose with a black reverse. Colonies on potato
refinements, Helminthosporium were segre- dextrose agar at 25 °C are initially white, soon
gated into several genera including Bipolaris, becoming dark gray to black with a black reverse.
Curvularia, Drechslera, and Exserohilum Microscopic morphology: Hyphae are septate
(Sivanesan 1987). The genera Drechslera, and dark. Conidiophores are brown, erect, multi-
Bipolaris, Curvularia, and Exserohilum are all celled, producing ellipsoidal, straight, or curved
220 A. S. Kantarcioglu
conidia with dark-brown, flat conidial scars. and Casadevall 2003). The function of melanin in
Conidia germinate from both poles. Conidia their natural habitat mostly is protection against
mostly curved, canoe-shaped, fusoid or obcla- solar irradiation because of growth on exposed
vate, rarely straight, 2–14 pseudoseptate (usually surfaces, such as natural rock, or against factors
more than 6), germinating only from the ends prevalent under conditions of stress. Melanin
(bipolar). The conidia of B. hawaiiensis have is believed to contribute to microbial virulence
a more overall delicate look and are generally by reducing a pathogen’s susceptibility to kill-
somewhat narrower than either B. spicifera or ing by host antimicrobial mechanisms and by
B. australiensis. Been developed for the direct influencing the host immune response to infec-
detection of Bipolaris species (Chowdhary et al. tion. Melanin has been shown to interfere with
2015; Shin et al. 2003; El-Morsy et al. 2010) numerous host defense mechanisms. Melanin
(Fig. 15.5). is an important mechanism to decrease phago-
cytosis and escape the oxidative burst of mac-
rophages, capable to alter cytokine responses
15.3.4 Virulence Factors (Nosanchuk and Casadevall 2003, 2006; Gómez
and Nosanchuk 2003; Nosanchuk et al. 2015).
Bipolaris is a melanized fungus. Melanin is Melanized cells are less susceptible to killing
believed to be a major virulence factor-enhanc- by oxygen- and nitrogen-derived radicals. B.
ing opportunism (Jacobson 2000; Nosanchuk hawaiiensis is a neurotropic fungus and may
a b
c d
Fig. 15.5 (a): Colony of B. hawaiiensis (10 days); (b) nomarsky-8 (100×, lactic acid preparation). (Courtesy
conidiophores (40×, lactic acid preparation); (c) conid- of Josep Guarro, Prof., MD, Unitat de Microbiologia,
iophores (100×, lactic acid preparation); (d) conidia Facultat de Medicina i Ciencies de la Salut, University
of Rovira i Virgilli, Reus, Spain)
15 Cladosporium spp., Fusarium spp., Bipolaris spp., Schizophyllum commune, and Scedosporium... 221
probably utilize catecholamines in the brain to et al. 1992; Latham 2000; Moore et al. 2001;
produce melanin and may be protected from oxi- Filizzola et al. 2003; Viola and Sutton 2010;
dative damage by scavenging free radicals. Rosow et al. 2011; Patel et al. 2012; Jelinek et al.
Melanin production provides survival advan- 2014; Teran et al. 2014; Frank et al. 2016). Two
tages to fungi in the environment and during infec- cases were due to B. hawaiiensis, eight were B.
tion of diverse hosts. There is conclusive evidence spicifera, one is with B. australiensis, and the
that many types of drugs, including antimicrobial remaining were reported due to Bipolaris spp.
drugs, bind to melanin. In particular, the melani- Seven patients died; outcome was not reported in
zation of certain fungi is associated with reduced one case.
susceptibilities to polyene and echinocandin-type
drugs in vitro. In contrast, melanization has not
been associated with reduced susceptibilities to 15.3.7 Signs and Symptoms
azole-type drugs, except at high concentrations.
Interestingly, the drug-binding properties of both Symptoms of CNS infection due to Bipolaris
host and microbial melanins could influence the species are nonspecific, involving headache, fas-
outcome of antimicrobial therapy (Nosanchuk cial pain, fever, neck stiffness, and vomiting.
and Casadevall 2006; Nosanchuk et al. 2015).
AmB and 5-FC or ITZ, and the prognosis mature fruiting bodies. Karyogamy and meiosis
reported was poor. occur in the basidia within the mature fruiting
body, and the resulting basidiospores can give
rise to new monokaryotic mycelia (Ohm et al.
15.4 Schizophyllum commune 2010).
Dikaryotic isolates of S. commune were char-
15.4.1 Introduction acterized by clamp connections, hyphal spicules,
and formation of basidiocarps (mushroom) with
Schizophyllum commune is a mold of phylum basidiospores. A monokaryon generates septate
Basidiomycota, subphylum Agaricomycotina, sterile aerial hyphae that form a fluffy white layer
order Agaricales, and family Schizophyllaceae, on top of the vegetative mycelium (Sigler et al.
with worldwide distribution that colonizes 1995).
diverse trees and rotting wood. The distinctive
feature of these fungi is the formation of a mac-
roscopic fructification, the basidiocarp, that con- 15.4.3 Ecology
tains basidiospores (sexual spores) developing
on the outside of a club-shaped or elongate struc- S. commune is one of the most commonly
ture called the basidium (De Hoog et al. 2000). found fungi and can be isolated from all conti-
S. commune is one of the most commonly found nents, except for Antarctica. S. commune has
mushroom-forming fungus (teleomorphic state) been reported to be a pathogen of humans and
and contains genes that encode enzymes for trees, but it mainly adopts a saprobic lifestyle by
degrading woody cell wall components (Schmidt causing white rot (Schmidt and Liese 1980). It
and Liese 1980). is predominantly found on fallen branches and
timber of deciduous trees (Ohm et al. 2010).
Schizophyllum basidiospores are readily airborne
15.4.2 Morphology and Biology and dispersed over long distances (James and
Vilgalys 2001; O’Gorman and Fuller 2008).
S. commune, like most basidiomycete fungi, The fungus is found so abundantly in some
have dikaryotic (binucleate condition) or parts of the world that in Hong Kong and
monokaryotic stages in their life cycle. In the Indonesia, it is used as a substitute for chew-
life cycle of S. commune (Wösten and Wessels ing gum; in Peru, the Congo, and Thailand, it is
2006), meiospores germinate to form a ster- cooked and eaten (Greer 1978).
ile monokaryotic mycelium, in which each
hyphal compartment contains one nucleus.
Initial growth of this mycelium occurs beneath 15.4.4 Epidemiology
the surface of the substrate, with formation of
aerial hyphae a few days after germination. CNS infection by the S. commune was first
Monokaryons that encounter each other fuse, reported in 1955 by Batista from Brazil (Batista
and a fertile dikaryon forms when the alleles of et al. 1955), who repeatedly isolated it from
the mating-type loci matA and matB of the part- cerebral spinal fluid of a patient with meningeal
ners differ. A short exposure to light is essen- symptoms (basidioneuromycosis). Other cases
tial for fruiting, whereas a high concentration were reported from the USA (Rihs et al. 1996),
of carbon dioxide and high temperatures (30– Austria (Hoenigl et al. 2013), and Japan (Tone
37 °C) are inhibitory. Mushroom formation is et al. 2018). Currently, there is no information
initiated with the aggregation of aerial dikary- on the isolation of S. commune from any clini-
otic hyphae. These aggregates form fruiting cal material in the African continent (Chowdhary
body primordia, which further develop into et al. 2013a).
224 A. S. Kantarcioglu
15.4.5 Clinical Presentations was performed and the fluid showed fungal ele-
ments suggestive of Aspergillus spp. on frozen
Infective propagules in this fungus are air trans- section. Histologic examination of biopsy tissue
ported (De Hoog et al. 2000). Although the from lungs and brain showed numerous septate,
worldwide distribution of S. commune, infections hyaline, hyphal elements of various widths, mea-
originating from this fungus other than chronic suring 2.5–5.5 μm in diameter with hematoxylin
or allergic sinusitis have been rarely reported in and eosin and Gomori methenamine silver stains.
humans. Four cases of CNS infections have been Many hyphae branched at acute as well as right
reported to date, including atypical meningitis angles but did not exhibit dichotomous branch-
(basidioneuromycosis) (Batista et al. 1955), epi- ing. Many hyphal elements seen in the tissue
dural (Tone et al. 2018) and cerebral abscesses slides of both the lung and brain showed clamp
extended from sinusitis (Hoenigl et al. 2013), and connections. Isolated fungus was identified by
a disseminated case from pulmonary infection to characteristic phenotypical features. According
the brain (Rihs et al. 1996) in immunocompetent to the in vitro antifungal susceptibility results,
and immunocompromised individuals (by diabe- fluconazole (600 mg twice daily) was substituted
tes mellitus or corticosteroid therapy). All cases for itraconazole. After the patient had received
involved men. 1.754 mg of AMB over 36 days, he died develop-
The four cases with central nervous system ing respiratory failure and sepsis.
involvement available in English literature are Case 3: A 59-year-old Australian man with
summarized below with diagnosis and treatment diabetes mellitus and severe headache was
processes. diagnosed as sinusitis. A MRI revealed three
Case 1: The patient was a 24-year-old abscess formations in the right frontal lobe of
Brazilian man who suffered from a disorder of brain. Functional endoscopic sinus surgery was
the central nervous system marked by mental performed. Histology of sinus tissue revealed
dullness and signs and symptoms indicating multiple branched and septate fungal hyphae.
increased intracranial pressure without any focal- Culture of brain abscess drainage grew S. com-
izing sign. An electroencephalogram seemed to mune. L-AMB (3 mg kg−1 body weight) was
point to a diffuse lesion in the anterior part of administered for total of 5 weeks. Control MRI
the brain. A basidiomycetes fungus of the genus showed complete resolution of cerebral abscess
Schizophyllum was repeatedly isolated it from formation. Therapy was changed to oral posacon-
cerebral spinal fluid samples (Batista et al. 1955). azole 400 mg twice daily for 4. The patient was
This was the first reported case. released in good clinical condition and without
Case 2: The patient was a 58-year-old any residual symptoms.
American man with progressive muscle weak- Case 4: A 53-year-old Japanese man with
ness and multiple lung and brain lesions but had history of bronchial asthma and pollen allergy
no respiratory tract symptoms. A magnetic reso- and no prior treatment, presented with headache
nance imaging scan (MRI) revealed an enhancing and was diagnosed with ethmoid and sphenoid
mass in the left pons. An empiric trial dexa- sinusitis by computed tomography (CT) of the
methasone was begun and continued 5 weeks cranium (Tone et al. 2018). He developed an epi-
(total dose 330 mg). Computed tomographic dural abscess after antibiotic treatment. Samples
examination of the head showed a new ring- were obtained from epidural abscess and from
enhancing lesion within the right frontal lobe. A sinuses by endoscopic sinus fenestration. Direct
wedge resection of a lung mass revealed necro- microscopical examination of the epidural
tizing granulomatous inflammation with hyphae abscess material showed filamentous fungal
consistent with an Aspergillus spp. Treatment of elements. Microscopic examination of slide
AMB and itraconazole was begun. An MRI of cultures showed white cottony sterile mycelia
the brain showed increase in size and number of without clamp connections. The isolated fungus
the frontal brain lesions. A stereotactic biopsy was identified by sequence analysis of the ITS of
15 Cladosporium spp., Fusarium spp., Bipolaris spp., Schizophyllum commune, and Scedosporium... 225
ribosomal DNA (rDNA) and D1/D2 domain of pale brown reverse after 2 weeks at 25 °C. It pro-
285 rDNA. Antifungal susceptibility tests were duces a strong disagreeable odor. Cycloheximide
done using E test strips, and he was treated with inhibits the growth on SDA.
liposomal amphotericin B (L-AMB, 300 mg/ Microscopic examination of the mold showed
day, titrated from 0.2 to 5 mg/kg/day). He was hyphae of various widths. Lactophenol cotton
discharged from the hospital on day 25, and no blue mount of the cultured fungus can reveal
recurrence has been observed for over 2 years. hyaline septate hyphae, often with clamp con-
nections or lateral pegs (O’Gorman and Fuller
2008).
15.4.6 Histopathology Most isolates readily produce basidiocarps
and clamp connections on Czapek’s, potato dex-
The biopsied tissue or surgical specimen trose, or corn meal agars and lateral tubercles on
should be submitted to laboratory for myco- their hyphae (Rihs et al. 1996). Slide cultures
logical as well as histopathological examination. showed hyaline, septate, branched hyphae of
Histological examination may be performed 2.0–4.5 mm in diameter with clamp connections.
using conventional hematoxylin-eosin, Gomori Many hyphae developed lateral, short, thin, trun-
methenamine silver and periodic acid-Schiff cate tubercles diagnostic of S. commune.
stains and may reveal an inflammation consist- Due to the presence of sterile filaments and
ing of mixed inflammatory cells and hyphae lack of specific structures in early stage of identi-
in the involved tissue. No angioinvasion was fication, for induction of sporulation, inoculated
reported by this fungus. Petri dishes containing malt extract agar, malt
The fungus was characterized by clamp con- yeast glucose agar may be incubated at 25 °C,
nections, hyphal spicules, and formation of Czapek Dox agar at 28 °C, and potato dextrose
basidiocarps with basidiospores (Sigler et al. agar at 30 °C for 4–20 days in different environ-
1995; Chowdhary et al. 2013a). In tissue sec- mental condition (dark, light, and UV exposure)
tions, the hyphae may not always exhibit clamp for further examinations (O’Gorman and Fuller
connections. Their differentiation from hyphal 2008).
elements of Aspergillus spp. could be readily Because monokaryotic isolates of S. com-
achieved by the use of serologic tests for the mune are difficult to identify, techniques such as
detection of Aspergillus antibodies and antigens mating (vegetative compatibility) tests or gene
(Rihs et al. 1996). sequencing may be required to identify S. com-
mune. However, the mating test is not easy to
perform in a conventional laboratory and may
15.4.7 Laboratory Diagnosis require prolonged time for identification of the
pathogen in most cases. Instead, a few studies
Direct microscopy of a KOH (10%) prepara- have shown that gene sequencing is an attrac-
tion of the biopsy or surgical specimen showed tive method to identify this fungus. Won et al.
the presence of hyaline septate hyphae. Giemsa (2012) recommended that gene sequencing of
stained preparations can show septate branched the ITS region and D1/D2 regions of the 26S
hyphae. ribosomal DNA was very helpful for accurate
Some portions of tissue samples can be inocu- identification of this fungus. In fact, given the
lated both on multipoints of SDA supplemented excellent specificity of this technique, gene
with chloramphenicol (0.5 μg/ml), or containing sequencing has been recognized as the gold
gentamycin, and on Brain Heart Infusion Agar standard for fungal identification. However, for
and can be incubated at 30 °C for 4–10 days. accurate identification of rare molds such as S.
Growth on SDA can be rapid, cottony, and whit- commune, morphological findings and molecu-
ish and raised in center. Colonies can become lar data should be compared to ensure consis-
slightly powdery and pale buff or tan having a tency (Balajee et al. 2007, 2009).
226 A. S. Kantarcioglu
The characteristic phenotypical features that histological similarity with Aspergillus infections
were indicative of S. commune were as follows: and of the difficult identification of the isolated
fungus with sterile mycelia.
(a) It grew well at 37 °C.
(b) It is easy to culture on media commonly used
in clinical laboratories and formed rapidly 15.5 Scedosporium apiospermum
growing, woolly colonies with septate hya-
line, branching hyphae of two widths. 15.5.1 Introduction
(c) It is susceptible to cycloheximide (400 μg/
ml). The fungus Scedosporium apiospermum, initially
(d) It tolerates to 2–10 mg of benomyl per ml. considered the anamorph or asexual state of asco-
(e) It forms a dense, tough (i.e., difficult to cut), mycetous fungus Pseudallescheria boydii, is a
woolly colony. filamentous fungus that can be commonly found
(f) It has a tart and pronounced odor (Won et al. as a saprophyte in soil, sewage, mud, and the pol-
2012; Premamalini et al. 2011). luted waters of streams and ponds with still water
(de Hoog et al. 1994). Recent molecular studies
Matrix-assisted laser desorption/ionization have demonstrated that P. boydii is a complex that
mass spectrometer (MALDI-TOF MS) makes it includes several phylogenetic species (Gilgado
possible to identify fungi whose macroscopic and et al. 2005; Chen et al. 2016).
microscopic morphological features are usually As a result of fundamental changes in the
uninformative. S. commune was identified using International Code of Nomenclature on the use of
MALDI-TOF MS, and identifications were con- separate names for sexual and asexual stages of
firmed via DNA sequence analysis (Chowdhary fungi, generic names of many groups should be
et al. 2013a; Michel et al. 2015). reconsidered. Members of the ECMM/ISHAM
working group on Pseudallescheria/Scedosp
orium infections proposed a novel nomencla-
15.4.8 Antifungal Susceptibility, ture for genera and species in Pseudallescheria,
Treatment, and Outcome Scedosporium, and allied taxa (Lackner et al.
2014).
Although the interpretive MIC breakpoints have The Scedosporium apiospermum species
not yet been defined for S. commune and the cor- complex, comprising five filamentous fungal
relations between clinical response and MIC val- species S. apiospermum sensu stricto, S. boy-
ues for a given strain are uncertain, MICs of the dii (= Pseudallescheria boydii, P. angusta), S.
isolates, treatment, and outcome reported in dif- aurantiacum, S. dehoogii, and S. minutispora,
ferent clinical cases and in an in vitro antifungal are important pathogens that cause a wide vari-
susceptibility study were shown in Tables 15.4 ety of infections (Giraud and Bouchara 2014).
(Rihs et al. 1996; Hoenigl et al. 2013; Tone et al. The species of genus Scedosporium can be
2018) and 15.5 (Chowdhary et al. 2013b). Delay distinguished with the primary fungal DNA
in initiation of appropriate antifungal treatment barcode, the ITS1/2 region of the rDNA gene
and the use of corticosteroids was associated with cluster (Chen et al. 2016). Chen et al. (2016)
treatment failure. stated that for reasons of absence of genetic
The occurrence of S. commune as a human separation, as well as absence of clinical rel-
pathogenic fungus may be much more frequent evance of individual lineages, the species S.
than previously assumed. Improved mycological apiospermum, P. angusta, and S. boydii should
identification methods may have given rise to a be referred to as the “S. apiospermum species
larger number of S. commune rhinocerebral cases. complex.” Because of the degree of involvement
Those cases would probably have been misdiag- of each individual species in human infections
nosed in the past because of the close clinical and has not been determined, the present chapter
Table 15.5 Antifungal susceptibility test results of S. Commune case isolates reported
MIC (μg/ml) Antifungal therapy
Reference AMB FLZ ITZ VRZ PSZ CAS Outcome
Rihs et al. (1996)a <0.03 8 ND ND ND ND AMB + ITZ (4 d), AMB (over 36 d) + FLZ (600 mg Died
twice daily)
Hoenigl et al. 0.25 12 >32 0.125 0.25 >32 L-AMB (5 w), PSZ (400 mg twice daily) Survived
(2013)b
Tone et al. (2018)b 0.75 ND 1 ND ND >32 L-AMB (300 mg/d, titrated from 0.2 to 5 mg/kg/d, Survived (2 y follow-up, no
3 w) recurrence)
Abbreviations: MIC minimal inhibitory concentration, AMB amphotericin B, FLZ fluconazol, ITZ itraconazol, VRZ voriconazole, PSZ posaconazole, CAS caspofungin, L-AMB
liposomal AMB, ND not determined, d day, w weeks, y years
a
Method unknown
b
E test
15 Cladosporium spp., Fusarium spp., Bipolaris spp., Schizophyllum commune, and Scedosporium...
227
228 A. S. Kantarcioglu
will maintain the name S. apiospermum in all or predisposing factors. Twenty-four of the pre-
disease entities. viously healthy patients (55%) had a history of
A computerized search of the MEDLINE data- aspiration of polluted water in association with
base (National Library of Medicine, Bethesda, near drowning. Nine of the previously healthy
MD, USA) was performed for cases reported in the patients were posttraumatic cases. A major-
literature between 1948 and mid-2006, with (by ity of patients with underlying disease and/or
cross-referencing) the terms “Pseudallescheria predisposing factor were transplant recipients.
boydii” and “Scedosporium apiospermum,” Apparent major risk factors for CNS infection
“Scedosporium apiospermum species complex,” were aspiration of polluted water in near drown-
“Pseudallescheria boydii/Scedosporium species ing episodes in immunologically intact patients
complex,” “cerebral,” “brain abscess,” “menin- and medical immunosuppression in the remain-
gitis,” “central nervous system infection,” “dis- ing patient groups (Kantarcioglu et al. 2008). The
seminated,” and “near-drowning.” Additional overall fatality rate was very high (76%).
search terms included were “Allescheria boydii,”
“Monosporium apiospermum,” and “Petriellidium
boydii” as referring to prior or other nomenclature 15.5.4 Portal of Entry and Routes
for this fungus. These keywords were used alone of Infection
and/or in combination with an “and” statement.
Additional cases were obtained by scanning the S. apiospermum shows a marked neurotropism
references cited in the original articles. Original and a high propensity to cause CNS infections.
full texts of all the relevant articles were obtained In most of the CNS associated disseminated
via MEDLINE, TUBITAK-ULAKBIM (Turkish cases, particularly the vascular organs such as
Academic Network and Information Center), or the kidney, the thyroid and heart have been also
personal communication of the authors and/or involved probably due to the particular tropism
other international libraries and were used for the for blood vessels and hematogenous spread of
analysis. this fungus. The usual portal of entry of CNS
infection by S. apiospermum was presumed to be
the respiratory tract with hematogenous spread to
15.5.2 Epidemiology the brain. However, isolation of S. apiospermum
from outside and indoor air was extremely infre-
This fungus shows a particular tropism for the quent. Remarkably, direct entry by aspiration of
CNS in both healthy and immunocompromised contaminated water from the pharynx, through
patients. The first report of S. apiospermum CNS sinuses close to the brain and ethmoid bone,
infection was by Benham et al. in 1948. The seemed the most frequent portal of entry regard-
majority of the CNS infections by S. apiosper- ing the 16 near-drowned cases in whom no pul-
mum were reported from the USA, Germany, monary involvement was observed (Kantarcioglu
France, and Canada, and sporadic cases were et al. 2008).
reported in other countries around the world
(Kantarcioglu et al. 2008).
15.5.5 Signs and Symptoms
behavior. Fifteen percent of the patients had molecular techniques involving genes such as
coma on admission, and 12% had no complaints β-tubulin and calmodulin and ITS regions 1 and
and/or had no abnormal neurological signs 2 had been recommended for species identifica-
(Kantarcioglu et al. 2008). tion (Lackner et al. 2012; Gilgado et al. 2008;
Lu et al. 2011).
MALDI-TOF MS can be used in the rou-
15.5.6 Clinical Presentations tine clinical laboratory in the identification of
members of the complex provided that valid
Main clinical types were brain abscess, coin- spectra libraries are developed. Species identi-
fection of brain tissue, and/or spinal cord with fication appears feasible with MALDI-TOF MS
meninges and meningitis. The mortality rate (Coulibaly et al. 2011).
was of 74% regardless of the patient’s immune
status or the infection type and/or location
(Kantarcioglu et al. 2008). 15.5.9 Treatment and Outcome
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Part III
Clinical Syndromes of Fungal Infections
Involving Central Nervous System and Its
Coverings
Cranial Vault Lesions
16
Ali Akhaddar
1950). Mucormycosis is habitually a skull base common cause of cranial osteomyelitis, while in
infection. However, two cases of mucormycosis developing nations, paranasal sinusitis and scalp
were reported in the calvaria following rhinocer- infections have become the predominant sources
ebral invasion (Fortún et al. 1995; Honeybul and (Akhaddar 2016).
Morrison 2012; Taj-Aldeen et al. 2017). Other The cranial vault has important and com-
fungi have been described involving the cranial plex anatomic relationships with extracranial
vault, but often much less frequently. The major- and intracranial structures. So, the problem of
ity of cases of calvarial mycosis are reported this disease is likely to get worse when intracra-
from developing countries. However, changes nial invasion occurs with their potentially life-
in the socioeconomic status and advances in threatening consequences (Akhaddar 2017a).
medicine are responsible for a relative increas- In general, the fungal agent reaches the skull
ing number of infections secondary to fungi vault by the outer table (local invasion) or through
especially in immunocompromised subjects and the diploe (hematogenously). Unlike fungal skull
those with fungal paranasal sinusitis and orbital base osteomyelitis, direct extension from parana-
sepsis (Patel et al. 2011). In the calvaria, pari- sal sinusitis or orbital infections is rare (Panda
etal and frontal bones are more usually affected and Ekambar Eshwara Reddy 2005; Patel et al.
than occipital and temporal which are less vas- 2011; Wolkow et al. 2017). CVM may be very
cularized. CVM are less commonly described invasive, involving both tables, and in extreme
in infants than in adults without apparent sex cases may destroy the galea, scalp, and dura with
predominance. Nevertheless, Rosanova et al. subdural and brain invasion (Fig. 16.1).
reported 3 children with CVM in a recent series Some comorbidities or patient-predisposing
of 12 children with burns and osteomyelitis. The risk factors should be taken into consideration.
youngest child described was only 18 months These include vulnerability due to metabolic
old (Wright et al. 1993). Unlike skull base osteo- states, compromised immune function, malig-
myelitis, CVM arises mostly from disseminated nancy, and advanced age (Agadi et al. 2010;
infection and skull trauma (Fortún et al. 1995; Amit et al. 2008; Effat et al. 2005; Pudipeddi
Honeybul and Morrison 2012; Rodríguez- et al. 2016; Qiu et al. 2015; Rerolle et al. 2005;
Hernández et al. 2001; Rosanova et al. 2018). Rootjes et al. 2016; Wolkow et al. 2017; Wright
One case of cranial Aspergillus fumigatus infec- et al. 1993). The use of broad-spectrum antibiot-
tion with intracranial epidural abscess follow- ics is known to be one major risk factor for fungal
ing craniotomy was previously reported in an infections (Akhaddar 2017b).
immunocompetent young patient (Letscher et al. The cranial vault may be infected by vari-
1997). ous fungal pathogens, mostly from Aspergillus
species which are ubiquitous in the environ-
ment and usually noninvasive. Several species
16.3 Pathogenesis and Pathology have been reported as pathogens especially A.
fumigatus and A. flavus. Apart from Aspergillus
Traditionally, cranial osteomyelitis is usually spp, Cryptococci, Coccidioidomycetes and
related to three main sources of infection: (1) Mucorales (Zygomycetes) are the most common
direct extension from contiguous site of infec- pathogens involved. Other reported fungi caus-
tion, (2) postsurgical or post-traumatic direct ing CVM include Blastomyces, Candida spe-
inoculation (from a contaminated wound), and cies, Trichosporon asahii, Penicillium marneffei,
(3) hematogenous dissemination from remote Pseudallescheria boydii, Apophysomyces varia-
source of infection. In developed countries, post- bilis, and Streptomyces somaliensis (Fernández-
surgical craniotomy infections remain the most Guerrero et al. 1987; Mateev et al. 1993;
16 Cranial Vault Lesions 239
Fig. 16.1 Staging
system for skull
osteomyelitis (3SO).
Five main types of
cranial osteomyelitis
based on the location of
infection and its
extension (Reproduced
from Akhaddar A (ed)
Cranial osteomyelitis
(2016); with permission)
Type 0 Type 1
Initial cranial bone involvement Limited to cranial bone
Type IV
Cranial, extracranial and
intracranial involvement
240 A. Akhaddar
Naim-Ur-Rahman et al. 1987; Patel et al. 2011; abscess and underlying frontal bone osteomyeli-
Qiu et al. 2015; Rosanova et al. 2018; Wolkow tis of frontal bone (Akhaddar 2016).
et al. 2017; Yocum and Seligson 1991). In more chronic forms, the onset of symptoms
is more insidious and the clinical course more
prolonged, masked in part by the primary ill-
16.4 Clinical Features ness. In fact, more immunocompromised patients
are less likely to exhibit florid systemic signs
Clinical features of CVM may vary greatly of infections. It is important to pay attention to
according to many factors such as age of onset, multifocal lesion in the body. Routine general
duration of disease, route of infection, underly- systemic examination should be performed to
ing etiologies, type of fungi, comorbidities, and identify apparent anomalies and possible other
anatomic location of infection and its extension. distal source of fungal infection (Mateev et al.
Generally, the most usually described clinical 1993; Qiu et al. 2015; Rerolle et al. 2005).
presentations are not too different from those Clinically, some patients with cranial vault
with bacterial cranial vault osteomyelitis: mainly osteomyelitis can present with signs and symp-
local signs of inflammation and/or infection toms that imitate other calvarial and scalp
(soft tissue swelling, pain, purulent discharge, pathologies (especially its tumoral or pseudo-
and exposed bone) (Patel et al. 2011). Fever was cystic presentations) such as many inflamma-
not a common feature in CVM (Akhaddar 2016; tory cutaneous lesions, vascular malformations,
Agadi et al. 2010). However, many patients pre- infected hematoma or scalp tumors, folliculitis,
sented skin tenderness and more rarely a drain- sebaceous cysts, skin and scalp abscesses, cellu-
ing fistulous tract (Mateev et al. 1993; Yocum litis, dermoid and epidermoid cysts, encephalo-
and Seligson 1991). When the infection spreads celes fibrous dysplasia, and different benign and
into the epidural space, headache worsens. In malignant tumors (Akhaddar 2016).
advanced conditions if the lesion increases in Any nonhealing ulcerations, spontaneous
size, altered mental status and occasionally focal scalp infections, and/or chronic sinus tracts
neurologic signs develop with or without signs of should raise a suspicion of a fungal infection
raised intracranial pressure (Letscher et al. 1997; (Beeram et al. 2008; Mateev et al. 1993).
Naim-Ur-Rahman et al. 1987). Focal neurologic
deficits are according to the site of the lesion in
the cranial vault such as hemiparesis, hemisen- 16.5 Diagnosis
sory deficit, cognitive disorder, visual deficit,
seizures, and meningismus. Rarely, more severe Diagnosis is often delayed and frequently con-
cerebral complications may arise with rapid neu- sidered only following failure of antibacterial
rologic deterioration, respiratory failure, hemo- therapy (Effat et al. 2005). A detailed history,
dynamic instability, loss of consciousness, and clinical evaluation, laboratory investigations,
death (Reining et al. 1984; Wright et al. 1993). culture, and imaging explorations help the diag-
When the duration of symptoms is less than nosis. However, the most significant finding for
one month, the cranial bone infection is consid- diagnosis should be made by histopathological
ered acute. In chronic infections, the duration of examination and culture of specimens obtained at
symptoms is several months (Akhaddar 2016). surgery (direct biopsy or surgical debridement).
Four cases of CVM were previously reported Neuroimaging data are usually indicative
with frontal sinusitis and forehead swelling mim- of osteomyelitis but are not specific for cranial
icking a Pott’s puffy tumor (Effat et al. 2005; vault mycosis. The diagnostic imaging features
Panda and Ekambar Eshwara Reddy 2005; Patel of osteolysis (irregular and well-defined lytic
et al. 2011). This is named after Sir Percival lesion), bone destruction, sometimes show-
Pott who described the association of forehead ing sequestrum (Fig. 16.2), and increased
localized swelling with overlying subperiosteal T2-weighted image on magnetic resonance
16 Cranial Vault Lesions 241
a b
Fig. 16.2 Chronic skull bone osteomyelitis with sequestrum. Axial CT scan of the skull in parenchymal (a) and bone
windows (b) showing isodense parietal bone lesion (arrows) with extracranial extension
imaging are compatible with any osseous infec- some difficult cases, definitive diagnosis requires
tion but without specificity for fungal patho- culture on enriched agars. The role of a histo-
gens. Dura matter involvement after contrast pathology is also to differentiate mycosis from
enhancement is not rare. In developing coun- tuberculosis and malignant tumors (Akhaddar
tries, tuberculosis should be considered as dif- 2016).
ferential diagnosis (Amit et al. 2008). Besides Morphologic and molecular diagnosis of fun-
computed tomography scan and magnetic gal infections of the central nervous system is
resonance imaging, nuclear medicine tech- discussed in the other chapters of the book.
niques are very sensitive for skull infection and
are an excellent positive indicator (Wood and
Miedzinski 1996). 16.6 Treatment Options
The results of routine laboratory investiga- and Outcomes
tions are usually within normal limits.
In aspergillosis, histopathologic study There are no specific treatment recommendations
shows vascular thrombosis that is surrounded for cranial bone osteomyelitis. Old publications
by extensive coagulative necrosis and hemor- have stressed the importance of radical surgery in
rhage. Granulomatous inflammation with septate combination with an antifungal agent in treating
hyphae branching at acute angle or dichoto- most osseous mycosis. Data on patient survival is
mously at irregular intervals is highly suggestive available only from case reports and small case
of the diagnosis. On the contrary, mucormyco- series.
sis is characterized by irregular hyphae without The antifungal agents of choice have changed
septae, branching at right-angle (90°). Histologic over the past two decades. For aspergillosis, vori-
features of cryptococcal infections correspond conazole now represents the first-line treatment
to chronic granulomatous inflammation and the for invasive aspergillosis and especially for bony
presence of numerous yeast-like organisms using invasion (Gamaletsou et al. 2014). Recent cases
hematoxylin and eosin, periodic acid Schiff, with cryptococcal cranial vault were treated suc-
Gomori methenamine silver and alcian blue cessfully with intravenous amphotericin B and
stains (Wood and Miedzinski 1996). However, in oral fluconazole. Coccidioidomycosis involving
242 A. Akhaddar
the skull is also treated with a combination of In some patients on antifungal therapy, disease
lipid amphotericin B and long-term fluconazole may progress slowly and intermittently, requiring
(Antony et al. 2015). Currently, itraconazole and multiple courses of antifungal drugs (Wolkow
ketoconazole are the best treatment options for et al. 2017).
bone mycetoma. Amphotericin B is the antifungal Immunocompetent patients tend to have better
drugs of choice for mucormycosis. Alternatively, outcomes as compared to those who are immu-
posaconazole can be considered. Treatment dura- nocompromised. Patients with intradural disease
tion depends on the condition of the patients, the carry the worst prognosis (Wright et al. 1993).
presence of complications, and the precocity of
diagnosis. A minimum of 3 months is necessary
to avoid recurrences. Some authors, suggest that 16.7 Conclusion
treatment duration of 6 months is mandatory
(Rodríguez-Hernández et al. 2001). More details Cranial vault fungal infection is a rare disease,
concerning chemotherapy for fungal infections infrequently encountered, and not always consid-
are discussed in another chapter of the book. ered as a first differential diagnosis. Pathology
Patients with partial extracranial soft tissue plays an important role for confirming the defini-
infection with mild cranial osteomyelitis can be tive diagnosis of CVM, especially if clinical find-
treated with antifungal drugs alone. Sometimes, ings, laboratory studies, and diagnostic imaging
simple cutaneous drainage or limited scalp inci- investigations are not conclusive.
sion are sufficient and may be used to collect The diagnosis should be considered in any
specimens for diagnosis. Surgical debridement insidious growing soft tissue mass or scalp
remains critical to eliminate areas of necrosis abscess of unertain origin in addition to any
and sequestrum. Infected bone with evidence of osteolytic calvarial lesion with granulomatous
necrosis should be removed (more or less exten- reaction.
sive craniectomy), followed by cranioplasty on
a second intervention (using methyl methacry-
late or titanium plate) (Antony et al. 2015). An
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Meningitis
and Meningoencephalitis 17
Alexa Bodman and Walter A. Hall
Infection with Aspergillus spp. in the CNS can cidioidal meningitis will often show eosinophilia
result in devastating neurological consequences. (Hall and Kim 2013). CSF analysis in coccidioi-
Aspergillus spp. gather near leptomeningeal ves- dal meningitis can show an elevated white blood
sels, forming fungal thrombi that can lead to cell count with a predominance of lymphocytes
cerebral infarction (Kleinschmidt-DeMasters and eosinophils, a decreased or normal glucose
2002). CNS aspergillosis is associated with a level, and a normal or elevated protein level (Zunt
poor prognosis and low survival rate (Hall and and Baldwin 2012). Cultures of CSF will only
Kim 2013). One study showed a mortality rate of demonstrate growth in 15% of coccidioides
57.5% in hematopoietic stem cell transplantation infections (Zunt and Baldwin 2012). Prior to the
patients that had invasive aspergillosis (Baddley advent of antifungal agents, the mortality for this
et al. 2010). Susceptibility testing to antifungal disease approached 100% (Drake and Adam
agents may be useful in guiding treatment 2009). Coccidioidal meningitis should be man-
(Ullmann et al. 2018). Voriconazole and isavuco- aged with oral fluconazole initially with doses of
nazole are recommended for the treatment of 400–1200 mg daily followed by lifelong mainte-
CNS aspergillosis where in those cases that are nance therapy (Galgiani et al. 2016). If patients
resistant to azoles, liposomal amphotericin B can fail management with an azole, intravenous
be used (Ullmann et al. 2018). amphotericin B can be used (Johnson and
Einstein 2007). Coccidioidal meningitis can
cause a vasculitis which can result in vascular
17.5 Coccidioidomycosis occlusion and stroke (Williams et al. 1992). In
patients who develop a stroke due to coccidioidal
Coccidioides immitis is a fungal species endemic meningitis, corticosteroids may reduce the inci-
to the southwestern United States that is resistant dence of experiencing a second adverse event
to extremes of climate and multiplies after rain- (Thompson III et al. 2017).
falls (Zunt and Baldwin 2012). Once inhaled, a
respiratory infection may develop that is known
as valley fever with disseminated disease which 17.6 Histoplasmosis
typically only occurs in immunocompromised
patients (Zunt and Baldwin 2012). Meningitis Disseminated disease from Histoplasma capsu-
occurs in roughly one-half to one-third of patients latum is usually associated with an immunocom-
with disseminated C. immitis (Zunt and Baldwin promised state and spreads to the CNS in
2012). Males of an African American or Asian 10–20% of cases (Hall and Kim 2013). This dis-
descent, specifically Pacific Islanders, are at ease is endemic to the Mississippi and Ohio river
higher risk for spread to the CNS (Drake and valleys in the United States (Zunt and Baldwin
Adam 2009). Clinical manifestations include 2012). Leptomeningeal involvement is usually
headache, altered mental status, nausea, vomit- basilar in nature presenting with headaches and
ing, nuchal rigidity, and focal neurologic deficits altered mental status but can also present with
(Zunt and Baldwin 2012; Drake and Adam 2009). multiple cranial neuropathies, hydrocephalus,
Vasculitis, cerebral infarction, and hydrocephalus and seizures (Hall and Kim 2013). CSF analysis
are common complications of C. immitis menin- shows a predominantly elevated mononuclear
gitis (Zunt and Baldwin 2012; Arsura et al. 2005). white blood cell count with elevated protein lev-
The presence of hydrocephalus was found in els and decreased glucose levels (Zunt and
51.6% of patients that had radiographic imaging Baldwin 2012). Histoplasmosis in the CNS
and was associated with a mortality increase of should be treated with 4–6 weeks of liposomal
12.5-fold (Arsura et al. 2005). Ventriculoperitoneal amphotericin B followed by itraconazole for
shunt placement may be necessary to treat hydro- 1 year. Histoplasma antigen levels can be fol-
cephalus in the setting of coccidioidal meningitis lowed to monitor the response to treatment
(Galgiani et al. 2016). The blood or CSF in coc- (Wheat et al. 2007).
17 Meningitis and Meningoencephalitis 249
Table 17.1 The common causative of fungal meningitis Boelaert JR, Fenves AZ, Coburn JW. Deferoxamine
and meningoencephalitis in humans therapy and mucormycosis in dialysis patients:
report of an international registry. Am J Kidney Dis.
Common causative agents of fungal meningitis
1991;18:660–7.
Aspergillus fumigatus Bongomin F, Gago S, Oladele R, Denning D. Global and
Blastomyces dermatitidis multi-national prevalence of fungal diseases—esti-
Candida albicans mate precision. J Fungi (Basel). 2017;3 https://doi.
Coccidioides immitis org/10.3390/jof3040057.
Cryptococcus gattii Chapman SW, Dismukes WE, Proia LA, Bradsher RW,
Cryptococcus neoformans Pappas PG, Threlkeld MG, Kauffman CA. Clinical
practice guidelines for the management of blastomy-
Histoplasma capsulatum
cosis: 2008 update by the Infectious Diseases Society
of America. Clin Infect Dis. 2008;46:1801–12.
Chen SCA, Slavin MA, Heath CH, et al. Clinical manifes-
fungal agent for C. bantiana. This fungal infec- tations of Cryptococcus gattii infection: determinants
tion is associated with a 65% mortality rate of neurological sequelae and death. Clin Infect Dis.
2012;55:789–98.
(Kantarcioglu et al. 2016). Cherian J, Atmar RL, Gopinath SP. Shunting in crypto-
coccal meningitis. J Neurosurg. 2015;125(1):177–86.
Concha-Velasco F, González-Lagos E, Seas C, Bustamante
17.10 Conclusion B. Factors associated with early mycological clear-
ance in HIV-associated cryptococcal meningitis. PLoS
One. 2017;12:e0174459–12.
Fungal meningitis and meningoencephalitis often van der Horst CM, Saag MS, Cloud GA, et al. Treatment
have a subacute or chronic presentation delaying of Cryptococcal meningitis associated with the
their diagnosis (Zunt and Baldwin 2012). acquired immunodeficiency syndrome. N Engl J Med.
1997;337:15–21.
Cultures can be slow to grow and may be nega- Dobre MC, Smoker WRK, Kirby P. A case of solitary
tive. Recognition of fungal meningitis and the Blastomyces dermatitidis meningitis. Clin Neurol
subsequent initiation of appropriate therapies is Neurosurg. 2011;113:665–7.
the key to decreasing the significant neurological Drake KW, Adam RD. Coccidioidal meningitis and brain
abscesses: analysis of 71 cases at a referral center.
morbidity and mortality associated with this dis- Neurology. 2009;73:1780–6.
ease (Table 17.1). Franco-Paredes C, Womack T, Bohlmeyer T, Sellers B,
Hays A, Patel K, Lizarazo J, Lockhart SR, Siddiqui W,
Marr KA. Management of Cryptococcus gattii menin-
goencephalitis. Lancet Infect Dis. 2015;15:348–55.
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Raised Intracranial Pressure
18
Hasan Emre Aydın and Ismail Kaya
Optic nerve sheath (ONS) diameter: In the In this context, various treatment strategies
study on monkeys by Hayreh in 1968, subjects have been defined for intracranial hypertension.
with high ICP were shown to have ONS dilation. Among these are medical treatment, series of
With the development of ultrasonography and fol- lumbar punctures and placing external lumbar
lowing the non-invasive evaluation of ICP through and ventricular drains. The literature is not clear
the ONS diameter method, ONS diameter has regarding which approach is indicated and when
been started to be evaluated with ultrasonography and how often the patients need neurosurgical
in many pathologies, which may have increased intervention. The importance of early diagnosis
ICP (Sekhon et al. 2014). The optic nerve is sur- and adequate treatment of cryptococcal meningi-
rounded by the dural sheath, which is a part of the tis and severe intracranial hypertension are
CNS, and there is an area between the sheath and emphasized (Cherian et al. 2016).
the white matter related to the subarachnoid A recently conducted autopsy study showed
space. In cases of increased ICP, this sheath wid- that there was a correlation between high con-
ening was shown to be correlated with ICP. centration of live and dead organisms in arach-
Involvement of brain parenchyma and menin- noid granulations and increased CSF pressure.
ges, observed in 40–86% of the patients, is specific Prevention of CSF resorption was suggested as
to CNS fungal infections and the predisposition of a cause for increased pressure and development
these areas to infection cannot be explained. It of hydrocephalus (Loyse et al. 2010). Cerebral
may be acute or chronic. Headache, fever, and herniation and brain death due to cryptococcal
neck stiffness, which indicate meningeal irritation, meningoencephalitis have been reported to be
manifest as acute (<3 days) or chronic rare. Many studies have shown that high CSF
(>30 days) symptoms. However, in human immu- fungal load (stated with high cryptococcal anti-
nodeficiency virus (HIV)-infected patients, head- gen titres) alters mental state, increases spread of
ache and fever are not always observed. Instead, infection (with the manifestation of fungaemia),
there may be lethargy, stupor, coma, and even and symptomatically increases ICP. Moreover,
dementia in these patients. Clinical symptoms are the low number of CSF leukocytes is a factor
not adequate in determining the etiology of menin- associated with poor outcome of acquired
gitis. Although papilloedema is observed, focal immunodeficiency syndrome (AIDS)-related
neurologic symptoms such as paralysis of cranial cryptococcal meningoencephalitis (Jarvis et al.
nerves are not observed. Intracerebral, cerebellar, 2014).
and spinal cord lesions are rarely reported. In most recent studies, non-invasive ICP mon-
Complications of CNS fungal infections are itoring methods have been shown to be systems
related to hydrocephalus, focal motor disorders, that provide ICP diagnostic findings, results of
mental changes, and increased ICP. interventional operations, and simultaneous
Increased ICP is a prevalent complication of monitoring of ICP. Currently, ICP monitoring is
fungal meningitis. It has been suggested that the carried out by placing invasive sensors inside the
reason for this is block in the outflow because of skull to detect pressure over time and its varia-
accumulation of fungal polysaccharide residues in tion. Invasiveness and high cost of this method
the arachnoid villus and subarachnoid spaces; thus, limit its use for most patients. Monitoring of ICP
CSF canals could be blocked (Bellner et al. 2004; with non-invasive methods is being developed by
Lee et al. 1996). Another important factor may be various groups around the world using technolo-
cytokine-induced inflammation and cerebral gies such as transcranial doppler and imaging
oedema, possibly due to the osmotic effect of the (Alperin et al. 2015). Non-invasive ICP sensors
fungal origin of mannitol (Denning et al. 1991). can monitor the pulses of ICP, which shows the
Increased CSF pressure is commonly observed morphologic changes consistent with the patient
among these patients. If the high CSF pressure is clinical state. Severe intracranial hypertension
inadequately managed, a significant neurologic can develop in patients with neuroinfection and
deficit may occur. even cause decrease in the cerebral perfusion
256 H. E. Aydın and I. Kaya
pressure connected to intracranial hypertension Different studies put forth that opening pres-
in patients with a normal opening pressure. sure must be measured as the first step in all
Monitoring of ICP and cerebral perfusion pres- patients with suspected fungal meningitis.
sure may be performed in patients with neuroin- Additional lumbar punctures including daily
fection and can be used in estimating cerebral lumbar punctures should be performed until the
hemodynamic changes. pressure is adequately controlled for patients
Intracranial hypertension is prevalent in fun- with high ICP (Day et al. 2013). Pharmacologic
gal meningitis and is related to increased mortal- agents such as mannitol and corticosteroids are
ity rates. CSF opening pressure in 60–75% of avoided, and neurologic interventions including
patients is >20 cmH2O. The effect of CSF hyper- ventriculoperitoneal (VP) or ventriculoatrial
tension on morbidity and mortality related to this (VA) shunt placements are usually avoided or
disease supports current suggestions with routine delayed. The most appropriate time to implant a
and frequent ICP measurement using spinal nee- permanent shunt (VP or VA) in patients with
dle manometry. When CSF opening pressure is uncontrollable ICP is controversial, but usually
>25 cmH2O, it is recommended for the CSF the device should be placed after starting the
drainage to be conducted with daily spinal nee- optimal antifungal treatment (Pappas 2005).
dles and the opening pressure to be lowered to A series of lumbar punctures is a prevalent
<20 cmH2O or to 50% of the starting value. application among clinicians for the treatment of
Usually there is a high level of uncertainty, and these patients. As a secondary choice, some clini-
the basis of clinical decisions regarding ICP cians prefer placing lumbar drainage, which has a
hypertension tends to be based on clinical symp- high risk of infections or complications.
toms (headache, nausea, and vomiting), low Permanent VP shunting is required after 4 weeks
Glasgow Coma Scale score, and/or fundoscopic of appropriate antifungal treatment in patients
papilloedema. Inadequate management of high who need a series of lumbar punctures (Corti
CSF pressure can result in neurologic decline et al. 2010).
(Bolela et al. 2017). ICP is associated with severe complications in
Optimal medical treatment consists of a com- patients with cryptococcal meningitis, and placing
bination of amphotericin-B and 5-flucytosine for a VP shunt can ensure adequate treatment even in
2 weeks, which is known as the fungal meningo- cases wherein neurologic imaging studies show no
encephalitis induction phase. Consolidation hydrocephalus or permanent CSF cryptococcus
phase is 8-week-long treatment period monitored infection. Although medical treatment of ICP
with fluconazole and is followed by care phase or among these patients is being discussed, placing a
secondary prophylaxis to prevent relapses. VP shunt should be the first therapeutic alterna-
Despite severe toxicity, triple treatment in accept- tive. However, post-operative meningitis and ven-
able doses is a superior treatment method in cryp- triculitis after shunt infection are well-documented
tococcal meningitis related and unrelated to HIV complications among these patients. Both compli-
(Xu et al. 2018). The management of increased cations frequently occur in post-operative early
ICP without hydrocephalus in patients with fun- period and are secondary to wound infection or
gal meningitis consists of CSF drainage with intraoperative contamination. Moreover, clinical
lumbar puncture with or without temporary per- status and potential or established complications
cutaneous lumbar drains in patients who require can still be considered for device implantation. In
repeated daily lumbar punctures. However, a harm–benefit evaluation, VP shunts can be ben-
although daily lumbar punctures provide quick eficial in patients who are independently selected
relief from headache, they may increase the risk from the permanence of cryptococcus infection to
of brain herniation (Jarvis et al. 2014; Antinori prevent sequels and to optimise the management
et al. 2000). of ICP (Perfect et al. 2010).
18 Raised Intracranial Pressure 257
a b c
Fig. 19.1 Magnetic resonance imaging (MRI) T1-weighted basal cisterns (arrows). (c) FLAIR image axial view show-
contrast imaging of the brain of fungal meningitis patient in ing marked enlargement of lateral ventricles periventricular
axial (a) and coronal (b) view showing marked leptomenin- ooze of cerebrospinal fluid (CSF) (arrows)
geal thickening with diffuse enhancement specially the
262 M. Jaiswal
2. Large pseudomycetes (Candida) producing sal sinuses, orbits, oral cavity, etc.) for a con-
cerebral abscesses and granulomas: These siderable period of time and then are capable of
fungi are larger than 20 microns and can invading contiguous cranial bones, meningeal
occlude cerebral arterioles. Such occlusions tissues, basal cerebral venous sinuses, etc., as
lead to focal cerebral ischemia and infarctions. well as intermediate- and large-sized intracra-
Depending upon the virulence of the fungi and nial arteries, and result in arterial thrombosis
host resistance, a variety of the cerebral fungal and occlusions which in turn causes extensive
lesions appear such as ischemic areas, focal cerebral infarctions. These patients clinically
infarctions, cerebritis, abscesses, granulomas, present with cerebral stroke. The evolving
and combinations of these lesions. The tissue hemorrhagic cerebral infarct is then converted
necrosis and highly virulent fungal infection to septic infarct with associated cerebritis and
rapidly convert infected cerebral areas into abscesses, whereas a good host defense results
micro-abscesses, whereas a good host resis- in granuloma formation. This granuloma may
tance but persistence of infection causes gran- block directly or most of the time direct the
ulomatous inflammatory reactions in adjacent way of the CSF flow pathway leading to hydro-
leptomeninges, neural parenchyma, or in both cephalus in some cases.
sites. This may result in hydrocephalus which
needs to be treated promptly (Fig. 19.2).
3. Septate (Aspergillus) and non-septate 19.3.2 Clinical Presentation
(Zygomycetes): Mycetes are very large in size of Fungal Hydrocephalus
and normally grow with large branched hyphae.
Usually they infect juxta-cranial sites (parana- Chronic fungal meningitis is common, whereas
subacute meningitis is relatively less common,
and acute fungal meningitis is distinctively rare
except in intensive care unit (ICU) patients or in
patients on ventilator for prolonged period or
patients with severely immunocompromised
states. Most of the pseudomycetes (yeasts) are
capable of producing meningitis or meningoen-
cephalitis. Clinical features of fungal meningitis
and meningoencephalitis usually are headaches,
nausea, vomiting, visual impairment, and papill-
oedema; later neck stiffness with fever, and per-
sonality changes may develop; and still later,
seizures, deterioration in sensorium, cranial
nerve palsies, and hydrocephalus may develop
(Shankar et al. 2007; Zarei Mahmoudabadi 2002;
Black and Baden 2007). In many patients, there
are no focal or generalized physical signs. Fungal
meningitis more frequently occurs with
Cryptococcus, Coccidioides, Blastomyces,
Paracoccidioides, Sporotrichum, Histoplasma,
and Candida as compared to filamentous fungi
such as Aspergillus, Cladosporium (phaeohypho-
mycosis), and Zygomycetes.
Fig. 19.2 Computed tomography (CT) scan head show-
ing fungal granuloma causing compression and obstruc-
Candida also reaches CNS via colonization of
tion of CSF flow through third ventricle leading to the ventricular drains, shunt tubing, and central
hydrocephalus venous lines. Therefore, candidal meningitis can
19 Hydrocephalus 263
occur spontaneously as a complication of dis- tricular width of the bilateral frontal horn to the
seminated candidiasis or as a complication of an maximum biparietal diameter.
infected wound or ventriculostomy via direct CSF opening pressure. The CSF opening pres-
inoculation of the organism into the CNS. Usually sure measured by lumbar puncture is recorded
chronic but infrequently subacute, basal fungal some time. There may be very high opening pres-
meningitis causes obliteration of intracranial sub- sure (>350 mmH2O) or a normal or moderately
arachnoid spaces and results in increased intra- high pressure (<350 mmH2O) (Hardesty et al.
cranial pressure with or without hydrocephalus 2014).
(Zarei Mahmoudabadi et al. 2006; Zarrin and CSF analysis. Samples of CSF obtained either
Najafi 2007; Hardesty et al. 2014; Pitisuttithum by lumbar puncture or by the ventricular route
et al. 2005). are sent to the laboratory for cell analysis and
Various neurological manifestations, includ- determination of glucose, lactate, and protein lev-
ing headache, vomiting, visual impairment, men- els and fungal antigen titer. In the analysis of the
tal change, gait ataxia, and deterioration of antigen titer, a titer of >1:1024 is considered to be
consciousness, may be associated with progres- high in Cryptococcus infection, whereas a titer of
sive hydrocephalus. These may be attributed to <1:1024 is considered to be low and a posi-
the infection itself, to hydrocephalus, or to both. tive correlation between CSF features and out-
These clinical findings are of little help in estab- come was observed by some researches (Zarei
lishing the presence of hydrocephalus (Shankar Mahmoudabadi 2002; Black and Baden 2007;
et al. 2007; Zarei Mahmoudabadi 2002; Black Zarei Mahmoudabadi et al. 2006; Zarrin and
and Baden 2007; Zarei Mahmoudabadi et al. Najafi 2007; Hardesty et al. 2014).
2006; Zarrin and Najafi 2007; Hardesty et al.
2014; Pitisuttithum et al. 2005). However, the
presence of headache, visual impairment, mental 19.4.2 Treatment
change, gait ataxia, and deterioration of con-
sciousness should raise the suspicion that hydro- 19.4.2.1 Drug Treatment
cephalus is present. Intravenous administration of antifungal drugs is
the standard treatment for fungal meningitis. In
Japan, miconazole and fluconazole have been used
19.4 Management since 1989 to treat Aspergillus infection. CNS infec-
tions due to Aspergillus have a poor prognosis,
19.4.1 Diagnostic Test because no drug has sufficiently effective activity
against aspergillosis. Fluconazole is absorbed well
Fungal meningitis is defined as isolation of fungal from the gastrointestinal tract and also penetrates
organism from CSF culture, positive CSF fungal the blood-brain barrier well so is frequently used to
antigen titer, or positive results of CSF pathologi- treat fungal meningitis. No definitive method for the
cal fungal-specific staining studies and clinical treatment of Aspergillus meningoencephalitis has
features of meningitis with typical CSF features. yet been established, but fluconazole is sometime
Brain computed tomography (CT) and magnetic effective in some cases. The long-term use of anti-
resonance imaging (MRI) are useful tools for fungal agents is thought to be effective for the treat-
rapid diagnosis of hydrocephalus (Baddley et al. ment of Aspergillus meningoencephalitis (Baddley
2002; Ito-Kuwa et al. 2008; Levitz 1991; Borha et al. 2002; Zarrin et al. 2010; Speed and Dunt 1995;
et al. 2009; Guarro and Gene 1995; Shankar et al. Kovoor et al. 2002; Pagano et al. 1997; Middleton
2007). Hydrocephalus is usually diagnosed by the et al. 1976; Shankar et al. 2007). Therefore, antifun-
presence of a dilated temporal horn of the lateral gal drugs should always be administered for the rec-
ventricle, without obvious brain atrophy, and/or ommended duration.
an Evans ratio of 10.3 on the initial and/or follow- Reliance only on Amphotericin B is not effec-
up CT or MRI. Evans ratio is the ratio of the ven- tive. Fortunately, during the same period, many
264 M. Jaiswal
useful antifungal drugs are discovered and intro- (VP) shunt or external ventricular drainage. The
duced initially, the lipid-based formulations of indications and timing for placement of a shunt in
the Amphotericin B, then the new triazoles, and, patients with fungal meningitis complicating
most recently, echinocandins. These medications hydrocephalus are not well understood or univer-
are used, more and more in combinations, in seri- sally accepted (Black and Baden 2007; Zarei
ously ill patients with invasive mycoses. Now Mahmoudabadi et al. 2006; Zarrin and Najafi
evidence-based data are gathering together in 2007; Hardesty et al. 2014; Pitisuttithum et al.
favour of their important roles in the manage- 2005). Most authors suggest early shunt place-
ment of invasive fungal infections (Dubey et al. ment for hydrocephalus to avoid irreversible neu-
2005; Black and Baden 2007; Zarrin and Najafi rological complications. However, in some
2007; Hardesty et al. 2014). patients with fungal meningitis and hydrocepha-
But still there are many unanswered questions lus, diversion of CSF through a VP shunt does
and controversies relating to their use. not result in any significant improvement. Thus,
Unquestionably, CNS fungal infections pose selecting patients who would benefit from a VP
serious challenges in their management with con- shunt becomes important. Some of the patients
troversies surrounding their medical and surgical who were successfully treated with temporary
therapies. Surgical options are less controversial external CSF drainage and medical therapy did
in cases of focal or localized superficial cortico- not undergo permanent shunting. Similarly, some
subcortical lesions (such as abscesses and granu- patients have been treated successfully with
lomas) in the non-eloquent areas of the brain. endoscopic third ventriculostomy; however, our
Currently the following classes of natural and personal experience is that third ventriculostomy
synthetic antifungal drugs are commonly used: is rarely successful in the setting of coccidioido-
mycosis (presumably due to basilar cisternal
1. Polyenes: Amphotericin B deoxycholate com- arachnoid scarring) (Hardesty et al. 2014;
plex (Fungizone registered, Bristol-Myers Pitisuttithum et al. 2005; Diamond and Bennett
Squibb), nystatin lipid formulations of 1974; Tang 1990; Park et al. 1999).
Amphotericin B.
(a) AmBisome. 19.4.2.3 Shunt Failure
(b) Amphocil (Amphotericin B colloidal dis- and Complication
persion {cholesteryl sulphate}, ABCD). The risk of shunt failure is high, and half of all
(c) Abelcet (Amphotericin B lipid complex, patients in few series required one or more shunt
ABLC). revisions during the follow-up period, and 9%
2. Pyrimidines: 5-Fluorocytosine (flucytosine). mortality rate during follow-up demonstrates the
3. Triazole drugs: Fluconazole, itraconazole,
severity of this often-fatal disease process despite
posaconazole, voriconazole. the best medical therapies (Black and Baden
4. Echinocandins: Caspofungin, anidulafungin, 2007; Zarei Mahmoudabadi et al. 2006; Zarrin
micafungin. and Najafi 2007; Hardesty et al. 2014; Pitisuttithum
5.
Miscellaneous: Imidazoles (clotrimazole, et al. 2005; Diamond and Bennett 1974; Tang
ketoconazole, etc.), oral polyenes (amphoteri- 1990; Park et al. 1999; Lu et al. 1999). The most
cin, nystatin), griseofulvin, etc. For dermal, common (50–81%) cause of shunt failure is due
oropharyngeal, esophageal, intestinal, and to mechanical clogging of the drainage system.
vaginal infections, terbinafine is effective for Coccidioidomycosis can lead to significant pro-
nail and ring worm infections. teinaceous debris; considerable biofilms or focal
abscesses have been observed on catheters or
19.4.2.2 Surgical Treatment within valve tapping chambers at the time of shunt
The treatment of patients with fungal meningitis revision. Unfortunately, no data suggest a solution
with hydrocephalus is essentially a diversion of to improve shunt longevity, as they did not observe
ventricular CSF through a ventriculoperitoneal any differences among shunt valve brands.
19 Hydrocephalus 265
Appropriate medical therapy with systemic with fungal meningitis. In one series, 30% of
anti-fungals is necessary in patients with hard- patients were found to have extremely high
ware placed in a chronically infected space, and intracranial pressure. Extremely high intracra-
all these patients must be maintained on systemic nial pressure is associated with increased early
antifungal medication. The role of intrathecal mortality. In one series, however, hydrocephalus
antifungals should be the focus of future study, and extremely elevated intracranial pressure did
and any benefit of intrathecal therapy on shunt not correlate with poor outcome. It is likely that
failure rate should be evaluated. Despite low VP shunts remove a large amount of CSF and
drainage pressures and adequate CSF diversion, lower the intracranial pressure, thereby signifi-
many patients with fungal hydrocephalus experi- cantly improving the outcome.
ence persistent ventriculomegaly after shunt sur- In fact, extremely high intracranial pressure
gery. The mechanism for this is not well without hydrocephalus associated with fungal
elucidated, but it may include an alteration in the meningitis can also be resolved using a VP shunt.
compliance of the ventricular wall or entire brain Patients with hydrocephalus who have poor con-
parenchyma due to infectious scarring or altered sciousness (GCS score, 8 or <8) at the time of sur-
extraventricular CSF circulation (Zarei gical intervention have poor outcome. Performing
Mahmoudabadi 2002; Black and Baden 2007; shunting procedures for these patients still does
Zarei Mahmoudabadi et al. 2006; Zarrin and not result in a good outcome. Perhaps patients
Najafi 2007; Hardesty et al. 2014; Pitisuttithum with poor consciousness and hydrocephalus
et al. 2005; Diamond and Bennett 1974; Tang already have irreversible brain tissue damage
1990; Park et al. 1999). The clinical importance before hydrocephalus is corrected by use of a VP
of this with regard to patient chronic headache or shunt (Chiou et al. 1994; Cruciani et al. 1992;
subtle cognitive function changes is unclear. Sanchez-Portocarrero et al. 1994). The duration of
However, it behoves the clinician treating patients altered consciousness is also an important predic-
with fungal hydrocephalus to assume that ven- tive factor of clinical outcome. A > 48 hours dura-
tricular size does not necessarily indicate shunt tion of deteriorated consciousness is associated
failure or a functional shunt system. with a poor response to VP shunting (Hardesty
et al. 2014).
Poor prognostic factors that influence the out- • Hydrocephalus is a serious cause of morbidity
come of fungal meningitis are low CSF glucose and mortality in patients with CNS fungal
level, high CSF lactate level, high CSF crypto- infection.
coccal antigen titer (>1:1024), level of con- • Patients with immunocompromised status are
sciousness, hydrocephalus, and increased at higher risk for the disease process.
intracranial pressure. Factors that influence the • Shunt failure rates are high, usually due to
outcome of VP shunt placement surgery to treat obstruction.
fungal meningitis with hydrocephalus have not • Not all patients’ ventriculomegaly resolves,
been discussed in few studies. Poor Glasgow even with low-normal draining pressures.
Coma Scale (GCS) score (<8) at the time of sur- • The use of VP shunts does not result in a good
gical intervention and prolonged duration response or outcome if it is associated with a
(>48 hours) of deteriorated consciousness GCS score of 8 and below and duration of
before surgery are associated with poor improve- altered consciousness of more than 48 hours.
ment and outcome after surgery (Hardesty et al. • Multidisciplinary teams of neurosurgeons and
2014). Extreme elevation of the intracranial infectious disease specialists are required to
pressure (>350 mmH2O) is common in patients treat these challenging patients.
266 M. Jaiswal
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Intracranial Space-Occupying
Lesions 20
Erdal Kalkan, Fatih Erdi, Yasar Karatas,
and Bülent Kaya
fibrosis. Frontal and temporal granulomas gener- tomical apertures such as orbital apex, and com-
ally occur due to spread from paranasal sinuses municating perivascular spaces act as a bridge
as in aspergillosis and mucormycosis, whereas for spreading to subarachnoid space. Retrograde
parietal granulomas occur due to hematogenous thrombophlebitis was also blamed for the devel-
spread of other fungi (Raman Sharma 2010). opment of cerebellopontine angle and cerebellar
masses. Although fungal pathogens can cause
meningitis, encephalitis, abscess formation, vas-
20.2 Epidemiology culitis, or granulomas, they tend to cause granu-
lomatous meningitis following an acute phase of
The incidence of CNS fungal infections is simi- infection and can affect the meninges, calvarium,
lar to that of systemic fungal infections and has brain, and intracranial vessels in distinct forms,
been rising due to increased life expectancy, large severity, and compositions (Naik et al. 2015).
ageing population, malignancy, extensive use of
immunosuppressive drugs, longer survival of
patients, increased frequency of acquired immu- 20.4 Diagnosis
nodeficiency syndrome (AIDS), and poor nutri-
tional status (Naik et al. 2015). The estimated A high index of suspicion is critical for early
annual incidences of invasive fungal infections diagnosis and avoidance from associated signifi-
caused by opportunistic pathogens per mil- cant morbidity and mortality. Past medical his-
lion of the population vary between 12 and 228 tory of the patient or the patient’s close relatives
infections according to causative agent (Raman should be taken into consideration. Like other
Sharma 2010). The Indian subcontinent has the intracranial space-occupying lesions, headache,
highest reported frequency of histologically nausea, vomiting, altered vision, and mental sta-
verified intracranial fungal mass lesions in geo- tus, focal neurological deficits are commonly
graphic distribution with one to two cases per encountered presentations. Invasion of the cav-
year. Hot and dry climate with high content of ernous sinus and orbital apex could cause visual
fungal spores in agrarian dust was blamed for symptoms, proptosis, ophthalmoplegia, superior
endemic form of the disease in India, Africa, and orbital fissure syndrome, and cranial nerve pal-
California (Naik et al. 2015). Predisposing fac- sies. Other uncommon manifestations include
tors include diabetes, tuberculosis, HIV, steroids, seizures, stroke, subarachnoid hemorrhage, and
immunosuppression, and chemotherapy (Naik mycotic aneurysm formation (Naik et al. 2015).
et al. 2015). Intracranial fungal mass lesions had
a 0.5–1% cumulative prevalence in transplant
recipients (Rajshekhar 2007). 20.5 Imaging Findings
a b
Fig. 20.1 Paranasal sinus computed tomography (CT) of a 45-year-old male patient with common variable immuno-
deficiency. Complete filling of maxillary (a) and frontal sinus with pus and pansinusitis appearance (c)
response. The wall of the fungal abscess is usu- of hemorrhage on gradient echo sequence gave
ally regular and thin. Irregularity of the rim can worthy supporting data (Fig. 20.2) (Naik et al.
vary depending on the aggressiveness of the 2015; Saini et al. 2010). The infarcts distribute
fungus and the host’s ability to exert an immune commonly at the gray-white matter junction as
response (Hadley et al. 2017). Infiltration of the well as basal ganglia, thalamus, corpus callosum,
orbit, extraocular muscles, or cranial nerve com- and perforating small artery territories in dis-
pression can be detected with MRI. Restricted seminated fungal infection (DeLone et al. 1999).
diffusion on diffusion-weighted imaging simi- The involvement of perforating arteries without
lar with pyogenic abscess, decreased perfusion involvement of the distal major arterial territories
on perfusion- weighted imaging, and presence stresses the pathophysiologic difference between
a b
c d
Fig. 20.2 T2-weighted pre-gadolinium (a), T1-weighted space-occupying lesion (SOL) at the left occipital lobe of
pre-gadolinium (b), and post-gadolinium (c) axial mag- the same patient. Note the irregular rim pattern (c) and
netic resonance imaging (MRI) sections showed a fungal restricted diffusion on diffusion MRI (d)
20 Intracranial Space-Occupying Lesions 273
septic fungal and thromboembolic infarction was reported in patients with sinocranial asper-
(Yamada et al. 2002). Corpus callosum involve- gillus granulomas with dense fibrous tissue
ment can be used for distinguishing fungal (Murthy and Sundaram 2014). Surgical clipping
infection from pyogenic infection and thrombo- or endovascular treatments with aggressive anti-
embolic infarction as the latter do not commonly fungal treatment are indicated for the patients
involve the corpus callosum. But other processes with mycotic aneurysms (Rajshekhar 2007).
such as glioblastoma, lymphoma, and multiple Prognosis after treatment depends on the prompt
sclerosis should keep in mind for differential recognition and management of the disease as
diagnosis (DeLone et al. 1999). well as preoperative neurologic status of patient,
immunocompromised state, contamination of
ventricular CSF during surgery, and renal fail-
20.6 Management ure (due to amphotericin B) (Naik et al. 2015).
Previously reported gloomy surgical results
Antifungal medications and surgery are the (Dubey et al. 2005; Sharma et al. 1997; Yanai
mainstay treatment options. Treatment modal- et al. 1985) were improving slowly (Naik et al.
ity preference depends on the primary loca- 2015). Naik et al. recommended preoperative
tion, size, and number of the lesions. General antifungal treatment for 1–2 weeks, followed by
medical status of the patient carries high impor- radical surgery and antifungal treatment for the
tance. Small fungal SOLs can be managed with following 6 weeks (Naik et al. 2015).
aggressive antifungal medications and support-
ive care, whereas significantly large lesions that
compress adjacent brain structures need radical 20.7 Conclusion
surgical excision. Urgent decompression can
be lifesaving in emergency situations. Surgical It is concluded that:
options include stereotactic procedures, craniot-
omy, shunt surgery, and mycotic aneurysm sur- • CNS fungal infections have been diagnosed
gery. Indications of the stereotactic procedures increasingly over the last few decades, due to
include deep-seated lesions and lesions in the the increase of immunocompromised patients
eloquent brain regions for the establishment of under risk.
tissue diagnosis (Murthy and Sundaram 2014). • Although some improvements have been
Surgical excision reduces infection load, reduces achieved, CNS fungal infections constitute a
mass effect, and improves efficacy of following diagnostic and therapeutic challenge.
medical treatment. Radical excision of lesions • Antifungal medications and surgery are the
without excessive contamination of cerebrospi- mainstay treatment options.
nal fluid (CSF) spaces was advocated (Naik et al. • Radical excision of SOLs without excessive
2015). Survival rates are much better in patients contamination of CSF spaces improves
treated with combined medical and surgical outcome.
treatment in patients treated with medical treat-
ment only (Gonzalez et al. 2002). Adherence/
invasion of the lesion to basal vessels and cranial
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Invasive Fungal Diseases
of the Skull Base 21
Manogaran Ravi Sankar, Mathialagan Arulalan,
Amit K. Keshri, Arun K. Srivastava,
Awadhesh K. Jaiswal, and Sanjay Behari
Fig. 21.4 An isolated sphenoidal pathology eroding the Fig. 21.6 MR T2 W image showing an isolated skull
skull base base aspergilloma
therapy and to identify the additional rare fun- immunoassay may be done. These tests are more
gal species causing skull base pathologies like specific for detection of aspergillosis infection.
the Fusarium, Alternaria and Pseudallescheria Molecular testing method that uses the oligonu-
species (Kalkanci et al. 2006) (Figs. 21.10 and cleotide probe and gene sequencing can be used
21.11). Sometimes, the routine evaluation does for rapid identification of the fungal species. In
not show any fungal infection; in these condi- case of frank intracranial involvement by inva-
tions, the newer modalities like identification sive aspergillosis, the sensitivity of cerebrospinal
of fungal cell wall markers in the serum like fluid (CSF) polymerase chain reaction (PCR) is
galactomannan (Chen et al. 2011; Schwartz 100% as compared to the sensitivity of galacto-
et al. 2005), beta-D-glucan and mannan enzyme mannan, which is 80% (Chen et al. 2011).
21 Invasive Fungal Diseases of the Skull Base 279
Fig. 21.9 Histopatho-
logical examination
showing Aspergillus and
its branching pattern
Fig. 21.11 Lacto-
phenol cotton blue
mount shows the septate
fungal hyphae bearing
conidiophores with
radiating conidial heads;
vesicles with biseriate
phialides with chains of
conidia are also seen
Clinical suspicion
Complete
Radiological Evaluation of
clinical
evaluation tissue (Biopsy)
evaluation
Complete Fungal
Neurologic Ophthalmological Fungal
ENT CT & MRI Histopathology smear for
evaluation evaluation culture
evaluation microscopy
Invasive Non-invasive
Acute Chronic
(< 4 weeks) (> 4 weeks)
Confirmation of diagnosis
Fig. 21.14 Extradural clearance of the disease from anterior skull base with CT correlation
282 M. Ravi Sankar et al.
Fig. 21.15 Pre- and post-operative (6 months follow-up with voriconazole) images of the skull base aspergilloma
anidulafungin), polyenes (amphotericin B) and rapid progression of the fungus into the parana-
flucytosine are available for the medical treat- sal sinus, orbit, vessels and nerves, the muscu-
ment of invasive fungal disease (Herbrecht et al. loskeletal system surrounding the skull base and
2002; Kontoyiannis 2012; Redmond et al. 2007). the brain parenchyma. The time course of less
Amphotericin B is the broad-spectrum fungicidal than 4 weeks’ duration differentiates this entity
agent, but its toxicity limits its widespread and from the CISBFD. AISBFD is almost always
prolonged usage. The newer liposomal formula- common in individuals suffering from an immu-
tions are comparatively less toxic and can be used nocompromised condition like a haematologi-
in higher dosages. Voriconazole, approved by the cal malignancy, uncontrolled diabetes mellitus,
US Food and Drug Administration (FDA) in prolonged steroid use, organ transplantation or
2002, is more effective than amphotericin in autoimmune deficiency syndrome (Abu El-Naaj
invasive aspergillosis (Gillespie and O’Malley et al. 2013; Kasapoglu et al. 2010). Although
2000). The duration of therapy is based on clini- rare, this condition is also reported in immu-
cal and radiological response and varies from 3 to nocompetent individuals (Chopra et al. 2006;
6 months. Posaconazole has shown its efficacy as Gillespie and O’Malley 2000; Marple 2001;
a salvage therapy in patients with end-stage renal Saravanan et al. 2006). AISBFD is a condition
disease caused due to diabetes (Mehta and that requires immediate management; other-
Langston 2009). wise the mortality can be as high as 50–80%
Shah et al. (2017) and Mohindra et al. (2008) (Gillespie et al. 1998; Kennedy et al. 1997).
have described the protocol for the management The most common organisms responsible are
of skull base invasive aspergillosis. Patient with the Aspergillus species and the Zygomycetes
skull base involvement with minimal invasion species (Süslü et al. 2009; Tarkan et al. 2012).
of the basal frontal lobe, cavernous sinus and Patients with uncontrolled diabetes mellitus are
the infratemporal fossa should be treated with more prone to developing mucormycosis infec-
extradural debridement followed by systemic tion because of their altered transferrin binding
antifungal therapy. Stable patients with massive capacity (Spellberg et al. 2012). Any patient
intracranial invasion of the frontal or temporal in an immunocompromised status with facial
lobes with cerebral oedema should be preloaded swelling, facial pain, headache, prolonged fever,
with liposomal amphotericin B of 2 g followed orbital symptoms and cranial nerve palsies
by debridement; and after debridement, they should be evaluated clinically, radiologically
should be given a cumulative dose of up to 6 g. and pathologically to confirm the diagnosis.
Patients who are not hemodynamically stable These patients require steps to revert the immu-
because of reasons like uncal herniation need nocompromised status, the surgical debridement
immediate debridement without the requirement and the administration of appropriate systemic
for preloading of antifungal medications. The antifungal therapy (Fig. 21.18).
debridement may followed by administration of
6–8 g of amphotericin B. After completion of
the desired dose of amphotericin B, the patients 21.2.3 Chronic Invasive
should be started on azole group of drugs. Granulomatous Fungal
The medicines are continued for 3–6 months Disease (CIGFD)
(Fig. 21.17).
CIGFD is seen in immunocompetent indi-
viduals, and it is most commonly caused by
21.2.2 Acute Invasive Skull Base Aspergillus flavus (Stringer and Ryan 2000).
Fungal Disease (AISBFD) The most common presentation is unilateral
proptosis. Other symptoms include nasal con-
Acute invasive fungal disease is also called as gestion, nasal obstruction, facial pain, headache
fulminant fungal disease. AISBFD results from and facial numbness (Stringer and Ryan 2000).
284 M. Ravi Sankar et al.
CISBFD
16 weeks systemic
antifungal
Progressing
therapy Preloading
Lesion Immediate
(newly with
decompression
developing amphotericin
Radiological
intracranial E B 2 gm
follow up
lesion)
Complete
Surgical
No cummulative
Persistant debridment
residual Resolving dose of
lesion lesion lesion amphotericine
Repeat B
Radiology (4–6 gm)
Biopsy
Serial Continue 8
follow weeks of Complete
up sysyemic cummulative 3–6 months
antifungal HPE, fungal dose of systemic
therapy smear, fungal amphotericine B azole
culture and 4–6 mg antifungal
sensitivity theraphy
Fig. 21.17 Management protocol for chronic invasive skull base fungal disease
Systemic
Surgical
Extradural antifungal Serial Follow Up
debridment
therapy
AISBFD
Same as
massive
Intradural
intracranial
CISBFD
Fig. 21.18 Management protocol for acute invasive skull base fungal disease
21 Invasive Fungal Diseases of the Skull Base 285
Fig. 21.20 A case of sphenoid fungal ball presenting with bilateral 6th nerve palsy and left-sided loss of vision
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Skull Base Clinical Syndromes
22
Jagarlapudi M. K. Murthy and Subhendu Parida
Table 22.1 Fungal infections of central nervous system: on precipitation patterns, humidity, temperature,
skull base syndromes (Murthy 2007)
and wind conditions (Panackal et al. 2010).
• Optic neuropathy Intracranial and orbital invasion from the
• Cavernous sinus syndromes: unilateral or bilateral juxta-cranial spaces, paranasal sinuses, nose,
• Orbital apex syndromes
and ear can be by direct extension. This mode of
• Proptosis with or without ocular palsies
spread is most commonly described in patients
• Polyneuritis cranialis
• Orbito-cranial syndromes with sino-cranial aspergillosis (Murthy et al.
2001; Murthy and Sundaram 2014; Shankar et al.
2007; Sundaram et al. 2006). The pathology of
22.3 Chronic Fungal Meningitis sino-cranial aspergillosis is well-formed granulo-
mas (Sundaram et al. 2006). Patients with sino-
Small-sized (diameter up to 20 μm) yeast fungi cranial aspergillosis may present with features of
(Cryptococci, Coccidioides, Paracoccidioides, intracranial focal mass lesions, skull base syn-
Blastomycetes, Histoplasma), access the dromes as follows: orbital apex syndrome, cav-
microcirculation from which they seed the ernous sinus syndrome, proptosis with or without
subarachnoid space and produce meningitis/ ocular nerve palsies, polyneuritis cranialis, and
meningoencephalitis. Chronic meningitis and orbito-cranial syndromes (Table 22.1) (Murthy
pachymeningitis can present with cranial neu- 2007) (Fig. 22.1).
ropathies, including optic neuropathy. Of the Other large-sized fungi, Mucorales spp.,
92 cases of Aspergillus meningitis reviewed, also infect juxta-cranial sites and invade the
chronic meningitis or pachymeningitis cranial cavity by contiguous spread. Skull base
accounted for 15 (16%) cases. Optic neuropa- syndromes are unusual presenting features of
thy, external ophthalmoplegia, lower cranial
neuropathy, and deafness were the clinical syn-
dromes (Antinori et al. 2013).
a b
Fig. 22.2 FLAIR axial (a) and T2 axial (b) magnetic yracea, there is intracranial extension into the left middle
resonance imaging (MRI) sections show mucosal thicken- cranial fossa with signs of cerebritis and focal abscess for-
ing and hyperintensity within the left sided ethmoidal air mation in the anterior aspect of left temporal lobe. There
cells, fat stranding is noted within the left orbit, probably is proptosis of left globe. The causative fungal pathogen in
due to extension of inflammation through the lamina pap- this patient was Mucorale spp.
mucormycosis. The common presenting clini- tral SBO cases are caused by microorganisms other
cal forms are cerebral and rhinocerebral (Roden than Pseudomonas aeruginosa including fungi
et al. 2005) (Fig. 22.2). Manifestations of the (Ridder et al. 2015). Of the SBO cases, fungal SBO
disease may reflect the sequential involvement of account for 10% (LeClerc et al. 2014; Spielmann
the nose, sinuses, eye, and brain. Manifestations et al. 2012). The true incidence of fungal SBO may
of cavernous sinus thrombosis include loss of be higher than what has been reported. Of the his-
vision and internal and external ophthalmople- tologically confirmed invasive aspergillosis, 80%
gia. Thrombosis of the internal carotid artery also were culture negative (Stoduslski et al. 2006). The
can occur and causes contralateral hemiplegia reported fungal pathogens associated with SBO
(Murthy and Sundaram 2014; Roden et al. 2005). include most commonly Aspergillus spp. and, less
commonly, Mucor spp. and Scedosporium spp.
(Blyth et al. 2011).
22.5 F
ungal Skull Base The most common symptoms at initial presen-
Osteomyelitis tation in patients with central SBO are headaches
and/or facial pain. Cranial nerve involvement most
Central SBO or atypical SBO refers to osteomy- commonly include VI, IX, X, and VII (Blyth et al.
elitis that affects the sphenoid and occipital bone, 2011) (Fig. 22.3). Only one-fourth of the patients
often centered on the clivus and can be sinugenic complain classic sinonasal symptoms, such as con-
or otogenic. Central SBO typically affects middle- gestion and discharge. Fever is relatively uncom-
aged males, with underlying diabetes mellitus and mon on presentation (Johnson and Batra 2014).
immunocompromised being predisposing factors There are some distinct differences between
(Johnson and Batra 2014). About half of the cen- bacterial SBO and fungal SBO. Fungal SBO is
292 J. M. K. Murthy and S. Parida
a b c d
e f g
Fig. 22.3 (a–g) Post contrast T1 coronal and axial MRI clivus with sclerotic changes in the rest of the clivus,
showing heterogenously enhancing lesion filling the sphe- enhancing craniovertebral junction with abscess in pre-
noid sinus spreading laterally to cavernous sinus and erod- vertebral space (e, f); Post contrast T1 MRI showing par-
ing floor of left middle cranial fossa (a–d); Post contrast tial resolution following treatment. The causative fungal
T1 MRI showing destruction of most anterior portion of pathogen in this patient was Aspergillus fumigatus
frequently associated with underlying chronic systemic aspergillosis. Case report and review of 92
sinusitis, sinonasal pain, facial/periorbital swelling cases. J Infect. 2013;66:218–38.
Blyth CC, Games L, Sorrell TC, da Cruz M, Sud A, Chen
and nasal stuffiness or discharge, and the absence SCA. Skull-base osteomyelitis: fungal vs. bacterial
of purulent ear discharge (Blyth et al. 2011). infection. Clin Microbiol Infect. 2011;17:306–11.
Johnson AK, Batra PS. Central skull base osteomy-
elitis: an emerging clinical entity. Laryngoscope.
2014;124:1084–8.
22.6 Conclusion LeClerc N, Verilaud B, Duet M, Gulchard JP, Herman
P, Kania R. Skull base osteomyelitis incidence
In countries with temperate climates, fungal of resistance, morbidity, and treatment strategy.
pathology should be considered in patients pre- Laryngoscope. 2014;124:2013–6.
Murthy JMK, Sundaram C, Prasad VS, Purohit AK,
senting with skull base syndromes, more so with Rammurti S, Laxmi V. Aspergillosis of central ner-
juxta-cranial infections in the paranasal sinuses, vous system: a study of 21 patients seen in a univer-
nose, and ears. The most common pathogen is sity hospital in south India. J Assoc Physicians India.
Aspergillus spp. 2000;48:677–81.
Murthy JMK, Sundaram C, Prasad VS, Purohit AK,
Rammurti S, Laxmi V. Sinocranial aspergillosis: a
form of central nervous system aspergillosis in south
India. Mycoses. 2001;44:141–5.
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Orbito-Rhinocerebral Syndrome
23
Gauri S. Mankekar, Rahul Mehta,
and Daniel W. Nuss
ists, and infectious disease specialists have been (Bae et al. 2012), and brain (Bae et al. 2012; Koc
shown to decrease mortality (Mankekar 2014; et al. 2007).
Palejwala et al. 2016). The role of the microbi-
ologist and pathologist in the early identification
of the type of fungus causing ROCS cannot be 23.2.2 Causative Agents
underestimated.
The fungi involved in causing AIFRS and, there-
fore, ROCS are often similar in particular forms
23.2 Classification of Paranasal of fungal disease, although there is a geographic
Sinus Fungal Infections variation (Montone et al. 2012). AIFRS is uni-
formly associated with A. fumigatus, A. flavus,
Paranasal sinus fungal infections have been and Rhizopus sp. worldwide (Epstein and Kern
classified by de Shazo et al. (deShazo et al. 2008; Panda et al. 1998; Das et al. 2009). In
1997; deShazo and Swain 1995) as invasive and 80% of patients with diabetes, Rhizopus spp.,
noninvasive. They further classified IFRS into Rhizomucor spp., Absidia spp., and Mucor spp.
three distinct entities based on clinical presen- are responsible for AIFRS (Gillespie et al. 1998).
tations, acute invasive (fulminant) fungal rhi- Aspergillus spp is responsible for 80% of AIFRS
nosinusitis (AIFRS), chronic invasive fungal cases in patients with neutropenia, e.g., post-
rhinosinusitis (CIFRS), and chronic granuloma- chemotherapy or steroids, hematologic malig-
tous fungal rhinosinusitis (CGFRS), while non- nancies, and organ transplantation, or in patients
invasive fungal rhinosinusitis was classified as with acquired immunodeficiency syndrome
allergic fungal rhinosinusitis (AFRS) and sinus (AIDS) (Gillespie et al. 1998). A few cases due to
mycetoma or fungal ball. Cunninghamella bertholletiae, Apophysomyces
IFRS, by definition, means demonstrable fun- elegans, and L. corymbifera have also been
gal hyphae within the mucosa, submucosa, bone, reported (Diwakar et al. 2007; Chakrabarti et al.
and blood vessels on histopathology (deShazo 2006). Apophysomyces elegans has been reported
et al. 1997). ROCS which follows the spread of to cause AIFRS in immunocompetent patients
paranasal sinus fungal infection to the orbit and (Sridhara et al. 2005; Garcia-Covarrubias et al.
intracranial structures is associated with ophthal- 2001).
mic and neurological manifestations. It is typi-
cally seen with the AIFRS.
23.3 Clinical Presentation
fests as cranial nerve palsies, especially involving (Idris and Lim 2012) (Fig.23.2). These muco-
cranial nerves II, III, IV, V, and VI with cavern- sal changes are commonly seen on the middle
ous sinus thrombosis. In advanced cases, hemi- turbinate, followed by the septum, hard palate,
paresis, hemiplegia, convulsions, delirium, and and inferior turbinate (Gillespie and O’malley
finally death can occur (Bae et al. 2012; Epstein 2000). It is important to watch for skip lesions
and Kern 2008; Yohai et al. 1994; Maheshwari which occur due to the spread of infection along
et al. 2013). the intima of blood vessels (Mankekar 2014).
Invasive mucormycosis of the maxillary sinus
presents with features which are often different
23.4 Diagnosis from ethmoid sinus mucormycosis (Gamaletsou
et al. 2012). While maxillary sinus infection
ROCS diagnosis is based on a high index of sus- extends into the oral cavity which is seen as a
picion and clinical examination, corroborated by black necrotic ulcer on the hard palate, ethmoid
microbiological and histopathological studies. sinus infection extends into the orbit or intracra-
Imaging studies are not diagnostic but provide nially. An oral examination may reveal gingival
information about the anatomy, extent of the dis- or palatal or lingual eschar which is clinically
ease, and complications. distinctive for AIFRS. In some cases, facial
An immunocompromised individual present- hyperesthesia or anesthesia may present even
ing with unexplained fever, cough, acute sinus- prior to the appearance of other symptoms.
itis, nasal congestion, or orbital apex syndrome An ophthalmological examination is manda-
or ophthalmoplegia should raise the suspicion of tory at the beginning and throughout the treat-
potential IFRS (Deshazo 2009) (Fig. 23.1). ment course in all patients suspected to have
A cursory nasal and oral examination may not AIFRS. It is important to test not only the visual
reveal any pathology. It is therefore essential for acuity but also document the field of vision to
an otolaryngologist to decongest the nose and exclude ophthalmoplegia and examine the retina
perform a rigid nasal endoscopy. Whitish dis- for chorioretinitis. Blurring of vision, diplopia,
coloration (due to tissue ischemia) of the nasal and ophthalmoplegia may be earliest manifesta-
mucosa with serosanguineous discharge may be tions of cavernous sinus thrombosis, even before
present. Often black eschar due to ischemic tis- the appearance of changes on imaging studies
sue necrosis is a distinct warning sign for AIFRS (Gamaletsou et al. 2012).
Fig. 23.1 Clinical appearance of patient with rhino- Fig. 23.2 Endoscopic appearance of black, devascular-
orbito-cerebral syndrome (ROCS) (Courtesy Dr. DW ized nasal mucosa in a patient with ROCS (Courtesy Dr.
Nuss) DW Nuss)
298 G. S. Mankekar et al.
Neurological examination for cranial nerve hybridization aid not only in the diagnosis but are
palsies, facial asymmetry, facial paresthesia or also the only methods to definitively identify the
anesthesia, motor and sensory weakness, and fungal species (Schwartz 2011).
signs of meningitis is essential during initial Thin axial and coronal computed tomogra-
evaluation as well as during the entire treatment phy (CT) scans of the paranasal sinuses must be
course. obtained in all patients with suspected AIFRS
Rapid diagnosis can be obtained with a potas- and ROCS to determine the extent of disease
sium hydroxide (KOH), calcofluor wet mount (Spellberg et al. 2005; Mankekar 2014; Epstein
(Figs. 23.3 and 23.4). Fungal culture and in situ and Kern 2008; Kim et al. 2015). Intravenous
contrast may be required to detect intra-orbital
and intracranial involvement. However, mag-
netic resonance imaging (MRI) is preferred to
CT in evaluating patients with altered senso-
rium, ophthalmoplegia, stroke, or cranial nerve
palsies with possible intra-orbital and intra-
cranial involvement. It can evaluate the spread
of disease to the retro-antral, intra-orbital
(Fig. 23.5), and intracranial regions (Fig. 23.6)
but can be time-consuming and could therefore
delay definitive diagnosis (Soler and Schlosser
2012). Ischemic tissue necrosis is seen as non-
enhancing lesions on gadolinium-enhanced MRI
Fig. 23.3 Potassium hydroxide (KOH) mount of
scans (Mankekar 2014) and is recommended for
Aspergillus showing septate hyphae (Reproduced from early diagnosis, determining the extent of sur-
Invasive Fungal Rhinosinusitis, ed. Mankekar, Springer, gical debridement required and for follow-up
India, 2014) (Kim et al. 2015).
The earliest imaging finding in rhinocerebral
mucormycosis is the infiltration of the periantral
fat planes. This may be accompanied by soft tis-
sue attenuation along the sinus walls. There is
23.5 Management
Posaconazole has also been reported to be useful bleeding tissue is visualized. So far, no specific
as salvage therapy in patients of invasive aspergil- criteria for the extent or range of debulking have
losis refractory to or intolerant to previous anti- been established. Reports of surgical debulking
fungal treatment (Walsh et al. 2014). However, it range from endoscopic debridement of affected
is important to monitor serum concentrations of sinus, nasal, and orbital tissues (Fig. 23.7) to radi-
the drug to reduce incidence of breakthrough or cal surgery such as enucleation of the eyeball and
relapsing mucormycosis due to inadequate serum maxillectomy (Dhiwakar et al. 2003a, b; Epstein
concentration. and Kern 2008; Songu et al. 2008) (Fig. 23.8).
The duration of systemic antifungal therapy
has not yet been defined and is determined by
the resolution of all associated clinical symptoms
and findings (Skiada et al. 2013). Maintenance
therapy or secondary prophylaxis has been
recommended in persistently immunocompro-
mised patients (Skiada et al. 2013).
Anticoagulant therapy has been considered
a standard part of cavernous sinus thrombosis
which can occur in rhinocerebral mucormy-
cosis. Advocates of anticoagulation believe
that it can prevent propagation of thrombus
and help improve blood flow. However, use
of anticoagulants is controversial as it could
potentially be associated with the risk of cerebral
and systemic hemorrhage. Fig. 23.7 Nasal endoscopic debridement of devitalized
orbital periosteum and tissues in a patient with ROCS
Granulocyte colony-stimulating factor is (Courtesy Dr. DW Nuss)
useful in reversing neutropenia in patients
with hematologic malignancies (Gillespie and
O’malley 2000; Spellberg et al. 2005; Skiada
et al. 2013). Adjuvant treatment with hyperbaric
oxygen has been reported to improve survival
in cases of ROCS as the higher concentration
of tissue oxygen increases neutrophil antifungal
activity and the oxidative killing mechanism
caused by polyenes (Walsh et al. 2014; Garcia-
Covarrubias et al. 2002; Price and Stevens
1980), although it has not been recommended
for routine use due to insufficient data (Skiada
et al. 2013).
23.7 Surgery
The dilemma of whether orbital exenteration is with invasive aspergillosis. Overall, the mortal-
indicated in ROCS continues (Songu et al. 2008; ity rate is less than 20% (Parikh et al. 2004) in
Hargrove et al. 2006; Nishimura et al. 2017) AIFRS and almost 100% in patients with intra-
with several authors recommending removal in cranial extension and symptoms of the disease
patients with orbital apex syndrome (Hargrove (Gillespie and O’malley 2000).
et al. 2006; Plowes Hernandez et al. 2015), while
others report successful treatment without it
(Nishimura et al. 2017; Kohn and Hepler 1985). 23.9 Conclusion
As outcomes of intracranial extension of disease
are poor, it is rarely treated with surgical resec- ROCS is associated with invasive fungal sinus
tion (Peterson et al. 1997). disease caused by A. fumigatus, A. flavus, and
Endoscopic debridement of the nose and Rhizopus spp. although a few other fungi have
paranasal sinuses, besides providing tissue for also been implicated. Its incidence is higher
microbiology and histopathological diagno- among immunocompromised individuals with
sis, decreases the fungal load, slows disease diabetic ketoacidosis or neutropenia secondary
progression (Gillespie and O’malley 2000), to hematologic malignancies, human immune
and controls local disease (Vironneau et al. deficiency virus, solid organ, and stem cell
2014). The pterygopalatine fissure and spheno- transplantation. A high index of clinical suspi-
palatine foramen are considered reservoirs of cion and early diagnosis are essential to prevent
invasive mucormycosis, and exposure of this the rapid spread of the disease from the nose and
fissure with removal of the posterior wall of paranasal sinuses to the orbit and brain. A mul-
the maxilla is recommended by several authors tidisciplinary approach with aggressive surgical
(Songu et al. 2008; Plowes Hernandez et al. debridement and systemic antifungal therapy is
2015) for adequate disease control. Nishimura required to achieve favorable outcomes.
et al. (Nishimura et al. 2017) have reported a
novel combined endoscopic-sinus approach to
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Cavernous Sinus Syndrome
24
Forhad Hossian Chowdhury,
Mohammod Raziul Haque,
Mohammod Zahed Hossain,
and Mainul Haque Sarker
Suprasellar Optic
cistern chiasm
Dura
infundibulum
Oculomotor
nerve
Pituitary Trochlear nerve
gland
Internal carotid
artery
Ophthalmic nerve
Abducent nerve
Maxillary nerve
Sphenoidal sinus
Fig. 24.1 Schematic drawing of cross-sectional (coronal) anatomy of both cavernous sinuses (CS) at midsellar region
showing boundary-related structures and contents
Superior
Superior
ophthalmic Optic Supraclinoid Oculomotor
vein (SOV) nerve (II) ICA nerve (III)
Trochlear
nerve (IV)
Eye
Posterior
Ophthalmic
CS
nerve (V1)
Anterior Superior orbital Abducent
fissure (SOF) nerve (VI)
Maxillary
Inferior ophthalmic
nerve (V2)
vein (IOV)
Trigeminal
nerve with motor roof
Foramen
rotundum Internal cartoid
artery (ICA)
Foramen
Inferior Mandibular
ovale
nerve (V3)
Fig. 24.2 Schematic drawing of left CS lateral view structures and boundaries with their relation and contents of CS
a b
c d
Fig. 24.3 (a–d) Sequential cadaveric temporal extradu- optic nerve, AN abducens nerve, 3 and 4 oculomotor and
ral dissection of left CS with drilling out of anterior cli- trochlear nerve, ICA internal carotid artery (cavernous
noid process. T temporalis muscle, TD&R temporal dura segment), SOF superior orbital fissure, SOV superior oph-
and retractor, TG trigeminal ganglion, MN mandibular thalmic vein, and RPt root of pterygoid
nerve, MXN maxillary nerve, OPN ophthalmic nerve, ON
308 F. H. Chowdhury et al.
blood transfusion can cause such infection. It has The acute variety rapidly progresses in few
also been reported to spread by surgery, especially hours to days and progresses to fulminant intra-
transsphenoidal surgery (Shah and Rathore 2009). cranial infections. Mostly, these are caused by
Mucor, Absidia, Fusarium, etc. Some authors
prefer to call such infections as rhinocerebral
24.3.4 Pathology (Shah and Rathore mucormycosis. These often occur in patients
2009) with diabetes having ketoacidosis. The chronic
one has an indolent course and could be granulo-
Fungal sinusitis is the inflammation of the matous also. The species implicated are
sinus mucosa, caused by a wide variety of Alternaria, Curvularia, Mucor, Bipolaris, etc.
fungi. Aspergillus spp is the most common, These are slow growing and cause slow tissue
and Rhizopus, Mucor, Cladosporium, Candida, destruction and subsequent invasion (Shah and
and Cryptococcus species are among the oth- Rathore 2009).
ers. The noninvasive ones are, generally, dema-
tiaceous molds including Curvularia, Bipolaris,
Alternaria, Fusarium, Aspergillus, etc. These 24.4 Clinical Presentation
cause intracranial complications in about 20% of
the patients. Cavernous sinus syndrome is characterized by
There are broadly two types of fungal infec- multiple cranial neuropathies. The clinical pre-
tion of the sinuses: sentation includes impairment of ocular motor
nerves leading to ophthalmoplegia, Horner’s syn-
• Noninvasive (extramucosal). drome, and sensory loss of the first or second
–– Allergic fungal sinusitis. divisions of the trigeminal nerve in various com-
–– Mycetoma. binations. The pupil may be involved or spared or
• Invasive. may appear spared with concomitant oculosym-
–– Acute. pathetic and parasympathetic involvement.
–– Chronic indolent. Various degrees of pain may be involved (Lee
–– Chronic granulomatous. et al. 2003).
A good history along with symptoms of any
Noninvasive: These generally do not invade systemic ailments should be elicited. A sinusitis
the bone or tissues and, more often, are a result of patient not responding to conventional antibiotics
hypersensitivity skin reactions. But a long-term should arouse the suspicion of fungal sinusitis.
disease can eventually erode the bone by pressure Diagnostic nasal endoscopy should be performed,
necrosis and hence cause an intracranial or intra- and one should look for allergic mucin, blackish/
orbital complication. It occurs in an immuno- brownish discharge, erosion of palate, pale/dark
competent host and is characterized by the nasal mucosa, etc.
presence of allergic mucin, Charcot-Leyden crys- The most common symptoms are nasal
tals and eosinophils, etc. obstruction, nasal discharge, headache, vomit-
Invasive: The more fatal variety is known to ing, nausea, epistaxis, periorbital pain, facial
penetrate the mucosa and cause tissue destruction pain, facial swelling, anosmia, altered senso-
and lead to intracranial extension. These are rium, seizures, and weakness of limbs, diplo-
known to occur in immunocompromised hosts in pia, visual disturbance, and fever. The signs
about 50% of the cases. The most common is depend upon the areas involved like ophthal-
Aspergillus spp followed by Mucor, Rhizopus, moplegia, chemosis, proptosis, lateral/medial
Cryptococcus, etc. The mortality rates are high in rectus palsy, and hemiparesis (Shah and Rathore
such infections (85–100%). 2009).
310 F. H. Chowdhury et al.
a b c
d e f
g h i
j k l
Fig. 24.5 Magnetic resonance imaging (MRI) of the with bilateral involvement of paranasal sinuses and orbits
head. (a–c) contrast coronal images; (d–f) axial contrast in a diabetic patient of 35 years (confirmed by histopa-
images; (g–i) axial T2W images; (j–l) sagittal contrast thology and fungal culture)
images showing bilateral CS aspergillosis (left > right)
Rhinocerebral mucormycosis is often an acute Goldberg et al. 1983; Mandava et al. 2001).
fulminant opportunistic infection, which may Extension into the CS may result in thrombosis
affect the orbits and paranasal sinuses. The and thickening of the ICA walls, with narrow-
MRI findings include enhancing soft tissue ing of its lumen. Its MRI features are nonspe-
masses in the orbital apex and CS, with thick- cific (Ohta et al. 2002; Pagliani et al. 2006).
ening and lateral displacement of the medial These infections should always be considered
rectus muscle and involvement of the neigh- in immunosuppressed patients who present
boring ethmoid sinus (Chan et al. 2000; with any type of paranasal sinus disease that
312 F. H. Chowdhury et al.
a b
c d
e f
Fig. 24.6 MRI of the head of a 35-year-old apparently showing aspergillosis of the left cavernous sinus, left
immunocompetent patient; (a–c) contrast axial images; orbital apex, and left sphenoidal sinus (confirmed by his-
(d–f) T1W coronal images; (g–i) contrast sagittal images topathology and culture)
24 Cavernous Sinus Syndrome 313
g h
Fig. 24.6 (continued)
extends beyond the bony walls of that sinus Aspergillus has uniform septate hyphae branch-
(Figs. 24.5, 24.6, and 24.7a–c) (Razek and ing at 45°. The Mucor shows nonseptate, nonuni-
Castillo 2009). form branching at 90°. The allergic mucin is
characteristic of allergic fungal sinusitis, and one
24.5.1.5 Radiological Differential has to stain deeply to look for fungal hyphae. The
Diagnosis of Fungal CS culture used is Sabouraud media. The biopsy can
Lesion be taken using endoscopic endonasal approach
Differential diagnoses of CS fungal infection are from PNS especially from sphenoid sinuses
tuberculosis, pyogenic chronic abscess (when sinuses are involved) and from CS. Where
(Fig. 24.9), sarcoidosis, lymphoma, myeloma, endonasal endoscopic biopsy is not available or
metastasis, meningioma, pseudotumor, inflam- suitable, then transcranial open biopsy may be
matory lesion, Tolosa-Hunt syndrome, etc. needed. All the material including soft tissue and
bones should be sent for histopathological exam-
ination (Shah and Rathore 2009). Aspergillus
24.5.2 Fungal Culture and Biopsy cultured optimally on Sabouraud agar demon-
strates characteristic conidiophores. However,
There are special stains required for detecting the blood and cerebrospinal fluid cultures, even in
fungus. These include Gomori methenamine sil- disseminated disease, are frequently negative
ver for Mucor, Rhizopus, and Absidia. The (Nadkarni and Goel 2005).
314 F. H. Chowdhury et al.
Aa Ab
Ad
Ac
Ae Af
Fig. 24.7 (a) MRI of the head of a young diabetic patient. moid sinuses, sphenoidal sinus, orbital apex, and CS with
(a–c) T1W sagittal images; (d–f) contrast coronal images; proptosis. (c) MRI of the head of patient of (a). (a–d)
and (g–i) contrast axial images showing mucormycosis Axial DW images showing multiple embolic infarcts in
involving right-sided maxillary sinus, ethmoid sinuses, the left cerebral hemisphere from left cavernous ICA ste-
sphenoidal sinus, orbital apex, and CS with proptosis nosis by invasion of mucormycosis. (f–i) MR angiogram
(confirmed by histopathology and culture). (b) MRI of the showing left ICA stenosis form invasion of mucormycosis
head of patient of (a). (a–d) Axial FLAIR images showing on arterial wall
mucormycosis involving right-sided maxillary sinus, eth-
24 Cavernous Sinus Syndrome 315
Ag Ah
Ai
Fig. 24.7 (continued)
316 F. H. Chowdhury et al.
Ba Bb
Bc Bd
Fig. 24.7 (continued)
24 Cavernous Sinus Syndrome 317
Ca Cd
Cd
Cc
Ce
Cf
Fig. 24.7 (continued)
318 F. H. Chowdhury et al.
Cg Ch
Fig. 24.7 (continued)
An initial biopsy and culture (endoscopic Once the infection is identified, aggressive man-
directed) is taken to confirm the presence and agement is necessary. Unfortunately, the response
type of fungal involvement (from PNS and/or rate to medical therapy is only 40–60% (Choi
CS). If sinus tissue is absent, open biopsy can be et al. 2008; Mauriello Jr et al. 1995). The mortal-
taken from cavernous sinus. In cases of obvious ity rate of invasive sino-orbital aspergillosis is
clinical fungal sinusitis, endoscopic sinus surgery also 40–75% despite maximal management
with removal of all fungal debris should be per- including surgical debridement and antifungal
formed even at the initial stage when sending therapy (Choi et al. 2008; Mauriello Jr et al. 1995;
material for histopathology and microbiologic Sivak-Callcott et al. 2004). Initially patient can be
studies. treated with antifungals including amphotericin,
itraconazole, voriconazole, and posaconazole, but
if the infection does not respond or progressed
24.6.1 Medical Treatment despite the therapies, surgical treatment is needed.
Surgery includes debridement in the form of
The mainstay of treatment is antifungal therapy resection of the cavernous sinus and a portion of
with amphotericin B or other antifungal drugs the petrous apex with or without
24 Cavernous Sinus Syndrome 319
a b
c d
Fig. 24.8 (a–d) Preoperative contrast axial computed Postoperative (2.5 months after operation and after anti-
tomography (CT) scan of the head in a 55-year-old female fungal therapy). CT scan of the head of the same patient
showing left CS and temporal lobe fungal (aspergillus) showing resolution of the lesion
lesion (confirmed by histopathology and culture). (e–h)
320 F. H. Chowdhury et al.
e f
g h
Fig. 24.8 (continued)
nerves and the carotid artery and reconstruction of beyond the foramen rotundum or ovale with
the skull base defect. For complete unilateral cav- resection of cranial nerve V). Similarly, manage-
ernous sinus resection, cranial nerves II–VI will ment of the carotid artery should be planned in
be resected. In such cases, a new cranial nerve detail prior to resection. In general, carotid artery
deficit is not a major consideration; for example, sacrifice will be necessary for cavernous sinus
in the present case, the patient had preoperative resection based on the pathology. Proceeding with
blindness and ophthalmoplegia in the affected revascularization is controversial. A selective
eye. In cases of infection, care should be employed approach including balloon occlusion testing can
to ensure that all infected tissue is removed, espe- be used (Taussky and Couldwell 2010). There is
cially if the infection involves proximal or distal limited literature regarding the risk of bypass spe-
cranial nerve tissue (e.g., the root entry zone or cific to carotid sacrifice for skull base lesions, but
24 Cavernous Sinus Syndrome 321
a b
c d
e f
Fig. 24.9 Contrast MRI of the head in an 80-year-old and CS with proptosis. It was thought to be due to fungal
lady. (a, b) Contrast coronal images; (c, d) contrast sagit- infection. But histopathology reported inflammatory
tal images; and (e–h) contrast axial images showing infec- lesion and culture confirmed Methicillin-resistant
tion involving left-sided ethmoid, sphenoid sinus, orbit, Staphylococcus aureus (MRSA) infection
322 F. H. Chowdhury et al.
g h
Fig. 24.9 (continued)
the published rates of morbidity are 3–20%, and allergic fungal sinusitis (McGinn et al. 2009) or
mortality rates of less than 6% have been reported non-invasive fungal infection in sphenoid sinus
(Abdulrauf 2005; Kalani et al. 2013; Lawton and (Devèze et al. 2005). Current recommendations
Spetzler 1996; Mendelowitsch et al. 2004). These for CST are controversial, especially regarding
risks should be weighed against the risk of carotid anticoagulation, secondary to the rarity of the
sacrifice in skull base lesions without bypass, for diagnosis. Early surgical debridement and intra-
which there are neurological morbidity of 17% venous antibiotics are crucial to prevent mortality
and mortality of 7% (Mendelowitsch et al. 2004). and decrease morbidity. Because thrombosis is
The middle cerebral artery (MCA) distal site thought to be caused by a bacterial superinfec-
ensures an anastomosis remote from the infection. tion, which follows a response to Aspergillus,
Previous experience with bypass grafts in patients antifungals may not be necessary. Despite the
with fungal infections with arterial involvement controversy, most physicians opt to treat with
has indicated that anastomosis sites even a short anticoagulation (McGinn et al. 2009).
distance from the infection may result in higher The most common clinical features are puru-
thrombosis (Neil et al. 2016). A regular follow-up lent or bloody rhinorrhea, mostly unilateral, with
(clinical and neuroimaging) and endoscopic fever, headache, general discomfort, blepharitis,
examination are essential. For sinusitis, the patient diplopia, decrease in visual acuity, unilateral or
is asked to do nasal wash. Postoperative neuro- bilateral rhinorrhea, proptosis, chemosis, perior-
logic follow-up and assessment are critical. Often bital cellulitis, alteration of the intrinsic and
serial scans are required to confirm the presence extrinsic ocular motoricity, and amaurosis.
or spread of disease postoperatively (Shah and Features of cerebral ischemia or infarction may be
Rathore 2009). present when ICA is involved (Haber et al. 2008).
The CST diagnosis is made both with clinical
signs and radiologic evaluation by CT and/or
24.7 S
pecial Situations in CS MRI, and the latter is more sensitive to the diag-
Fungal Infection nosis. This exam may reveal direct signs of CST
such as changes in the signal intensity, size and
24.7.1 Cavernous Sinus Thrombosis contour of the CS and indirect signs like the
(CST) Syndrome thickening and enhancement of the contrast on
the lateral wall of the CS (Haber et al. 2008).
It is a rare and serious complication secondary to CTA or MRA may show ICA narrowing or
invasive fungal sinusitis but rarer still in cases of occlusion.
24 Cavernous Sinus Syndrome 323
Treatment is surgery and aggressive antifun- blade, or through arteries and veins in the orbital
gal therapy. wall (Ferry 1961). The fungal invasion may com-
mit the ocular globe and the retina artery and
produce amaurosis (Yohai et al. 1994). The ini-
24.7.2 Mucormycosis and CST tial complaints of the CST are retro-orbital pain,
periorbital edema, chemosis, proptosis, palpebral
The first description of mucormycosis was ptosis, and diplopia. Such symptoms are not spe-
made by Paultauf in 1885 (Paulltauf 1885). cific and may be present in other affections as in
This is a necrotizing disease caused by fungi the orbital cellulitis. However, the presence of
of the Zygomycetes class and Mucorales sepsis, paralysis of cranial nerves, and bilateral
order. From these, the most common genders ocular involvement are important signs for the
found are Rhizopus (about 70% of the cases), CST. The early visual loss favors the suspicion
Absidia, Mucor, Rhizomucor, Apophysomyces, of the retina artery involvement by mucormyco-
Saksenaea, Cunninghamella, Cokeromyces, and sis, but the amaurosis caused by the CST occurs
Syncephalastrum (Yohai et al. 1994). The main more lately (Bray et al. 1987; Van Johnson et al.
route of infection is inhalatory. The mucormy- 1988). The patient is treated clinically with sys-
cosis is a rare and opportunist disease in immu- temic antifungal agents (amphotericin B), with
nocompromised person, specially in poorly intravenous antibiotics (initially ceftriaxone and
controlled diabetes mellitus. Before the 1960s the after with vancomycin), with heparinization,
mucormycosis was almost always fatal, and with and with intranasal and paranasal sinus surgery
the discovery of amphotericin B and its wide use with debridement of the nasal mucosa necrosis
associated with surgical debridement, the mortal- areas. In the suspicion of acute invasive fungal
ity rate was reduced to approximately 40% (Yohai rhinosinusitis (mucormycosis), the treatment
et al. 1994). This potentially fatal evolution is due must be carried out quickly and aggressively, by
to a certain specific characteristic of these fungi, surgery for removal of the necrotic areas of the
which is vascular tropism, that initially invades nasal mucosa and the use of intravenous systemic
the arteries and causes thrombosis and ischemic antifungal agents. Even so, there is a high rate of
injuries (Fig. 24.7a–c). Then there occurs vein mortality and morbidity (Haber et al. 2008).
and lymphatic node invasion (Van Johnson et al.
1988). The mucormycosis may present as rhino-
orbito-cerebral form which is the most common 24.7.3 Internal Carotid Artery
presentation (Prabhu and Patel 2004). The infec- Involvement with Fungal
tion may be disseminated to the CNS through Infection
the orbital apex and the cribriform plate or cause
thrombosis in arteries that supply the CNS. The Sphenoid sinus fungal infection results in inva-
CNS manifeatations characterized by a change in sion to ICA and worsens the patient’s prognosis
the conscience level, convulsion, and/or hemiple- by cerebral infarction (Hase et al. 2013), espe-
gia are associated with a worse prognosis (Ferry cially mucormycosis which has vascular tropism
1961). A potential intracranial complication of that initially invades the arteries and causes
mucormycosis is the CST. In the mucormycosis thrombosis and ischemic injuries (Haber et al.
and CST, the diagnosis must be early with aggres- 2008). So early diagnosis and treatment are
sive therapy. The mucormycosis is a more fatal important. For diagnosis CTA, MRA or DSA is
acute fungal infection for mankind, with mortal- needed (Fig. 24.7a–c). Systemic antifungal with
ity rate from 15 to 34% (Fairley et al. 2000). The anticoagulant with EC-IC bypass and trapping/
mucormycosis rhino-orbito-cerebral form, when excision of ICA may be needed with or without
present in the nasal cavity or in the PNS, may CS excision specially in ICA occlusion with
spread to the orbit through the nasolacrimal duct, malignant cavernous sinus fungal infection (Neil
through natural dehiscences in the papyraceous et al. 2016).
324 F. H. Chowdhury et al.
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Rao SP, Kumar KR, Rokade VR, Khanna V, Pal C. Orbital factors in rhino-orbital-cerebral mucormycosis. Surv
apex syndrome due to mucormycosis caused by Ophthalmol. 1994;39(1):3–22.
Intracranial Fungal Aneurysms
25
Ali Akhaddar and Sylma Diabira
Abbreviations
pathogen, most infectious intracranial aneurysms
CNS Central nervous system are bacterial (Choi et al. 2014; Ducruet et al. 2010).
CSF Cerebrospinal fluid It is well known that intracranial fungal aneu-
CT Computed tomography rysm (IFA) is among the most serious complica-
ICA Internal carotid artery tions of central nervous sytem (CNS) fungal
IFA Intracranial fungal aneurysm infections with a high mortality rate (Azar et al.
IIA Intracranial infectious aneurysm 2016; Kannoth et al. 2007; Kunimatsu et al.
MR Magnetic resonance 2015). The first publication of a histologically
verified IFA was reported in 1965, when Stehbens
described a 35-year-old woman with a history of
acute leukemia who was found to have an intra-
25.1 Introduction cranial internal carotid artery (ICA) fusiform
aneurysm due to proliferation of mucormycosis.
Infectious intracranial aneurysms (IIA) or so-called This patient presented with an episode of menin-
mycotic aneurysms or microbial aneurysms are gitis (Stehbens 1965). Since Stehbens’s descrip-
rare cerebrovascular lesions that occur through tion, reports of IFAs are very rare and largely
microbial infection of the brain arterial wall confined to isolated case reports and small series.
(Akhaddar 2017a). The term “mycotic” was first For this chapter we will focus on IIA second-
used by Osler in 1885 because of the resemblance ary to fungal infections. Although there is a pau-
of such lesion with “fungal vegetation” (Osler city of data in the current literature regarding IFA,
1885). Although they can be secondary to fungal we will provide in this report a practical review
regarding these potentially fatal cerebrovascular
A. Akhaddar (*) lesions and their management strategies.
Department of Neurosurgery, Avicenne Military
Hospital of Marrakech, Mohammed V University of
Rabat, Rabat, Morocco
25.2 Epidemiology
S. Diabira
Department of Neurosurgery, Avicenne Military
Hospital of Marrakech, Mohammed V University of Among IIA, fungal forms are known to be rare
Rabat, Rabat, Morocco representing about 1% of all infectious aneu-
Spine Surgery Unit, Private Hospital of Saint- rysms (Brown et al. 1984). But, in the past few
Grégoire, Rennes, France years, the literature in this field has increased in
© Springer Nature Switzerland AG 2019 327
M. Turgut et al. (eds.), Fungal Infections of the Central Nervous System,
https://doi.org/10.1007/978-3-030-06088-6_25
328 A. Akhaddar and S. Diabira
complexity and quantity owing to the recent mary forms could be cryptogenic without obvi-
increase in the number of immunocompromised ous definite source of fungal infection
patients and those with fungal paranasal sinusitis (Abecassis et al. 2013). Direct invasion from
(Azar et al. 2016; Muraoka et al. 2016; Ogawa adjacent skull base structures (such as paranasal
et al. 2015; Radotra et al. 2015; Shinya et al. sinuses, orbit, or the ear) is more likely encoun-
2015; Yamaguchi et al. 2016). In Kannoth’s series tered involving proximal segments of the intra-
published in 2007, IFA represented 16 % (4 cranial arterial vasculature (Corvisier et al.
cases) of all 25 cases of IIA (Kannoth et al. 2007). 1987; Gulshan et al. 1978; Morriss and Spock
More recently, a literature review performed by 1970; Takeda et al. 1998; Watson et al. 1999). In
Choi et al. estimated that 4% of all IIA were fun- the opposite, hematogenous dissemination is
gal in origin (Choi et al. 2014). At least, half of likely associated with distal branching aneu-
these patients were immunocompromised. rysm (Choi et al. 2014).
Knouse et al., reported that 14% of all carotid IIA Most invasive skull base fungal infections
were fungal in origin (Knouse et al. 2002). The have a propensity to invade the ICA, cavernous
difficulty in estimating the exact incidence of IFA sinus, and the adjacent brain vasculature
is due to the undefined natural history of these (Akhaddar 2017b, 2008). This can result in
vascular lesions. thrombosis, stenosis or aneurysmal formations
IFA are less common in infants than in adults including their own cerebrovascular complica-
without apparent sex predominance (Ahmad tions (Hurst et al. 2001; Muraoka et al. 2016).
et al. 2006; Choi et al. 2014; Goldman et al. 1979; Also, IFA may be associated with a granuloma-
Loeys et al. 1999; Marazzi et al. 2008). Unlike tous inflammation of the brain and meninges,
the bacterial ones, IFA arise mostly in the setting causing granuloma formation, meningitis, or
of an extravascular infection (paranasal sinusitis, meningoencephalitis (Kannoth et al. 2007;
orbital cellulitis, meningitis, and post-craniotomy Negoro et al. 2013). In a study of 11 patients with
infections) especially in an immunocompromised fungal ICA aneurysms, 7 were confirmed to be
host (Azar et al. 2016; Corvisier et al. 1987; secondary to direct invasion from a paranasal
Hurst et al. 2001; Morriss and Spock 1970; sinus, and the sphenoid sinus was the most fre-
Negoro et al. 2013; Okada et al. 1998; Piotrowski quently involved sinus (4 cases among 7) (Hot
et al. 1990; Takeshita et al. 1992; Yip et al. 2012). et al. 2007).
Fungal infections are unusually encountered in Unlike the non- infectious aneurysm, size
the context of endocarditis (Ahmad et al. 2014; seems not a reliable predictor of potential rupture
Takeda et al. 1998). for IFA. It was observed that even small IIA can
rupture and bleed fatally, while many IIA may
enlarge and produce progressive symptoms (Choi
25.3 Pathogenesis and Pathology et al. 2014).
Aspergillus species, Candida albicans, and
As is the case for all other forms of IIA, three Mucor species are the three most common
recognized sources of fungal intracranial aneu- pathogens causing IFA (Abecassis et al. 2013;
rysmal formation were previously suggested Azar et al. 2016; Hot et al. 2007). Aspergillosis
(Akhaddar 2017a; Kannoth and Thomas 2009): may be involved in more than 2/3 of all fungal
(1) from a remote extracranial site (hematoge- forms. Other reported fungi include
nous dissemination), (2) through a contiguous Pseudallescheria boydii, Coccidioides, and
cranial focus (direct invasion), or (3) from a Scedosporium species (Kannoth and Thomas
contamination secondary to improper neurosur- 2009; Ong et al. 2011). Aspergillosis has a spe-
gical procedures, especially following pituitary cial vasocentric tropism in deep fungi. Elastase
surgery (Horten et al. 1976; Kasliwal et al. produced by this fungus can induce aneurysm
2009; Okada et al. 1998; Radotra et al. 2015; formation. Elastase decomposes elastin, a major
Visudhiphan et al. 1973). However, some pri- component of the vascular wall, and induces
25 Intracranial Fungal Aneurysms 329
Fig. 25.1 Computed tomography (CT) scan of the brain (ICA) and eroding into the sphenoid sinus [Reproduced
shows a 1.2 × 0.8 cm bilobed aneurysm (arrows) arising from Azrar MM et al. Mycopathologia (2016)181:425-33;
from the cavernous part of the right internal carotid artery with permission]
25 Intracranial Fungal Aneurysms 331
a b
Fig. 25.2 Angiography of the right carotid artery and parent vessel (c) is shown. Completion angiography
cerebral circulation. The aneurysm (arrow) is shown aris- shows successful exclusion of the aneurysm (c)
ing from the horizontal segment of the cavernous right [Reproduced from Azrar MM et al. Mycopathologia
ICA (a). Coil embolization of the aneurysm (b) and the (2016)181:425-33; with permission]
26.1 Introduction
26.2 Epidemiology
Acute stroke is a medical emergency; it is
the leading cause of adult physical disabil- The exact incidence and prevalence of acute
ity and second most common cause of death ischemic and hemorrhagic stroke syndromes
worldwide (Murray et al. 2012). According to related to fungal infections are unknown (Fugate
the World Health Organization (WHO), there et al. 2014). They occur rarely. Case reports and
are 15 million people in the world who suffer case series have been reported (Kalita et al.
1999; Calli et al. 1999; Goel et al. 1999; Murthy
et al. 2000). Ischemic strokes related to fungal
A. Anita (*)
University Clinic of Neurology, Medical Faculty, infections are more common compared to hem-
University of Ss. Cyril and Methodius, orrhagic strokes.
Skopje, Republic of North Macedonia
A. Zoran
University Clinic of Pulmonology and Allergy,
Medical Faculty, University of Ss. Cyril and
Methodius, Skopje, Republic of North Macedonia
fungal endocarditis (Horten et al. 1976; Iihara Systemic fungal infection can also lead to septic
et al. 1990; Piotrowski et al. 1990; Chou et al. embolic showers to the brain.
1993; Radhakrishnana et al. 1994; Kurino et al. Molds: Aspergillus spp. are the most impor-
1994; Suzuki et al. 1995; Takeda et al. 1998; tant human pathogenic molds associated with
Watson et al. 1999; Ho and Deruytter 2004). stroke. Aspergillosis is the most common form
Rarely, they can occur following surgery and of angioinvasive mycosis. Most of the published
rarely from contiguous spread from the para- cases describe aspergillosis in patients with
nasal sinuses (Takeshita et al. 1992; Hurst AIDS, neutropenia, tuberculosis, cancer, or alco-
et al. 2001). In the first two settings, the host is holism, causing cerebral thrombosis, intracranial
immunocompromised, and in the latter settings, mycotic aneurysms, and subarachnoid hemor-
the host is otherwise immunocompetent. The rhage. Aspergillus enzyme elastase digests the
majority of intracranial fungal mycotic aneu- internal elastic lamina of cerebral arteries leading
rysms are in the proximal portion of the major to focal micro-hemorrhages. Mycotic aneurysm
arteries at the base of the brain and have been formation may occur in the weakened walls of
described with Aspergillus spp., Candida spp., the cerebral arteries causing their rupture and
and Zygomycetes spp. infections (Horten et al. consecutive intracerebral and/or subarachnoid
1976; Iihara et al. 1990). hemorrhage. Aspergillus spp has a predilection
Histoplasma capsulatum, Rhizopus spp., for the small perforating arteries, causing their
Cryptococcus neoformans, and Exserohilum occlusion, with consecutive brain infarction
rostratum have been related to the occurrence with hemorrhagic transformation and embo-
of ischemic stroke, while Mucormycetes and lism. Commonly involved brain structures are
Rhizopus spp. have been related to hemorrhagic the thalamus, basal ganglia, and corpus callosum
stroke. Aspergillus spp. and Coccidioides immitis (Ashdown et al. 1994; Dayal et al. 1974; Seton
have been associated with the occurrence of both et al. 2008).
types of stroke (Murthy 2007). Rhinocerebral mucormycosis, caused by
Sometimes ischemic and hemorrhagic stroke Mucor spp. or Rhizopus spp., is a craniofa-
can present simultaneously in the same patient. cial infection that can cause ischemic stroke or
Endo et al. described a case of fatal subarach- hemorrhage. The highly destructive organisms
noid hemorrhage, with brain stem and cerebellar can invade the walls of major intracranial arter-
infarction, caused by Aspergillus infection after ies, including the basilar artery, predisposing to
cerebral aneurysm surgery (Endo et al. 2002). stroke and aneurysmal rupture (Rangel-Guerra
Extremely rarely intracerebral hemorrhage can et al. 1996). Fu et al. published a case report of a
be the complication of fungal infections of the 38-year-old man with a basilar artery stroke due
CNS. to invasive fungal sphenoid sinusitis caused by
There are three types of fungi that are impli- rhinocerebral form of mucormycosis (Fu et al.
cated in stroke: single-celled yeasts, hyphae- 2015). The rhinocerebral form can be rapidly pro-
forming molds, and dimorphic fungi that take the gressive and invasive with a high mortality rate.
form of molds at ambient temperature and yeasts The lumbar puncture revealed eosinophilic pleo-
at human body temperature. Yeasts usually cause cytosis with a mildly elevated total protein and
fungal meningitis and may cause large vessel borderline low glucose level. Computed tomog-
vasculopathy, obstruction of the venous outflow, raphy (CT) scan revealed a left medullary and
and endarteritis leading to stroke (Rosario et al. cerebellar infarct confirmed by magnetic reso-
2012; Ludmerer et al. 1993; Saul et al. 1986). nance (MR) imaging. MR imaging also displayed
They can also form focal brain parenchymal a diffuse marrow signal abnormality in the clivus
abscesses associated with hemorrhage. Molds with contiguous sinus disease. Endoscopic sinus
carry enzymes that invade the wall of the cerebral surgery confirmed that the fungal sinusitis was
blood vessels and tend to cause strokes hematog- mucormycosis of the Rhizopus genus, which had
enously. Molds directly invade the cerebral vas- affected the left sphenoid sinus, invaded through
culature, causing mycotic arteritis or aneurysms. the skull base, and involved the basilar artery. He
338 A. Anita and A. Zoran
was given liposomal amphotericin (500 mg i.v.) in immunocompetent patients (Saccente 2008).
with posaconazole (400 mg i.v. twice daily). Due Coccidioidomycosis can cause cerebral ischemia
to the severity of the invasion and poor progno- from septic emboli and meningeal inflammation,
sis, the patient was discharged with comfort care particularly around the basal meninges, and can
measures. be complicated by vasculitis and stroke (Williams
Exserohilum rostratum (Setosphaeria rostrata), et al. 1992). In a retrospective study of 62 patients
a ubiquitous pigmented mold, is a rare cause of with CNS coccidioidomycosis, nearly 40% had
human disease but was implicated in hundreds of ischemic infarction; however only 1 case had con-
cases of iatrogenic nervous system infections from comitant basilar meningitis (Arsura et al. 2005).
contaminated epidural steroid injections. Twelve
percent of meningitis cases presented with pos-
terior circulation strokes (Bell et al. 2013; Smith 26.4 Diagnosis
et al. 2013). Stroke types were ischemic or hemor-
rhagic and caused by mycotic arteritis. Diagnosis is established with the detailed history,
Yeasts: Cryptococcus spp. and Candida spp. clinical examination, neuroimaging methods (CT
are the most important yeast infections in the and MR imaging of the brain, CT angiography,
CNS. Stroke, either ischemic or hemorrhagic, MR angiography, conventional pancerebral angi-
might arise as a rare complication of candida ography) and laboratory testing (specific antibod-
infection (Grouhi et al. 1998; Wasserman et al. ies in blood), lumbar puncture (cerebrospinal fluid
2009). Disseminated candidiasis can also pro- (CSF) culture and microscopy), and if possible
voke arteritis, subarachnoid hemorrhage, and brain biopsy. Diagnostic criteria to distinguish
multiple brain microabscesses. infectious vasculitis from other causes of stroke
Cryptococcus is the most common yeast have not been well established in the literature and
infection in the CNS and usually happens in need further review and standard guidelines.
immunocompromised conditions, although
immunocompetent hosts exist (Browne et al.
2012). Cryptococcus spp. invade the meninges 26.4.1 When to Investigate
and parenchyma to result in meningitis and, sub- for Infectious Causes
sequently, abscesses. Strokes can occur by irrita-
tion of subarachnoid blood vessels, resulting in Patients who have had strokes caused by infec-
vasospasm and ischemic damage (Rosario et al. tion might be misdiagnosed if lumbar puncture
2012). Alternatively, endarteritis from inflamma- is not performed. However, lumbar puncture is
tion can result in small vessel ischemic strokes, not indicated when evaluating a typical stroke
usually in the basal ganglia, internal capsule, and patient, except in the setting of a subarachnoid
thalamus (Lan et al. 2001). hemorrhage. The clinical symptoms that might
Dimorphic Fungi: Major dimorphic fungi are indicate infectious causes include a history of
Histoplasma capsulatum (Ajellomyces capsula- fever, rash, and known prior infections. For
tus), Blastomyces dermatitidis (Ajellomyces der- immunocompromised patients, the suspicion
matitidis), Coccidioides immitis, Paracoccidioides should be higher, and lumbar puncture should be
brasiliensis, Sporothrix schenckii, and Penicillium performed. Cerebrospinal fluid (CSF) analysis is
marneffei (Talaromyces marneffei). CNS involve- indicated when focal neurological symptoms and
ment by these infections is unusual; stroke is signs appear gradually, rather than hyperacutely,
reported in only a few cases. The only reported and if other classic features of CNS infection
cases of stroke were caused by blastomyco- are present, such as fever, neck stiffness, and
sis (Elias et al. 2005; Bariola et al. 2010; Bush impaired consciousness. In patients with focal
et al. 2013). Histoplasmosis of the nervous sys- neurologic deficits, brain imaging should be
tem usually manifests as meningitis, but pon- obtained if possible before a lumbar puncture is
tine stroke secondary to small vessel vasculitis done. Brain imaging findings might assist in the
has been reported as a result of septic emboli decision to do a lumbar puncture. We should also
26 Acute Ischemic or Hemorrhagic Stroke Syndromes 339
take into account possible contraindications to lesions with perilesional edema, enhancing or
perform lumbar puncture. Meningeal enhance- nonenhancing lesions of the meninges, choroid
ment or multifocal infarctions, particularly ones plexus or ependyma, hydrocephalus, white mat-
that do not respect traditional arterial or venous ter edema, and cerebral infarcts with or without
territories, could raise the suspicion for infectious hemorrhage. Invasive Zygomycetes/Phycomycetes
or inflammatory pathologies. and Aspergillus may cause characteristic lesions at
Besides the clinical picture of stroke, the the inferior area of frontal lobes adjacent to poste-
occurrence of headache, seizures, and encepha- rior sinuses (Arsura et al. 2005). Fungal microab-
lopathy in a patient who has multiple cerebral scesses may appear hypointense on T2-weighted
ischemic involvement, hemorrhages, or multiple images and may show ring enhancement after con-
white matter lesions on a brain scan should lead trast administration.
to the suspicion of cerebral vasculitis. An infec- Enhancement of contrast, wall thickening, and
tious origin should be considered in those patients lumen narrowing may be seen on CT angiogra-
with progressive headache, seizures, recurrent phy, MR angiography, and cerebral angiography,
strokes, fever, infectious risk factors and findings in patients with meningitis complicated with
of increased cells and proteins in CSF, and the stroke. Different vascular abnormalities have
detection of multiple cerebral artery stenoses or been reported, such as narrowing of supraclinoid
mycotic aneurysms on neuroimaging. internal carotid artery, middle cerebral artery
The clinical presentation varies, depending (MCA), anterior cerebral arteries, etc. Nowadays,
on the severity, location, and extension of stroke. cerebral angiography, as the invasive technique,
Usual symptoms and signs include conscious- is only used in selected cases; CT angiography
ness impairment, focal motor neurological deficit and MR angiography are preferred methods.
(hemiparesis, hemiplegia, quadriparesis, quad- Also, it is important to know that these vascular
riplegia), visual field impairment (hemianopia), abnormalities seen in infectious vasculitis are not
cerebellar symptoms, brain stem signs, and epi- specific. Unfortunately, these neuroimaging tech-
leptic seizures. Primary infections of the CNS niques cannot provide a diagnosis of vasculitis,
may be further complicated with cerebritis, cere- but only vasculopathy. Vascular abnormalities
bral edema, and hydrocephalus. Infectious vascu- can also be observed in noninfectious vasculitis
litis may be complicated with recurrent ischemic and in other conditions, including intracranial
strokes and multiple brain hemorrhagic lesions. atherosclerosis, cerebral reversible vasoconstric-
Initial brain CT scan helps to rule out intrace- tion syndrome, hypercoagulable states, and intra-
rebral hemorrhage, but it has a lower sensitivity vascular lymphoma. For this reason, the analysis
for visualization of subarachnoid hemorrhage. of inflammatory markers, blood cultures, and
MR imaging is a preferred diagnostic method; CSF analysis, in the appropriate clinical con-
it can detect the size and distribution of deep text, may be needed. Brain biopsy should also be
ischemic lesions and signs of mural inflamma- considered in the diagnosis in those cases with
tion. Unenhanced axial T1-weighted images severe, progressive, and even life-threatening
may depict luminal narrowing and wall thicken- presentation. In many cases, a brain biopsy may
ing compared to contralateral arteries. Contrast- be the only way to make certain of a vasculitis
enhanced T1-weighted MR imaging is a sensitive diagnosis, and pathological examination of brain/
test to detect large vessel vasculitis. Gadolinium meningeal tissue may be necessary to distinguish
enhancement is a classical sign of inflammation of between infectious CNS vasculitis and other dis-
vessel wall, and wall enhancement can be seen in eases and to begin appropriate treatment.
the intracranial vessels of circle of Willis in vari- Transcranial Doppler ultrasound is a noninva-
ous forms or arteritis. Gadolinium-enhanced MR sive tool that may be helpful to detect focal accel-
imaging may also help during the follow-up of eration of cerebral blood flow in stenotic vessels
patients, since decrease of contrast enhancement and may detect changes in arteries velocity in the
is expected to be seen during convalescence. MR circle of Willis (Topcuoglu 2012). It can be used
images can show enhancing intraparenchymal for long-term follow-up of the patients.
340 A. Anita and A. Zoran
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Seton M, Pless M, Fishman JA, Caruso PA, Hedley- Wasserman AM, Sarantopoulos GP, Khanna D. Fungal
Whyte ET. Case records of the Massachusetts General leukocytoclastic vasculitis as a presentation of
Hospital. Case 18-2008. A 68-year-old man with systemic vasculitis in a patient with systemic
headache and visual changes after liver transplanta- lupus erythematosus. J Clin Rheumatol. 2009;15:
tion. N Engl J Med. 2008;358:2619–28. 383–6.
Sharma RR, Lad SD, Pawar SJ, Gurusinghe NT, Bhagwati Watson JC, Myseros JS, Bullock MR. True fungal mycotic
SN, Mahapatra AK. Surgical management of fungal aneurysm of the basilar artery: a clinical and surgical
infections of the central nervous system. In: Schmidek dilemma. Cerebrovasc Dis. 1999;9:50–3.
HH, Roberts DW, editors. Schmidek Sweet’s operative Williams PL, Johnson R, Pappagianis D, et al. Vasculitic
neurosurgical techniques, indications, methods and and encephalitic complications associated with
results. 5th ed. Philadelphia, PA: Saunders Elsevier; Coccidioides immitis infection of the central nervous
2006. p. 1633–71. system in humans: report of 10 cases and review. Clin
Smith RM, Schaefer MK, Kainer MA, et al. Fungal Infect Dis. 1992;14:673–82.
infections associated with contaminated methyl- Wolf PA, D’Agostino RB, Belanger AJ, Kannel
prednisolone injections. N Engl J Med. 2013;369: WB. Probability of stroke: a risk profile from the
1598–609. Framingham study. Stroke. 1991;22:312–8.
Spinal Syndromes
27
Álynson Larocca Kulcheski, Xavier Soler I. Graells,
and André Luís Sebben
Spinal infection is an ancient entity with some In 1928, the first case of a fungal osteomyelitis
descriptions dating from the Iron Age (Tayles was described by Conor. He reported a patient
and Buckley 2004). Historically, they were dev- with Monilia psilosis osteomyelitis (Conner
astating diseases with exceedingly high morbid- 1928). Since then, the incidence of fungal infec-
ity and mortality rates. With the advent of new tions in general has increased substantially over
diagnostic techniques, multidrug antimicrobial the past several decades but is still extremely rare
chemotherapy, and improvements in surgical (Broner et al. 1996; Van den Bergh et al. 1999).
techniques, the prognosis has improved dramati- Consequently, the number of the spinal infections
cally in recent years. also rose. Fungal spinal infections can range
Spinal infections are rare and comprise from a relatively simple discitis to more com-
approximately 1% of bone infectious involve- plicated and destructive osteomyelitis with large
ment (Ghanayem and Zdeblick 1996). Most of abscesses and sometimes marked deformity.
these infections are of pyogenic or tuberculous Numerous risk factors for their development have
origin. been described, generally related to immunosup-
Staphylococcus aureus is the most common pressive states (Frazier et al. 2001). The main
cause of spinal infections in general (Sapico and risk factors are diabetes mellitus, corticosteroid
Montgomerie 1979). usage, chemotherapy, human immunodeficiency
virus ( HIV), organ transplantation, malnour-
Á. L. Kulcheski (*) · X. S. I. Graells · A. L. Sebben ished people, intravenous drug users, alcohol
Orthopedics and Traumatology Service, Hospital de abuse, long-term catheters, sepsis, oncological
Clínicas, Universidade Federal do Paraná (UFPR), therapies, extensive use of antibiotics, and oth-
Curitiba, Paraná, Brazil
ers (Frazier et al. 2001; Salvalaggio et al. 2003; Spinal meningitis, spinal arachnoiditis,
Salloum et al. 2004). radiculopathy, and fungal myelitis may occur.
The most common fungal organisms caus- Progressive myelopathic syndromes may be due
ing spondylodiscitis include Aspergillus spp., to mass effect producing compressive spinal
Candida spp., and Cryptococcus. neoformans. lesions such as granulomas or intramedullary
These species are ubiquitous. Coccidioides abscess (Sharma 2010).
immitis and Blastomyces dermatitidis are less In fungal spondylodiscitis, back pain is the
frequent and are restricted to some specific most frequent complaint, while neurological
geographical areas. Blastomyces mainly lives impairment appears to be relatively infrequent.
in areas of the United States and Canada sur- Kyphosis is also uncommon due to the indolent
rounding the Ohio and Mississippi River valleys nature of the infection. Involvement of the ver-
and the Great Lakes, but cases have also been tebral bodies can lead to vertebral compression
reported in Central and South America, Africa, fractures and deformity of the spine. In addition,
and the Middle East. The first is most commonly spread of infection along the anterior longitudi-
seen in the desert regions of the Southwestern nal ligament can lead to psoas muscle or paraver-
United States and in Central and South America tebral abscesses (Skaf et al. 2010).
(Kim et al. 2006). Hematogenous dissemination of fungal
infections causes constitutional symptoms
like fever, headache, malaise, anorexia, night
27.4 Pathogenesis sweats, lethargy, musculoskeletal pains, etc.
(Sharma 2010).
Fungi profoundly grow in the environment with Kulcheski et al. reported an atypical case
abundance of organic matter and water. The of Candida albicans spondylodiscitis. That
fungus exists in its mycelia l phase in the soil. occurred after blunt chest contusion and pro-
Humans become infected most commonly by gressed with necrotizing fasciitis of the ante-
inhalation of the spores (conidia) or less com- rior region of the chest and osteomyelitis of the
monly through abrasions of the skin. Person-to- sternum. Through contiguity, it also affected
person transmission is rare (Crum et al. 2004). the upper thoracic spine. The patient evolved
Spinal infections can occur mainly in two with neurological alterations and recovered
ways. The first is the hematogenous spread due satisfactorily after appropriate treatment with
to fungemia, and the second is the contiguous surgical decompression of the spinal cord and
dissemination from a soft tissue infection or by specific antibiotic therapy (Kulcheski et al.
direct inoculation of the organism at the time of 2015).
a surgical intervention (Frazier et al. 2001; Kim
et al. 2006).
27.6 Differential Diagnosis
Fig. 27.1 Candida albicans spondylodiscitis. Computed tomography (CT) scan images in sagittal, axial, and coronal
348 Á. L. Kulcheski et al.
Table 27.1 Preferred pharmacotherapeutic options for fungal infections of the spine (Kim et al. 2006)
Disease Treatment regimen
Coccidioidomycosis Preferred treatment
(1) Fluconazole 400–1000 mg/day postoperatively
(2) Itraconazole 400–600 mg/day postoperatively for up to 6 months
Alternative
Amphotericin B 1–2.5 g/day intravenously
Blastomycosis Preferred treatment
Amphotericin B in a 1.5–2.5 g/day for lite threatening diseases
Intraconazole 200–400 mg/day for 6–12 months
Alternative treatment
(1) Ketoconazole or fluconazole 400–800 mg/day postoperatively for 6–12 months
Cryptococcosis Preferred treatment
(1) Amphotericin B 0.7–1 mg/kg/day and flucytosine 100 mg/kg/day for 2 weeks: then
fluconazole 400 mg/day for minimum 10 weeks
(2) Amphotericin B 0.7–1 mg/kg/day; and flucytosine 100 mg/kg/day for 10 weeks
(3) Amphotericin B 0.7–1 mg/kg/day tor 6–10 weeks
(4) Lipid formulation of amphotericin B 3–6 mg/kg/day for 6–10 weeks
Candidiasis Preferred treatment
(1) Fluconazole 6 mg/kg/day for 6–12 months: or
(2) Amphotericin B 0.5–1 mg/kg/day for 6–10 weeks
Alternative
(1) Amphotericin B 0.5–1.0 mg/kg/day for 2–3 weeks: then fluconazole 6 mg/kg/day
for 6–12 months
Aspergillosis Preferred treatment
(1) Amphotericin B deoxycholate 1–1.5 mg/kg/day for 2–3 years
(2) Lipid formulation of amphotericin B 3–6 mg/kg/day
(3) Itraconazole 200–400 mg/day
27 Spinal Syndromes 349
Fig. 27.3 Postoperative
image of a patient with
Candida albicans
spondylodiscitis that had
instability due to
destruction of vertebral
bodies and kyphosis
Kulcheski AL, Graells XS, Benato ML, Santoro PG, Skaf GS, Kanafani ZA, Araj GF, Kanj SS. Non-pyogenic
Sebben AL. Espondilodiscite fúngica por Candida infections of the spine. Int J Antimicrob Agents.
albicans: um caso atípico e revisão da literatura. Rev 2010;36:99–105.
Bras Ortop. 2015;50(6):739–42. Tayles N, Buckley HR. Leprosy and tuberculosis in
Mc Cormack HM, Horne DJ, Sheather S. Clinical appli- iron age southeast Asia? Am J Phys Anthropol.
cations of visual analogue scales: a critical review. 2004;125(3):239–56.
Psychol Med. 1988;18:1007–19. Tsiodras S, Falagas ME. Clinical assessment and medical
Salloum A, Rao S, Havasi A. Aspergillus rib and vertebral treatment of spine infections. Clin Orthop Relat Res.
osteomyelitis in a former intravenous drug user. Am J 2006;444:38–50.
Med. 2004;116:208–9. Van den Bergh MF, Verweij PE, Voss A. Epidemiology
Salvalaggio PR, Bassetti M, Lorber MI. Aspergillus verte- of nosocomial fungal infections: invasive aspergillo-
bral osteomyelitis after simultaneous kidney-pancreas sis and the environment. Diagn Microbiol Infec Dis.
transplantation. Transpl Infect Dis. 2003;5:187–90. 1999;34:221–7.
Sapico FL, Montgomerie JZ. Pyogenic vertebral osteo- Yeo SF, Wong B. Current status of nonculture methods for
myelitis: report of nine cases and review of the litera- diagnosis of invasive fungal infections. Clin Microbiol
ture. Rev Infect Dis. 1979;1:754–76. Rev. 2002;15:465–84.
Sharma RR. Fungal infections of the nervous system: cur-
rent perspective and controversies in management. Int
J Surg. 2010;8:591–601.
Part IV
Radiologıcal Findings of Fungal Infections
Involving Central Nervous System and Its
Coverings
Imaging of Fungal Infections
of the Brain 28
Subhendu Parida
CNS Central nervous system Fungal pathogens are ubiquitous organisms and are
CSF Cerebrospinal fluid found worldwide. Most of the systemic fungal infec-
CT Computed tomography tions are associated with involvement of the central
DSA Digital subtraction angiography nervous system (CNS). Increasing recognition of
DTI Diffusion tensor imaging fungal infections over the last two decades is due to
DWI Diffusion-weighted imaging expansion of the immunosuppressed population at
FDG Fluorodeoxyglucose risk and due to improved diagnostic facilities. This
FLAIR Fluid-attenuated inversion recovery chapter reviews the radiological and imaging fea-
HIV-AIDS Human immunodeficiency virus- tures of various fungal infections of the CNS. Certain
acquired immunodeficiency fungal infections have very characteristic image
syndrome morphology, thus helping the clinicians in early
MRI Magnetic resonance imaging diagnosis. The imaging features may also help in
MRS Magnetic resonance spectroscopy predicting the outcomes (Kourbeti and Mylonakis
MT Magnetization transfer 2014; Scully et al. 2008; Schwartz et al. 2018).
PET-CT Positron emission tomography- The radiological features partly depend on the
computed tomography type of fungal pathogen and the route of spread.
ppm Parts per million The relevant CNS fungal pathogens are of three
SWI Susceptibility-weighted imaging categories: yeasts, molds or filamentous fungi
VR Virchow-Robin (septate, aseptate, or minimally septate), dema-
tiaceous or pigmented fungi, and dimorphic
fungi. Small-sized yeast fungi (Blastomyces,
Coccidioides, Paracoccidioides, Histoplasma,
Cryptococcus, Candida) access the microcircu-
lation from which they seed the subarachnoid
space and produce meningitis and subpial isch-
emic lesions. Intermediate-sized yeast fungi
S. Parida (*)
(Candida) occlude the small vessels and produce
Department of Radiology, CARE Hospitals,
Hyderabad, India local tissue necrosis and subsequent abscess
formation. Larger-sized fungi septate and asep-
Department of Neurology, CARE Hospitals,
Hyderabad, India tate, Aspergillus, Cladosporium, and Mucorales,
e-mail: subhendu@neuroimaging.in obstruct large- and intermediate-sized arteries
© Springer Nature Switzerland AG 2019 353
M. Turgut et al. (eds.), Fungal Infections of the Central Nervous System,
https://doi.org/10.1007/978-3-030-06088-6_28
354 S. Parida
and on occasions veins, resulting in large infarcts strate the pathology optimally. Meningeal and
(Chimelli and Mahler-Araújo 1997; Shankar parenchymal pathologies can be demonstrated
et al. 2007). CNS fungal infections are usually using various magnetic resonance imaging
secondary to infections elsewhere in the body (MRI) sequences including contrast studies.
by hematogenous spread, most commonly from
the lung. CNS invasion can be by direct exten-
sion from the adjacent structures; sinuses, nose, 28.2.1 Meningitis
and ear canal. This mode of spread is most com-
monly described in patients with sino-cranial Leptomeningeal enhancement refers to the super-
aspergillus granulomas (Chimelli and Mahler- ficial enhancement that closely follows the sur-
Araújo 1997; Shankar et al. 2007; Murthy et al. face of the cerebral and cerebellar hemispheres
2001). Rhinocerebral infection of Mucorales spp. and brain stem. Such enhancement may be uni-
is from the nasal infection. Spread can also be form, predominantly basal, or more prominent
via penetrating wounds and neurosurgical pro- along the cerebral convexities. Enhancement of
cedures. Often the CNS infections occur in the the basal cisternal meninges, sometimes referred
immunocompromised individuals. Rarely infec- as leptomeningeal thickening, is seen in patients
tion may occur in immunocompetent individu- with basal meningitis, typically cryptococcal
als (Sundaram et al. 2006; Mathur et al. 2012; meningitis. An etiological diagnosis may not be
Starkey et al. 2014; Lee et al. 1996). possible from the demonstration of leptomenin-
geal enhancement, but in a population at risk,
fungal meningitis should be considered (Lee
28.2 Techniques and General et al. 2016; Smirniotopoulos et al. 2007; Kamran
Features in Imaging et al. 2004). Magnetization transfer (MT) imag-
ing has demonstrated differing MT ratios within
The presenting clinical syndromes in patients the leptomeningeal thickening both in tubercular
with CNS fungal infections include meningitis, and fungal meningitis but has been inconsistent
meningoencephalitis, sino-cranial syndrome, as to which has higher values; furthermore it is
sino-orbital syndrome, rhinocerebral syn- difficult to reliably calculate MT ratios within the
dromes, parenchymal mass lesions, and vascu- regions of interest (Kamran et al. 2004; Gupta
lar syndromes. Depending upon the presentation, et al. 1999). The sensitivity to detect leptomenin-
the imaging protocol can be tailored to demon- geal enhancement increases with the use of
a b c
Fig. 28.1 Contrast enhanced fluid-attenuated inversion ter identified on contrast enhanced FLAIR images.
recovery (FLAIR) images in a patient of Candidial lepto- Enhancement is seen along the cerebral sulci upto their
meningitis, leptomeningeal enhancement is not seen in depths (a), on the surface of brainstem (b) and along the
most patients, however some may show enhancement bet- cerebellar foliae (c)
28 Imaging of Fungal Infections of the Brain 355
contrast-
enhanced fluid-attenuated inversion lated or crenated margins as compared to smooth-
recovery (FLAIR). FLAIR is very sensitive to walled pyogenic and tubercular abscesses. They
minor changes in composition in cerebrospinal may also have non-enhancing projections along
fluid (CSF); it does not show enhancement in their inner walls unlike pyogenic or tubercular
normal vascular structures and hence may be abscesses; these projections have been demon-
superior to routinely used postcontrast T1 images strated to show diffusion restriction (Luthra et al.
(Fig. 28.1) (Lee et al. 2016; Smirniotopoulos 2007). On susceptibility-weighted imaging (SWI)
et al. 2007). Parenchymal spread occurs through images fungal abscesses may show complete
the Virchow- Robin (VR) (perivascular) spaces smooth or irregular rim and occasionally low sig-
from the basal cisterns in Cryptococcus. nal at the center (Antulov et al. 2014). Parenchymal
Production of excess amounts of mucoid material fungal granulomas have been commonly reported
leads to cystic enlargement of the perivascular with Aspergillus spp. and Mucor spp. Frontal lobe
spaces. Intensity remains similar to CSF and is the most common location of intra-axial granu-
hence is very well demonstrated on T2 sequences lomas (Mathur et al. 2012; Starkey et al. 2014).
(Starkey et al. 2014; Lee et al. 1996). The lesions mostly show nonspecific imaging fea-
tures; clues to fungal ethology include isointense
to hypointense signal on T2-weighted images. A
28.2.2 Parenchymal Lesions thin rim of contrast enhancement may be seen
(Michael et al. 1985).
Parenchymal lesions include acute infarcts, cere- There are few reports of magnetic resonance
britis, focal abscesses, and fungal granulomas spectroscopy (MRS) findings in fungal parenchy-
(Sundaram et al. 2006; Mathur et al. 2012). mal lesions; abscesses have been reported to show
Cerebritis refers to an area of focal cerebral peaks of cytosolic amino acids such as valine, leu-
inflammation due to fungal invasion either by cine, and isoleucine and lactate at 0.9 ppm and
hematogenous dissemination or by direct invasion multiple peaks of trehalose between 3.6 and 4 parts
from an extra-axial source. It is often seen as an per million (ppm) (Fig. 28.2) (Oner et al. 2006;
ill-defined area of focal T2/FLAIR hyperintense Gupta et al. 2013). Diffusion tensor imaging (DTI)
parenchymal swelling with varying amounts of metrics have been evaluated for usefulness in pyo-
surrounding edema. Lesions may demonstrate het- genic abscesses; however such evidence for fungal
erogeneous diffusion restriction. Only minimal or lesions is lacking (Nath et al. 2007).
no postcontrast enhancement has been reported
(Starkey et al. 2014; Tung and Rogg 2003; Marzolf 28.2.2.1 Extra-axial: Sino-nasal
et al. 2016). Host immune system restricts the and Skull Base Syndromes
infection, further evolving to fungal abscess. On MRI with a dedicated “skull base” protocol
imaging fungal abscesses are seen as defined should be the imaging modality in patients with
lesions with T2 hypointense rim and hyperintense sino-nasal and skull base syndromes. The proto-
center with surrounding vasogenic edema. col should include thin section fat-saturated T1
Diffusion restriction may be seen in these and T2 sequences in coronal, axial, and sagittal
lesions; the foci of restriction can be central and planes and postcontrast fat-saturated T1 images
heterogenous, peripheral, or diffusely punctate; in all planes (Herrera et al. 2009; Hudgins and
in contrast pyogenic abscesses generally have Baugnon 2017). These sequences can optimally
uniform high degree of diffusion restriction at the demonstrate sinus disease, bone infiltration in
center of the lesion (Starkey et al. 2014; Tung and skull base, and perineural spread.
Rogg 2003; Marzolf et al. 2016). Thin section skull base computed tomography
Poor contrast enhancement is noted; contrast (CT) offers better resolution and demonstration of
enhancement depends on the host immune status. signs of focal bone destruction. Maxillary and eth-
In the immunocompetent patients, the contrast moidal sinuses are the sinuses most commonly
enhancement may be homogenous and dense. affected. Sinus lesions appear as center hyper-
Fungal abscesses have been reported to have lobu- dense surrounded by hypodense thickened
356 S. Parida
Fig. 28.2 Magnetic resonance spectroscopy (MRS) (TE 135 ms) in a case of left frontal aspergilloma, shows a promi-
nent lipid peak at 1.3 parts per million (ppm) and characteristic Trehalose peak at 3.6–3.8 ppm
mucosa. Extra-sinus spread may be fat stranding lyses elastin in the arterial walls resulting in loss
or mass-like lesion even without frank destruction in integrity. The consequences of angioinvasion
of the sinus walls; this is due to spread through include stenosis, thrombosis, or aneurysm forma-
blood vessels (Mathur et al. 2012; Starkey et al. tion (Mathur et al. 2012). Stenosis and thrombo-
2014; Laine et al. 1990). Skull base foramina such sis may result in ischemia and infarction in the
as foramen ovale, foramen rotundum, Vidian area of the arterial territory. T2/FLAIR imaging
canal, etc. may appear enlarged in case of perineu- show infarct as hyperintense lesion with diffu-
ral spread along the branches of fifth cranial nerve sion restriction in the acute stage. Hemorrhagic
passing through them (Kandpal et al. 2008). transformation is common with CNS aspergillosis
MRIs show mucosal thickening in the affected (Marzolf et al. 2016; Oner et al. 2006).
sinuses, with central signal loss which is due to Fungal mycotic aneurysm is a rare complica-
the high mineral content of the fungal concre- tion; the possible pathogenetic mechanisms
tions. In case of extra-sinus extension, soft tissue include compromise of vasa vasorum, direct inva-
intensity is noted in the surroundings such as sion of vessel wall, immune complex deposition
masticator space in case of maxillary sinus dis- in the vessel wall, and subsequent damage (Ahuja
ease. Postcontrast may show heterogeneous et al. 1978; Hurst et al. 2001). Bacterial mycotic
mucosal enhancement (Mathur et al. 2012; aneurysms may be single or multiple, are usually
Starkey et al. 2014). Normal bright enhancement small, and located in the distal segments of the
of the mucosa may not be seen due to angioinva- vessels, whereas fungal mycotic aneurysms are
sion, “black turbinate” sign described in nasal proximal, fusiform, and larger (Ahuja et al. 1978).
cavity disease (Safder et al. 2010).
2014; Fennelly et al. 2013). Candida cerebral bleeding. Most often the lesions show periph-
abscess is common in infants and young indi- eral ring enhancement. Contrast study may be
viduals (Ahuja et al. 1978). Brain involvement useful in delineation of smaller lesions. MRS
in candida infection is hematogenous; blood findings may be nonspecific. Diagnosis is by
cultures isolate the organism in about half the biopsy and/or CSF analysis (Gupta et al. 2013;
cases (Fennelly et al. 2013). The clinical pre- Fennelly et al. 2013; Figueiredo et al. 2014).
senting features of CNS candida infection Diagnosis of leptomeningitis diagnosis is diffi-
include microabscesses; macroabscesses; lepto- cult on imaging; FLAIR hyperintensity on sulcal
meningitis with or without complications such spaces and leptomeningeal pattern of enhance-
as hydrocephalus, arteritis, and related infarcts; ment on postcontrast are suggestive of leptomen-
and mycotic aneurysms (Figueiredo et al. 2014). ingitis, though it is not specific for any etiology
Microabscesses are innumerable, most often (Fig. 28.1) (Lee et al. 2016; Smirniotopoulos et al.
less than 3 mm in size, and indistinctly demar- 2007; Kamran et al. 2004). Vascular involvement
cated from the rest of the cerebral parenchyma. due to arteritis causes luminal compromise or
They may show mild perilesional edema and focal weakening of the vessel wall resulting in the
diffusion restriction. Nodular or discoid post- formation of mycotic aneurysms. Luminal steno-
contrast enhancement is the feature (Fig. 28.3) sis may cause ischemic stroke, predominantly
(Mathur et al. 2012; Starkey et al. 2014; Hurst localized to basal ganglia. In the acute stage, the
et al. 2001). Macroabscesses can be single or lesions appear hyperintense on T2 or FLAIR with
multiple of different sizes and peripheral uniform diffusion restriction (Mathur et al. 2012;
in location. These can be variably hypointense Starkey et al. 2014). Mycotic aneurysms have
on T1 images and hyperintense on T2 images. been rarely reported in cerebral candida infection.
Some of the lesion may show intralesional Subarachnoid hemorrhage and intraventricular
a b
Fig. 28.3 A 65 years old diabetic patient presented with Contrast could not be administered to the patient due to
altered consciousness. Axial FLAIR image (a) reveals poor renal function. Candida spp was isolated from cere-
multiple rounded lesions in the periventricular and deep brospinal fluid (CSF), consistent with the diagnosis of can-
white matter, showing diffusion restriction on the diffu- didial micro and macrobascesses
sion-weighted imaging (DWI) (b), suggestive of abscesses.
358 S. Parida
hemorrhage can be identified as hyperattenuation these regions are supplied by perforating arteries.
on unenhanced CT scan or as hyperintensity Aspergillus abscesses are seen in deeper regions
within the subarachnoid space on FLAIR. SWI such as basal ganglia and thalami and also at the
images may show blooming (Ahuja et al. 1978; gray-white junction. In immunocompromised
Hurst et al. 2001). Aneurysms are demonstrated patients, the lesions are ill-defined with minimal
often by CT angiography; digital subtraction angi- perilesional edema. The lesions are usually T1
ography ( DSA) is usually reserved for endovascu- hypointense, and hyperintensity seen in some of
lar treatment planning. the lesions is due to blood or minerals such as
manganese and iron found abundantly in fun-
gal hyphae. Central areas of necrosis seen are of
28.3.2 Aspergillosis nearly fluid intensity. T2 images show heterog-
enous intensity; the periphery can be T2 hypoin-
Aspergillus spp. can affect both immunocompe- tense with central areas approaching fluid signal
tent and immunocompromised individuals. Skull (Shaikh and Sundararajan 2015; Muraoka et al.
base and sino-orbital syndromes are mostly 2016; Tempkin et al. 2006; Almutairi et al. 2009).
described from countries with temperate cli- Peripheral diffusion restriction may be noted
mates and in otherwise immunocompetent indi- (Fig. 28.4). SWI shows peripheral blooming due
viduals. Hematogenous dissemination is more to blood or minerals. Postcontrast enhancement
often in immunocompromised patients (Marzolf is marked in immunocompetent patients and may
et al. 2016). be absent or minimal in immunocompromised
Angioinvasion is the primary pathology in patients (Figs. 28.3 and 28.5) (Sundaram et al.
cerebral involvement. Perforator arteries at the 2006; Gupta et al. 2013; Shaikh and Sundararajan
base of the brain are primarily involved. This 2015; Tempkin et al. 2006; Almutairi et al. 2009).
results in infarcts in the corresponding arterial ter- Parenchymal lesions can however be mistaken
ritories and subsequent fungal invasion. Findings for lymphoma or glioblastoma due to their vary-
on imaging depend on the stage of involvement ing appearances. It may be difficult to prospec-
and hence reflect the progression (Sundaram et al. tively diagnose a fungal granuloma in such
2006; Shaikh and Sundararajan 2015; Muraoka atypical lesions; imaging in such patients is use-
et al. 2016). The location of infarcts in aspergil- ful in planning biopsy and for subsequent follow-
losis include basal ganglia, thalami, brain stem as up (Sidani et al. 2013; Kumar et al. 2018).
a b c
Fig. 28.4 A case of cerebellar aspergillus abscess. The istic restricted diffusion along the wall of the abscess on
axial T2 images (a) reveals a hyperintense left cerebellar DWI images (b). Mild perilesional edema is noted. Post
lesion with thin hypointense wall and showing character- contrast study shows uniform peripheral enhancement
28 Imaging of Fungal Infections of the Brain 359
a b
Fig. 28.5 A case of parenchymal aspergillus granuloma. isointense rim and central hyperintensity surrounded by
Image (a) reveals a heterogenous ill defined lesion in the edema. The post contrast scan (image b) reveals heteroge-
left temporal-occipital region, showing peripheral T2 neous enhancement of the lesion
Infiltration of skull base leading to skull base and ischemia and infarction in the correspond-
osteomyelitis and focal granuloma formation ing arterial tertiaries, and this includes sinuses
may be seen as a direct extension from sino-nasal and cerebral parenchyma (Moll et al. 1994;
disease or from an infection in the ear. They pres- Mandava et al. 2001; Koc et al. 2007). Imaging
ent as focal lytic lesions or an area of permeative is reflective of these changes. Sinusitis appears
pattern of bone destruction on unenhanced CT as mucosal thickening; the involved sinuses may
scan. The lesion may be hypointense on T2 have internal iso-attenuating or hypo-attenuating
images, heterogeneous postcontrast is often seen, areas. Inspissated secretions may appear hyper-
and there may be contiguous extension into the dense on non-contrast scans. Spread beyond
cavernous sinuses and orbits (Hudgins and maxillary sinuses appears as fat stranding or
Baugnon 2017; Laine et al. 1990). soft tissue attenuation in the masticator space
or subcutaneous region of face. Further spread
into the pterygopalatine fossa appears as oblit-
28.3.3 Mucormycosis eration of the normal fat attenuation seen within
it (Horger et al. 2006; Orguc et al. 2005; Raz
Rhinocerebral-orbital mucormycosis is the et al. 2015). Focal bone erosions may be seen
most common localized form of invasive dis- in m ucormycosis; however, bone erosions are
ease caused by Mucorales sp. and is more com- more common with suggestive of an Aspergillus
mon in patients with poorly controlled diabetes. infection (Nithyanandam and Correa 2010). MRI
The infection usually starts in the sinuses (more is more helpful to demonstrate the extent, espe-
commonly maxillary sinus) and ethmoidal air cially in depicting soft tissue involvement, intra-
cells, and further spread occurs commonly cranial extension, and perineural spread. Involved
through macroscopically intact sinus walls via sinuses show mucosal thickening. Sinus cavities
angioinvasion of vessels, perineural invasion,
often contain concretions that show signal loss
small dehiscences, or foramina (Sundaram et al. and require examination on multiple sequences
2006). Extension into orbits, cavernous sinus, to differentiate from normal signal void of air.
and middle ear – petrous mastoid region – and Extra-sinus extension appears as fat stranding
intradural intraparenchymal extension can occur or replacement of fat by soft tissue intensity
(Herrera et al. 2009; Moll et al. 1994). Similar to (Fig. 28.6) (Raz et al. 2015).
Aspergillus sp., angioinvasion is a pathologic fea- Perineural spread has been documented in
ture, this leads to occlusion of the blood vessels 70–90% of cases in pathological case series
360 S. Parida
a b
c d
Fig. 28.6 A 55 years old diabetic female patient with rhi- of the right cavernous sinus. The DWIs (d) reveal acute
nocerebral mucormycosis. The computed tomography right basal ganglia and anterior thalamic infarct. The time-
images (a) revealed ill definded soft tissue attenuating of-flight magnetic resonance angiography images reveal
lesion at right ethmoidal sinus and right medial extra- (e) irregularity of supraclinoid right internal carotid artery
conal orbital space. The T2 (b) & post contrast T1 (c) and the M1 segment of right middle cerebral artery. Basilar
images reveals extension of lesion to bilateral ethmoidal, artery flow signal is normal, loss of signal at its apex is
nasopharynx and right parasellar region with infiltration artifactual
28 Imaging of Fungal Infections of the Brain 361
(Sravani et al. 2014; Cornely et al. 2014). This base sign.” Communicating hydrocephalus may
mode of spread is uncommonly demonstrated on be seen (Katchanov et al. 2016). A small pro-
imaging. The imaging findings include thickness portion of patients with cryptococcal meningitis
of the nerves, postcontrast enhancement, and may present with acute infarcts in the territory of
atrophy of muscles supplied by the nerves. The the lenticulostriate arteries predominantly in the
foramina through which the nerves egress the basal ganglia and thalami. It may be due to direct
skull are often enlarged. The nerve involved in invasion of the lenticulostriate arteries; vasculitis
the perineural spread depends on the lesion loca- has been demonstrated angiographically in pre-
tion: infraorbital nerve in maxillary sinus lesions; vious studies. Other mechanisms include vessel
the nerves involved depend on the tissue exten- spasm, vessel entrapment by exudate, and throm-
sion of the lesion, for example, a lesion in the bus formation (Starkey et al. 2014; Katchanov
maxillary sinus may involve the infraorbital et al. 2016; Offiah and Naseer 2016).
nerve, and for lesion in the masticator space or Parenchymal lesions include gelatinous pseu-
middle cranial fossa, floor 2nd division of trigemi- docysts, multiple small ill-defined T2 hyperin-
nal nerve may be involved. Cases have been tense lesions found predominantly in the vicinity
reported where perineural spread of the nerve of VR spaces, miliary lesions, and cryptococco-
reached up to the lateral aspect of the pons in its mas. Gelatinous pseudocysts are a common man-
lateral aspect (McLean et al. 1996). ifestation of parenchymal lesions, as the organism
enters the parenchyma through the VR spaces,
and the expansion of the VR spaces is due to the
28.3.4 Cryptococcosis abundant mucoid material of the capsule of
Cryptococcus. These appear as multiple closely
Cryptococcosis of the CNS mostly occurs in spaced cystic lesions in the basal ganglia, thal-
immunocompromised subjects, and the spread ami, or midbrain with signal intensity similar to
is by hematogenous route. Neurological mani- CSF with no perilesional edema. These lesions
festations include leptomeningitis and brain usually do not show contrast enhancement
parenchyma lesions (Lee et al. 1996). Imaging (Fig. 28.7). One should have a high index of sus-
of leptomeningitis is nonspecific, as the basal picion while interpreting the images of patients
exudates either poorly enhance or do not enhance with human immunodeficiency virus-acquired
postcontrast. T2 sequence may show increased immunodeficiency syndrome (HIV-AIDS) infec-
signal of the basal regions of the brain near the tion since prominent VR spaces may be dis-
anterior perforated substance, the “hazy brain missed as an innocuous or age-related finding.
a b c
Fig. 28.7 A 34 years old immunocompromised patient or post contrast enhancement (c), suggestive of gelati-
presented with fever and headache. Axial T2 images (a) nous pseudocysts. CSF study confirmed the presence of
reveal multiple rounded hyperintense lesions at bilat- Cryptococcus
eral basal ganglia showing no diffusion restriction (b)
362 S. Parida
Smaller parenchymal lesions may be seen festations include multiple cerebral abscesses
appearing hypointense on T1 images and hyper- or granulomatous encephalitis. The lesions may
intense on T2 with variable amounts of perifocal mimic high-grade glial tumors. Leptomeningitis
edema and postcontrast enhancement. In situa- and ischemic stroke probably due to arteritis have
tions of very poor immune response, e.g. those in also been reported (Filizzola et al. 2003; Moja
patients with HIV-AIDS, these lesions may be et al. 2000; Hauck et al. 2008; Buxi et al. 1996).
numerous resembling a miliary pattern of disease
(Lee et al. 1996; Katchanov et al. 2016; Offiah
and Naseer 2016; Tore et al. 2010; Costa et al. 28.3.6 C
urrent Role
2013). Cryptococcomas are usually seen in of Fluorodeoxyglucose-
immunocompetent subjects; it appears as a lobu- Positron Emission Tomography
lated mass within the parenchyma, both in supra- in Fungal Infections
and infratentorial spaces; and it may be
multiloculated T1 hypointense and T2 hyperin- Positron emission tomography-computed tomog-
tense lesion. These lesions show central diffusion raphy (PET-CT) has been reported to be useful in
restriction and perifocal edema. Enhancement is the diagnosis and follow-up of invasive fungal
peripheral and can be smooth and/or irregular infections including the assessment of treatment
nodular. It may mimic a cystic necrotic tumor response; however owing to the high physiologi-
(Liu et al. 2015). cal uptake of fluorodeoxyglucose (FDG) by the
brain parenchyma, the role of FDG-PET in the
evaluation of fungal infections of brain is limited
28.3.5 Blastomycosis, (Ankrah et al. 2016; Sharma et al. 2014). There is
Histoplasmosis, a report of PET-CT use in the diagnosis, biopsy
Coccidioidomycosis, planning, and follow-up in a case of rhino-orbital
and Phaeohyphomycosis mucormycosis. The lesion could be correctly
identified as an FDG avid area in the orbit, peri-
Blastomycosis is caused by Blastomyces derma- orbital tissues, and ethmoidal air cells; treatment
titidis and is endemic in certain parts of North was adjusted according to response until follow-
America. CNS manifestations include menin- up PET-CT revealed resolution of lesions (Liu
gitis, intracranial mass lesions, and epidural et al. 2013). The routine use of FDG-PET in the
abscess. The mass lesions may be single or mul- evaluation of CNS fungal infections is however
tiple (Bariola et al. 2010). MRI is the imaging rare and needs further evaluation.
modality for demonstration of leptomeningeal
enhancement. Intracranial mass lesions have
intense peripheral enhancement with internal 28.4 Conclusion
foci of diffusion restriction. It is difficult to dif-
ferentiate such lesions from other fungal lesions. Fungi cause meningeal and parenchymal dis-
Brain parenchymal lesions of Histoplasma, eases predominantly in immunocompromised
histoplasmomas present as peripheral contrast- individuals and occasionally in immunocom-
enhancing multiple small rounded lesions. They petent patients. The imaging findings are rela-
may be widely scattered in the brain parenchyma. tively nonspecific. Yeasts like Cryptococcus
Appearance on T1 and T2 images is nonspecific. spp. and Candida spp. cause leptomeningitis
CNS manifestation of Coccidioides immitis infec- predominantly; hyphal organisms cause para-
tion (coccidioidomycosis) is basal meningitis nasal sinus, skull base, and brain parenchymal
and rarely parenchymal mass lesions (Stavrakis disease. Angioinvasion may lead to infarcts
et al. 2015). Phaeohyphomycosis of the CNS is in the basal ganglia, thalami, and areas sup-
caused by phaeoid fungi such as Bipolaris sp. plied by lenticulostriate arteries. Parenchymal
and Cladophialophora sp. Neurological mani- lesions of Aspergillus spp. include granulomas
28 Imaging of Fungal Infections of the Brain 363
and abscesses; peripheral diffusion restriction is Gupta RK, Kathuria MK, Pradhan S. Magnetization
transfer MR imaging in CNS tuberculosis. Am J
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Hauck EF, McGinnis M, Nauta HJ. Cerebral phaeohy-
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Herrera DA, Dublin AB, Ormsby EL, Aminpour S,
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Imaging Findings of Fungal
Infections of the Cranial 29
and Peripheral Nerves
Ahmet T. Turgut, Elif Başbay Gündoğdu,
Can Başaloğlu, and Mehmet Turgut
of fungal infections of the CNS is necessary for alitis. Rarely, it has been reported that some
accurate diagnosis because sometimes immune clinical syndromes are specific for some fungal
response is intact in immunocompromised infections: the rhinocerebral form in patients
patients (Khandelwal et al. 2011). with zygomycosis, skull base syndromes in
cases with sinocranial aspergillosis (Fig. 29.2),
and meningitis in cases with cryptococcal infec-
29.2 Fungal Infections tion (Borges 2005; Murthy 2007).
As a rule, the size of the size of the fungus is
In contrast to other microorganisms, the term critical for the involvement of CNS because only
fungus is used for eukaryotic saprotrophic organ- small fungal pathogens may enter the circulatory
isms with membrane-bound nuclei that derive system and then they cause meningitis and
nutrition from decomposition of organic matter abscess formation, while hyphal forms produce
(Shih and Koeller 2015). As a rule, the fungal ischemic or hemorrhagic infarcts by invading
infections of the CNS are caused by the systemic vessels (Shankar et al. 2007). Microbiologically,
fungal pathogens via: (a) the hematogenous common causes of fungal acute and chronic men-
route; (b) direct extension from colonized sinuses ingitis are Cryptococcus neoformans, Candida
or ear canal (sinonasal) (Fig. 29.1); or (c) direct albicans, Coccidioides immitis, and Histoplasma
inoculation during neurosurgical procedures capsulatum (Chakrabarti 2007). However,
(Chakrabarti 2007; Fockaert et al. 2014; Murthy Aspergillus spp., Candida spp., and Zygomycetes
2007). In general, fungal infections of the CNS usually cause space-occupying lesions in the
are opportunistic infections with hematogenous CNS (Chakrabarti 2007).
dissemination in susceptible subjects with immu- In this chapter, we will review radiological
nodeficiency such as human immunodeficiency characteristics of fungal diseases involving the
virus (HIV)/AIDS, systemic neoplasia, and organ CNS, according to morphologic features of fun-
transplantation (Shih and Koeller 2015). Fungi in gal pathogens: (a) yeast, (b) mold, and (c) dimor-
the nature find a nidus in the human body and phic fungus (Mathur et al. 2012).
become pathogenic in hosts with immunocom-
promised states; thus, they increase the frequency
of fungal infections (Shankar et al. 2007). Unlike 29.2.1 Yeasts
tuberculosis, fungus is not transmitted from per-
son to person by respiratory droplets but is In fungal pathogens known as yeasts such as
acquired through inhalation of fungal spores Cryptococcus neoformans and Candida albi-
from environment and soil (Shih and Koeller cans, only 1% of fungal species generally cause
2015). They reach to the CNS from pulmonary opportunistic infections in immunocompro-
focus with hematogenous spreading. mised patients with HIV/AIDS with suppressed
Clinical manifestations of fungal infections cell-mediated immunity (Shankar et al. 2007;
of the CNS are related to the growth of the fungi, Shih and Koeller 2015). On the other hand,
the antigenic feature of the capsule and the pro- Cryptococcus gattii may cause disseminated
teases secreted and may result in a clinical course infections even in immunocompetent hosts
ranging from acute fulminant course with sig- (Suchitha et al. 2012). Cryptococcus neofor-
nificant morbidity and mortality to a chronic mans is found in bird feces and transmitted by
indolent form (Khandelwal et al. 2011; Shankar inhalation. It has a unique protective polysac-
et al. 2007). Initial symptoms such as fever, nau- charide capsule that produces a characteristic
sea, vomiting, headache, seizures, lethargy, halo with India ink stain (Shih and Koeller
altered mental status, mass effect, or meningeal 2015). Cryptococcal meningitis is the most
irritation findings are not specific and indistin- common fungal disease of the CNS, due to the
guishable from other causes of meningoenceph- presence of essential nutrients and the absence
29 Imaging Findings of Fungal Infections of the Cranial and Peripheral Nerves 369
A
a b
R L
c d
Internal
carotid
artery
Oculomotor
nerve
Fig. 29.1 (a) T2-weighted image at the level of Dorello’s of the abducens nerve is slightly higher at the site where it
canal. The non-aerated sphenoid sinus has a very low sig- is cutting the dura (arrow). (c) Gd-enhanced T1-weighted
nal intensity in the anterior part (white arrow), being com- image with fat suppression. The invaded clivus on the left
patible with fungal material. The fungus has invaded the side protruding in the prepontine cistern is enhancing
clivus resulting in a low signal intensity replacing the nor- (black arrow). Non-aerated sphenoid sinus (white arrow).
mal marrow in the clivus on the left side, where the abdu- (d) Dorello’s canal. The 6th cranial nerve enters the cav-
cens nerve enters Dorello’s canal (black arrow). The bone ernous sinus at the Dorello’s canal, formed by the petrous
marrow on the right side has a normal high signal inten- apex, the superolateral part of the clivus, and the petro-
sity (gray arrow). (b) 3D-balanced FFE image at the level sphenoidal ligament or Gruber’s ligament (A anterior, P
of Dorello’s canal. The clivus is slightly protruding in the posterior, L left, R right) (from Fockaert et al. (2014),
prepontine cistern on the left side, and the signal intensity with permission)
370 A. T. Turgut et al.
a b
c d
Fig. 29.2 (a, b) Axial computed tomography (CT) sec- petrous carotid arteries bilaterally and infiltrating the
tions obtained through the skull base (bone algorithm) left cavernous sinus. The abnormal thickening of the
depicting bilateral opacification of the middle ear cav- cisternal segment of the right trigeminal nerve and the
ity and mastoid associated with bony destructive changes soft tissue filling in Meckel’s cave should be noted (skull
involving the petrous apices and central skull base. Note base osteomyelitis, presumably of fungal etiology, in a
the destruction of the petrous carotid canals. (c, d) Axial diabetic patient presenting with Gradenigo’s syndrome)
SE T1W images revealing a soft tissue mass replacing (from Borges (2005), with permission)
the bone marrow of the central skull base, encasing the
of inhibitor substance in the CSF (Igel and et al. 2017). Neuroimaging findings include
Bolande 1966). This fungus can be seen world- small or large cryptococcomas especially in
wide, and not endemic. Candida albicans is midbrain or basal ganglia when infection passes
found in the gut flora; c andidiasis may manifest through the brain parenchyma (Fig. 29.4)
as invasive systemic disease (Shih and Koeller (George et al. 2009; Shih and Koeller 2015).
2015). Cryptococcomas have low signal in T1-weighted
Cryptococcus is small enough to enter the magnetic resonance images (MRIs) and high
meningeal microcirculation through hematoge- signal in T2-weighted MRIs and
nous dissemination (Shih and Koeller 2015). FLAIR. Contrast enhancement is variable. In
There are mainly three forms of this disease: some patients with cryptococcal infection
meningitis, cryptococcomas, and gelatinous involving the CNS, yeast typically produces
pseudocysts. The infection likes to involve the “gelatinous pseudocysts” in the thalamus, mid-
perivascular spaces. In meningitis form, nodular brain, cerebellum, basal ganglia, and the regions
leptomeningeal enhancement is observed post- adjacent to ventricular regions (Fig. 29.5)
contrast T1-weighted images (Fig. 29.3) (Zhong (Kovoor et al. 2002; Shih and Koeller 2015).
29 Imaging Findings of Fungal Infections of the Cranial and Peripheral Nerves 371
a b
c d
e f
Fig. 29.3 The abnormal magnetic resonance findings in enhancement of the mass lesion. (d) Axial T2-weighted
non-human immunodeficiency virus (HIV) patients with image showing multiple dilated hyperintense Virchow-
cryptococcal meningitis. (a) Sagittal T1-weighted post- Robin spaces in bilateral centrum semiovale. (e) Axial T2
gadolinium image showing apparent nodular meningeal sequence revealing bilateral thick-walled, septated pseudo-
enhancement. (b) Axial T2-weighted image depicting mass cysts in the region of basal ganglia with a proteinaceous
lesion in the white matter of the left lateral ventricle trigone. content. (f) Post-gadolinium MRI sequences (from Zhong
(c) Post-gadolinium MRI depicting remarkable ring et al. (2017), with permission)
372 A. T. Turgut et al.
a b
Fig. 29.5 (a) T2-weighted MRI showing dilated hemispheres corresponding to focal dilatation of Virchow-
Virchow-Robin spaces in the central and periventricular Robin spaces or gelatinous pseudocysts. Note that no
parietal white matter. (b) Adjacent to the perivascular intraparenchymal lesions are detectable outside the CSF
cerebrospinal fluid (CSF) spaces, small, CSF-isointense spaces (from Berkefeld et al. (1999), with permission)
cyst-like lesions are seen in both cerebral and cerebellar
29 Imaging Findings of Fungal Infections of the Cranial and Peripheral Nerves 373
a b
Fig. 29.6 Cryptococcosis: 36-year-old with axial T2 Diffusion-weighted imaging (DWI) (b) reveals areas of
sequence (a) showing multiple hyperintense punctate lesions restricted diffusion correlating with the areas of signal abnor-
within the ganglia representing gelatinous pseudocysts. mality (from Bhatia and Pruthi (2016), with permission)
Fig. 29.7 (a) Eye movements. Bilateral symmetrical near- ganglia and thalamus (white arrows). Ill-defined area of sig-
complete ophthalmoplegia. (b) Fluid-attenuated inversion nal intensity change in the left temporoparietal region
recovery (FLAIR) (right) and T2-weighted (left) images of (black arrows) (from Liyanage et al. (2005), with
MRI of the brain. Bilateral multiple nodular lesions in basal permission)
374 A. T. Turgut et al.
In patients receiving immunosuppressive ratory tract after spore inhalation (Shih and
therapy or with indwelling catheters, C. albicans Koeller 2015). They then can invade pulmonary
infection is the most common nosocomial fun- arteries and access to systemic circulation and
gal infection (Shih and Koeller 2015). Candida hematogenously spread to the CNS. Importantly,
usually causes more invasive and disseminated multicellular hyphae cause invasive parenchy-
parenchymal disease called a “pseudohyphal mal disease in immunocompromised patients
form” characterized by microabscesses, throm- because they are big pathogens for the menin-
bosis, and hemorrhage, although macroab- geal microcirculation in contrast to yeasts (Shih
scesses, cerebral vasculitis, and meningitis are and Koeller 2015).
rarely seen (Shih and Koeller 2015; Tyc et al. Infections due to molds such as aspergillosis
2014). C. albicans has small size, so it can pass and mucormycosis involving the CNS may (a)
microcirculation and leads to microabscesses originate from hematogenous dissemination from
which is typical for this disease (Fig. 29.8) a distant pulmonary infection, (b) spread from a
(Starkey et al. 2014). They are less than 3 mm paranasal sinus or orbital infection known as
in size, multiple, and most commonly located “rhinocerebral disease” (Fig. 29.9), or (c) direct
at the gray-white matter junction, basal ganglia, implantation during traumatic event (Fig. 29.10)
and cerebellum. They have same imaging find- (Dhirawani et al. 2015; McLean et al. 1996). It
ings with other cause of abscesses. In addition, has been reported that aspergillosis frequently
they can contain small hemorrhagic foci which presents with ring-enhancing cerebral abscesses
is better seen on gradient-echo (GRE) or sus- from hematogenous dissemination (Ashdown
ceptibilityweighted imaging (SWI) MRIs. And et al. 1994). In contrast to other abscesses caused
also, because of hemorrhage, rim-like low signal by pyogenic organisms or M. tuberculosis, intra-
is seen on T2-weighted MRIs. When infection cavitary hypointense projections on T2-weighted
affects leptomeninges, nonspecific enhance- MRI and apparent diffusion coefficient MRI
ment may be seen, rarely. In differential diagno- without associated enhancement are seen in fun-
sis of candidal microabscesses, infections with gal abscesses (Luthra et al. 2007). And also, they
Staphylococcus aureus and Mycobacterium have peripheral low signal intensity on
tuberculosis infection, metastatic disease, and T2-weighted images which is better appreciated
multiple sclerosis should be considered (Lai on GRE or SWI images, possibly due to hemor-
et al. 1997). rhage (Fig. 29.11). They are often multiple and
randomly localized (Starkey et al. 2014).
Aspergillosis may cause life-threatening cere-
29.2.2 Molds bral infarction which is often multiple, present in
a random distribution because of invasion to
Molds grow as multicellular filaments called small or large blood vessels (Almutairi et al.
“hyphae” and then macroscopic networks 2009; Hurst et al. 2001; Negoro et al. 2013).
called “mycelia” (Shih and Koeller 2015). Hemorrhage and mycotic aneurysms may accom-
Microbiologically, the most common pathogens pany infarcts (Fig. 29.12).
producing hyphal forms are Aspergillus and The involvement of the cranial nerves in these
Zygomycetes (Shankar et al. 2007). Some molds localizations in the invasive disease caused by
such as Aspergillus and Mucorales are patho- Aspergillus may cause the initial symptoms of
genic, although the vast majority of molds are the disease. Trigeminal neuralgia secondary to
beneficial (Shih and Koeller 2015). Molds are the involvement of the maxillary branch of the
ubiquitous in the soil and can infect the respi- 5th cranial nerve may occur in invasive fungal
29 Imaging Findings of Fungal Infections of the Cranial and Peripheral Nerves 375
a b
c d
Fig. 29.8 Cerebral candidiasis in a 6-year-old boy with for Disease Control (CDC)/Sherry Brinkman). (b, c)
meconium ileus who developed lethargy. The entity usu- Axial and sagittal T1 post-gadolinium sequences show
ally presents as microabscesses measuring less than punctate subcortical foci of enhancement. (d) Axial DWI
3 mm. (a) Photomicrograph (original magnification, shows restricted diffusion of multiple lesions, including
×600; PAS stain) shows rounded bodies with some pseu- those that are not detectable on contrast-enhanced
dohyphae typical of Candida species (courtesy of Centers sequence (from Starkey et al. (2014), with permission)
376 A. T. Turgut et al.
a b
Fig. 29.9 (a) CT scan demonstrating a large heteroge- lesion in retro-orbital planes engulfing the optic nerve
neously enhancing mass lesion, invading the right maxil- leading to proptosis. (c) Histologic section confirmatory
lary sinus, eroding its medial and lateral walls and of Aspergillosis (from Dhirawani et al. (2004), with
extending up to right pterygoid plate. (b) Extension of the permission)
sinusitis (Fig. 29.13), whereas paralysis of 10th, ary to multiple cranial nerve involvement
11th, and 12th cranial nerve may develop due to (Fig. 29.15) and may be responsible for the
involvement of the skull base by invasive fungal symptoms associated with isolated involvement
osteomyelitis (Fig. 29.14) (Maschio et al. 2012; of the cranial nerves (Fig. 29.16) (Bakshi 2016;
Ridder et al. 2015). Jiang et al. 2016).
Mucormycosis is more likely to manifest as Osteomyelitis, local dural enhancement, and
rhinocerebral disease in immunocompromised or subdural empyema may also be present
diabetic patients because of a locally aggressive (Fig. 29.17) (Hassler et al. 2015). There is no
sino-orbital infection (Luthra et al. 2007). doubt that early diagnosis, appropriate antifungal
In aggressive course, it may spread to the orbit therapy, and surgical debridement are very
and cranium in days and may result in death in important for these infections (Shih and Koeller
turn. It may cause orbital apex syndrome second- 2015).
29 Imaging Findings of Fungal Infections of the Cranial and Peripheral Nerves 377
a b
Fig. 29.10 Fungal central nervous system (CNS) infec- meningitis resulting in ventricular wall enhancement and
tion may develop via hematogenous spread, CSF seeding, subtle frontal and occipital leptomeningeal enhancement.
or direct extension. (a) Axial T1 post-gadolinium image (c) Axial T1 post-gadolinium image depicts mucormyco-
shows typical lesions of multifocal angioinvasive asper- sis with intracranial extension and enhancement at the
gillosis at the gray-white junction (arrowheads). (b) Axial inferior frontal lobe developing after a sinus infection
T1 post-gadolinium image reveals typical cryptococcal (from Starkey et al. (2014), with permission)
378 A. T. Turgut et al.
a b
c d
Fig. 29.11 Aspergillus species are usually angioinvasive, and hemorrhage. (c) Axial diffusion-weighted sequences
multiplying within blood vessels and producing artery- show magnetic susceptibility artifact implying microhem-
destroying elastases and resulting in microhemorrhage orrhage and reduced diffusion representing infarction in
and infection of adjacent brain parenchyma in turn. the lesions located at the gray-white junction. (d) Gross
Fungal elements clogging vessels cause downstream ster- pathologic examination shows corresponding lesion with
ile infarction. (a) Illustration showing angioinvasive peripheral hemorrhage and central necrosis. The patient
nature of Aspergillus with fungal elements in the vessel was a 56-year-old woman with acute myelogenous leuke-
lumen and hyphae invading the vessel wall. (b) Axial T2 mia who developed widely disseminated multiorgan
sequence shows characteristic peripheral low signal inten- aspergillosis (from Starkey et al. (2014), with
sity (arrowheads), which may be secondary to hemor- permission)
rhage and increased iron associated with fungal elements
29 Imaging Findings of Fungal Infections of the Cranial and Peripheral Nerves 379
a b
Fig. 29.12 (a) Gadolinium-enhanced T1-weighted MRI tours and branched at 45°. Aspergillus species was sug-
scans of the brain showing low intensity in the sphenoid gested by microscopic morphology (periodic acid Schiff
sinus. (b) The fungus in the sphenoid sinus had a long staining). (c) A mycotic aneurysm in the left anterior cere-
uniformity and septate hyphae which had parallel con- bral artery (from Negoro et al. (2013), with permission)
Fig. 29.13 CT scans show a hypodense tissue associated with an inflammatory disease in the left maxillary sinus with
a fluid level and in the left ethmoid sinus, without bone erosion (from Maschio et al. (2012), with permission)
a b
c d
Fig. 29.15 MRI scans of mucormycosis presenting with lesion in the left orbital apex; (d) coronal contrast-
orbital apex syndrome. (a) Axial T1WI MRI shows an enhanced T1WI MRI shows an enhancing lesion in the
isointense lesion in the left orbital apex; (b) axial T2WI left orbital apex. Nasal biopsy shows mycelial filaments
MRI shows a lesion with low signal intensity lesion in the of variable thickness and necrosis (PAS; ×200) (from
left orbital apex and high signal in the sphenoid sinus; (c) Jiang et al. (2016), with permission)
axial contrast-enhanced T1WI MRI shows an enhancing
382 A. T. Turgut et al.
a b
Fig. 29.16 (a) Female patient presenting with mucor- Microscopic picture revealing broad, irregular, ribbon-
mycosis and ulcer over the left cheek and facial nerve like fungal hyphae which are aseptate with a background
palsy (L) side. (b) CT scan showing soft tissue opacifica- showing necrosis (HE, ×100) (from Bakshi (2016), with
tion of the left nasal cavity with fluid collection in the permission)
left maxillary sinus and edema over left cheek. (c)
29 Imaging Findings of Fungal Infections of the Cranial and Peripheral Nerves 383
a b
Fig. 29.17 10-year-old girl with aplastic anemia present- low signal intensity in the right maxillary and in the right
ing with mucormycosis. (a) CT shows the destruction of ethmoidal sinus (straight arrows) (from Hassler et al.
the frontobasis in the area of the cribriform plate (curved (2015), with permission)
arrow). (b, c) Axial T2-weighted images show areas of
a b
Fig. 29.18 The rare Coccidioides CNS infections gener- den onset of acute neurologic deficits following 3 days of
ally occur only in endemic regions of the Southwestern nausea, vomiting, and dehydration with persistent head-
United States. (a) Photomicrograph (original magnifica- aches and “fuzzy” vision and had a history of recurrent
tion, ×400; GMS stain) shows rounded spherules of rashes. The patient developed low-grade fevers. CSF fun-
Coccidioides immitis (arrowhead) (courtesy of CDC/ gal cultures grew Coccidioides immitis. The patient living
Martin D. Hicklin). (b) Axial T2-weighted sequence in the Midwestern United States had no history of travel to
reveals hyperintensity in the right globus pallidus and left endemic areas. Later she was diagnosed with hyper-IgE-
putamen with mild narrowing of the left lateral ventricle. related immunodeficiency, developed frank ventriculitis
(c) Axial DWI shows reduced diffusion in a similar ana- requiring shunt placement, and benefited from antifungal
tomical distribution. Contrast-enhanced imaging was not therapy (from Starkey et al. (2014), with permission)
performed. The patient was a 4-year-old girl having a sud-
29 Imaging Findings of Fungal Infections of the Cranial and Peripheral Nerves 385
29.3 Conclusion Erly WK, Bellon RJ, Seeger JF, Carmody RF. MR imag-
ing of acute coccidioidal meningitis. AJNR Am J
Neuroradiol. 1999a;20:509–14.
Fungal pathogens involving the CNS, including Fockaert N, D’Hooghe L, Casselman J, Dycke AV. Sixth
the yeast Cryptococcus and the dimorphic fungi cranial nerve palsy in isolated sphenoid sinusitis: a
Coccidioides, Histoplasma, and Blastomyces, are case report. Acta Neurol Belg. 2014;114:335–7.
Galgiani JN, Ampel NM, Blair JE. Coccidioidomycosis.
important sources of morbidity and mortality Clin Infect Dis. 2005;41:1217–23.
worldwide, especially in immunocompromised George R, Andronikou S, Plessis J, Plessis A, Toorn
patients in less developed countries. Today, it is RV, Maydell A. Central nervous system manifesta-
well-known that they usually start with inhalation tions of HIV infection in children. Pediatr Radiol.
2009;39:575–85.
of spores, followed by hematogenous dissemina- Hassler A, Porto L, Lehrnbecher T. Cerebral fungal infec-
tion in susceptible individuals. Immunodeficient tion in pediatric cancer patients. Curr Fungal Infect
patients are at risk for parenchymal disease for Rep. 2015;9:6–14.
aspergillosis and mucormycosis, whereas micro- Hirschmann JV. The early history of coccidioidomycosis:
1892-1945. Clin Infect Dis. 2007;44:1202–7.
abscesses are seen in invasive candidiasis. Hughes AD, Lorusso GD, Greer DL. Cost-effective
Radiologically, they have similar appearances to method for identification of dimorphic fungi. J Clin
other granulomatous diseases, such as tuberculo- Microbiol. 2004;42:4408–9.
sis. Therefore, a high degree of suspicion is Hurst RW, Judkins A, Bolger W, Chu A, Loevner
LA. Mycotic aneurysm and cerebral infarction result-
required for correct diagnosis and appropriate ing from fungal sinusitis: imaging and pathologic cor-
antifungal treatment in patients with fungal infec- relation. AJNR Am J Neuroradiol. 2001;22:858–63.
tions of the CNS. Computed tomography may Igel HJ, Bolande RP. Humoral defense mechanisms
show hydrocephalus, mass effect, and parenchy- in cryptococcosis: substances in normal human
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mal or subarachnoid hemorrhage, but MRI is growth of Cryptococcus neoformans. J Infect Dis.
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Imaging Findings of Fungal
Infections of the Sinuses 30
Extending into the Brain
Ahmed Abdel Khalek Abdel Razek
treatment is essential, sometimes combined with carotid artery in the cavernous sinus (Abdel Razek
antifungal treatment. The invasive forms require et al. 2011; Razek et al. 2009).
aggressive course of antifungal treatment, com-
bined with surgery in some forms, particularly
mucormycosis (Raz et al. 2015; Velayudhan 30.5 Imaging Appearances
et al. 2017). of Fungal Sinusitis
30.4.2 MR
30.5.2 Mycetomas
in the course of the disease (Ghegan et al. 2006; 30.6.4 Perineural Spread
Holbrook et al. 2014; Lafont et al. 2017). This
is attributed to the angioinvasiveness of the fungi Rarely, invasive form of fungal infection of
and their propensity to extend into the soft tis- PNS that often progresses to the orbit and
sues of the orbit and deep face and into the brain cavernous sinus may be complicated by
by way of vessels penetrating through partitions perineural invasion and spread (Orguc et al.
in the skull base (Marfani et al. 2010; Montone 2005).
2016; Stewart et al. 2011).
30.7 Conclusion
human immunodeficiency virus (HIV) infection, Gallium-67 scan to improve the specificity for the
injectable drug use, surgery, immunosuppression, diagnosis of osteomyelitis (Palestro 2015). 18fluo-
debilitating disease and neutropenia. It can cause rodeoxyglucose positron emission tomography
vertebral osteomyelitis, epidural abscess menin- (18F-FDG-PET) is especially helpful if the MRI is
gitis, spinal arachnoiditis and syringomyelia indeterminate due to degenerative disc disease
(Derkinderen et al. 2000; Phanthumchinda and (Palestro 2015).
Kaoropthum 1991; Miller and Mejicano 2001). Vertebral collapse and neurological deficits usu-
Candida infection reaches the spine most ally appear within 3–6 months from the onset of
commonly through haematogenous route (Miller symptoms (Stumpe et al. 2002; Khazim et al.
and Mejicano 2001). Infection is usually centred 2006). Neurological symptoms may result from
around the intervertebral disc space, leading to spinal epidural abscess, vertebral collapse, spinal
narrowing of the disc cartilage and destruction of cord infarct or meningitis. Indications for surgical
the vertebral endplates and underlying vertebral treatment are presence of neurological deficit, to
bone (Gathe et al. 1987). The low virulence of the provide a diagnosis, failure of medical therapy and
Candida species and the poor vascularization of presence of spinal instability (Ugarriza et al. 2004).
the disc space result in a slowly progressive Spinal cord lesions in candidal spondylodisci-
inflammatory destruction and thus a delay in the tis are often caused by vertebral collapse and/or
diagnosis (Ugarriza et al. 2004). spinal cord infarct rather than by associated epi-
Typically, the patient presents with back pain, dural abscess (Derkinderen et al. 2000). MRI is
fever, focal tenderness and neurological deficits. an excellent way of demonstrating the presence
However, the absence of fever does not rule out of epidural or paraspinal extension of the infec-
the diagnosis of candidal vertebral osteomyelitis tion (Fig. 31.1) and thus may be useful for early
(Hennequin et al. 1996). Presence of point ten- diagnosis and starting early medical treatment
derness over the affected area is the most useful (Torres-Ramos et al. 2004).
clinical sign (Hennequin et al. 1996; Smith and
Blaser 1991). Only 20% of the patients with can-
didal vertebral osteomyelitis develop neurologi- 31.3 Aspergillosis
cal defects (Miller and Mejicano 2001).
Laboratory findings may show elevated erythro- Aspergillus is the most common cause of skeletal
cyte sedimentation rate and C-reactive protein. mycosis, and the vertebral column is the most com-
Blood and urine cultures are rarely positive. monly involved bony structure in mycotic osteo-
Diagnosis of candidal vertebral osteomyelitis is a myelitis. Infection spreads through haematogenous
diagnostic problem due to the insidious progres- route; however, direct spread from the adjacent
sion of disease, the non-specific clinical and labo- organ most commonly the lung and iatrogenic
ratory findings and the failure to recognize Candida infection during spine surgery are other routes of
as a potential pathogen (Ozdemir et al. 2008). infection. Haematogenous spread occurs from
There are no typical radiological findings in infections primarily located in pulmonary, gastro-
candidal vertebral osteomyelitis (Ozdemir et al. intestinal or cerebral locations. The most common
2008). Magnetic resonance imaging (MRI) is more causative species is Aspergillus fumigatus with A.
sensitive, specific and accurate than radioisotope flavus, A. niger, A. nidulans and A. terreus species
bone scan or computed tomography (CT) scan for being other rare causes (Kim et al. 2006).
early recognition and localization of infectious dis- On MRI, spinal aspergillosis shows relatively
ease. MRI features include absence of disc hyper- non-specific findings like vertebral marrow
intensity and preservation of the internuclear cleft hypointensity on T1-weighted images (WIs) and
on T2-weighted images and enhancement on gado- iso- or minimal hyperintensity on T2-WIs with
linium-enhanced T1-weighted images (Williams intervening disc involvement. Relative preserva-
et al. 1999). If the clinical suspicion is high and tion of disc height, signal intensity and retained
MRI features are not definitive, or if MRI is contra- intranuclear cleft are features which may be seen
indicated, a bone scan can be combined with a in Aspergillus infection of the spine (Fig. 31.2).
31 Imaging Findings of Fungal Infections of Spine and Spinal Cord 395
a b c
e f
Fig. 31.1 Candida spondylitis. Sagittal and axial posterior epidural soft tissue (a–d). Sagittal and axial post-
T2-weighted image (WI) and T1-WI show D5–7 vertebral contrast T1-WI show heterogeneous enhancement with cen-
body signal changes with relative preservation of interverte- tral necrotic area well (e, f)
bral disc, posterior elements signal changes and large
Epidural and rarely intradural abscess may be invasion may be due to a blunted immune
seen in spinal Aspergillus infection (Kwon et al. response in immunocompromised patients.
2011; Winterstein et al. 2010; McCaslin et al. Factors intrinsic to fungi like presence of para-
2015). Paraspinal inflammation is minimal to magnetic and ferromagnetic elements within
moderate in comparison to the vertebral fungi may contribute to an absence of disc
destruction which is a hallmark of tubercular hyperintensity on T2-WIs. Similar mechanism
disease in which large paraspinal abscesses are for T2 hypointensity in fungal sinusitis has been
seen. Minimal disc changes despite fungal disc proposed previously (Winterstein et al. 2010).
396 J. Saini et al.
a b
Fig. 31.2 Aspergillus spondylitis: Sagittal T2-WI, T1-WI b). Post contrast T1-WI shows heterogenous enhance-
and post contrast T1-WI of the cervical spine show ment within the abnormal soft tissue and relative preser-
destructive lesion involving C6 and C7 vertebral bodies vation of the intervertebral disc (c)
with large prevertebral and epidural soft tissue (a and
Mollahoseini et al. described a case of cerebral brain MRI. One month later the patient developed
aspergillosis in an immunocompetent patient who features of spinal cord involvement, and MRI
was treated successfully for Aspergillus infection showed T2 hyperintensity involving the entire
of the brain (Mollahoseini and Nikoobakht 2011). cord with no significant enhancement to suggest
Brain lesion resolved after antifungal therapy that myelitis. Brain MRI was unremarkable at this time
was confirmed by follow- up contrast-enhanced to suggest postinfective demyelination of the
31 Imaging Findings of Fungal Infections of Spine and Spinal Cord 397
s pinal cord. The patient was treated with cortico- ing sclerosis and periosteal reaction (Wang et al.
steroids and showed complete recovery to suggest 2014). Meningoradiculitis (Fig. 31.3) and spinal
post-Aspergillus infection demyelination. cryptococcal granulomas are other rare forms of
spinal involvement seen in cryptococcal infec-
tion (Grosse et al. 2001; Deus-Silva et al. 2004).
31.4 Cryptococcosis Diffusion-weighted MRI in a case of spinal
Cryptococcus infection shows hyperintensity of
Spinal cord disease is a rare presentation of involved vertebral body, epidural space and pos-
cryptococcosis. Bony involvement is seen in 5% terior elements (Chhem et al. 2001).
of disseminated cryptococcosis. The neuroim- Asanuma et al. described an unusual case of
aging findings are remarkable, although not spe- isolated extradural cryptococcoma involving the
cific, and are characterized by involvement of left S1 and S2 roots (Asanuma et al. 2014). MRI
the vertebral body with involvement of the pos- showed enhancing extradural mass with enhance-
terior elements and paraspinal and perivertebral ment of the right S2 nerve sheath. On surgery
soft tissues with relative preservation of the fibrous tissue was removed and S1 and S2 nerve
disc. The vertebral body may show osteolytic roots were decompressed and histopathology and
lesions with discrete margins, absent surround- microbial examination showed cryptococcosis.
a b
Fig. 31.3 Cryptococcal myelitis with arachnoiditis. enhancement (c). Cerebrospinal fluid (CSF) was positive
Sagittal and axial T2-WI of the cervical spine show hyper- for Cryptococcus neoformans (Courtesy of Dr. Aruna
intense signal in the spinal cord with irregular margins Pallewatta, Radiologist, Hemas Hospital, Thalawathugoda
(a, b). Axial post-contrast T1-WI shows leptomeningeal Sri Lanka)
398 J. Saini et al.
Fig. 31.4 Cervical
blastomycosis: Sagittal
a b
section of computed
tomography shows
near-complete
destruction of C6
vertebral body (arrow)
(a). Sagittal and axial
T2WI at the C6 level
show C6 osteomyelitis
with significant vertebral
body destruction (arrow)
and vertebral abscess at
C6 and C7 levels with
posterior sub-
ligamentous extension
into the spinal canal
including mild posterior
displacement and
compression of cervical
spinal cord (b, c). In
addition, extensive c
prevertebral collection
noted extension from the
D1 vertebral level to the
skull base level. (With
permission Patel KR
et al. Journal of Medical
Case Reports (2015)
9:271)
lumbar epidural lesion heterogeneously enhanc- The galactomannan and (1,3)-β-d-glucan antigen
ing on contrast-enhanced T1-WI. In addition, assays were both negative in the serum. Review
enlargement and edema of the thoracolumbar of the literature suggested that involvement of the
spinal cord appeared as intramedullary hyperin- spine due to Triadelphia pulvinata has not been
tensity and showed intramedullary and leptomen- previously reported. There are only two reported
ingeal enhancement on post-contrast T1-WIs. cases of human infection involving the skin and
Histopathologic examination of the obtained blood in the patient of acute myeloid leukaemia
contents showed marked chronic inflammatory on autologous bone marrow transplant (Edathodu
process and fibrosis. Culture of the material et al. 2013).
obtained from the epidural inflammatory collec-
tion revealed infection by S. prolificans.
We observed a case with multiple thoracic 31.8 I atrogenic Fungal Infections
vertebral involvements with sparing of the disc in of the Spine
a 63-year-old female patient who was found to
have Triadelphia pulvinata on culture. MRI Postoperative fungal spondylodiscitis:
showed T2 hyperintense lesion involving the tho- Postoperative fungal spondylodiscitis is a rare
racic vertebrae with paravertebral soft tissue that cause of infection. In the literature, 14 cases of
is enhanced on post-contrast study (Fig. 31.5). postoperative fungal spondylodiscitis have been
400 J. Saini et al.
a b c
d e
Fig. 31.5 Triadelphia pulvinata fungal infection. A 60-year- imaging through the D4 vertebra show abnormal signal of the
old female presented with pain in the cervicodorsal region. vertebra with no compression of the spinal cord, diffuse
MRI of the cervicodorsal region shows T2 hyperintense sig- enhancement of the vertebral body with perivertebral soft tis-
nal from D2 to D6 vertebral bodies which appear hypointense sue enhancement and restricted diffusion (c–f). Biopsy from
on T1-WI with sparing of intervertebral discs (a, b). Axial vertebral body revealed non-specific infective etiology. The
T2-WI, sagittal and axial post-contrast T1-WI and diffusion tissue culture revealed Triadelphia pulvinata fungal infection
31 Imaging Findings of Fungal Infections of Spine and Spinal Cord 401
described (Gerometta et al. 2012), nine caused tebral and epidural abscesses. Ohtori et al. (Ohtori
by molds and five by Candida species. The most et al. 2010) who analysed the utility of the
common organism was Aspergillus spp followed 18F-FDG-PET for patients with suspected spon-
by Candida and Scedosporium prolificans, dylitis showing Modic change concluded that the
Rhizopus rhizopodoformis and Trichosporon rate of detecting spondylodiscitis infection was
asahii. All the cases have been reported in lum- very high if FDG-PET was used along with other
bosacral region. The most common symptom diagnostic methods. FDG-PET can successfully
was back pain followed by fever, while neuro- distinguish between common Modic change and
logical deficits were seen in two patients. Onset spinal infection.
of symptoms was delayed, and the first symptom Contamination of the steroid with Exserohilum
appeared average 6 weeks after the primary pro- rostratum resulting in infections: Moudgal et al.
cedure (Gerometta et al. 2012). MRI showed reported a large series of patients who received
osteomyelitis and/or discitis in all cases, compli- methylprednisolone epidural injection for pain
cated by paravertebral abscess in three cases and and who developed pain and/or spinal/paraspinal
by epidural abscess in four cases. All patients infection following treatment and underwent at
received antifungal drugs. least one MRI study (Moudgal et al. 2014).
MRI remains the most sensitive and specific Abnormalities were most often noted in the lum-
imaging modality for the diagnosis of postopera- bosacral region which was the site of injection of
tive spondylodiscitis (Kim et al. 2006; Gerometta contaminated methylprednisolone (New England
et al. 2012). MRI findings of postoperative disci- Compounding Center, Framingham,
tis include decreased signal intensity on T1-WIs Massachusetts). In few patients noncontiguous
and increased signal intensity on T2-WIs in the sites of involvement were also noted. Initial study
disc space, secondary to inflammation and infec- was able to identify infection in majority of
tion. Post-gadolinium T1-WIs showed disc and patients; however, in approximately 15% of
adjacent bone marrow enhancement. patients, additional follow-up MRI was needed to
In 2011, Li et al. (Li et al. 2011) reported identify the presence of infection. The imaging
radiographic findings in 34 patients with postop- findings included an epidural or paraspinal
erative intervertebral discitis and found disc abscess or phlegmon, arachnoiditis and osteomy-
space narrowing in 29 patients and destructive elitis (Fig. 31.6).
and sclerotic vertebral body changes in 14
patients. Nielsen et al. (Nielsen et al. 1990)
reported disc space loss and vertebral fusion in 31.9 Conclusion
17% of patients following postoperative discitis.
In the study of Hsieh et al. (Hsieh et al. 2011), all Fungal infections of the spine are rare and are
patients were found to have loosening of the ped- usually diagnosed late due to slow onset of the
icle screws. Kulkarni et al. (Kulkarni and Hee disease and should be evaluated with MRI even if
2006) reported a case of adjacent-level discitis the imaging features are non-specific. Once the
after anterior cervical discectomy and fusion, and lesion is detected on MRI, it should be immedi-
the radiographs showed segmental kyphosis, ately biopsied to confirm the diagnosis using cul-
decreased disc height, erosion of endplates, halo ture and histopathology which should result in
around the screws and enlarged prevertebral soft early institution of antifungal treatment. Delay in
tissue shadow. MRI showed discitis with prever- diagnosis results in poor prognosis.
402 J. Saini et al.
a b c
d e
f g
Fig. 31.6 Exserohilum rostratum fungal meningitis after of enhancement within the spinal canal with decreased dural
epidural injection of methyl prednisone from New England and leptomeningeal enhancement, decreased size of enhanc-
Compounding Center, Framingham, Massachusetts. Sagittal ing multiloculated fluid collection in the left posterior epi-
T2-WI, T1-WI and post-contrast T1-WI of the lumbosacral dural space at the S1 level and continued clumping and
spine show enhancement and clumping of the nerve roots waviness of the cauda equina, in keeping with arachnoiditis
with CSF loculation (a–c). Repeat study following treatment (d–g) (Courtesy: Dr. Suyash Mohan, Department of
with antifungal agent shows interval decrease in the amount Neuroradiology, University of Pennsylvania, USA)
31 Imaging Findings of Fungal Infections of Spine and Spinal Cord 403
Smith AS, Blaser SI. Infectious and inflammatory processes Wang C, Jia N, Zhang L, Liu K, Liu H, Yu H. Imaging
of the spine. Radiol Clin North Am. 1991;29(4):809–27. findings of cryptococcal infection of the thoracic
Stumpe KDM, Zanetti M, Weishaupt D, Hodler J, Boos N, spine. Int J Infect Dis. 2014;29:162–5. Available from:
Von Schulthess GK. FDG positron emission tomography https://www.ncbi.nlm.nih.gov/pubmed/25449251
for differentiation of degenerative and infectious end- Williams RL, Fukui MB, Meltzer CC, Swarnkar A,
plate abnormalities in the lumbar spine detected on MR Johnson DW, Welch W. Fungal spinal osteomyelitis
imaging. AJR Am J Roentgenol. 2002;179(5):1151–7. in the immunocompromised patient: MR findings in
Torres-Ramos FM, Botwin K, Shah CP. Candida spon- three cases. AJNR Am J Neuroradiol. 1999;20(3):
dylodiscitis: an unusual case of thoracolumbar pain 381–5.
with review of imaging findings and description of the Winterstein AR, Bohndorf K, Vollert K, Wagner T,
clinical condition. Pain Physician. 2004;7(2):257–60. Gnekow A, Roemer FW. Invasive aspergillo-
Ugarriza LF, Cabezudo JM, Lorenzana LM, Rodríguez- sis osteomyelitis in children—a case report and
Sánchez JA. Candida albicans spondylodiscitis. Br J review of the literature. Skeletal Radiol. 2010;39(8):
Neurosurg. 2004;18(2):189–92. 827–31.
Part V
Therapy of Fungal Infections Involving
Central Nervous System and Its Coverings
Surgical Therapy
32
Mehmet Turgut
a b
c d
Fig. 32.2 (a) Photograph illustrating angioinvasive fea- gal elements. (c) Axial diffusion-weighted MRI showing
ture of Aspergillus with fungal elements in the lumen of microhemorrhage and reduced diffusion caused by infarc-
the vessel, with hyphae involving the vessel wall. (b) tion in the lesions at the gray–white junction region. (d)
Axial T2-weighted magnetic resonance imaging (MRI) Gross specimen photograph illustrating hemorrhage with
demonstrating typical peripheral hypointensity (arrow- central necrosis caused by fungi (from Starkey et al.
heads) owing to high iron caused by hemorrhage and fun- (2014), with permission)
together with medical treatment, in addition to long-term antifungal treatment, it has been sug-
removal of immunosuppressive agents, if possible gested that only medical therapy with conventional
(Kural et al. 2018). In patients with fungal infec- AmB or combination of liposomal AmB and oral
tions involving the CNS, duration of systemic anti- itraconazole is the choice of treatment for this
fungal agents and morbidity/mortality of fungal infection, even if complete surgical excision of the
diseases treated without oral or intravenous anti- multiple mass lesions, granulomas, or abscesses is
fungal agents, including liposomal amphotericin not possible in complicated cases (Turgut et al.
B, fluconazole, and voriconazole, however, are 2008). In cases with meningoencephalitis caused
still among unsolved questions in neurosurgery by fungi, induction therapy including fungicidal
(Turgut et al. 2008). Based on experience from our regimens with flucytosine and then suppressive
case of multiple fungal granulomas involving the regimens with fluconazole are advised, while sur-
brain due to aspergillosis, which was treated by gery may be necessary in the most of the patients
410 M. Turgut
a b
c d
Fig. 32.3 (a) Photographs demonstrating fungal forms linium administration demonstrating presence of
with broad, pleomorphic hyphae within a necrotic brain enhancement. (d) Axial diffusion-weighted MRI reveal-
tissue (original magnification, 400 ×; PAS stain). (b) Axial ing a reduced diffusion (from Starkey et al. (2014), with
FLAIR image demonstrating hyperintensity areas involv- permission)
ing both gyrus rectus. (c) Axial T1 MRI following gado-
32 Surgical Therapy 411
with fungal granulomas and abscesses involving are located in accessible regions of the brain or
the CNS (Muzumdar 2011). Furthermore, ampho- spinal cord, it has been reported that complete/
tericin B regimens with lipid formulations may be incomplete excision of space-occupying lesions
used as an alternative agent in patients with renal via open surgical craniotomy/laminectomy is a
disease (Muzumdar 2011). Moreover, in some feasible and safe procedure, as mentioned above.
cases with refractory infections, use of adjunctive On the other hand, open surgical craniotomy/lam-
recombinant cytokine, growth factor, or cortico- inectomy procedure is also performed in the pres-
steroid may be used as an alternative approach ence of a suspected fungal granuloma and abscess
(Panackal and Williamson 2015). (Rajshekhar 2007; Panackal and Williamson
2015; Thurnher and Olatunji 2016).
Thurnher and Olatunji 2016; Sharma et al. infections involving the CNS (Salama et al. 1997;
1997). Arunkumar et al. 2000; Casey et al. 1994;
Goodman and Coffey 1989; Kerkmann et al.
32.6.1.2 Local/Intracavitary 1994). In patients whose general health condition
Antifungal Adjuvant is not good for open surgery, minimal invasive
Therapy After Surgical stereotactic procedures may be used for total
Excision excision of the fungal granulomas and abscesses,
Interestingly, it has also been reported that local even under local anesthesia (Rajshekhar 2007).
antifungal therapy with amphotericin B has been Importantly, Siddiqui et al. (2004) suggested that
used in patients with incomplete removal of the the periphery of the mass lesions, granuloma, or
fungal granulomas and abscesses during open abscess, in addition to its center, should be tar-
surgery craniotomy procedure in a previous study geted because the fungal hyphae are frequently
(Langmayr et al. 1993). Most recently, it has been located in the periphery region of the lesion.
suggested that intracavitary amphotericin B ther-
apy may be given as an adjunct following the sur- 32.6.2.2 Stereotactic Craniotomy
gical excision of granuloma or abscess in patients To reduce the morbidity and mortality of an open
with intracranial aspergillosis because the admin- surgery with craniotomy, stereotactic craniotomy
istration of antifungal drugs through the blood may be used in some patients with fungal granu-
brain barrier (BBB) is limited, with aim of the lomas and abscesses of the CNS (Rajshekhar
regression of mass lesion or avoid from the recur- 2007; Middelhof et al. 2005).
rence (Kural et al. 2018).
32.6.2.3 Placement of Ommaya
Reservoir
32.6.2 Minimal Invasive Stereotactic Surgically, stereotactic placement of Ommaya
Procedure reservoir may be used for the injection of ampho-
tericin B into fungal granulomas or abscesses
As expected, minimal invasive stereotactic sur- involving the CNS (Rajshekhar 2007; Young
gery procedure is less invasive than open surgical et al. 1985). In a previous study, Camarata et al.
craniotomy procedure, and it can be performed (1992) reported that intracavitary drug adminis-
even in patients with poor general condition, in tration of amphotericin B for fungal infections
contrast to that of open craniotomy procedure, as involving the CNS, granuloma or abscess, pro-
an alternative. vide a better outcome in selected cases. Then, it
has been suggested that such therapy avoids high
32.6.2.1 Stereotactic Biopsy systemic toxicity of the antifungal agents by
and Aspiration bypassing the BBB (Jamjoom et al. 1995;
Minimal invasive stereotactic biopsy and aspira- Siddiqui et al. 2004).
tion procedure for the fungal mass lesions of
brain such as granuloma and abscess is used if
the lesions are multiple or located in deep or elo- 32.6.3 Other Surgical Procedures
quent brain regions such as brain stem, basal gan-
glion, thalamus, motor strip, or Broca and 32.6.3.1 Ventriculoperitoneal Shunt
Wernicke areas (Rajshekhar 2007). Using a mini- for Hydrocephalus
mal invasive procedure, it is possible to obtain In some patients with fungal mass lesions involv-
infected tissue or pus for microbiological exami- ing the CNS, such as granuloma and abscess, a
nation for definitive diagnosis (Rajshekhar 2007). communicating type of hydrocephalus may
However, some authors suggested that minimal develop due to blockage of cerebrospinal fluid
invasive stereotactic biopsy and aspiration proce- flow as a result of arachnoiditis of the basal cis-
dures, in addition to systemic antifungal therapy, terns, and they may require the surgical placement
provide a good outcome in patients with fungal of a ventriculoperitoneal shunt either in the early
32 Surgical Therapy 413
Kural C, Ozer MI, Ezgu MC, Mehtiyev R, Yasar S, Kutlay Perfect JR, Dismukes WE, Dromer F, Goldman DL,
AM, Daneyemez MK, Onguru O, Erdogan E, Izci Graybill JR, Hamill RJ, Harrison TS, Larsen RA,
Y. Intracavitary amphotericin B in the treatment of Lortholary O, Nguyen MH, Pappas PG, Powderly
intracranial aspergillosis. J Clin Neurosci. 2018;51:75– WG, Singh N, Sobel JD, Sorrell TC. Clinical practice
9. https://doi.org/10.1016/j.jocn.2018.02.018. guidelines for the management of cryptococcal dis-
Langmayr JJ, Schwarz A, Buchberger W, Hochleitner ease: 2010 update by the infectious diseases society of
W, Twerdy K. Local amphotericin for fungal brain America. Clin Infect Dis. 2010;50:291–322.
abscess. Lancet. 1993;342:123. Rajshekhar V. Surgical management of intracranial fungal
Li Q, You C, Liu Q, Liu Y. Central nervous system cryp- masses. Neurol India. 2007;55:267–73.
tococcoma in immunocompetent patients: a short Salama AD, Rogers T, Lord GM, Lechler RI, Mason
review illustrated by a new case. Acta Neurochir. PD. Multiple Cladosporium brain abscesses in a renal
2010;152:129–36. transplant patient: aggressive management improves
Mazumder SA, Cleveland KO. Cryptococcal meningitis outcome. Transplantation. 1997;63:160–2.
after neurosurgery. Am J Med Sci. 2010;339:582–3. Sharma BS, Khosla VK, Kak VK, Banerjee AK, Vasishtha
Mehta VS, Bhatia R, Mahapatra LN, Banerji AK. RK, Prasad KS, Sharma SC, Mathuriya SN, Tewari
Intracranial mycotic infection in non-immunocompro- MK, Pathak A. Intracranial fungal granuloma. Surg
mised individuals. J Indian Med Assoc. 1985;83:185–8. Neurol. 1997;47:489–97.
Menon S, Bharadwaj R, Chowdhary A, Kaundinya DV, Siddiqui AA, Shah AA, Bashir SH. Craniocerebral asper-
Palande DA. Current epidemiology of intracranial gillosis of sinonasal origin in immunocompetent
abscesses: a prospective 5 year study. J Med Microbiol. patients: clinical spectrum and outcome in 25 cases.
2008;57:1259–68. Neurosurgery. 2004;55:602–13.
Middelhof CA, Loudon WG, Muhonen MD, Xavier C, Starkey J, Moritani T, Kirby P. MRI of CNS fungal
Greene CS Jr. Improved survival in central nervous infections: review of aspergillosis to histoplasmo-
system aspergillosis: A series of immunocompro- sis and everything in between. Clin Neuroradiol.
mised children with leukemia undergoing stereotac- 2014;24:217–30.
tic resection of aspergillomas. Report of four cases. J Thurnher MM, Olatunji RB. Infections of the spine and
Neurosurg. 2005;103:374–8. spinal cord. Handb Clin Neurol. 2016;136:717–31.
Muzumdar D. Central nervous system infections and the Turgut M, Ozsunar Y, Oncü S, Akyüz O, Ertuğrul MB,
neurosurgeon: a perspective. Int J Surg. 2011;9:113–6. Tekin C, Gültekin B, Sakarya S. Invasive fungal gran-
Nadkarni T, Goel A. Aspergilloma of the brain: an over- uloma of the brain caused by Aspergillus fumigatus: a
view. J Postgrad Med. 2005;51:37–41. case report and review of the literature. Surg Neurol.
Panackal AA, Williamson PR. Fungal infections of the 2008;69:169–74.
central nervous system. Continuum (Minneap Minn). Young RF, Gade G, Grinnell V. Surgical treatment for
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Prognosis of Fungal Infections
Involving the Central Nervous 33
System and Its Coverings
Kartik Munta and Jay Dip Ray Chaudhuri
acquired immunodeficiency syndrome (AIDS), rates may range around 10% for neurosurgical
malignancy, immunological defects (which patients, whereas it increased up to 30% in HIV-
might arise due to hereditary factors), organ infected patients (Nguyen and Yu 1995; Casado
transplant recipients, or patients receiving et al. 1997). Candida meningitis in premature
immunosuppressive medications. (Gavito- infants has a higher rate of mortality and is asso-
Higuera et al. 2016). ciated with neurodevelopmental abnormalities
(Lee et al. 1998).
Aspergillus spp. is the commonest causative been the leading cause of death in HIV-infected
fungal pathogen accounting for 56–69% of the patients in Thailand, Uganda, Malawi, and South
intracranial fungal mass lesions (Sharma et al. Africa, with an estimated mortality risk of 17% at
1997b; Dubey et al. 2005). Early institution of 2 weeks and 34% at 10 weeks (Jarvis et al. 2014).
voriconazole therapy, immunosuppressive ther- Chronic neuropsychiatric sequelae and altered
apy, transfusion of granulocytes, and hyper- brain imaging are common after cryptococcal
baric oxygen may improve outcomes in these meningitis (Lu et al. 2011).
patients (Mattiuzzi and Giles 2005; Antinori Patient treated with amphotericin B and flucy-
et al. 2013). tosine carried better prognosis than those who
received only amphotericin B (Perfect et al.
2010). Development of an apparent paradoxical
33.4 Cryptococcus neoformans immune response (immune reconstitution inflam-
matory syndrome) while on treatment was asso-
The two varieties of C. neoformans are var. neo- ciated with cerebral edema and worsening of
formans (serotypes A and D) and var. gattii (sero- neurological symptoms. Many of these patients
types B and C). The most prevalent agent causing required treatment with steroids and it leads to
chronic fungal meningitis is C. neoformans var. increased morbidity and prolonged hospitaliza-
neoformans, and 90% of infections happen in tion (Singh et al. 2005). Acetazolamide and man-
immunocompromised patients (Speed and Dunt nitol therapy for increased intracranial pressure
1995). Cryptococcus incidence is higher among was also associated with poor outcomes (Newton
AIDS survivors in Africa and Southeast Asia than et al. 2002).
in the population of the west (Levitz 1991). Both Cryptococcoma or brain mass lesions because
cryptococcal species neoformans- and gattii- of C. neoformans are less frequently seen in com-
related meningitis outcomes remained the same. parison to meningitis caused by serotypes A and
But a non-outbreak gattii-related CNS infection D. In contrast, serotype B, commonly seen in
seemed to produce worse outcome when com- non-immunocompromised hosts, frequently
pared to outbreak-related gattii infections causes a pseudotumor mass in the brain. Rarely
(Lockhart et al. 2012). does such a mass lesion occur without meningitis
Important markers of poor prognosis in HIV- (Kovoor et al. 2002).
associated cryptococcal meningitis include
altered mental status at presentation and high
organism load, (as determined by cerebrospinal 33.5 Dimorphic Fungi
fluid (CSF) culture or CSF antigen titer) (Brouwer
et al. 2004). Low CSF white cell count, high CSF Coccidioides immitis and Histoplasma capsula-
lactate, and raised CSF opening pressure are tum are the commonest organisms of this class
associated with a poor outcome. Mortality in causing infections of the CNS. The most frequent
non-HIV-associated cryptococcal meningitis is cause of meningitis among both organisms is C.
associated with factors like chronic renal failure, immitis which is geographically limited to
liver failure, hematological malignancy, absence Southwest United States and South America
of headache, and altered mental status (Pappas countries (Dupont 2003; Singh and Husain
et al. 2001). 10-week mortality rates due to cryp- 2001). Coccidioides meningitis occurs in 30–50%
tococcal meningitis remain high at around 10%. of patients with disseminated infection. HIV-
Few have reported higher rates, up to 26% in positive individuals, solid organ transplant recipi-
selected groups. Studies from Africa and Asia ents treated with steroids, and pregnant patients
have shown between 20% and 40% where are at a high risk of dissemination and developing
amphotericin B therapy has been available CNS infections. Prognosis depends on the host
(Kambugu et al. 2003; Imwidthaya and response. The disease may cause unique symp-
Poungvarin 2000). Cryptococcal meningitis has toms among HIV-positive patients. Patients who
418 K. Munta and J. D. R. Chaudhuri
developed hydrocephalus, infarcts due to vasculi- penetrating traumatic brain injury leading to
tis generally had poorer outcomes. Despite CNS infections. Outcome may not be influenced
advances in antifungal therapy, the morbidity and by host immune status in scedosporium infec-
mortality associated with Coccidioides meningi- tions. Prognosis depends upon early diagnosis,
tis remain high, with a current mortality rate at early surgical resection of the abscess, and early
around 30%. Due to high risk of relapse if ther- use of voriconazole (Gopinath et al. 2010).
apy is stopped, treatment should be given lifelong Concomitant usage of terbinafine with voricon-
in these individuals (Dewsnup et al. 1996). azole has shown to improve outcomes in CNS
Histoplasmosis caused by H. capsulatum is infections caused by Scedosporium prolificans
endemic in the United States, South America, (Cooley et al. 2007).
Southeast Asia, and Africa. This fungus can cause
meningitis in 5–25% of AIDS patients which is
similar to its incidence in non-AIDS patients 33.6.1 Melanized Fungi
(Wheat et al. 1990). Occasionally, brain abscesses
are caused due to Histoplasma (Venger et al. Exophiala dermatitidis, Ramichloridium mack-
1987). Approximately 20–40% of these patients enziei, and Cladosporium bantiana mainly cause
who fail initial therapy may have relapse (Wheat the primary cerebral infections. E. dermatitidis
et al. 2005). has been described as the major neurotropic fungi
of East Asia though it is isolated worldwide in
environment. An uncommon presentation of
33.6 Dematiaceous Fungi brain involvement is formation of abscess with-
out meningitis. Otherwise, meningitis can be the
These pigmented soil-based fungi are known to only manifestation (Middleton et al. 1976).
cause CNS infections in both immunocompro-
mised and immunocompetent individual.
Hematogenous spread of this fungus is known to 33.7 Zygomycetes
cause brain abscesses. Mortality is not dependent
on host immune status. Outbreak of Exserohilum CNS zygomycosis is a worldwide fungal infection
rostratum infection was reported after contami- caused by class Zygomycetes. Zygomycetes class
nation of methyl prednisolone containers in 2013 includes genera Rhizopus, Rhizomucor, Absidia,
which had reported 9% mortality (White and Mucor, Cunninghamella, Apophysomyces, and
Barnes 2014). Various types of CNS pathologies Saksenaea. Zygomycetes thrive in a highly acid
including meningitis, abscess, stroke, epidural condition that has rich carbohydrate. Therefore a
abscess, and cauda equina syndrome have been diabetic ketoacidosis person has a more risk due to
documented (Larone and Walsh 2013). Patients defective phagocyte function and offers an envi-
who had cauda equina syndrome or posterior cir- ronment for quick invasion (Chakrabarti et al.
culation stroke secondary to infection had worst 2006). Zygomycetes proliferate in neutropenic
outcomes. Low CSF sugars are seen universally patients whose serum iron concentration is
in these infections. Complete resection of mass increased by deferoxamine (Pagano et al. 1997).
lesion surgically was associated with better out-
comes than partial resection (Katragkou et al.
2014). 33.8 Conclusion
Scedosporium apiospermum affects individ-
uals in drowning and near-drowning situations Fungal neuroinfections are known to be associ-
(Munta et al. 2015). It can cause brain abscess, ated with high morbidity and mortality. More
meningitis, vasculitis, and stroke secondary to than other pathogens involving the CNS, fungal
infection. Spinal cord involvement in these fun- ones require timely diagnosis and early appropri-
gal infections is uncommon. It also may follow ate treatment in improving the outcomes.
33 Prognosis of Fungal Infections Involving the Central Nervous System and Its Coverings 419
Prognosis will also depend on the host, the under- Hassan A, Poon W, Baker M, Linton C, Mühlschlegel
FA. Confirmed Candida albicans endogenous fungal
lying disease, the virulence of the fungal patho- endophthalmitis in a patient with chronic candidiasis.
gen implicated, the extent of infection, and the Med Mycol Case Rep. 2012;1(1):42–4.
response to treatment. Imwidthaya P, Poungvarin N. Cryptococcosis in
AIDS. Postgrad Med J. 2000;76(892):85–8.
Jamjoom A, al-Abedeen Jamjoom Z, al-Hedaithy S,
Jamali A, Naim-Ur-Rahman MT. Ventriculitis and
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Part VI
Further Insights into Fungal Infections
Fungal Infections of the Spine
Mimicking Tuberculosis 34
Turan C. Dülgeroğlu and Mehmet Turgut
34.2 Epidemiology
Fig. 34.1 Nonseptate hyphae with long branched sporan-
Fungal infections of the spine are considered as giophores bearing terminal round sporangia filled with
uncommon clinical entity, comprising only 1% of spores. Rhizoids absent—Mucor species. Lactophenol
all spinal infections, and they occur more fre- cotton blue mount 40× (courtesy of L. Vemu, M.D.)
quently in the fifth decade of life and in the
immunosuppressed patients with history of and Histoplasma capsulatum have regional pre-
immune disorders, diabetes, surgical interven- dilections, and thus they may affect immunocom-
tion, malignancy, solid organ transplantation, use petent patients with the proper exposure history
of immunosuppressive drugs, parenteral nutri- (Sundaram and Doshi 2016).
tion, prolonged intravenous access, or travel to
any endemic regions of the world (Caldera et al.
2016; Kim et al. 2006; Schmiedel and Zimmerli 34.3 Clinical Findings
2016; Pahlavan and Bhatia 2016; Gouliouris
et al. 2010). In both fungal and tuberculous infections of the
Today, spinal tuberculosis is seen in up to 5% spine, the most frequent initial symptom is limi-
of all cases with tuberculosis (Weinberg and tation of the motion of the spinal column and
Silber 2004) being more frequently in the first localized pain in contrast to pyogenic infections
three decades of life, particularly in the underde- of the spine, though nonspecific symptoms such
veloped countries, as a result of various predis- as night sweating, weight loss, fever, and malaise
posing factors such as immunocompromised may be seen in some patients (Sugrue and Koski
status, poor nutrition, social deprivation, AIDS, 2011; Sundaram and Doshi 2016; Pahlavan and
renal failure, and increased immigration (Clamp Bhatia 2016). As a general rule, a detailed history
and Grevitt 2009; Sugrue and Koski 2011; James and physical examination are very important for
and Davies 2006; Moorthy and Prabhu 2002). In diagnosis (Sugrue and Koski 2011). Furthermore,
particular, M. tuberculosis is the main pathogen existence of predisposing factors such as exis-
responsible for tuberculous spine infections in tence of immunosuppression or travel history
human immunodeficiency virus (HIV)-positive should raise the index of suspicion for various
patients (Duarte and Vaccaro 2013; Sugrue and infections, fungal or tubercular, involving the
Koski 2011). On the other hand, the risk of spinal spine (Sugrue and Koski 2011). Recently, Caldera
infections from fungi such as Aspergillus species, et al. (2016) suggested that presence of weight
Candida species, and Mucor species (Fig. 34.1) loss and dermal sequelae of disease suggests a
is higher in immunocompromised patients, but fungal infection in an older patient. On the other
Coccidioides species, Blastomyces dermatitidis, hand, tuberculous infections involving the spine
34 Fungal Infections of the Spine Mimicking Tuberculosis 425
have a predilection for the thoracic spine in a tensity up to severe destruction, while an exten-
younger age (Sundaram and Doshi 2016). sive loss of vertebral body height with severe
Nonetheless, fungal infections of the spine may kyphotic angulation, called “gibbus deformity,”
mimic tuberculous infections involving the spine, may be observed in very advanced cases of tuber-
thus complicating the differential diagnosis (Hali culosis (Duarte and Vaccaro 2013; Sundaram and
2004). Doshi 2016; Acharya and Gibbs 2016; Rankine
2004). Moreover, “skip lesions” with multiple
level involvement is also a feature of tuberculous
34.4 Imaging Findings infections of the spine (Duarte and Vaccaro 2013;
Sundaram and Doshi 2016; Acharya and Gibbs
In conventional radiographs and computerized 2016; Rankine 2004). Nevertheless, tendency for
tomography (CT) of the spine, vertebral body sparing of the IVD and existence of “skip lesions”
involvement with severe kyphotic angulation with multiple level involvement are also common
(gibbus deformity) is frequently seen in advanced imaging features of fungal infections involving
cases of both tuberculous and fungal spine infec- the spine (Duarte and Vaccaro 2013; Sundaram
tions (Sundaram and Doshi 2016). Specifically, and Doshi 2016; Acharya and Gibbs 2016;
however, fungal infections have a predilection for Rankine 2004). It has been reported that fungal
paraspinal encroachment involving the adjacent infections involving the spine tend to show more
ribs with a typical intervertebral disc sparing subtle changes on MRI compared to those in cases
(Sundaram and Doshi 2016). with tuberculous spondylodiscitis (Duarte and
In the differential diagnosis of fungal and Vaccaro 2013; Sundaram and Doshi 2016;
tuberculous infections of the spine from pyo- Acharya and Gibbs 2016; Rankine 2004). In con-
genic infections caused by bacteria, the sparing trast, fungal infections of the spine also include a
of the intervertebral disc tissue on magnetic reso- “small” paraspinal abscess demonstrating thick
nance imaging (MRI), affection of the posterior and irregular rim enhancement, and they have a
spinal elements, and the formation of paraspinal tendency to involve posterior elements and adja-
masses are important findings (Sundaram and cent ribs (Duarte and Vaccaro 2013; Sundaram
Doshi 2016; Sharma 2010). From two different and Doshi 2016; Acharya and Gibbs 2016;
kinds of infections involving the spine, fungal Rankine 2004) (Table 34.1).
ones produce destructive lesions affecting “mid- Interestingly, it should be noted that certain
dle/center” of the vertebral bodies of the entire imaging findings in patients with fungal infec-
spine, while tuberculous ones involve “anterior” tions of the spine suggest certain fungal causative
of the vertebral body in the thoracic spine agents as follows: (1) swelling of the paraverte-
(Sundaram and Doshi 2016; Sharma 2010). bral soft tissue and affection of the posterior ele-
Radiologically, the thoracic part of the spinal ments of the spine with Coccidioides infections
column is the most frequently affected part in (Fig. 34.2); (2) the vertebral body lytic lesions
cases with Pott’s disease (Duarte and Vaccaro with Cryptococcus and Coccidioides infections
2013; Sundaram and Doshi 2016; Acharya and (Fig. 34.3); (3) vertebral body collapse and gib-
Gibbs 2016; Rankine 2004). In cases with tuber- bus formation with Blastomyces infections
culous infections involving the spine, there is a (Fig. 34.4); and (4) the presence of high T2 signal
heterogeneous enhancement of the corpus of the vertebral body microabscesses or vertebral, para-
vertebra and a “large” paraspinal abscess demon- vertebral macro-abscess with Candida infections
strating rim enhancement (thin and/or smooth) on (Fig. 34.5) (Sugrue and Koski 2011; Sundaram
MRI (Duarte and Vaccaro 2013; Sundaram and and Doshi 2016; Hali 2004; Chen et al. 2013;
Doshi 2016; Acharya and Gibbs 2016; Rankine Chemm et al. 2001; Hadjipavlou et al. 1998;
2004). Also, involvement of intervertebral space Kathuria and Gupta 2001; Lai et al. 2017; Lindner
ranges from IVD sparing with lack of T2 hyperin- et al. 1995).
426 T. C. Dülgeroğlu and M. Turgut
Table 34.1 Differential diagnosis of fungal vs. tuberculous infections involving the spine according to imaging
findingsa
Imaging features
Tuberculous spinal infection Fungal spinal infection
Spine region Thoracic Lumbar
Vertebral body Early stage: anterior part of vertebral body Serrated margins of CEPs without
Late stage: T1 variable intensity with bone severe destruction of vertebral body
healing
Involvement of IVD Variable: from IVD sparing up to severe Typically spared; lack of T2
destruction hyperintensity
Involvement of “Large” paraspinal abscesses and thin/smooth “Small” paraspinal abscesses and
paraspinal/epidural space rim enhancement thick and irregular rim enhancement
Posterior elements May be involved May be involved
Spread to anterior May be more extensive than vertebral Common
subligamentous structures involvement
Involvement of adjacent High bone destruction Uncommon
vertebral levels
Multi-level involvement Common Common
(skip lesions)
IVD intervertebral disc, CEP cartilage end plates
a
Adapted from: Eur Spine J. 2013;22(12):2787–99. https://doi:10.1007/s00586-013-2850-1 (Duarte and Vaccaro 2013)
a b c
Fig. 34.2 Spondylitis caused by coccidioidomycosis. (a) within the IVD and adjacent CEPs with narrowing of the
Sagittal T1-weighted magnetic resonance imaging (MRI) spaces and prevertebral region (white arrows). (c)
shows hyperintensity within intervertebral disc (IVD) and Postcontrast T1-weighted sagittal MRI demonstrates mild
adjacent cartilage end plates (CEPs) with bone marrow enhancement of the diseased vertebrae and prevertebral
edema in the vertebral bodies of L3 and L4, with involve- region near to IVD (white arrow) (from Kathuria and
ment of anterior and posterior ligamentous structures. (b) Gupta (2001), with permission)
Sagittal T2-weighted MRI demonstrates hyperintensity
for differential diagnosis between fungal infec- There is no doubt that microscopic identifica-
tions and tuberculous lesions (Sugrue and Koski tion of the organism by histopathologic examina-
2011). tion of the surgical specimen is essential for
correct diagnosis and definite treatment of fungal
infections involving the spine (Sugrue and Koski
2011; Gupta et al. 2003; Prapruttam et al. 2014).
Accordingly, a high index of suspicion is of
utmost importance in differential diagnosis of
fungal infections from tuberculous ones and
timely starting of appropriate medical therapy for
fungi to reduce the rates of mortality and morbid-
ity (Sugrue and Koski 2011; Gupta et al. 2003;
Prapruttam et al. 2014). Unfortunately, in cases
with fungal infections of spine misdiagnosed as
tuberculous spine infection, the delay in diagnosis
of fungal etiology results with a high mortality
rate (Gupta et al. 2003, 2012; Houda et al. 2011;
Jain et al. 1999).
In a previous report, Jain et al. (1999) reported
a case of cryptococcosis involving T6 vertebra
mimicking tuberculosis in an old diabetic patient
with diagnosis of Cryptococcus neoformans
Fig. 34.3 Preoperative sagittal MRI demonstrates pres-
ence of bone destruction and vertebral abnormalities in L1 based on a CT-guided fine-needle aspiration
(upper red arrow), S1, and S2 (lower red arrow) levels biopsy (FNAB). Gupta et al. (2003) reported a
(From Lai et al. (2017), with permission) young patient presented with cervical
a b
Fig. 34.4 (a) Sagittal T1-weighted MRI shows replace- enhancement of the L2 vertebra spreading to right and left
ment of the normal fatty signal of L2 vertebra. (b and c) psoas muscles. The IVDs are spared with involvement of
Following gadolinium administration with fat saturation the L1 vertebra (black arrow) (from Hadjipavlou et al.
sagittal and coronal MRIs sections demonstrate abnormal (1998), with permission)
428 T. C. Dülgeroğlu and M. Turgut
a b c
Fig. 34.5 MRI of the lumbar spine reveals presence of in affected vertebrae and paravertebral regions following
erosion of the CEP and diffuse bone marrow infiltration at gadolinium administration (from Chen et al. (2013), with
L3 and L4 levels, which is characterized by enhancement permission)
niques have increasingly been used even in more tiating fungal infections from tuberculous ones to
severe cases (Caldera et al. 2016; Pahlavan and reduce the rates of mortality and morbidity. To
Bhatia 2016). The necessity and safety of using make a correct diagnosis, it is necessary to do a
instrumentation in the surgical treatment of fun- detailed investigation by microscopy, culture of
gal infections involving the spine still remains the specimen, and antigen detection for fungal
inconclusive (Caldera et al. 2016). agents.
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Bogdahn U, Toyka KV. MRI findings of spinal intra- review of clinical and imaging findings. Appl Radiol.
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Fungal Infections in Cancer
Patients 35
Yaşar Barış Turgut, Gökhan Sargın,
and Özgür Tanrıverdi
a b
Fig. 35.1 (a) Axial T1-weighted magnetic resonance occipital leptomeninges in a case with cryptococcal men-
imaging (MRI) after administration of gadolinium show- ingitis. (c) Axial T1-weighted MRI after administration of
ing typical lesions of aspergillosis localized in the junc- gadolinium showing mucormycosis with intracranial
tion of gray–white junction (arrowheads). (b) Axial extension involving the inferior frontal lobe (from Starkey
T1-weighted MRI after administration of gadolinium et al. (2014), with permission)
showing enhancement of ventricular wall, frontal, and
35 Fungal Infections in Cancer Patients 433
a b c
d e f
g h i
Fig. 35.2 (a–c) Axial T1-weighted MRI following con- restricted diffusion in the cavity of the lesion (c).
trast demonstrating a heterogeneous lesion in the right T2-weighted MRI demonstrating a heterogeneous lesion
parieto-occipital region of the brain in a 7-year-old girl in the right parieto-occipital region with surrounding
patient with acute lymphoblastic leukemia complicated edema (d, f). Follow-up post-contrast MRIs demonstrat-
with cerebral aspergillosis (a). The fungal lesion is ing a lesion as an isointense mass (g) and an abscess for-
hypointense on T2-weighted MRI (b). Diffusion-weighted mation having peripheral contrast enhancement (arrows)
MRIs demonstrating a hyperintensity lesion with (h and i) (from Haßler et al. (2015), with permission)
spp., and Zygomycetes from different hospitals CNS in immunocompromised patients with can-
over the world during the last decade (Fig. 35.2) cer has a different distribution in some parts of the
(Chitasombat et al. 2012; Douglas et al. 2016; world owing to various geographical and meteo-
Haßler et al. 2015; Malani and Kauffman 2007; rological factors affecting the spread of fungal
Pfaller et al. 2006; Peghin et al. 2016). On the spores, in addition to the lack of appropriate
other hand, incidence of fungal infections of the health conditions in the hospitals (Chamilos et al.
434 Y. B. Turgut et al.
2006). Today, an increasing incidence of invasive count called “neutropenia” as a toxicity of che-
fungal infections of the CNS in immunocompro- motherapy, which is limiting the dose, may result
mised cancer patients has also been observed, in a high risk of infection (Infectious Diseases
possibly due to an increased use of antifungal Working Party (AGIHO) of the German Society
prophylaxis and new chemotherapeutic agents, of Hematology and Oncology (DGHO) et al.
an increased use of new chemotherapeutic and 2009). Recently, Stanzani et al. (2016) suggested
immunosuppressive agents, an increase in the various patterns of invasive fungal disease with
numbers of rare fungal pathogens, and prolonged different radiographic findings in patients with
survival of immunocompromised cancer patients neutropenia (Fig. 35.3). Even today, end results
(Krishnan 2016). of “neutropenia” and its effects still remain
unknown (Crawford et al. 2004). Considering
the presenting signs and symptoms, neutropenic
35.3 Cancer Types patients are usually categorized as those at “high
risk” or “low risk” for infection, underlying can-
In general, it is well-known that the frequency of cer, form of therapy, and associated comorbidi-
fungal infections among cancer patients is high. ties (Freifeld et al. 2011).
In particular, stem cell and solid organ trans-
planted patients or those who have a hemato-
logic malignancy such as lymphoma, myeloma, 35.6 Types of Fungal Infections
or leukemia are at high risk for development of
fungal infections than individuals with other Invasive fungal infections are common causes of
types of cancer (Fig. 35.2) (Haßler et al. 2015; high rates of mortality and morbidity in immuno-
Bodey et al. 1992; Pagano et al. 2011). In a pre- compromised patients with cancer, especially in
vious study, fungal infections were diagnosed patients with leukemia, receiving various chemo-
in approximately 25% of leukemic patients therapeutical agents (Bodey et al. 1992; Pagano
and transplant recipients for each, respectively et al. 2011; Infectious Diseases Working Party
(Bodey et al. 1992). (AGIHO) of the German Society of Hematology
and Oncology (DGHO) et al. 2009; CDC et al.
2001; Center for International Blood and Marrow
35.4 Chemotherapy Protocols Transplant Research (CIBMTR) et al. 2009).
From a clinical perspective, there are different
Risk of infection may be different according to forms of fungal infections, ranging from mild to
the strength of chemotherapy protocol of the severe forms, and they mimic many bacterial or
patient. Some types of cancer may need stronger viral infections. Despite appropriate therapy, rare
chemotherapy protocol, known as “aggressive but important fungal pathogens causing invasive
chemotherapy,” than others, especially the blood fungal infections of the CNS in immunocom-
cancers. In particular, such chemotherapy proto- promised cancer patients are as follows: yeast-
col weakens the immune system seriously and like fungi (Trichosporon spp., Rhodotorula spp.,
may increase the risk for development of a fungal Malassezia furfur, Geotrichum capitatum), mold
infection (Ribaud 1997). filamentous fungi (Aspergillus spp., Scedosporium
spp., Fusarium spp., Zygomycetes), and phaeo-
hyphomycetes (cryptococcus) (Figs. 35.2, 35.4,
35.5 Severity of Neutropenia 35.5 and 35.6) (Haßler et al. 2015; Kappagoda
et al. 2017). Among these fungal pathogens, in
Recent studies revealed that “neutropenia” has particular, some fungi including Aspergillus spp.,
a serious problem associated with a high rate of Candida spp., and molds (e.g., Zygomycetes and
morbidity, mortality, and cost (Crawford et al. Fusarium spp.) are the most important fungal
2004). As a rule, a reduced low white blood cell pathogens infecting susceptible individuals who
35 Fungal Infections in Cancer Patients 435
Fig. 35.3 Various forms of invasive fungal disease and their typical radiographic features in severe neutropenic
patients (from Stanzani et al. (2016), with permission)
Chamilos G, Luna M, Lewis RE, Bodey GP, Chemaly Malani AN, Kauffman CA. Changing epidemiology of
R, Tarrand JJ, Safdar A, Raad II, Kontoyiannis rare mold infections: implications for therapy. Drugs.
DP. Invasive fungal infections in patients with hema- 2007;67(13):1803–12.
tologic malignancies in a tertiary care cancer center: Mcneil MM. Trends in mortality due to invasive aspergil-
an autopsy study over a 15-year period (1989–2003). losis in the United States 1980-1997. Clin Infect Dis.
Haematologica. 2006;91:986–9. 2001;33:641–7.
Chen K, Wang Q, Pleasants RA, Ge L, Liu W, Peng K, Pagano L, Akova M, Dimopoulos G, Herbrecht R,
Zhai S. Empiric treatment against invasive fungal Drgona L, Blijlevens N. Risk assessment and prog-
diseases in febrile neutropenic patients: a systematic nostic factors for mould-related diseases in immu-
review and network meta-analysis. BMC Infect Dis. nocompromised patients. J Antimicrob Chemother.
2017;17:159. 2011;66(Suppl 1):i5–14.
Chitasombat MN, Kofteridis DP, Jiang Y, Tarrand J, Peghin M, Monforte V, Martin-Gomez MT. Epidemiology
Lewis RE, Kontoyiannis DP. Rare opportunistic of invasive respiratory disease caused by emerging
(non- Candida, non-Cryptococcus) yeast blood- non-Aspergillus molds in lung transplant recipients.
stream infections in patients with cancer. J Infect. Transpl Infect Dis. 2016;18:70–8.
2012;64:68–75. Pfaller MA, Pappas PG, Wingard JR. Invasive fungal
Crawford J, Dale DC, Lyman GH. Chemotherapy-induced pathogens: current epidemiological trends. Clin Infect
neutropenia: risks, consequences, and new directions Dis. 2006;43:S3–S14.
for its management. Cancer. 2004;100:228–37. Pu S, Niu S, Zhang C, Xu X, Qin M, Huang S, Zhang
Douglas AP, Chen SC, Slavin MA. Emerging infections L. Epidemiology, antifungal susceptibilities, and risk
caused by non-Aspergillus filamentous fungi. Clin factors for invasive candidiasis from 2011 to 2013 in
Microbiol Infect. 2016;22:670–80. a teaching hospital in southwest China. J Microbiol
Freifeld AG, Bow EJ, Sepkowitz KA, Boeckh MJ, Ito JI, Immunol Infect. 2017;50:97–103.
Mullen CA, Raad II, Rolston KV, Young JA, Wingard Ribaud P. Fungal infections and the cancer patient. Eur J
JR, Infectious Diseases Society of America. Clinical Cancer. 1997;33(suppl 4):S50–4.
practice guideline for the use of antimicrobial agents Salmeron G, Porcher R, Bergeron A, Robin M, Peffault de
in neutropenic patients with cancer: 2010 update by Latour R, Ferry C, Rocha V, Petropoulou A, Xhaard A,
the Infectious Diseases Society of America. Clin Lacroix C, Sulahian A, Socié G, Ribaud P. Persistent
Infect Dis. 2011;52:e56–93. poor long-term prognosis of allogeneic hematopoi-
Haßler A, Porto L, Lehrnbecher T. Cerebral fungal infec- etic stem cell transplant recipients surviving invasive
tion in pediatric cancer patients. Curr Fungal Infect aspergillosis. Haematologica. 2012;97:1357–63.
Rep. 2015;9:6–14. Schwartz IS, Kenyon C, Feng P, Govender NP, Dukik
Infectious Diseases Working Party (AGIHO) of the K, Sigler L, Jiang Y, Stielow JB, Muñoz JF, Cuomo
German Society of Hematology and Oncology CA, Botha A, Stchigel AM, de Hoog GS. 50 years of
(DGHO), Böhme A, Ruhnke M, Buchheidt D, Emmonsia disease in humans: the dramatic emergence
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H, Heinz W, Junghanss C, Karthaus M, Krüger W, 2015;11:e1005198.
Krug U, Kubin T, Penack O, Reichert D, Reuter Sekhon AS, Jackson FL, Jacobs HJ. Blastomycosis: report
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Kappagoda S, Adams JY, Luo R, Banaei N, Concepcion 2016;10:78–86.
W, Ho DY. Fatal Emmonsia sp. infection and fungemia Starkey J, Moritani T, Kirby P. MRI of CNS fungal
after orthotopic liver transplantation. Emerg Infect infections: review of aspergillosis to histoplasmo-
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patients- a brief overview. Med Mycol. 2016;2:16. Wang H, Liu N, Yin M, Han H, Yue J, Zhang F, Shan T,
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Invasive Fungal Infections
in Patients with Hematologic 36
Malignancies
Yaşar Barış Turgut, Gökhan Sargın,
and Gökhan Pektaş
ous factors affecting the spread of fungal spores rare molds or yeasts may be observed as a cause
have an important role in the incidence of IFIs in of infection in these patients (Pagano et al. 2010a,
patients (Chamilos et al. 2006). During the last b; Jahagirdar and Morrison 2002; Ruhnke and
half-century, the incidence of IFIs in patients with Maschmeyer 2002; Segal et al. 2002). From myco-
hematologic malignancies was underestimated logical view of point, various fungal pathogens
due to non-specific clinical manifestations, diffi- such as Fusarium, Scedosporium, Zygomycetes,
culty in diagnosis from biological samples, and it Trichosporon, Malassezia, Penicillium, and
has increased from 3% to 30% (Hsiao et al. 2006; Paecilomyces are well-known causative agents in
Martino and Girmenia 1999). these patients (Jahagirdar and Morrison 2002).
Unfortunately, the incidence of invasive Candida albicans among yeasts is an impor-
mycoses has increased in the early period of the tant pathogen, while other Candida species such
hematologic diseases, due to various difficulties as C. glabrata, C. guilliermondii, C. parapsilo-
in the clinical and therapeutic approach (Martino sis, C. tropicalis, and C. krusei have also been
and Girmenia 1999). Thus, even today, IFIs are described (Pagano et al. 2006; Montagna et al.
frequent causes of morbidity and mortality in 2012; Caggiano et al. 2008; Girmenia et al. 2006;
these patients. Marr 2004; Neofytos et al. 2009). On the other
hand, Aspergillus spp. among molds is a serious
mortal complication in cases with hematologi-
36.3 Pathogenesis cal malignancies, while other molds, including
Zygomycetes and Fusarium spp. have also been
Majority of IFIs are caused by Aspergillus previously reported (Montagna et al. 2012).
(Fig. 36.1) and Candida species but unusual fun- Moreover, infections of other filamentous fungi
gal pathogens such as Zygomycetes, Trichosporon, are also rarely seen (Pagano et al. 2006). In clini-
Fusarium, and Scedosporium species, and other cal practice, the IFIs in patients with hematologi-
cal malignancies are caused by yeasts than by
filamentous fungi (Montagna et al. 2012).
Risk factors for fungal infections involving the
central nervous system (CNS) are neutropenia for
patients with underlying acute myeloid leukemia
(AML), use of steroid or cytarabine, use of various
medicines), advanced age, genetic predisposition,
and other systemic comorbidities (Lass-Flörl 2009;
Sainz et al. 2007; Viscoli et al. 2005). Importantly,
it has been reported that Cryptococcus neoformans
and Pneumocystis carinii may produce serious
infections in patients with severe T-cell suppres-
sion (Segal et al. 2002) (Fig. 36.2).
Nowadays, the patients are classified into
three groups depending on the risk for IFIs: (a)
high-risk group, (b) intermediate-risk group, and
(c) low-risk group (Rambaldi et al. 2017). In par-
ticular, patients with AML those treated with an
allogeneic HSCT and some conditions like neu-
tropenia and use of steroids at high doses are clas-
Fig. 36.1 Thin frequently septate hyphae with acute
angled branching of Aspergillus species (courtesy of
sified as high risk for developing IFI (Rambaldi
L. Vemu, M.D.) et al. 2017).
36 Invasive Fungal Infections in Patients with Hematologic Malignancies 443
Fig. 36.2 Capsulated, narrow-based budding yeast—Cryptococcus neoformans (courtesy of L. Vemu, M.D.)
36.4 Hematological Malignancies suppressive therapy (Pagano et al. 2010a, b). In
addition to Aspergillus species, other molds such
In addition to patients with aplastic anemia as Zygomycetes and Fusarium are encountered
(Fig. 36.3) (Haßler et al. 2015), and patients with increasingly in clinical practice, possibly due to
allogeneic HSCTs the hematological malignan- the use of aggressive chemotherapy protocols in
cies including AML or non-Hodgkin’s lymphoma recent years (Pagano et al. 2006).
are at highest risk of development of various
fungal infections (Pagano et al. 2006; Montagna
et al. 2012). In particular, among patients with 36.5 Clinical Manifestations
AML and those who receive HSCT, IFIs are fre-
quent with prolonged neutropenia and/or fever, Clinically, many of fungal infections involving
unresponsiveness to broad-spectrum antibacte- the CNS have a characteristic clinical disease
rial therapy in cases with chronic lymphocytic spectrum (Jahagirdar and Morrison 2002). The
leukemia (30%) compared to those with AML clinical findings are usually non-specific and
(1.5%), possibly due to the wide use of mono- symptoms are seen late in the course of the
clonal antibodies therapy and presence of severe infection (Chen et al. 2017). Clinical features
immunosuppression (Pagano et al. 2010a, b). of chronic pulmonary aspergillosis include
Some authors reported that the incidence of IFIs prolonged and recurrent cough, shortness of
due to molds increases following an immuno- breath, and hemoptysis. The most common
444 Y. B. Turgut et al.
a b
Fig. 36.3 (a) Computerized tomography of a 10-year-old T2-weighted magnetic resonance imaging (MRI) demon-
girl with aplastic anemia suffering from mucormycosis strating hypointensity areas in the maxillary and eth-
demonstrating the destruction of the anterior skull base moidal sinus on the right side (straight arrows) (from
near the cribriform plate (curved arrow). (b, c) Axial Haßler et al. (2015), with permission)
(Montagna et al. 2012). In general, the detection 2014). Bronchopulmonary disease, lymphadenitis,
of the (1,3)-β-d-glucan antigen is made using abscesses of skin, and soft tissue are clinical mani-
calorimetric methods (Montagna et al. 2012). festations of tuberculosis. The diagnosis of brucel-
Morphologically, fungi are diagnosed with stan- losis depends on clinical features, serological, and
dard techniques based on their macroscopic and histopathologic findings (Zumla and James 1996).
microscopic features (de Hoog et al. 2009). It is widely accepted that IFI is a frequent com-
a b
Fig. 36.5 (a) Photo micrograph demonstrating rounded strating hyperintensity in the right globus pallidus and left
spherules of Coccidioides immitis (arrowhead) (original putamen. (c) Axial diffusion-weighted MRI demonstrat-
magnification, 400 ×; GMS stain) (courtesy of CDC/ ing a reduced diffusion in same regions of the brain (from
Martin D. Hicklin). (b) Axial T2-weighted MRI demon- Starkey et al. (2014), with permission)
446 Y. B. Turgut et al.
with caspofungin or amphotericin B is adminis- Görük M, Dal MS, Dal T, Karakus A, Tekin R, Özcan N,
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Fungal Infections of Central
Nervous System and Their 37
Relationship to Neuropsychiatric
Disorders
Onur Gökçen, Nermin Gündüz,
and Mehmet Turgut
tions other than AIDS such as diabetes mellitus, 2005; Thienhaus and Khosla 1984; Sa’adah et al.
cancer, hematological malignancy, solid organ 1995; Goeb et al. 2007; Prakash and Sugandhi
transplantation, autoimmune hemolytic ane- 2009; Holikatti and Kar 2012; Chou et al. 2016).
mia, sarcoidosis, and chronic steroid treatment Some of these cases are human immunodefi-
(Kumar et al. 2011; Satishchandra et al. 2007). ciency virus (HIV) positive. In association with
It often presents with chronic or subacute menin- the pathophysiology of brain infection, HIV has
gitis symptoms. Clinical picture including head- been reported in literature for mania secondary
ache and fever is a characteristic in these patients. to AIDS (Holikatti and Kar 2012). From a clini-
In addition, altered sensorium has been noted in cal aspect, HIV-positive patients with second-
the vast majority of these patients (Satishchandra ary mania at first episode have severe mania
et al. 2007). and psychosis symptoms, severe cognitive
The most frequent neuropsychiatric symptoms dysfunction, and serious immunosuppression
of CNM in the literature are manic episodes, and (Nakimuli-Mpungu et al. 2008). However, a rela-
it is followed by delirium (Satishchandra et al. tion was determined between psychiatric symp-
2007; Johannessen and Wilson 1988; Johnson toms and CNM with the cases, and patients have
and Naraqi 1993; Spiegel et al. 2011; Tang et al. responded to CNM treatment (Table 37.1) (Jacob
2005). The retrospective study of 142 patients et al. 2013; Kumar et al. 2011; Johannessen and
who meet the diagnostic criteria of cryptococcal Wilson 1988; Johnson and Naraqi 1993; Spiegel
meningitis and severe immunosuppression (CD4, et al. 2011; Tang et al. 2005; Thienhaus and
b100 cells/mm3) has defined the neuropsychiat- Khosla 1984; Sa’adah et al. 1995; Goeb et al.
ric symptoms of the disease (Ibanez-Valdes et al. 2007; Prakash and Sugandhi 2009; Holikatti and
2005). Delirium (47%) and manic episode (33%), Kar 2012; Chou et al. 2016).
followed by depression and anxiety (19% and Another controversial topic is the time of
16%) have been determined in this series, and no initiating highly active antiretroviral therapy
dementia was determined (Ibanez-Valdes et al. (HAART) in AIDS patients who have devel-
2005). Approximately 25% of women and 20% oped CNM. When an AIDS patient who has
of men were determined to have mania (Ibanez- not received HAART develops CNM, HAART
Valdes et al. 2005). Manic symptoms identified is required for long-term survival. Early admin-
in CNM are elated and decreased need for sleep, istration may promote immune reconstitution
irritable mood, flights of ideas, increased libido, inflammatory syndrome; however, delay in start-
impulsive behavior, grandiose delusions, per- ing therapy has been associated with significant
secution delusions, and various hallucinations mortality in the developing world (Sloan et al.
(auditory and/or visual ones) (Tang et al. 2005; 2009). Nevertheless, other studies suggest that
Ibanez-Valdes et al. 2005). Although lower than the reason of immune reconstitution inflamma-
delirium and mania, a number of cases have been tory syndrome may be organism load rather than
reported with acute psychosis symptoms such as time of starting HAART (Bicanic et al. 2010).
persecution delusions, suspiciousness, and hal- There is a need for more studies on the time of
lucinations, auditory or visual, without elated starting HAART in AIDS patients with CNM.
mood (Jacob et al. 2013; Kumar et al. 2011). In general, infectious factors were not con-
A summary of literature data upon neuropsy- sidered at first because most of the patients did
chiatric manifestations of fungal infections of the not have symptoms such as fever and headache
CNS from several selected publications are given at the beginning (Table 37.1) (Jacob et al. 2013;
in Table 37.1 (Jacob et al. 2013; Kumar et al. Kumar et al. 2011; Johannessen and Wilson
2011; Johannessen and Wilson 1988; Johnson 1988; Johnson and Naraqi 1993; Spiegel et al.
and Naraqi 1993; Spiegel et al. 2011; Tang et al. 2011; Tang et al. 2005; Thienhaus and Khosla
37 Fungal Infections of Central Nervous System and Their Relationship to Neuropsychiatric Disorders 453
Table 37.1 Summary of selected case series from the current literature regarding fungal infections and their relation-
ship to neuropsychiatric disordersa
Medical condition
Author(s), Age, gender, and neurological
year country Psychiatric symptoms symptoms Treatment and follow-up
Thienhaus 63, male, Restless, irritable, No fever and stiff Chlorpromazine and lithium carbonate
and Khosla USA agitation, social neck. Occasional were started first. Amphotericin B was
(1984) disinhibition, urinary incontinence started after cryptococcus meningitis
insomnia over the past diagnosis
months
Johannessen 35, male, Insomnia, grandiose HIV positive Mania symptoms disappeared, then
and Wilson USA delusion, Fever paranoia and disorientation. Patient
(1988) hyperactive, died due to respiratory arrest after
hypersexual 3 months
32, male, Paranoid delusions, HIV positive Positive response to haloperidol
USA auditory and visual treatment
hallucinations,
combative, trouble
concentrating,
insomnia
Johnson and 38, male, Restless, auditory No immune Mental state improved, but patient
Naraqi Papua and visual deficiency. Facial died due to meningitis in a follow-up
(1993) New hallucinations, weakness, of 1 year
Guinea aggressiveness, blindness,
distractibility, convulsions
logorrhea, grandiose
delusion, elated
mood
Sa’adah et al. 22, male, Psychosis symptoms Confusion Recovery with amphotericin B and
(1995) Kuwait 5-fluorocytosine
Tang et al. 25, female, Manic symptoms, HIV negative. Olanzapine, clonazepam, and sodium
(2005) China grandiose delusions Weight loss, fever, valproate treatment was started first
and disrupted chills, rigor, bone Amphotericin B was started after
orientation pain, insomnia, lack CNM diagnosis. Lithium carbonate
of appetite was added to treatment. With the
decrease of manic symptoms, patient
was discharged. After 6 months, all
psychotropic drugs were stopped with
disappearance of all manic symptoms
Goeb et al. 27, male, Confusion, HIV negative. He Amphotericin and 5-fluorocytosine;
(2007) France psychosis, affective was a sarcoid recovered
disorder, but not patient
hallucination or Fever, disrupted
delusion. Social orientation in the
withdrawal follow-up
suggesting
schizophrenia
Prakash and 28, female, Insomnia, irritability, She had no immune Olanzapine treatment was started first.
Sugandhi India confusion, deficiency. Fever Amphotericin B was started after
(2009) psychosis, disrupted CNM. Psychotropic treatment was
reality stopped. She recovered after the
12-week treatment. Psychosis did not
recur in the 1-year follow-up
(continued)
454 O. Gökçen et al.
Table 37.1 (continued)
Medical condition
Author(s), Age, gender, and neurological
year country Psychiatric symptoms symptoms Treatment and follow-up
Kumar et al. 49, male, Psychosis symptoms A case with Olanzapine and clonazepam treatment
(2011) India hemolytic anemia was started. IV amphotericin B was
with positive direct started after CNM. Treatment was
Coombs test. HIV changed to oral fluconazole.
negative. Headache Psychotropic drugs were stopped at
for 2 months, and discharge. There were no psychiatric
no fever signs in the 7-month follow-up
Spiegel et al. 53, male, He had signs of HIV-positive patient Manic symptoms have decreased with
(2011) USA mania, thoughts with 14-day azole treatment and 10 mg
grandiose content, olanzapine, and olanzapine treatment
lack of attention and was stopped, and HAART was started
concentration at discharge
Jacob et al. 30, male, Hallucination and History of severe Firstly, 5 mg olanzapine was started
(2013) Singapore acute psychosis headache, high and after HIV-positive result was
symptoms fever during determined during monitoring, CNM
monitoring was diagnosed
IV amphotericin and oral flucytosine
treatment was initiated, and later
treatment was changed to oral
fluconazole. Antipsychotic treatment
was stopped. Clinical status has
recovered, and psychotic symptoms
have disappeared
Holikatti and 28, male, Manic symptoms, HIV-positive Divalproex, trifluoperazine,
Kar (2012) India cognitive symptoms, patient. Fever, escitalopram, venlafaxine, and
persecution headache, blurred quetiapine treatments were used in the
delusions, nihilistic vision, cognitive follow-up. After being diagnosed with
delusions, hostility dysfunction CNM, amphotericin B treatment was
and insomnia administered, and then cognitive
disruption was decreased, and
suspiciousness was reduced, but
auditory hallucinations persisted.
Quetiapine treatment was continued
Chou et al. 78, male, Manic symptoms Oriented at first, but Manic symptoms decreased after start
(2016) China and grandiose then impaired of 2 weeks treatment with quetiapine
delusions consciousness. and lorazepam. After CNM diagnosis
HIV-positive at fourth week, psychotropic drugs
patient. Fever were stopped, and amphotericin B and
HAART were started. Manic
symptoms have subsided, but he was
infected with pneumocystis carinii at
eighth week and died due to sepsis
associated with pneumonia
CNM Cryptococcus neoformans meningitis, HAART highly active antiretroviral therapy, HIV human immunodeficiency
virus, IV intravenous
a
Adapted from: Gen Hosp Psychiatry. 2005;27:301–3. doi: 10.1016/j.genhosppsych.2005.03.003 (Tang et al. 2005) and
Cases J. 2009;2:9084. doi: 10.1186/1757-1626-2-9084 (Prakash and Sugandhi 2009)
1984; Sa’adah et al. 1995; Goeb et al. 2007; tiation of antifungal treatment in CNM (Spiegel
Prakash and Sugandhi 2009; Holikatti and Kar et al. 2011; Tang et al. 2005). Therefore, a posi-
2012; Chou et al. 2016). It has been reported that tive response to treatment does not exclude the
psychiatric symptoms decreased with the use of presence of underlying infection. Underlying
various psychotropic drugs even before the ini- infection can be recurring and fatal as a result of
37 Fungal Infections of Central Nervous System and Their Relationship to Neuropsychiatric Disorders 455
delayed diagnosis, though psychiatric symptoms sory disorder, and agitation. These symptoms
generally respond to treatment (Spiegel et al. may show fluctuations within the day. While
2011; Tang et al. 2005). Due to lack of neuro- delirium may be the first symptom of CNM, it
logical symptoms and symptoms suggesting may be due to other neuro-AIDS factors like
infectious factors such as fever, most cases in lit- HIV encephalopathy, progressive multifocal
erature have been misdiagnosed due to psychiat- leukoencephalopathy, herpes or cytomegalovi-
ric symptoms at first, and they were treated with rus encephalitis, neurosyphilis, toxoplasmosis,
psychotropic drugs which extend the disease or lymphoma (Ibanez-Valdes et al. 2005). It was
course further. It has been reported that CNM is a indicated in a case in literature that CNM was
fatal disease resulting in death in 83% of patients identified a 70-year-old male with treatment-
without neuropsychiatric symptoms and 76% resistant endogen depression 10 months follow-
of patients with neuropsychiatric symptoms, if ing administration of antidepressant drugs and
appropriate treatment is not started without delay that his depressive symptoms were significantly
(Kumar et al. 2011; Ibanez-Valdes et al. 2005). recovered following successful treatment of
So far, olanzapine was preferred for treat- infection of the CNS (Hsueh and Lin 2010).
ment in most of the cases (Table 37.1). This was In brief, CNM may manifest with mania,
attributed to the limitations in the use of conven- delirium, and acute psychosis without any other
tional mood regulators in HIV-infected patients neurological symptoms or signs favoring infec-
(Spiegel et al. 2011). Furthermore, treatment tion. Caution should be exercised in risk groups,
of patients infected with HIV using dopamine and this should be considered upon signs indicat-
receptor-2 antagonists may cause development ing CNM in the follow-up.
of various extrapyramidal symptoms (EPS) since
HIV causes neuronal damage in basal ganglia
(Aylward et al. 1993). Risperidone, ziprasidone, 37.3 F
ungal Infections in Drug
and olanzapine were used safely in patients with Abusers
AIDS in the previous case reports and series
(Spiegel et al. 2011; Rummel-Kluge et al. 2012). Systemic fungal infections like infections of the
Nevertheless, olanzapine was preferred among CNS and fulminant infective endocarditis may
these antipsychotics since it was recognized that cause a high mortality, although occurrence of
it poses lower risk of EPS (Spiegel et al. 2011). fungal infections in drug users is infrequent in
Likewise, quetiapine was preferred in a case due comparison with bacterial and viral infections
to low risk of EPS (Chou et al. 2016). (Badiee and Hashemizadeh 2014).
Case reports of patients with CNM, who have There are some predisposing factors related
applied with acute psychosis, are very rare (Jacob with encountering microbial pathogens. Some of
et al. 2013; Kumar et al. 2011). If there are risk them are associated with substance use itself such
factors for identifying opportunistic infections as using unsterile needles or syringes, contami-
and early treatment particularly in patients with- nated drug paraphernalia, and drug adulterants
out premorbid history for family history and (Kaushik et al. 2011). In addition, substance use
psychosis, HIV test is important for patients especially cannabis sativa or marijuana, opiates,
presenting with acute psychosis. Another neu- cocaine, morphine, and heroin adversely affects
ropsychiatric manifestation in CNM is delirium. the patient’s immune system and nutrition.
It may appear especially in elderly people and Patient gets prone to infectious diseases due to
young individuals with disrupted metabolic pic- immune deficiencies and malnutrition (Kaushik
ture (Satishchandra et al. 2007; Ibanez-Valdes et al. 2011; Friedman et al. 2006). On the other
et al. 2005). Delirium may appear due to many hand, some predisposing factors are directly asso-
reasons disrupting metabolic picture. Different ciated with preparation of the drug; For example,
from psychosis and mania, place, time, and per- dilution of black tar heroin, which is a form of
son orientation is disrupted, and there is impaired raw and impure opium derivative (Bucardo et al.
consciousness. There may be distractibility, sen- 2005), with farina, lidocaine, and even shoe-pol-
456 O. Gökçen et al.
ish-saturated paper (Kaushik et al. 2011); addi- of cocaine mixed with lemon juice in the related
tion of levamisole, which can cause infectious literature (Melnychuk and Sole 2017; Keyashian
contaminations leading to reversible neutropenia, and Malani 2007). One of them was reported by
to cocaine for enhancing the cocaine’s euphoric Melnychuk and Sole (2017). This 23-year-old
effects and using colored methamphetamines male patient with the history of IV brown heroin
obtained by adding adulterants to increase their use reported to the Department of Emergency with
effects (Lee et al. 2012; Strathdee et al. 2008). the complaints of visual disturbances and left-
Drugs are generally dissolved within mild acids, sided visual loss. Fundoscopic examination of the
like tartaric or citric acid, to increase tissue degra- patient was reported to be suggestive of possible
dation on the injection site. One of the fruit juice Candida endophthalmitis (Melnychuk and Sole
used for dissolving heroin or cocaine before the 2017). Another interesting and rarely reported
injection is preserved lemon juice. Unfortunately, endogenous Candida endophthalmitis case as a
it is known to be a good culture medium for endog- result of the use of buprenorphine was presented
enous endophthalmitis caused by Candida albi- by Aboltins et al. (2005). Authors suggested that
cans (Albini et al. 2007). Lemon juice is often used endophthalmitis could be associated with IV injec-
to dissolve solid brown heroin which is not only tion after sublingual diversion of the drug from the
used after heating and inhaling but also used for oropharyngeal cavity following its dispensation
injection (Melnychuk and Sole 2017). Sometimes (Aboltins et al. 2005).
the drug users put a tablet in their mouth and Aspergillus endophthalmitis and Candida
lick the injection syringe to facilitate the dissolv- endophthalmitis have also been reported among
ing process. It may result as a potential source of IV drug users associated with preparation of the
Candida inoculum (Deutscher and Perlman 2008). drug such as using unsterile injection needles or
Also, severe damage to the nasal epithelium during syringes, dissolving solid drugs in impure tap
the inhalation of the drugs or damages on the skin water and/or or filtering the mix through ciga-
during injection might play a predisposing role for rette filters (Hirst et al. 2005). While evaluating
infection (Kernt and Kampik 2010). the literature, we noticed a rare case presenting
Several clinical manifestations of fungal as bilateral keratitis because of Rhizopus infec-
infections such as endophthalmitis and chorio- tion in a crack cocaine user, reported by Zhou
retinitis; infections of the CNS such as cerebral et al. (2016). This 33-year-old male patient with
microabscesses, meningitis, and cerebral mac- the history of regularly using crack cocaine pre-
roabscesses; vascular complications; and infec- sented to the Department of Emergency with
tive endocarditis may be observed as a result of significant eye pain, redness, and progressively
intravenous (IV) drug use (Kaushik et al. 2011; decreased vision in the right eye. He had been
Melnychuk and Sole 2017; Kernt and Kampik treated with antibiotics with no improvement.
2010; Aboltins et al. 2005; Hirst et al. 2005; After culture results had been found to be posi-
Keyashian and Malani 2007; Zhou et al. 2016). tive for Rhizopus species and starting antifungal
treatment, significant clinical improvement had
been observed (Zhou et al. 2016).
37.3.1 Endophthalmitis and Keratitis In a case series including a total of 14 patients
with crack cocaine-related corneal symptoms such
Endogenous form of endophthalmitis is usu- as corneal ulcer or infectious keratitis, a total of 10
ally related to IV drug use. Hematogenous dis- patients were defined to have both bacterial and
semination of the fungus especially Candida and fungal corneal ulcers (Sachs et al. 1993). One of
Aspergillus species to the eye may result in endog- the possible predisposing causes may be the anes-
enous endophthalmitis (Melnychuk and Sole 2017; thetic properties of cocaine causing the decreased
Kernt and Kampik 2010; Keyashian and Malani corneal sensation and weakened blink reflex
2007). There are some informative endogenous (Mantelli et al. 2015). Also the alkaline character
Candida endophthalmitis cases caused by injection of crack cocaine fumes can increase the risk of
37 Fungal Infections of Central Nervous System and Their Relationship to Neuropsychiatric Disorders 457
minimal chemical burns and can facilitate fungal with or without mycotic aneurysms caused by
infection on the corneal e pithelial defects by pro- Candida endocarditis (Sánchez-Portocarrero
moting rubbing the eyes (Sachs et al. 1993). et al. 2000). Unfortunately, there is no specific
Endogenous endophthalmitis and keratitis diagnostic indication tool for fungal infections
should be kept in mind by the psychiatrists because of the CNS regarding laboratory tests, sero-
of its high morbidity and mortality among IV logic tests, and hematologic parameters. So, it
drug users. Once any visual change among these is important to suspect fungal infections (Badiee
patients is noticed, because of early diagnosis and and Hashemizadeh 2014).
treatment’s importance, ophthalmology consulta- In a review conducted by Kim et al. (1993),
tion should be requested immediately. Second it is it was reported that drug addiction is one of the
important to consider the fungal agents especially predisposing factors for cerebral aspergillosis. The
with a lack of clinical recovery after initial treat- cerebral infections, which are less frequent than
ment with broad-spectrum antibiotics. candidiasis, were also reported with Aspergillus
species among drug users (Hadley et al. 2017;
Morrow et al. 1983). It has been reported that in
37.3.2 Infections Involving immunocompromised patients with aspergillosis
the Central Nervous System of the CNS, CT and MRI may reveal multiple
septic infarcts involving the basal ganglia, inter-
Unfortunately, fungal infections of CNS can nal capsule, and corpus callosum (Hadley et al.
often be underdiagnosed or overlooked among IV 2017). Also one of the less frequent but high mor-
drug users because of the lack of specific clinical tal fungal infection among IV drug users is cere-
symptoms for these populations. These patients bral mucormycosis which is known to particularly
were usually diagnosed after death (Kaushik affect the basal ganglia (Roden et al. 2005).
et al. 2011).
Several clinical presentations of candidia-
sis such as cerebral micro- or macroabscesses, 37.3.3 Infective Endocarditis
meningitis, and vascular complications can be
seen as result of fungal infections of the CNS In a review conducted by Yuan (2014), it has
after systemic candidiasis (Henao and Vagner been suggested that approximately in one-third
2011). There are no “specific clinical manifesta- of the patients with right-sided infective endocar-
tions” of CNS candidiasis especially for cerebral ditis, drug abuse was shown to be predisposing
microabscesses which is defined as non-specific factor (Yuan 2014). IV drug user patients with
diffuse encephalopathy (Kaushik et al. 2011; infective endocarditis are frequently males and
Henao and Vagner 2011; Neves et al. 2014; younger than non-drug user infective endocar-
Sánchez-Portocarrero et al. 2000). Although ditis (Colville et al. 2016). Also the most com-
cerebral microabscesses are frequently diag- mon fungal agent is Candida albicans (Ellis et al.
nosed only after death of the patient because of 2001).
being clinically silent, cerebral macroabscesses It has been suggested that drug paraphernalia
may be diagnosed easily with patient’s clinical and drug adulterants used for preparing the sub-
manifestations like fever, headache, diminished stance can impair the endocardium particularly on
consciousness, and focal neurological signs and the right side and the tricuspid valve (Cole et al.
regarding neuroimaging studies such as com- 2011). One of the predisposing factors for infec-
puted tomography (CT) and magnetic reso- tive endocarditis due to drug particularly cocaine
nance imaging ( MRI) and also with culture of is causing damage of the tissues or skin by vaso-
cerebrospinal fluid (Neves et al. 2014; Sánchez- constriction (Wurcel et al. 2015). Clinicians must
Portocarrero et al. 2000). Vascular complications be aware of infective endocarditis among IV drug
of fungal infections of the CNS can be observed users because of its high morbidity and mortality
as cerebral infarction, subarachnoid hemorrhage (Colville et al. 2016).
458 O. Gökçen et al.
in obese patients, risk of fungal infections was Kivac Altunay 2006). Some of patients may even
determined to be lower in patients using clozapine be unaware of these disorders due to negative
compared to patients using typical antipsychotics symptoms (Wu et al. 2014). Moreover, there are
(Wu et al. 2014). The authors attributed this to the studies showing that schizophrenic patients have
fact that clozapine shows an immunomodulator higher pain threshold, and this may prevent them
effect by inducing several cytokines such as inter- from seeking help until their symptoms become
leukin-6 and tumor necrosis factor-α, and they severe (Jeste et al. 1996). Fungal infections of the
have indicated that this immunomodulator effect CNS may cause secondary acute bacterial celluli-
may alter the immune response of the patient tis and increase morbidity in this patient group, if
against the fungal infection, and thus it may form they are undetected and untreated in early period
a protective effect (Wu et al. 2014). Skin disorders (Bristow and Spruce 2009).
were also determined to be much higher in schizo- Similar to the fact that psychiatric disorders
phrenic patients compared to general population in increase the risk of infectious skin disorders and
this study (Wu et al. 2014). some fungal infections, some fungal infections
Today, there are studies which determined may have an effect on the mental state (Chacon
an increased risk of fungal infections and et al. 2013). Psychological and social param-
other infectious skin disorders in subjects with eters affect function, well-being, and quality of
schizophrenia and other psychiatric disorders. life. Chacon et al. suggested that the prevalence
According to studies, this risk is higher par- of major depression is increased in patients with
ticularly in patients with obesity and diabetes. onychomycosis due to negative psychosocial rea-
This may be due to several different reasons. sons such as shame, low self-esteem and social
To begin with, metabolic syndrome and diabe- withdrawal (Chacon et al. 2013). Accordingly, in
tes are common comorbid disorders in schizo- a study performed in 258 patients with onycho-
phrenia and some psychiatric disorders by the mycosis in 1998, 74% of patients have reported
effect of the nature of psychiatric disorders, life- they are ashamed of their state (Drake et al. 1998).
style of patients, and psychotropic medications Also, it has been stated that the mental health of
used. Considering the predisposition of diabetes onychomycosis patients is under higher risk com-
patients toward infectious diseases, this relation pared to normal population due to reasons such
may be secondary (Mookhoek et al. 2010, 2011). as appearance anxiety and disrupted social func-
Another reason of the increased risk in tionality (Chacon et al. 2013; Lubeck et al. 1993).
schizophrenic patients may be related to immune
response. In addition to decreased levels of
natural killer cytotoxicity/lymphocyte prolifera- 37.5 Conclusion
tion in some psychiatric patients, disruption of
monocytic system in schizophrenic patients may In recent years, an increased incidence of fungal
increase sensitivity toward infectious skin disor- infections involving the CNS is worldwide due to
ders (Cohen et al. 2001; Krause et al. 2012). considerable rise in the number of immunosup-
Another reason may be the disruption of self- pressed patients. In some cases, fungal lesions
care and decreased seeking of medical care due may present with various neuropsychiatric symp-
to symptoms such as cognitive impairment, apa- toms; therefore microscopic identification of the
thy, anergy, and lack of interest in schizophrenia organism is very important for definite diagnosis
and some psychiatric disorders (Moftah et al. of fungal infection in these cases. We stress that
2013; Mohamed et al. 1999). Inability of patients a high index of suspicion is of utmost importance
to perform daily cleaning routines increases the in differentiating fungal infections from certain
risk of fungal infections and other infectious neuropsychiatric disorders to reduce the rates of
skin disorders (Moftah et al. 2013; Mercan and mortality and morbidity.
460 O. Gökçen et al.
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Real-Time PCR: Advanced
Technologies and Applications 38
Güliz Uyar Güleç and Yaşar Barış Turgut
(PCR) techniques could facilitate diagnostic con- to the amplicon, the light emission of a fluores-
firmation (Schwartz et al. 2018). Real-time PCR cent dye coupled to an oligonucleotide changes.
has emerged as a suitable and faster technique for This is the basis for the amplicon-specific detec-
identification and quantification of fungal agents. tion of fluorescence (Kaltenboeck and Wang
In this chapter we aim to discuss general char- 2005). In real-time PCR, several examples of
acteristics and clinical applications of real-time amplicon detection methods are shown in
PCR technique based on clinical trials. Fig. 38.1 (Yadav and Singh 2017).
In the conventional PCR, qualitative informa-
tion may be obtained by detection of a specific
38.2 General Characteristics dsDNA product using gel electrophoresis at the
of Real-Time PCR end of amplification (Kaltenboeck and Wang
2005). In real-time PCR, however, quantitative
The PCR assay has found extensive use in micro- information may be obtained by plotting the
biology laboratories since its introduction in intensity of the fluorescence signal versus cycle
1983. Improvements in technology and chemis- number (Kaltenboeck and Wang 2005).
try developed PCR methods in time (Stephenson Melting curve analysis is performed to under-
2016). Firstly, as quoted by Kaltenboeck and stand whether the obtained fluorescence is
Wang (2005) Higuchi et al. introduced real-time achieved by amplification of the desired target
PCR by to analyze the kinetics of PCR by con- region or is a nonspecific product (Loftis and
structing a system for detection of PCR products Reeves 2012). After the amplification is com-
in the amplification process. The most prominent plete, samples are cooled and then slowly heated,
feature of real-time PCR assays is that products while fluorescence is monitored to determine the
of a reaction are measured in “real time,” as the temperature at which the dsDNA-binding dye is
PCR reaction is being performed, rather than fol- released (Loftis and Reeves 2012). In Fig. 38.2,
lowing the completion of the reaction (Loftis and an example of melting curve analysis is shown
Reeves 2012). (Buil et al. 2017).
In real-time PCR, two general chemistries are As filamentous fungi have complex cell wall
available for amplicon detection. These are DNA- that is difficult to disrupt, rigorous extraction
binding dyes and fluorescent probes (Arya et al. methods are required. It has been reported that
2005). The assays using DNA-binding dyes are the most important factors for the sensitivity of
simple and cost-effective. Ethidium bromide was any PCR assay are complete lysis of fungal cells
used in the earliest form of real-time PCR. SYBR from various biological samples and purification
Green 1, less toxic and sensitive dye, binds to only of DNA without inhibitors (Espy et al. 2006;
double-stranded DNA (dsDNA) (Loftis and Francesconi et al. 2008). There are manual and
Reeves 2012; Arya et al. 2005). The progress of automated or semiautomated extraction methods.
PCR amplification may be followed in real time by The use of a standardized, efficient, and rapid
measuring the fluorescence of sample during each DNA extraction method is a main component for
stage of amplification reaction cycle (Kaltenboeck both the optimization and reproducibility of
and Wang 2005; Loftis and Reeves 2012). quantitative PCR assays (Espy et al. 2006;
The other one uses fluorescently labeled oli- Francesconi et al. 2008).
gonucleotide probe that anneals to one of the The PCR primer must be highly efficient and
template strands. Specific hybridization between specific for the target primer sequences in the
probe and template generates signal. Among the specimen of interest (Espy et al. 2006). For
probe formats, the most common are fluores- molecular identification of fungi, the DNA-
cence resonance energy transfer (FRET), TaqMan coding sequences appear as an excellent target
or 5′ nuclease, scorpions, and molecular beacons for PCR amplification, while RNA is very rarely
(Stephenson 2016; Arya et al. 2005; Espy et al. used (Somogyvari et al. 2012). The vast majority
2006). After hybridization of the oligonucleotide of assays target the ribosomal DNA (rDNA)
38 Real-Time PCR: Advanced Technologies and Applications 465
nucleotides
a R
Free primers
c
Primer
Target specific nucleotides
R Q
Primer PCR product
Fluorescent
reporter dye
R Q Quencher dye Primer Primer
Fig. 38.1 Three representative examples of amplicon method, (c) Molecular beacon method (Reproduced, with
detection methods in real-time polymerase chain reaction permission, from Yadav and Singh: Molecular Markers in
( PCR). (a) TaqMan probe, (b) SYBR Green 1 dye Mycology. Fungal Biology. Springer, Cham, 2017)
Fig. 38.2 A representative example of melting curve analysis (Reproduced, with permission, from Buil et al.: Molecular
Diagnostics. Springer, Singapore, 2017)
genes 18S, 28S, and 5.8S and the intervening used as reference standards for the validation of
internal transcribed spacer (ITS) regions (ITS1 some methods such as repetitive-sequence PCR
and ITS2) on rDNA (Lau et al. 2007). Owing to (Somogyvari et al. 2012). Studies regarding iden-
the presence of multicopy genes, they have uni- tification of fungi showed that the most useful
versal fungal primers and contain highly variable targets are the ITS1 are the ITS2 regions which
regions for identification of species (Lau et al. are followed by the D1 and D2 regions of the 28S
2007). In addition, the ITS sequences may be rDNA (Lau et al. 2007).
466 G. U. Güleç and Y. B. Turgut
Significant application areas of real-time PCR Alborzi 2011). In this study, a total 152 samples
are detecting and quantifying DNA and RNA of from 38 patients were examined; 10 patients had
microorganism, genotyping and following treat- positive real-time PCR result in CSF samples; and
ment efficacy, monitoring the transcription of 4 of them had positive serum results for Aspergillus
genes, and typing of genetic polymorphisms or Candida spp (Badiee and Alborzi 2011).
(Kaltenboeck and Wang 2005; Espy et al. 2006; A study from Sweden evaluated clinical appli-
Bretagne 2003). The fast turnaround time of less cability of real-time PCR targeting 18S rRNA in
than 2 h and minimal false-positive results the detection of Candida spp. and Aspergillus spp.,
because of no postamplification handling are using a total of 1330 blood samples, 295 samples
advantages of real-time PCR for routine mycol- of other body fluids, and 25 samples of biopsy
ogy laboratory (Bretagne 2003). specimen (Klingspor and Jalal 2006). In this study,
Nevertheless, there are some disadvantages of a positive result for Candida spp was obtained in
real-time PCR assays: (1) false-negative results only 4 of 24 CSF samples (17%) (Klingspor and
due to their excess sensitivity to various inhibi- Jalal 2006). However, it has been reported that the
tors in the environment, (2) lack of any informa- technique used identified fungal DNA in various
tion about infectivity of pathogen because the samples with a high sensitivity and specificity in a
assays give only number of total pathogens, and short time (Klingspor and Jalal 2006).
(3) the presence of inconsistent results from vari- Cultural isolation of fungus is time-consuming
able assay designs for same samples (Johnson and not always possible. PCR-based diagnostic
et al. 2012). approaches may be considered for formalin-fixed,
paraffin-embedded material if there is no alterna-
tive sample material available. Nowadays, many
38.3 Clinical Applications of Real- studies have underlined the advantages of PCR
Time PCR technology for identification of fungal pathogens,
viable or nonviable, in different biopsy samples.
Fungi are important as emerging pathogens. The However, sample age and environmental contami-
diagnostic approach for invasive fungal infec- nants are important factors to be considered in
tions is to detect the strain as early as possible these studies (Frickmann et al. 2015).
(Liu et al. 2012). To date, many real-time PCR In a retrospective study, a total of 151 biopsy
protocols have been suggested for Aspergillus specimens including lung, skin, liver, and brain
and Candida species, but there are also protocols samples were examined; fresh (n = 92) and
for other less frequent species (Klingspor and paraffin-embedded (n = 52) tissues were ana-
Jalal 2006; Schabereiter-Gurtner et al. 2007). It lyzed, and a total of 28 different fungal species
may be difficult to obtain CNS samples due to the were detected: Aspergillus spp. (47%), endemic
poor conditions of the patients. Samples are valu- mycoses (21%), Mucormycetes (10%), Candida
able and may be small in amount. spp. (8%), and other rare species. (14%) (Buitrago
Morton et al. (Morton et al. 2011) used real- et al. 2014). In another study, the ability of a
time PCR in blood, brain, cerebrospinal fluid quantitative real-time PCR assay targeting the
(CSF), and spinal cord samples for detection of A. ITS region of rDNA of Aspergillus, Fusarium,
fumigatus in neutropenic mice; fungal DNA was Scedosporium, and Mucormycetes was retrospec-
detected at highest concentrations in the brain tively evaluated in a total of 102 paraffin-embed-
(96%) and spinal cord (92%). Their extraction ded tissue specimens following formalin fixation
protocol allowed PCR detection of fungal DNA (Salehi et al. 2016). It has been confirmed that
from small sample volumes (10 μl CSF, 200 μl real-time PCR assay is useful for rapid and accu-
blood) (Morton et al. 2011). In another study, CSF rate identification of various fungal pathogens
samples and sera from patients suspected of fun- (Salehi et al. 2016).
gal meningitis were evaluated for Aspergillus and A real-time PCR that could simultaneously
Candida DNA by real-time PCR (Badiee and detect bacteria and fungi in same CSF by one
38 Real-Time PCR: Advanced Technologies and Applications 467
PCR reaction was designed. Among 137 CSF study using serum and tissue samples from
specimens, 20 bacterial strains and 7 fungal patients with invasive mucormycosis because
strains were detected with higher sensitivity than biopsy procedure in a patient may cause various
conventional methods. The authors concluded complications. This PCR-based method has a
that this TaqMan probe-based real-time PCR can high sensitivity (91%) in paraffin-embedded tis-
be applied to other biological fluids as well as sue samples, and more importantly Mucorales
CSF (Han et al. 2014). DNA was detected in the sera of all probable/
When the clinical picture does not point out a proven patients (100%), with an earlier diagnosis
specific agent, the utility of species-specific than tissue samples (Springer et al. 2016).
approaches remains limited. Therefore, fungal Cryptococcal disease has become a major
PCR assays with a broad range as an alternative infection, especially in HIV-infected individuals.
method have been developed to overcome this Conventional nested and real-time PCR were
problem. Landlinger et al. (Landlinger et al. compared in murine model of cryptococcal men-
2010) introduced a real-time PCR with panfungal ingitis. Real-time PCR was found more sensitive
range for detection of more than 80 pathogenic and rapid method (Bialek et al. 2002). For
fungi. In this study, more than 600 peripheral endemic mycosis the role of real-time PCR is
blood specimens, 11 CSF, and 2 lung biopsies unclear and limited in clinical experience or
were investigated (Landlinger et al. 2010). They remains investigational (McCarthy et al. 2017).
found the sensitivity of the assay as 96% (95%
CI, 82–99%) and the specificity as 77% (95% CI,
66–85%), while the negative and positive predic- 38.4 Conclusion
tive values were 98% (95% CI, 90–100%) and
62% (95% CI, 47–75%), respectively (Landlinger Real-time PCR is highly sensitive and rapid tech-
et al. 2010). A real-time PCR assay with panfun- nique for fungal identification. Quantification of
gal range using the combination of a DNA- fungal burden is one of the important steps for
binding dye and specific molecular beacon management of fungal infections of CNS. With
probes following by a melting curve analysis was developments in molecular technologies, real-
designed by Valero et al. (2016). In this study, a time PCR methodology is improving. Nucleic
total of 44 fresh or paraffin-embedded biopsy acid extraction method, primers, targets, and
specimens, 8 respiratory samples, 5 CSF, 1 aque- PCR detection chemistries must carefully be cho-
ous humor, and 1 nail sample were tested, with a sen for reliable results. Usefulness of real-time
sensitivity of 83.3% (Valero et al. 2016). PCR in clinical laboratory should strengthen with
Specifically, a fast identification is of great prospective clinical trials.
importance for effective treatment for mucorales
(Spellberg et al. 2009). Therefore, a real-time
PCR assay using the FRET probes was devel-
oped for detection of mucorales, and the sensitiv-
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Next-Generation Sequencing:
Current Technologies 39
and Applications
39.3 Clinical Mycology analysis and SNPs are commonly used, ribo-
somal ITS and whole genome sequencing are the
In addition to data obtained from population-scale most sensitive in distinguishing various fungal
projects such as HNP and MetaHIT, Bittinger pathogens (Araujo 2014; Dannemiller et al.
et al. has demonstrated that the relative proportion 2014). Decker et al. showed that a NGS-based
of bacteria and mycobiota in the lung can be used diagnostic approach was the best in patients with
to differentiate infection from colonization septic shock. They also demonstrated that inva-
(Bittinger et al. 2014). However, fungal species siveness mycoses could be distinguished from
constitute only 0.1–1% of the human microbiome colonization, thus indicating the need for antifun-
and even less so in the CNS. This would imply that gal therapy (Decker et al. 2017). Alonso et al.
a level of 1012–1014 nucleotides per sequencing run suggested that amyotrophic lateral sclerosis
per sample is required to even detect subspecies of (ALS) may be a fungal disease and demonstrated
the fungi. This means that current available a variety of fungal species in each case of ALS
sequencers such as Illumina Miseq, Ion Torrent with the help of NGS. They even suggest that
PGM, and HiSeq are far from the desired sensitiv- severity and evolution of the disease may vary
ity required to diagnose fungal infections. from patient to patient in accordance with the
Using mycobiomes obtained by sequencing fungal species (Alonso et al. 2017).
only amplicons of internal transcribed spacer Azole resistance has been demonstrated in
(ITS) of the fungal ribosomal genes, using fungal infections including itraconazole, voricon-
platforms like Illumina NextSeq, it may be possi- azole, posaconazole, and isavuconazole, espe-
ble to extract information in clinical samples. This cially after long antifungal azole treatment (Bueid
would provide a relative composition of the micro- et al. 2010; Lockhart et al. 2011; Howard et al.
biological flora and thus provide an opportunity 2009; Snelders et al. 2008; Verweij et al. 2009a).
for application to clinical mycology. Especially in Mechanisms include efflux pumps, which reduce
the CNS, the relative proportion of fungi would intracellular drug concentration, increased azole
also give an indication of the severity of the fungal target enzyme production, and adaptation of tar-
infection. Salzberg et al. in a prospective pilot get site of demethylases active in sterol synthetic
study applied NGS in combination with computa- pathways. Forward and reverse genetic approaches
tional analysis to detect the presence of pathogenic have demonstrated genes involved in Cryptococcus
microbes in brain and spinal cord biopsies from neoformans life cycle. Ianiri and Idnurm evalu-
ten patients with findings suggestive of infection ated 35 genes required for viability in ascomyce-
but clinical and microbiological studies yielding tes and drug resistance, demonstrating genes
inconclusive results (Salzberg et al. 2016). They involved in ergosterol biosynthetic pathway
were able to detect infectious processes in eight of (Ianiri and Idnurm 2015). Similarly, Candida iso-
the ten cases, thus providing evidence that NGS lates resistant to azoles have shown mutations in
can dramatically improve our ability to detect or genes involved in formation of demethylases and
rule out a wide range of CNS pathogens. This is efflux pumps (Garnaud et al. 2015).
especially applicable to cases where conventional Aspergillus fumigatus is an opportunistic fun-
microbiology has been negative or takes too much gus causing a variety of diseases in immuno-
time to be clinically viable (Salzberg et al. 2016). compromised hosts, including invasive
aspergillosis. Azole resistance is widespread in
this species with resistant species being isolated
39.4 Research Mycology form the environment (Snelders et al. 2009;
Verweij et al. 2009b). Insight into the methods of
NGS has been used frequently for research pur- azole resistance in this group has been investi-
poses. Options include single-nucleotide poly- gated with the help of whole genome sequencing
morphisms (SNPs), microsatellite analysis, on isolated strains of Aspergillus from patients
amplicon sequencing of ribosomal ITS, and with long-term azole resistance. Elucidated mech-
whole genome sequencing. While microsatellite anism points to mutations in Cyp51 A protein.
39 Next-Generation Sequencing: Current Technologies and Applications 473
Cyp51 A protein is a demethylase involved in stress and is regulated with epigenetic mecha-
ergosterol synthesis which is a component of the nisms like DNA hydroxylation-methylation.
fungal cellular wall and the main substrate for Cytosine methylation or hydroxylation can be
demethylase inhibitors like azoles (Latgé 1999). studied by the chemical conversion using bisul-
Mutations like amino acid substitution including fate of cytosine into uracil.
G54A, P216L, M220V, Y121F, and duplication Computational analysis along with NGS is
of 34 and 46 bp nucleotides in the promoter powerful tool to study/diagnose fungal infec-
region of CYP51A gene have been found to be tions. Transcriptome analysis studies gene
responsible for azole resistance. While former expression by growing fungal cells after messen-
mutations are just a marker for azole resistance, ger RNA are extracted from cells and sequenced
duplication in the promoter region leads to after conversion to complementary DNA. Thus,
increase in Cyp51 A protein synthesis. the relative abundance of messenger RNA grown
Other more obscure mechanisms have been after and before exposure to azoles provides evi-
reported. Camps et al. reported four Aspergillus dence to the change in gene expression because
fumigatus isolates in which two species devel- of these compounds.
oped azole resistance after prolonged therapy and Thus, while whole genome sequencing over-
could not be explained by Cyp51 A protein muta- comes much of the limitations of currently
tions (Camps et al. 2012). They followed these available sequencers like Illumina NextSeq and
changes with Whole genome sequencing of the HiSeq and provides insight into mechanisms of
isogenic Aspergillus fumigatus species and azole resistance, transcriptome analysis and
revealed several non-synonymous mutations. To DNA methylation provide proof of gene expres-
correlate mutations with phenotypes, sexual sion changes with azole exposure. These are the
crossing experiments were done on the progeny tools currently available for research in
of azole resistance phenotype. These revealed mycology.
that azole resistance was associated with a P88L
amino acid substitution in the CCAAT-binding
transcription factor complex subunit HapE. Also, 39.5 Conclusion
the HapE P88L mutation caused an increased
CYP51A gene expression thus causing Accuracy, speed, and sensitivity are the USP of
resistance. NGS. It is especially valuable in fungal infec-
Similar study done by Fraczek et al. in tions as these are notoriously difficult to grow in
Aspergillus fumigatus species lacking Cyp51 A culture and thus offer “culture-independent”
mutation revealed 20 potential azole transporter mechanisms of diagnosis. In addition, these offer
genes (Fraczek et al. 2013). In one, CYP51A similar if not better information considering the
expression was increased 500 times in the pres- application of NGS to detect azole resistance,
ence of azoles. Others demonstrated an increase distinguishing infection and colonization and
in CDR1B efflux transporter gene and in one out guiding treatment. Thus, “next” generation needs
of five isolates, a P216L amino acid substitution to be incorporated to current diagnostic
was found in Cyp51A in conjunction with several methodologies.
other non- synonymous mutations and deletions
of clusters of genes (Hagiwara et al. 2014). These
authors thus concluded that the cdr1B efflux
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Current Innovations and Future
Trends 40
Neeraj Kumar, Ravindra Kumar Garg,
and Hardeep Singh Malhotra
fungal agents are different, and newer stains are sis. Manual DNA extraction is having risk of con-
required to identify the actual fungal species. tamination as compared to fully automated DNA
Advances in the field of adaptive optics in extraction. Utilizing automated DNA extraction
scanning will be helpful (Lagree et al. 2018). and real-time PCR will provide result in few hours.
New imaging modalities like cryo-scanning Multiplex PCR is helpful in distinguishing
transmission electron tomography (CSTET), various types of fungi, thus guiding appropriate
focused ion beam scanning electron microscopy treatment. Many commercial PCR-based panels
(FIB-SEM), and soft X-ray tomography will be are available. Two USFDA (United States Food
the future in diagnostics. Super resolution fluo- and Drug Adminstration) approved panels are
rescence microscopy helps in structural and func- being used: (1) T2Candida Panel (2) SeptiFast
tional delineation of cellular and subcellular (Ostrosky-Zeichner and Al-Obaidi 2017).
structures. Combination of CSTET with fluores- Quantitative PCR methodology requires eval-
cent light microscopy will be an additional boon uation for its utility in monitoring treatment
(Briegel and Uphoff 2018). Culture requires spe- response and virulence. Fungal ribosomal gene
cial media and takes a long time. The incubation identification can also help in identification of
period may range from 1 to 4 weeks. Augmented different species. Luminex xMAP is a new
culture and new culture media is the need of the DNA-based identification method using color-
hour. Combination of fluorescence tagging with coded microspheres to detect specific gene
media substrate may expedite diagnosis. sequence (Kozel and Wickes 2014). Increasingly,
non-rDNA loci are being identified to recognize
the particular fungal species. Future innovation
40.2.2 Serology requires techniques to detect and identify differ-
ent species with more sensitivity and specificity.
Serological measurement of mannan antigen and
antimannan antibodies is approved in Europe for
diagnosis in fungal infections. The sensitivity and 40.2.4 Loop-Mediated Isothermal
specificity is more than 80% in combined analysis Amplification
(Ostrosky-Zeichner and Al-Obaidi 2017). Testing
of 1,3-β-D-glucan levels in serum and cerebrospi- Loop-mediated isothermal amplification (LAMP) is
nal fluid (CSF) is also a sensitive marker of fungal a newer technique that amplifies target DNA rapidly,
infection. In abdominal candidiasis, it has been efficiently, and specifically (Malhotra et al. 2014).
found to be better than culture and polymerase
chain reaction (PCR) testing (Colombo et al. 2017).
More biomarkers in CSF and serum are required to 40.2.5 Fingerprinting/Proteomic
be discovered and tested for clinical efficacy. Profiling
Medical advances owe heavily to innovations in Drug adjuvants like iron chelators have been
imaging technology. X-ray, computerized tomog- shown good action as a synergist. A study by Lai
raphy, and magnetic resonance imaging (MRI) et al. showed increased efficacy of amphotericin
are widely available and help in diagnosis. when combined with iron chelators (Lai et al.
Though some imaging features are characteristic 2016). In the future, more such adjuvants are
in identifying the fungal organism type, many a required to be discovered and tested.
times the radiological picture is inconclusive.
Positron emission tomography is now increas-
ingly being advocated in fungal diseases too. It 40.4 Current Innovations
has been shown to be able to detect fungal in Vaccines and Future
involvement in the lung, bone, and other organs. Trends
Additionally, it shows promise in deciding the
treatment completion end point. More research is Aging population, industrialization, and
required to fully utilize this technique in identify- increased antibiotics use are causing resurgence
ing different type of fungal organism. of fungal infections. Vaccination may be helpful
Magnetic resonance spectroscopy can be in preventing such infections. Research in this
improvised to detect various chemical differ- area is at its naïve stage and requires a major
ences in imaging signal, thus assisting in species thrust. Combined bacterial and fungal vaccine
identification. may be a reality in future.
In future, improved imaging modalities will be
increasingly required. Higher Tesla machines with
greater resolution will eventually be as good as 40.5 Conclusion
microscopy. Innovations in software and linking
with MRI machine will lead to species identifica- Advancement in research is leading to new diag-
tion and characterization in a noninvasive manner. nostic and therapeutic tools, such as FIB-SEM,
LAMP, etc. in the field of fungal infections of the
CNS. Newer innovation in diagnosis will greatly
40.3 Current Innovations reduce diagnostic time and specificity. Also,
in Treatment and Future some new promising drugs are being tested. We
Trends hope that an upcoming vaccine may be a reality
in the future.
40.3.1 Antifungals
Lai YW, Campbell LT, Wilkins MR, Pang CN, Chen S, Ostrosky-Zeichner L, Al-Obaidi M. Invasive fungal infec-
Carter DA. Synergy and antagonism between iron tions in the intensive care unit. Infect Dis Clin N Am.
chelators and antifungal drugs in Cryptococcus. Int J 2017;31(3):475–87.
Antimicrob Agents. 2016;48(4):388–94. Revie NM, Iyer KR, Robbins N, Cowen LE. Antifungal
Malhotra S, Sharma S, Bhatia NJ, Kumar P, Bhatia NK, drug resistance: evolution, mechanisms and impact.
Patil V, Hans C. Recent diagnostic techniques in Curr Opin Microbiol. 2018;45:70–6.
mycology. J Med Microbiol Diagn. 2014;3(3):1.
Conclusion
Although the most common causes of neuro- not effective, and severe complications needing
infections are bacteria and viruses, the role of surgical intervention may occur.
fungi should not be ignored especially since With these in mind, the main purpose of this
these potentially devastating infections are richly illustrated book is to provide the reader
encountered world wide. Fungal infections of the with a frame of symptoms and signs of this par-
central nervous system (CNS) have a large spec- ticular infectious disease in an effort to suspect
trum of presenting features, and though most and confirm the diagnosis of CNS fungal infec-
patients are immunocompromised hosts, immu- tions at and early stage and therefore prevent
nocompetent subjects can also be affected. damage to the brain parenchyma and meninges.
Unfortunately, the diagnosis is often delayed or Education and training of medical person-
not considered in routine clinical practice. nel to recognize the disease early forms the
Diagnosis and management of CNS infections pedestal of successful management along with
due to fungi is a true challenge because they careful infection control practices. Also, con-
often occur in a clinical context that is neither comitant research to understand the patho-
specific nor alarming. Neuroimaging data may be genesis of the disease, genetic risk factors for
indicative but are not specific. However, pathol- invasive fungal infections in humans, and the
ogy plays an important role for confirming the advance in diagnostic tools in addition to new
definitive diagnosis, especially if clinical find- therapeutic modalities can improve outcomes
ings, laboratory studies, and diagnostic imaging in the future.
investigations are not conclusive. Systemic anti-
fungal medications remain the cornerstone of Aydın, Turkey Mehmet Turgut
management. However, in many patients diagno- Hyderabad, India Sundaram Challa
sis remains uncertain, or medical treatments are Marrakech, Morocco Ali Akhaddar