+ Models

MYCMED-702; No. of Pages 4

Journal de Mycologie Mdicale (2017) xxx, xxxxxx

Available online at

ScienceDirect
www.sciencedirect.com

CASE REPORT/CAS CLINIQUE

First report of two cases of cryptococcosis in

Tripoli, Libya, infected with Cryptococcus
neoformans isolates present in the urban
area
M.S. Ellabib a, Z.A. Krema a, A.A. Allafi b, M. Cogliati c,*

a
Department of Medical Microbiology and Immunology, Faculty of Medicine, Tripoli University, P.O. Box
13555, Tripoli, Libya
b
Tripoli Medical Center, Department of Infectious Disease, P.O. Box 13555, Tripoli, Libya
c
Lab. Micologia Medica, Dipartimento di Scienze Biomediche per la Salute, Universita degli Studi di Milano,
Via Pascal 36, 20133, Milano, Italy

Received 6 February 2017; received in revised form 20 April 2017; accepted 30 April 2017

KEYWORDS Summary Cryptococcosis is a potentially fatal fungal disease caused by the basidiomycetes
Cryptococcus yeasts Cryptococcus neoformans and C. gattii with high predilection to invade the central
neoformans; nervous system mainly in immunocompromised hosts. Skin can be secondarily involved in
HIV; disseminated infection or be exceptionally involved as primary cutaneous infection by inocula-
Skin involvement; tion with contaminated materials. We report the first two Libyan cases of cryptococcal
Meningitis; meningitis in HIV patients, in which one of them presented a secondary cutaneous involvement
Libya due to systemic dissemination. The first patient was a 17-year-old female, had fever, cough,
headache and intractable vomiting as well as itchy water bumps on her skin and upper limbs. The
cutaneous eruption prompted the accurate diagnosis. Cultures were positive for C. neoformans
in both cerebrospinal fluid and skin specimens, as well as cryptococcal antigen was detected in
serum. The isolate was identified, by molecular analysis, as C. neoformans AD-hybrid belonging
to molecular type VNIII and mating type aAAa, the same genotype found for some environmental
isolates recovered from olive trees in Tripoli. The second patient was a 36-years-old male with a
long history of HIV on irregular treatment. Cryptococcal antigen in serum was positive and
cultures yielded the growth of C. neoformans var. grubii, molecular type VNI and mating type
aA. Both patients did not respond adequately to treatment and died of impaired central nervous
system function and respiratory failure, respectively.
# 2017 Elsevier Masson SAS. All rights reserved.

* Corresponding author.
E-mail address: massimo.cogliati@unimi.it (M. Cogliati).

http://dx.doi.org/10.1016/j.mycmed.2017.04.104
1156-5233/# 2017 Elsevier Masson SAS. All rights reserved.

Please cite this article in press as: Ellabib MS, et al. First report of two cases of cryptococcosis in Tripoli, Libya, infected with Cryptococcus
neoformans isolates present in the urban area. Journal De Mycologie Mdicale (2017), http://dx.doi.org/10.1016/j.mycmed.2017.04.104
+ Models
MYCMED-702; No. of Pages 4

2 M.S. Ellabib et al.

Introduction LAM) test, which was positive in urine, whereas other

Cryptococcosis is caused by two yeast species, Cryptococcus
neoformans and C. gattii, which are now considered two
species complex including several cryptic species [1,2].
Cryptococcus neoformans comprises molecular types VNI,
VNII, VNB, VNIII and VNIV, is found worldwide and causes
cryptococcosis primarily in AIDS patients [3]. Cryptococcus
gattii, including molecular types VGI, VGII, VGIII and VGIV,
has an endemic distribution and often infects immunocom-
petent hosts [4].
Cryptococcus neoformans is the most frequent cause of
meningitis in HIV-infected patients and can infect any organ
in the human body. Central nervous system is commonly
involved, and is mostly associated with VNI molecular type,
which has the most widespread distribution worldwide
accounting more than 90% of the cases in immunocompro-
mised patients [3]. Skin is the most common external site of
infection, affecting 1020% of those with systemic involve-
ment [5]. Cryptococcus neoformans var. neoformans (mole-
cular type VNIV) has been more commonly isolated from
these skin lesions, which could be related to dermotropism
[5,6]. Cutaneous cryptococcosis lesions vary greatly in mor-
phology and mimic other dermatological entities and are
often the first presenting symptom of systemic disease [7]. In
this study, we report for the first time two cases of crypto-
coccosis in HIV patients living in Tripoli, Libya.
investigations including venereal disease research labora-
tory tests, hepatitis B surface antigens and anti-hepatitis
core, were all negative. The initial CD4+ count was 17 cells/
mm3, significantly lower than the patient CD4+ baseline
(350 cells/mm3) detected six months before. The patient
started an antifungal therapy with amphotericin B (50 mg/
d) and fluconazole (750 mg/d), and two weeks later, an
anti-tuberculosis therapy with isoniazid, rifampicin,
ethambutol and pyrazinamide. After 25 days from admis-
sion, the patient vision decreased. Fundoscopic examina-
tion and serology by detection of IgM antibodies allowed
diagnosing cytomegalovirus retinitis. Septrin, dexametha-
sone and ganciclovir were then administered. The patient
response to therapy was very poor and she became unres-
ponsive after 35 days from admission. Later, she was trans-
ferred to the intensive care unit where she died after one
day with suspected severely impaired central nervous sys-
tem function.

Case 1

The patient was a 17-year-old Libyan female HIV infected

since birth. Antiretroviral treatment was discontinuous
depending on the availability of the drugs in the depart-
ment. She presented to Tripoli Medical Center, in August
2016, with a three-week history of itchy water bumps that
ruptured upon scratching and subsequently crusted over.
This was also associated few days later with fever, heada-
che and vomiting. No previous chronic medical condition or
drug allergy was reported. Physical examination revealed
that patient was malnourished with fever at 38.3 8C, good
conscious level and no sign of meningitis. Upon inspection
of skin, numerous skin-colored vesicles and crusted lesions
were seen on the upper limbs (Fig. 1), on the face (Fig. 2),
lower limbs and trunk. Laboratory tests as well as renal and
hepatic function were within normal ranges. Direct micro-
scopic examination of cerebrospinal fluid (CSF) and scraped
crusts using India ink test revealed encapsulated yeast
resembling Cryptococcus. The serum tested for cryptococ-
cal antigen by lateral flow assay (CrAg LFA, IMMY, Norman,
OK, USA) was positive. Culture of both CSF and skin lesions
on sunflower seed agar yielded brown yeast colonies sug-
gesting the presence of C. neoformans. Capsule observed in
India ink preparation, urease production and ability to grow
at 37 8C as well as Vitek2 compact system (bioMerieux,
Firenze, Italy) confirmed C. neoformans identification.
Molecular type and mating type allelic pattern were deter-
mined performing two specific multiplex PCRs as described
elsewhere [8,9], which revealed that the isolate was a
C. neoformans AD-hybrid belonging to molecular type VNIII
and with mating type aAAa [10]. Disseminated tuberculosis
was also diagnosed by tuberculosis lipoarabinomannan (TB- Figure 1 Cryptococcus skin-colored vesicles on the upper limbs.

Please cite this article in press as: Ellabib MS, et al. First report of two cases of cryptococcosis in Tripoli, Libya, infected with Cryptococcus
neoformans isolates present in the urban area. Journal De Mycologie Mdicale (2017), http://dx.doi.org/10.1016/j.mycmed.2017.04.104
+ Models
MYCMED-702; No. of Pages 4
Cryptococcosis cases in Libya
Figure 2 Cryptococcus vesicles on the face.
Case 2
The second patient was a 36-year-old Nigerian male with HIV
infection for long time and antiretroviral discontinuous
treatment (efavirenz, lamivudine, zidovudine). He was
admitted, in May 2015, to Tripoli Medical Center, with a
three-week history of headache, vertigo and blurred vision.
Vital signs as blood pressure, body temperature, pulse and
respiratory rate were normal. Neurological examination
revealed weakness of both lower limbs and no signs of
meningeal irritation. Fundoscopic examination revealed a
normal bilateral optic disc. Serum glucose, renal and liver
function tests were normal. Hepatitis B surface antigen was
positive, whereas TB-LAM and anti-hepatitis C were both
negative. Initial CD4+ count was less than 100 cells/mm3
(baseline 350 cells/mm3). A lumbar puncture was performed
and cryptococcal antigen in serum was positive as well as
India ink test for capsule examination of CSF. CSF culture was
positive and molecular typing identified the pathogen as
C. neoformans, molecular type VNI and mating type
Please cite this article in press as: Ellabib MS, et al. First report of two cases of cryptococcosis in Tripoli, Libya, infected with Cryptococcus
neoformans isolates present in the urban area. Journal De Mycologie Mdicale (2017), http://dx.doi.org/10.1016/j.mycmed.2017.04.104
3
aA. The patient started a therapy with intravenous ampho-
tericin B 50 mg/d (the only drug available at time). Initially,
the patient seemed to respond to the therapy with impro-
vement in his headache, then, on the eleventh day of
treatment (day 13 from admission), he became unresponsive
with decreased respiratory rate. Patient was transferred to
intensive care unit but he died two days later of suspected
respiratory failure.
Discussion
At present, data from Libya concerning occurrence and
incidence of cryptococcosis as well as species and molecular
type identification of clinical isolates has not been published.
Therefore, this study represents the first report describing
two cases of cryptococcal meningitis from Libya with mole-
cular characterization of the isolates. Current recommenda-
tions consider use of antiretroviral therapy (ART) at least
after the initial two-week consolidation phase and within the
next eight weeks of the maintenance phase of cryptococcal
therapy [11]. In the two cases here described, ART was not
implanted due to unavailability of drugs before and during
the admission of patients and this might explain poor pro-
gnosis to antifungal treatment in both cases. Both patients
were HIV infected for long time and were on irregular
treatment as indicated by their CD4+ cell count and other
positive laboratory tests. This might have also contributed to
poor prognoses response in both cases, particularly in case
one who has both cryptococcal meningitis and disseminated
cryptococcosis involving skin, and who did not respond to all
treatment regimen applied to AIDS patients in the depart-
ment setting. In both cases, despite all measures taken, both
patients could not survive and died of severely impaired
central nervous system function in case one, and respiratory
failure in case two. In Libya, current practices for anti-
cryptococcal therapy in HIV patients generally include
amphotericin B alone or combined with fluconazole [12].
In case one, despite treatment using both drugs, the patient
did not respond at all to the therapy. This might be due to the
late and short course of cryptococcal disseminated menin-
gitis symptoms. Therefore, whether the patients are immu-
nocompromised or not, the outcome can be fatal unless the
disease is diagnosed early in course of illness. Molecular
typing revealed that patient one was infected by a
C. neoformans AD hybrid, molecular type VNIII, mating type
aAAa, the same isolated in some olives trees in the area of
Tripoli in a recent environmental survey [13]. In the other
case, the isolation of C. neoformans var. grubii, molecular
type VNI, mating type aA is consistent with many previous
studies worldwide [14], as well as with our recent study
which showed that VNI molecular type is the most prevalent
cryptococcal isolate from pigeon droppings and other envi-
ronmental samples in Tripoli [13]. Therefore, in both cases
the infection was likely acquired from the Tripoli area
environment, although we cannot exclude that patient 2
acquired the infection in Nigeria because we have no Nige-
rian isolates available for comparison. More extensive stu-
dies to obtain isolates from clinical and environmental
sources should be carried out in the future to establish
the geographical distribution and the genetic relationship
between clinical and environmental genotypes in Libya.
+ Models
MYCMED-702; No. of Pages 4

4 M.S. Ellabib et al.

Disclosure of interest [7] Kamble R. Cutaneous cryptococcosis in an immunocompetent

host. Int J Curr Microbiol App Sci 2015;4:5937.
[8] Cogliati M, Allaria M, Tortorano AM, Viviani MA. Genotyping
The authors declare that they have no competing interest. Cryptococcus neoformans var. neoformans with specific
primers designed from PCR-fingerprinting bands
References sequenced using a modified PCR-based strategy. Med Mycol
2000;38:97103.
[9] Esposto MC, Cogliati M, Tortorano AM, Viviani MA. Determina-
[1] Hagen F, Khayhan K, Theelen B, Kolecka A, Polacheck I, Sionov
tion of Cryptococcus neoformans var. neoformans mating type
E, et al. Recognition of seven species in the Cryptococcus
by multiplex PCR. Clin Microbiol Infect 2004;10:10924.
gattii/Cryptococcus neoformans species complex. Fungal Gen-
[10] Cogliati M, Esposto MC, Tortorano AM, Viviani MA. Cryptococcus
et Biol 2015;78:1648.
neoformans population includes hybrid strains homozygous at
[2] Kwon-Chung KJ, Bennett JE, Wickes BL, Meyer W, Cuomo CA,
mating type locus. FEMS Yeast Res 2006;6:60813.
Wollenburg KR, et al. The case for adopting the species
[11] Perfect JR, Dismukes WE, Dromer F, Goldman DL, Graybill JR,
complex nomenclature for the etiologic agents of cryptococ-
Hamill RJ, et al. Clinical practice guidelines for the manage-
cosis. mSphere 2017;2:e00357416.
ment of cryptococcal disease: 2010 update by the Infectious
[3] Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas
Diseases Society of America. Clin Infect Dis 2010;50:291322.
PG, Chiller TM. Estimation of the current global burden of
[12] Mustafa AA, Kowalski SR. A comparative analysis of the Libyan
cryptococcal meningitis among persons living with HIV/AIDS.
national essential medicines list and the WHO model list of
AIDS 2009;23:52530.
essential medicines. Libyan J Med 2010;5:5403.
[4] Chen SC, Meyer W, Sorrell TC. Cryptococcus gattii infections.
[13] Ellabib MS, Aboshkiwa MA, Husien WM, DAmicis R, Cogliati M.
Clin Microbiol Rev 2014;27:9801024.
Isolation, identification and molecular typing of Cryptococcus
[5] Dromer F, Mathoulin S, Dupont B, Letenneur L, Ronin O. Indi-
neoformans from pigeon droppings and other environmental
vidual and environmental factors associated with infection due
sources in Tripoli, Libya. Mycopathologia 2016;181:6038.
to Cryptococcus neoformans serotype D. French Cryptococcosis
[14] Cogliati M. Global molecular epidemiology of Cryptococcus
Study Group. Clin Infect Dis 1996;23:916.
neoformans and Cryptococcus gattii: an atlas of the molecular
[6] Martinez LR, Garcia-Rivera J, Casadevall A. Cryptococcus neo-
types. Scientifica (Cairo) 2013;2013:675213.
formans var. neoformans (serotype D) strains are more suscep-
tible to heat than C. neoformans var. grubii (serotype A)
strains. J Clin Microbiol 2001;39:33657.

Please cite this article in press as: Ellabib MS, et al. First report of two cases of cryptococcosis in Tripoli, Libya, infected with Cryptococcus
neoformans isolates present in the urban area. Journal De Mycologie Mdicale (2017), http://dx.doi.org/10.1016/j.mycmed.2017.04.104