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Abstract
Poster Abstracts
P001
Cryptococcosis in hematological patients in SaintPetersburg, Russia
S. N. Khostelidi,1 M. Ignatyeva,1 T. S. Bogomolova,1
M. Sorokina,1 V. Fillipova,1 V. Borzova,1 O. Popova,2
G. Potapenko,3 S. Zubarovskaya,2 V. Afanasyev,2 N. V. Vasilyeva1
and N. Klimko1
1
I.I.Metchnikov North-Western State Medical University, SaintPetersburg, Russia; 2I.P. Pavlov Saint Petersburg State Medical
University, Saint-Petersburg, Russia and 3City Hospital No. 31,
Saint-Petersburg, Russia
Objectives To analyze demographic parameters, underlying diseases,
etiology, treatment and survival rate of hematological patients with
cryptococcosis in St. Petersburg, Russia
Methods The prospective study was conducted during the period
20102013. Diagnosis of cryptococcosis was made according to EORTC/MSG criteria (2008).
Results We observed 8 hematological patients with cryptococcosis.
The mean age of patients was 24 years (range 2.660), male/female
ratio 1/1, adults were 62.5%. Main underlying conditions were:
acute myeloid leukemia 25%, acute lymphoblastic leukemia 25%,
non-Hodgkins lymphoma 25%, chronic lymphoblastic leukemia
12.5% and acute leukemia 12.5%.
Test Pastorex Crypto-Plus (Bio-Rad) was positive in 87.5%
patients (in serum 2, BAL 1, CSF 5). Cryptococcus neoformans
were isolated from 25% patients. Diagnosis of cryptococcosis was
confirmed by histology and direct microscopy of biopsy samples in
25% of patients. The main sites of infection were CNS 62.5%, lungs
37.5%. In 50% patients after cryptococcosis had invasive aspergillosis. The median of 4+ in these patients was 0.298.
Antifungal therapy of cryptococcosis was performed in all patients:
fluconazole 62.5%, voriconazole 50%, amphotericin B deoxycholate 37.5%. Combination therapy was performed for 37.5% patients
(amphotericin B + fluconazole). Surgical treatment (resection of the
affected lung) was conducted in 25% patients. Duration of antifungal
therapy was 72360 days (median 199). Overall survival at
12 weeks was 87.5%, 1 year 25%.
Conclusion The main underlying diseases in hematological patients
with cryptococcosis acute leukemia (62.5%). Main locations CNS
and lungs. Twelve weeks overall survival was 87,5%.
dearth of published article about the pathogen diversity, epidemiology and virulence potential in Nigeria.
Aim This paper represents a compendium on this disease in the
Nigerian clinical setting, highlighting areas of need for future
research and collaborative structures within the country as well as
the technical difficulties affecting Cryptococcus research in Nigeria.
Methods and results Analysis of published articles on PubMed,
AJOL and Google were made using keywords like Cryptococcosis in
Nigeria, Cryptocococcus neoformans + Nigeria, Cryptococcus gattii +
Nigeria, and Cryptococcus + Nigeria regardless of date of
publication.
Less than 20 papers were found reporting few data concerning
Cryptococcus and cryptococcosis in Nigeria. A study carried out in
2010 reported a frequency of 36% of cryptococcal meningitis in a
cohort of 100 HIV positive patients. In another study a prevalence of
18.8% of pulmonary cryptococcosis among HIV patients was
reported. Other papers reported a prevalence of 13% of cryptococcosis in HIV pregnant and 4% among other HIV patients. Cryptococcus
species were not identified in the results and none of the papers
reported C. gattii isolation. There is also a need for comprehensive
reports on the antifungal susceptibility patterns of Nigerias clinical
isolates of C. neoformans as there are no information on the susceptibility profile. In the environment, some studies have reported isolation of C. neoformans serotype A and D from pigeons and other
healthy birds including bats.
Conclusion On the basis of the few results reported in the literature
it is clear that cryptococcosis epidemiology in Nigeria is far to be elucidated. Therefore, a great effort of collaboration between hospitals and
research centers of the country connected with international reference
centers is needed. The main goals in the next future to improve the
knowledge of this life-threatening disease are the following:
(1) to establish a network involving clinician, microbiologists and
researchers of the main hospitals and research centers in Nigeria
under the coordination of a local reference center connected with an
international Cryptococcus reference laboratory;
(2) to report clinical and microbiological data concerning each
case of cryptococcosis in a standard form collected by the coordinator
center;
(3) to collect Cryptococcus isolates for biochemical and molecular
analysis as well as determination of their antifungal susceptibility;
(4) to analyze clinical and microbiological data to improve cryptococcosis diagnosis and to determine distribution of Cryptococcus
genotypes in Nigeria;
(5) to survey the environment by sampling trees, soil, and bird
excreta;
(6) to compare clinical and environmental data.
P003
P002
Cryptococcosis in Nigeria: the neglect of a giant
E. Nnadi,1 M. Cogliati2 and I. B. Enweani3
1
Plateau State University, Bokkos, Bokkos, Nigeria; 2University of
Milan, Milan, Italy and 3Nnamdi Azikiwe University, Nnewi,
Nigeria
Background Cryptococcosis is one of the most severe opportunistic
infections in patients with HIV/AIDS and other underlying immunocompromising disease conditions having grave complications and
prognosis especially in developing countries. It is estimated to cause
about one million cases of meningitis per year globally, prevalently
in HIV infected subjects. It has been associated with 17% of all
deaths among HIV-infected patients in sub-Saharan Africa and therefore dubbed a sleeping disease emerging as an awakening giant.
Although cryptococcosis incidence has risen dramatically in the last
two decades especially within the HIV positive population, there is a
doi:10.1111/myc.12196
Poster Abstracts
P004
Epidemiological and clinical features of aids patients with
cryptococcosis confirmed or diagnosed at necropsy in a
teaching hospital in Brazil
M. L. Silva Vergara, R. G. Garcia Torres, L. M. Da Silva Junior,
R. M. Etchebehere, D. J. Mora, K. F. Ferreira Paim, L. A. Andrade
Silva and A. R. Rua Micheletti
Tria^ngulo Mineiro Federal University, Uberaba, Brazil
Background Yearly, one million cases of cryptococcosis in AIDS
patients are diagnosed around the world, of which two thirds die
during the second week on therapy. Commonly, cryptococcosis is the
first AIDS defining condition in these patients and concerns about
the high mortality rates observed are intimately associated to late
AIDS diagnosis and advanced immunodeficiency in most patients at
admission. Consequently, a severe and disseminated fungal infection
is commonly seen, mainly in poor-resource settings where diagnosis
and specific therapy are far to be ideal.
Aim To evaluate epidemiological, clinical and anatomopathological
features of AIDS patients with cryptococcosis confirmed or diagnosed
at necropsy in a Teaching Hospital in Brazil.
Methods Retrospectively, medical and necropsy records of 45 AIDS
patients with cryptococcosis diagnosed from 1993 to 2012 in the
Teaching Hospital in Uberaba, Brazil, were reviewed. Demographic,
epidemiological, clinical, laboratory and necropsy data of these cases
were obtained and compared to those of 24 AIDS patients with cryptococcosis who presented a good outcome over the same period.
Results A total of 45 AIDS patients with cryptococcosis who performed necropsy were included. Of these, 35 (77.8%) were male with
mean age of 31.7 years. Cryptococcosis was the first AIDS defining
condition in 30 (66.7%). The evolution of symptoms in 33 (73.3%)
34
P005
Hydrocephalus as the first clinical feature of cryptococcosis
in an aids patient on antiretroviral therapy
M. L. Silva Vergara, G. B. Borges Machado, G. H. Machado,
R. G. Garcia Torres, L. A. Andrade Silva, D. J. Mora, L. M. Da
Silva Junior and K. F. Ferreira Paim
Tria^ngulo Mineiro Federal University, Uberaba, Brazil
Background Clinical signs and symptoms of cryptococcosis are commonly related to meningeal and or encephalic involvement. Globally,
most cases of Cryptococcal meningitis occur in AIDS patients who
usually present a subacute clinical picture and are severely immunocompromised. Hydrocephaly is a common complication of intracranial increased pressure, but to our knowledge, it is uncommon as a
first clinical presentation of this infection.
Aim To describe an AIDS patient with uncommon clinical picture of
central nervous system cryptococcosis.
Case report A 30-year-old Brazilian woman was admitted at the
teaching hospital in March 2013. She complained of holocranial
headache for several months which became more intense persistent
during the last month. Besides, she referred ataxia and lack of balance. No other general symptoms or neurological complains were
described. She was found HIV positive in 2004 during a pregnancy
and remained without medical care until 2010 when she was admitted with Pneumocystis jirovecii pneumonia, Herpes zoster infection and
lymphocytic meningitis which were adequately treated. The skull CT
was normal at the time. Her CD4 T cell baseline count was
19 cells mm3 and the viral load of 356.322 RNA copies/ml She
started antiretroviral therapy with AZT + 3TC + efavirenz and was
Poster Abstracts
P006
Ocular cryptococcosis in an AIDS patient with
disseminated infection: case report
M. L. Silva Vergara, R. G. Garcia Torres, L. M. Da Silva Junior,
R. M. Etchebehere, D. J. Mora, L. A. Andrade Silva, K. F. Ferreira
Paim and A. R. Rua Micheletti
Tria^ngulo Mineiro Federal University, Uberaba, Brazil
Background As opportunistic infection in immunocompromised
patients, cryptococcosis can potentially involve any organ or system.
Ocular primary cryptococcosis seems to be a rare event, and most
cases already reported occurred associated to disseminated infection
in patients with or not underlying immunodeficiency due to corticosteroids, hematologic malignancy, vascular collagen disease and AIDS
, among others. According to the literature reviewed, most cases presented a presumptive clinical diagnosis of ocular cryptococcosis.
Aim To describe an AIDS patient with cryptococcal choroiditis confirmed at necropsy.
Case report A 20-year female Brazilian patient was admitted at the
Teaching Hospital with fever, progressive dyspnea, dry cough and
weight loss of 10 kg during the last two months. Moreover she complained of holocranial headache during the last two weeks. Due to the
symptoms related to severe anaemia, she had been previously
P007
Cryptococcosis in ferrets from Spain and Portugal: the
veterinarians role
M. F. Colom Valiente,1 C. Juan Salles,2 R. Patricio,3 C. Artigas,4
J. I. Serra,4 F. Hagen5 and N. Morera6
1
University Miguel Hernandez, Sant Joan dAlacant, Spain;
2
Noahs Path, Elche, Spain; 3Clnica Veterinaria Alcabidechevet,
Alcabideche, Portugal; 4Clnica la Vileta, Palma De Mallorca,
Spain; 5Canisius-Wilhelmina Ziekenhuis, Nijmegen, The
Netherlands and 6Clinica Exotics, Barcelona, Spain
Background Cryptococcus gattii is considered to be emerging in different areas of the world being the Mediterranean basin one of them.
Exposure to infection might be more likely in animals than in human
beings, given their closer relationship with the natural habitat of the
yeast. Animals and especially pets, can act as indicators of the presence of this yeast in a determined area. We present a review of cryptococcosis in ferrets in Spain and Portugal, and its relationship with
the findings of Cryptococcus in environmental samples in the same
locations. The work emphasizes the importance of suspecting the disease in ferrets, which can act as sentinels, and performing an
35
Poster Abstracts
P008
Cryptococcosis gattii: a poor recognized fungal infection in
Brazil
F. Queiroz-Telles and V. A. Vicente
Federal University of Parana, Curitiba, Brazil
Background Infection by Cryptococcus gattii may result in serious,
disseminated and life-threatening disease. Cryptococcosis gattii (CG)
is an emerging infection in many in many parts of the world including Brazil where cases occur all over the country
Aim To review proved CG cases in an University Hospital
Methods We reviewed charts and collected the clinical, epidemiological and therapeutic data from patients with documented C. gattii
infection. The patients were observed in a tertiary 600 beds University Hospital in Curitiba, South of Brazil, from 1985 to 2013. Diagnosis was demonstrated by culture and CGB agar test. Data from
twenty cases of CG were evaluated.
36
Results Most of the cases (60%) were diagnosed in the last decade
period. The male-to-female ratio was 3:1 and the median age was 48
years (17-76). Seven patients (44%) had a recent history of contact
with trees or manipulation of woods. Five patients (31,3%) were
immunosuppressed: 04 HIV+ and one under high dose steroid regimen. The average time of diagnosis since the onset of symptoms was
33 days (560). The most prevalent symptom was headache, found
in 18 patients, and followed by other neurological symptoms, vomiting, hyporexia, weight loss, fever and respiratory manifestations. CG
suspected radiological images were detected in 11 (55%) patients. In
only one patient, the pulmonary involvement by C. gattii was exclusive. The most used induction therapeutic regimen was the combination of deoxycholate amphotericin B (D-AMB) (0.7-1 mg/kg/day) and
fluconazole (400 to 800 mg per day), since flucytosine is unavailable
in Brazil. In the last decade period, most of the patients were submitted to therapeutic lumbar punctures. Maintenance treatment was
with fluconazole 200 - 600 mg per day. Thirteen patients (65%)
developed CAMB renal toxicities and in 08 of them, therapy was
changed to liposomal amphotericin B (L-AMB) or voriconazole. Two
patients were included in a non-comparative open clinical trial with
an investigational triazole. Six patients died (30%), but only 03
(15%) due to CG.
Conclusion Our case series may not reflect the real incidence of CG
in Brazil as well as in other Latin American countries. Unfortunately
several cryptococcal meningitis patients are diagnosed by the India
ink test only and the CGB agar test is available in less then 10% of
the Latin America Hospitals. Our clinical and epidemiological findings are similar to the literature: most of the patients are apparently,
non -immunocompromised and in almost half of them a history of
wood or tree contact was obtained. Most of our cases presented
simultaneous neurologic and pulmonary involvement but only one
third of them presented with respiratory manifestations. So, C. gattii
lung involvement should be investigated by radiological exams specially tomography. As most of our patients developed renal toxicities,
L-AMB should be the first line therapy but costs are still an outstanding barrier for most of the patients. The second-generation triazoles,
especially voriconazole and isavuconazole may have a potential hole
in the therapy patients with CG.
[Correction added on 5 June 2014, after print publication: Erroneous
abstract has been replaced with the correct abstract.]
P009
Investigation of environmental sources of a cryptococcosis
Island, Para
state, Brazil
outbreak in Marajo
L. Trilles,1 A. L. S. Ferreira,2 C. Garcia,2 M. R. Pureza,2
I. C. F. Bonna,1 R. Reis,1 A. C. Souto,1 R. Medeiros,3 L. Luz,3
M. S. Lazera1 and B. Wanke1
1
Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; 2SESPA,
Belem, Brazil and 3Federal University of Para, Belem, Brazil
Cryptococcosis by Cryptococcus gattii in Brazil is endemicin the North
(N) and Northeast (NE) of the country. In these regions, noteworthy
was the emergence of cryptococcosis in immunocompetent (HIV-negative) children in about one fifth of the reported cases, suggesting that
natural infection occurs early in life. This study was motivated by the
occurrence, in a 3 months period (2013) of two laboratory confirmed
cases and one not confirmed case of cryptococcal meningitis in S~
ao
Sebasti~
ao da Boa Vista, a municipality on the Maraj
o Island at the
estuary of the Amazon River in Brazil. This locality at the coordinates
01430 04S 49320 27W has an estimated population of 22 890 people (2010). To investigate possible sources of infection that caused the
outbreak, 27 environmental samples from the wooden houses where
the patients live and surrounding areas where they work or study
were collected. The samples, constituted mostly of indoor dust and
decaying wood from the houses and surrounding hollow trees, were
diluted in sterile saline and plated on bird seed agar. Dark brown
Poster Abstracts
P010
Molecular characterization of Cryptococcus gattii genotype
AFLP6/VGII isolated from woody debris of divi-divi
(Caesalpinia coriaria), Bonaire, Dutch Caribbean
F. Hagen,1 A. Chowdhary,2 A. Prakash,2 J. B. Yntema3 and
J. F. Meis1
1
Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands;
2
Vallabhbhai Patel Chest Institute, Delhi, India and 3Radboud
University Medical Centre, Nijmegen, The Netherlands
Background The basidiomycetous yeast Cryptococcus gattii is a primary pathogen that is mainly restricted to subtropical and tropical
climate zones. It is an important cause of cryptococcosis among
immunocompetent subjects and it has emerged as a significant pathogen in Canada and the Pacific Northwest, USA. There is a lack of
information about its environmental presence outside Mediterranean
Europe, India, North and South America. Environmental sampling
for C. gattii and molecular characterization of the obtained isolates
will provide an insight into the global spread of the various genotypes. Therefore, environmental sampling was carried out on the
island of Bonaire, Dutch Caribbean.
Material and methods Woody debris, collected from inside trunk
hollows of living divi-divi trees in April 2013, were cultured on simplified niger seed agar as described. The sampled divi-divi trees were
located in Lagun Goto (N12 140 3.13440 , E-68 220 6.3660 ), Rincon
village (N12 140 24.11160 , E-68 190 32.56620 ) and neighboring
surroundings of Hato village (N12 100 11.87340 , E-68 170
1.23660 ). Plates were incubated at 28 C and periodically observed
for chocolate brown colonies of C. gattii and C. neoformans for up to
7 days. Suspected colonies of Cryptococcus spp. were subcultured by
dilution plating and identified by their morphologic and biochemical
profiles, including development of blue color on L-canavanine-glycine
bromothymol blue medium and identification by matrix-assisted laser
desorption ionization-time of flight mass spectrometry.Isolates identified as C. gattii were subjected to amplified fragment length polymorphism genotyping, mating-type analysis and multi-locus sequence
typing.
Results Ten colonies of C. gattii were cultured from different trunk
hollows of the same divi-divi tree, molecular characterization showed
that all isolates were genotype AFLP6/VGII and mating-type a.
Multi-locus sequence typing revealed that all isolates were genetically
indistinguishable from each other and that these isolates are genetically closely linked to strain CBS1930 (a strain isolated from a goat,
after inoculation of clinical material from a fatal pediatric case from
Aruba, Dutch Caribbean in the 1950s).
Conclusions Cryptococcus gattii is present in the environment of Bonaire, which suggests that C. gattii will be present in the environment
of other Caribbean islands too. Puerto Rico is the only Caribbean island
where C. gattii has been found to date in the environment.
P011
Clinical and epidemiological profile of Cryptococcal
meningoencephalitis cases in a Reference Center Teresina
Piaui, Brazil
H. Alves da Silva Machado,1 J. Noronha Vieira Junior,1
A. V. Guimar~aesde Miranda Correia,1 M. A. Salmito Cavalcanti,1
L. Soares Martins2 and K. Dantas Eulalio1
1
Instituto de Doencas Tropicais Natan Portella, Teresina/Piaui,
Brazil and 2Universidade Federal do Piau, Teresina/Piaui, Brazil
Background Cryptococcosis is an important opportunistic fungal infection, with considerable morbidity and mortality in patients with HIV. It
is the second most common cause of opportunistic CNS infections.
Aim of the study To establish an epidemiological clinical profile of
cryptococcal meningoencephalitis patients, and to assess a possible
relationship between CSF cellularity with immunosuppression and to
correlate the findings with the literature.
Methods A descriptive, retrospective cohort, quantitative study
based on a review of the records of patients admitted to a referral
hospital over a period of 7 years.
Results In the sample analyzed, 67% were men, with a mean age of
33.8 years. Most patients came from the states of Piau, Maranh~
ao
and Par
a, where the disease is considered to be endemic; 28.5 %
were HIV negative and 57.1% HIV positive. It was observed that the
patients analysed showed lymphocytic pleocytosis (<500 cells mm3
, with a normal CSF glucose and high protein concentration. Of the
91 patients, 35% died. The correlations performed showed no statistical significance.
Conclusion In this paper we conclude that there is a high frequency
of cryptococcosis patienst in adult young men and HIV-infected
patients, mainly coming from the states of Piau and Maranh~
ao.
P012
Lethality of Cryptococcal meningitis in a Reference
Hospital in Teresine, Piau, Brazil
H. Alves da Silva Machado,1 J. Noronha Vieira Junior,1
A. V. Guimar~aesde Miranda Correia,1 M. A. Salmito Cavalcanti,1
L. Soares Martins2 and K. Dantas Eulalio1
1
Instituto de Doencas Tropicais Natan Portella, Teresina/Piaui,
Brazil and 2Universidade Federal do Piau, Teresina/Piaui, Brazil
Background Cryptococcosis is a systemic mycosis caused by species
of the fungus genus Cryptococcus genus. C. gattii occurs mainly in
areas of tropical and subtropical climate affecting immunocompetent
hosts, and C. neoformans is a cosmopolitan species affecting mainly
immunocompromised individuals. The infection is often progressing
to pulmonary asymptomatic infection that can spread to various
other organs and organ systems. In the systemic form of the disease
the most commonly affected organ is the central nervous system
(CNS) with meningoencephalitis as the most common clinical manifestation in our environment.
Aim of the study Analysis of clinical cryptococcosis patients during
a 7-year period in Piau, Brazil.
Methods This study analysed data from 92 patients hospitalized for
cryptococcal meningitis in a referral centre for infectious diseases in
Teresina, Piau, Brazil for a period of 7 years.
Results Sixty two of the patients were male and 30 were female,
56.53% (52) were HIV-positive and 43.47% (40) were HIV-negative.
The overall mortality was 39.13% (36/92) and 46.15% (24/52) in
patients with retrovirus and 30% (12/40) in non-retrovirus carriers.
Discussion Cryptococcal meningitis remains a disease of high mortality despite the new strategies used for early diagnosis and adjuvant
treatment developed in recent years.
37
Poster Abstracts
P013
P014
38
Poster Abstracts
P015
Report of filamentous forms in a mating type VNI clinical
sequential isolates from a single AIDS patient
L. Oliveira,1 M. A. Martins,1 M. W. Szeszs,1 J. E. Vidal2 and
M. S. C. Melhem1
1
Instituto Adolfo Lutz, Sa~o Paulo, Brazil and 2Instituto de
Infectologia Emilio Ribas, Sa~o Paulo, Brazil
Background Molecular type and antifungal resistance profiles vary
depending on the causative strain and could result in different clinical presentation and outcome. Of note, it is recognized that multiple
molecular types can infect a single patient and serial samples yield
accurate determination of the etiological(s) agent(s).
Aim We reported two distinct molecular profiles in the causative
agent of case of meningeal Cryptococcosis that showed filamentous
cells in cerebral spinal fluid (CSF) direct examination.
Methods and results A male patient 43-old, HIV-positive, native of
La Paz, Bolivia, living in S~ao Paulo City, Brazil for 15 years, was
attended at a tertiary public hospital. He presented umbilicated papules skin lesions on the trunk and limbs, besides pulmonary and
meningeal symptoms. CD4 counting was 43 cells mm3 and CSF
showed qualitative positive latex agglutination for Cryptococcus and
618 fungal cells per mm3. Treatment was started on day 1 with
1 mg kg1 day1 of amphotericin B desoxicolate (AmB) associated
with 400 mg of fluconazole (FCL) twice/day/4 weeks. Relief punctures were performed daily in the first seven days of treatment. The
blood culture, CSF and skin tissue cultures yielded the recovery of
typically cryptococci encapsulated yeasts. CFS samples from day 1, 7,
14 and 21 were referred to the Public Health Reference Laboratory
(Adolfo Lutz Institute) for quantitative culture, species identification,
molecular type determination and antifungal susceptibility pattern.
Multiple colonies from quantitative cultures related to each CSF sample were collected to speciation, sexual mating typing, PCR-RFLP
genotyping and molecular diversity analysis. Morphological and biochemical characteristics, CGB test were performed to speciation.
URA5gene RFLP was conducted using primers URA5 and SJ01 followed by digestion with Sau96I and HhaI enzymes. The minisatellitespecific core sequence of the wild-type phage M13 was used in the
PCR-fingerprinting of 19 distinct colonies. Molecular type was
assigned, according to the major bands in the patterns. All visible
bands were included for analysis, independent of their intensity,
using Bionumerics software. Antifungal susceptibility test was performed by broth micro dilution reference method (E.Def 7.2,AFST-EUCAST) for FCL and voriconazole (VCL), and Etest method for AmB.
Results The patient presented good clinical course before hospital
discharge. The initial quantitative CSF culture showed high initial
fungal burden (880 000 CFU ml1) dropping to 270 and
180 CFU ml1 at day 7 and 14, respectively, followed to negative
results at day 21. The PCR-RFLP results of 18 tested colonies indicated C neoformans molecular type VNI and a mating type. Homogeneous high MIC-values of FCZ (16 mg l1) and voriconazole
(0.25 mg l1), but low AmB-MIC (0.064 mg l1) were found.
Discussion and conclusion All colonies showed identical band pattern by PCR fingerprinting and RFLP analysis, suggesting the occurrence of just one single molecular pattern of the etiologic agent even
in distinct samples. The antifungal susceptibility to AmB, FCL,voriconazole was identical for all colonies in concordance with the homogeneous molecular type. The combined therapy resulted in
improvement suggesting the in vivo efficacy of AmB, confirmed by in
vitro results. Otherwise, the primary and sequential isolates showed
high FCL-MIC values with no correlation with the good outcome.
The success of the therapy was demonstrated by serial quantitative
culture, which showed considerable decrease in fungal load during
treatment. Little is known about the occurrence of filamentation in
C.neoformans cells and no relationship to antifungal susceptibility was
described, although low virulence in filamentous forms was prior
reported in murine model. One hypothesis is that hyphal production
is an adaptive mechanism to environmental pressures to yeast development. There is insufficient data to confirm if morphologic alterations could result in different molecular pattern as we observed in
this study. Further studies are necessary to explain the clinical relevance of multiple molecular profiles of the etiologic cryptococcosis
agent and its relationship with morphological changes.
P016
Childhood Cryptococcosis in Colombia, and literature
review
J. L. Lizarazo,1 P. Escandon,2 C. A. Agudelo2 and
E. Castaneda2
1
Hospital Erasmo Meoz, Cucuta, Colombia and 2Instituto
Nacional de Salud, Bogota, Colombia
Background In the world literature less than 1000 cases of cryptococcosis in children have been described including those occurring in
immunosuppressed patients. It has not been established the cause of
the low presentation of cryptococcosis in children and it is thought
that it is not because of the lack of exposure.
Aim To do an analysis of the epidemiological manifestations of cryptococcosis in Colombian children and compare them with data published in the literature.
Methods Epidemiological data (19932010) were obtained by
means of a survey processed by Colombian clinicians and microbiologists. A search was performed in the Medline database from 1996 to
September 30, 2013, additionally, we searched for articles in Spanish
and Portuguese in the databases Scielo and Lilacs.
Results The average age 8.4 years found in this study together with
a slight predominance of the male sex (58.5%), is similar to that
found in other studies: South Africa, Thailand, China, Brazil and the
United States. Almost half (46.3%) of the studied patients did not
have a known risk factor, 24.4% had HIV infection and the rest
other conditions. Series in USA, South Africa and Thailand have
been described where all or almost all the patients had AIDS; likewise, series exist with patients without AIDS but with a percentage
of underlying disease and series of only immunocompetent patients.
It is important to highlight the high percentage of immunocompetent
patients in our series, which is similar to that described in China,
Taiwan and Brazil. C. neoformans var. grubii was recovered in 94.1%
and C. gattii in 5.9%. A few of the published series determined the
species responsible for cryptococcosis. In South Africa, 7% of the
pediatric infections are by C. gattii while in Brazil the percentage is
much higher, 29.6%. The prevalence of 2.6% found in Colombian
children is low compared with the prevalence reported in the Northern and Northeastern regions of Brazil, 32% in the State of Bahia,
24% in the State of Par
a and 9.5% in Piau. However, Colombian
prevalence is similar to those reported in Uruguay (1.3%), Venezuela
(0.91%) and USA (0.851.4%). In South Africa it has been estimated
that between 0.9% and 2% of the cryptococcosis cases occur in children <15 years. In Ghana, cryptococcosis is responsible for 6.9 % of
the meningitis with positive culture in children <18 years and in
Botswana, 2.36% of the meningeal cryptococcosis with positive culture occur in <13 years. The incidence of 0.017 cases 9 100 000
children under 16 years reported for Colombia is very low, however
in one department, the mean annual incidence is 7 times higher
(0.122 9 100 000). In South Africa it was calculated, for the year
2007, an incidence of 1 case 9 100 000 children in the general
population and 47 cases 9 100 000 children HIV+. In the Gauteng
province, it is estimated an incidence of 38 cases 9 100 000 children HIV+. In USA an annual incidence of 0.1 % among the pediatric population HIV+ was reported. Recently, China reported an
incidence of 0.43 cases 9 100 000 children <18 years.
Comments In recent years, interest in cryptococcosis in children has
been on the rise. However, epidemiological data are still scarce; factors that determine its relative rarity in the child population are still
poorly understood. Undoubtedly, new studies are needed to improve
the approximation to the handling of children affected by
cryptococcosis.
39
Poster Abstracts
P017
Molecular epidemiological and phylodynamic analyses of
Cryptococcus neoformans -Cryptococcus gattii species
complex in Taiwan
H. K. Tseng,1 W. L. Cho,2 C. P. Liu1 and Y. C. Chen3
1
Mackay Memorial Hospital, Taipei, Taiwan; 2Mackay Medical
College, New Taipei City, Taiwan and 3National Taiwan University
Hospital, Taipei, Taiwan
Background Cryptococcosis is a systemic mycosis caused by the
encapsulated, basidiomycetous yeast-like fungi Cryptococcus neoformans
P018
Isolation and identification of Cryptococcus Species from
Eucalyptus trees in Shiraz, Southern Iran
K. Pakshir, K. Zomorodian, M. Mahmoodi, A. Gharavi, H. Fakhim
and S. Ansari
Shiraz University of Medical Sciences, Shiraz, Iran
Introduction Cryptococcus gattii is an important pathogenic Cryptococcus species that has ecologically relationship with eucalyptus trees
and could affected lungs and the central nervous system in healthy
peoples.
Aim of the study To isolate and identify Cryptococcus species from
Eucalyptus trees in Shiraz, Southern Iran, by conventional and molecular method.
Material and methods A total of 406 samples were collected from
trunks (200), flowers (100) and leaves (106) of eucalyptus trees
around the city of Shiraz. Suspicious colonies were purified and subcultured. Initial identification of the isolates was performed using the
urease test, the chlamydoconidia test, presence of capsule, colony
color on Niger seed agar, and ability to growth at 37 C. For DNA
sequence analysis, the ITS regions of DNA were amplified by universal ITS1 and ITS4 primers and the PCR products were sequenced.
Results More than 100 yeasts isolated from the samples and a total
of 54 Cryptococcus species were identified as: Cryptococcus albidus 44,
Cryptococcus adeliensis 1, Cryptococcus friedmannii 3 and Basidiomycete
sp 6. Cryptococcus gattii was not isolated in this study,
Conclusion This is the first study about isolation of Cryptococcus species from Eucalyptus trees and identification by molecular methods in
Shiraz, Southern Iran. Unfortunately we could not isolate Cryptococcus gattii and more study needs to confirm our results.
Figure 1. Dendrogram of the neighbor-joining tree based on the concatenated seven loci of MLST.
40
Poster Abstracts
P019
Molecular identification of Cryptococcus species isolated
from pigeon dropping in Shiraz, Southern Iran
K. Pakshir, K. Zomorodian, H. Fakhim, A. Gharavi, M. Mahmoodi
and S. Ansari
Shiraz University of Medical Sciences, Shiraz, Iran
Introduction Cryptococcus species are usually isolated from birds
dropping and may be acquired by inhalation of the organism from
the environment.
Aim of the study Isolation and identification of Cryptococcus species
from birds guano in Shiraz, southern Iran.
Material and methods 139 pigeon droppings samples were collected from different areas in Shiraz city over a period of 1 year. The
samples were cultivated on Staib agar and suspicious colonies were
purified and subcutured. Identification of Cryptococcus species was
done using the urease test, yeast form growth on chlamydospore
agar, presence of capsule in india ink, colony color on Staib agar,
ability of growth at 37 C and internal transcribed spacer sequencing
analysis. Genomic DNA was extracted by the boiling method and
sequence analysis of the ITS regions was used for species
identification.
Results Forty seven (33.8%) out of 139 samples were positive for
Cryptococcus species. Result of sequence analysis revealed C. neoformans var. grubii 24 (17.2%), C. albidus 16 (11.5%), C. uzbekistansis 5
(3.5%), C. saitoi 1 (0.7%) and C. adelienisis 1 (0.7%).
Conclusion Our result revealed that most of the investigated places
were contaminated by different Cryptococcus species and this is a public health concern. Pigeons play an important role in the spread of
these organisms.
P020
Effect of adjunctive sertraline on QTc interval in patients
with Cryptococcal meningitis
S. Velamakanni,1 R. Kiggundu,2 J. Rhein,1 E. K. Butler,1
D. B. Meya2 and D. R. Boulware1
1
University of Minnesota, Minneapolis, MN, USA and 2Infectious
Disease Institute, Makerere University, Kampala, Uganda
Background The first prospective survey on cryptococcosis in Italy
carried in 1997-1999 drew a picture of the epidemiology of this lifethreatening disease confirming that it was mainly associated to AIDS
patients. Cryptococcus neoformans var. grubii, C. neoformans var. neoformans, and AD-hybrids were isolated with a similar frequency but
with a different geographical distribution.
Aim In the present study we carried out a new prospective survey
on cryptococcosis in Italy with the aim to compare the clinical and
microbiological data collected to those obtained ten years ago. The
study represents an update of epidemiological changing of cryptococcosis in the recent period.
Methods An Italian network including 18 hospitals was established
and cryptococcosis cases were recorded from January 2010 to
December 2013. Clinical information were collected using the same
form adopted 10 years ago to enable data comparison. One or more
isolates from each case were collected for molecular type and mating
type identification.
Results During the survey 58 cases of cryptococcosis were recorded.
Males represented 74% of the cases. The median age was 47.5 years
ranging from 24 to 81 years. Twenty-four (41%) patients were HIV
negative with different predisposing conditions. Diagnosis of cryptococcosis was mainly made by positive GXM antigen and cultures. In
three cases diagnosis relied only on positive antigen in serum and/or
cerebrospinal fluid. Sixty-four isolates were available from 58 cases.
Molecular typing showed that 22 patients were infected by C.
P021
Clinical analysis of Non-HIV, non-transplant cryptococcal
meningitis patients treated with amphotericin B and
flucytosine plus fluconazole in Western China from 2006
to 2013
41
Poster Abstracts
P022
Coupling of nucleotide homeostasis and mRNA decay in
Cryptococcus neoformans virulence and Amphotericin B
susceptibility
D. Banerjee and J. C. Panepinto
State University of New York at Buffalo, Buffalo, NY, USA
Background Nucleotide biosynthesis pathways are common therapeutic targets for cancer therapy, antiviral therapy, and anti-parasite
therapy. A combination of Amphotericin B (AmB) and a single
nucleoside analogue, 5-fluorocytosine (5-FC) is the gold standard for
anti-cryptococcal therapy. Cryptococcus neoformans encodes the necessary enzymes for de novo synthesis and salvage of purine and
pyrimidine nucleotides. Mutation of de novo uridine and guanosine
synthesis has been demonstrated to reduce virulence in mouse models of cryptococcosis. Given the requirement of nucleotide and nucleotide sugars for growth and pathogenesis of C. neoformans,
disrupting nucleotide metabolic pathways might thus be an effective
mechanism for the development of novel antifungal drugs. When de
novo biosynthesis is inhibited, salvage of nucleotides occur by utilizing recycled bases endogenously within the cell or from exogenous
sources. One such source of endogenous precursor is nucleoside
monophosphates (NMPs), the end products of mRNA degradation, a
process initiated by the deadenylase Ccr4p. Our overall hypothesis is
that nucleotide homeostasis modulates virulence and AmB susceptibility in C. neoformans and this balance is maintained partially by
mRNA turnover.
Aim To demonstrate the role of nucleotide metabolism on virulence
and AmB susceptibility of C. neoformans. To investigate the role of
mRNA turnover in the maintenance of nucleotide pools.
Methods E-tests, MIC checkerboard and time-kill assays were performed with AmB and mycophenolic acid (MPA) together to perturb
purine synthesis and investigate the drug interactions. Similar experiments were performed with a ura- FOA mutant and a ura1-delta
(dihydroorotate dehydrogenase) mutant and respective complemented strains in addition to capsule detection assay by India ink
staining. In vitro virulence assays- to determine growth at 37 C,
capsule production by India ink staining, cell wall integrity by growing in presence of 0.03% SDS and melanin synthesis by growing in
Asparagine agar supplemented with L-DOPA were performed. mRNA
stability assays were performed with ccr4-delta mutant during
carbon starvation and levels of ribosomal protein transcripts were
measured by Northern blot analyses. Time-kill and growth assays
were done with ccr4-delta mutant in presence of MPA and exogenous guanine. Levels of de novo and salvage genes were measured
by qRT-PCR.
Results C. neoformans ura- and ura1-delta mutants were more susceptible to AmB, and this was reverted by complementation. Addition
of uracil/uridine to the medium did not reverse this hypersensitivity
to AmB but the supplementation restored capsule formation in the
otherwise acapsular strains. ura1-delta mutant also demonstrated
growth sensitivity at 37 C and in presence of SDS. Treatment with
MPA in wild type also showed increased susceptibility to AmB, displaying synergistic interactions in checkerboard MIC and killing
42
assays. Results with ccr4-delta mutant showed stabilization of ribosomal protein (RP) transcripts during carbon starvation. Analysis of
gene expression revealed an up-regulation of the nucleotide synthesis
machinery. Time-kill assays in the presence of MPA demonstrated
enhanced killing of the mutant compared to wild type that was rescued by exogenous guanine. It also exhibited increased sensitivity to
AmB.
Discussion Nucleotide depletion, either due to a drug or a mutation,
potentiates the antifungal efficacy of AmB. Our data also suggests a
role for mRNA turnover in maintaining cellular nucleotide homeostasis, as a ccr4-delta mutant is both AmB sensitive and nucleotidestarved. Future studies will aim to quantify and compare the intracellular nucleotide pools in wild type and ccr4-delta mutant during carbon replete and deplete conditions. We will also conduct experiments
to investigate the mechanism of enhanced AmB efficacy during
nucleotide depletion that may aid in identifying novel antifungal
targets.
P023
The novel fungal Cyp51 inhibitor VT-1129 demonstrates
potent in vivo activity against Cryptococcal meningitis
with an improved formulation
L. K. Najvar,1 N. P. Wiederhold,1 A. Alimardanov,2 J. Cradock,2
X. Xu,2 M. Behnke,2 E. A. Ottinger,2 W. J. Hoekstra,3
E. P. Garvey,3 S. R. Brand,3 R. J. Schotzinger,3 W. R. Moore,3
R. Bocanegra,1 W. R. Kirkpatrick1 and T. F. Patterson1
1
University of Texas Health Science Center at San Antonio, San
Antonio, TX, USA; 2NIH/Therapeutics for Rare and Neglected
Diseases, Bethesda, MD, USA and 3Viamet Pharmaceuticals, Inc.,
Durham, NC, USA
Background Cryptococcal meningitis is a significant cause of morbidity and mortality in immunocompromised patients. Even with
appropriate therapy, morbidity and mortality associated with this disease remains unacceptably high. Thus, there is a need for new antifungal therapies against this invasive mycosis. VT-1129 is a novel
fungal-specific Cyp51 inhibitor with potent in vitro and in vivo activity against Cryptococcus species (Najvar et al. 8th ICCC, 2011 &
accompanying abstract).
Aim The aim of this study was to evaluate the in vivo efficacy of a new
solid-state formulation of VT-1129 against cryptococcal meningitis.
Methods ICR mice were inoculated intracranially with C. neoformans
USC 1597. Treatment with oral VT-1129 (VT-1129 MIC
0.12 lg ml1, 100% inhibition), oral fluconazole (MIC 1 lg ml1,
50% inhibition), or placebo began 1 day later. In the fungal burden
arm (N greater than equal 10 mice/group), treatment consisted of
VT-1129 0.1 to 20 mg kg1 day1 or fluconazole 10 and
20 mg kg1 twice daily. Treatment continued for either 7 or
14 days, and brains and plasma were collected on day 8 or 15,
1 day after therapy had stopped. In the survival arm (N = 10 mice/
group), treatment consisted of VT-1129 at 10 and 20 mg kg1 once
daily by oral gavage, fluconazole at 10 and 20 mg kg1 twice daily
by oral gavage, or placebo. Treatment continued until day 10 after
which mice were monitored off-therapy until day 30 to assess survival. Fungal burden was assessed by colony-forming units per gram
of brain tissue (CFU per gram). VT-1129 plasma and brain concentrations were measured by LC/MS-MS. Survival was assessed by
Kaplan-Meier analysis and differences in brain tissue fungal burden
were assessed for significance by ANOVA with Tukeys post-test for
multiple comparisons. Non-linear regression analysis was used to
assess the relationship between VT-1129 concentrations and tissue
burden.
Results VT-1129 at doses of greater than equal 0.3 mg kg1 day1
and each dose of fluconazole significantly reduced brain tissue fungal
burden compared to placebo control after both 7 and 14 days of dosing. Reductions in fungal burden in mice dosed with VT-1129
Poster Abstracts
ranged between 0.69 to 6.06 log CFU g1 in mice dosed for 7 days,
with a 3.48 log CFU g1 reduction achieved with a VT-1129 mean
plasma trough concentration of 1.4 lg ml1. Cryptococcal CFUs
were not detected in animals treated with VT-1129 20 mg kg1 with
a mean trough concentration of 21 lg ml1. After 14 days of dosing, reductions in fungal burden from 0.39 to 6.67 log CFU g1 were
observed, with a 4.44 log CFU g1 reduction achieved and a VT1129 mean trough concentration of 0.95 lg ml1. Cryptococcal
CFUs were not detected in mice treated with VT-1129 at 10 or
20 mg kg1, with a mean trough concentration of 15 lg ml1 in
the 10 mg kg1 group. Non-linear regression analysis demonstrated
an inverse relationship between VT-1129 concentrations on days 8
and 15 and reductions in fungal burden (R2 values 0.790.92).
VT-1129 brain tissue concentrations were ~2-fold higher than those
achieved in the plasma in all experiments. A survival advantage was
also observed with VT-1129. Median survival and percent survival
with VT-1129 20 mg kg1 (>30 days and 100%) were significantly
improved compared to placebo (10.5 days and 40%; P < 0.01). In
addition, the percent survival with VT-1129 20 mg kg1 was also
markedly improved compared to either fluconazole dose group (range
4060% survival).
Conclusions The new formulation of the fungal Cyp51 inhibitor
VT-1129 demonstrated potent efficacy in this murine model of cryptococcal meningitis. Both reductions in brain tissue fungal burden
and improvements in survival were observed, with undetectable CFUs
at the higher doses. These data demonstrate the potential utility of
VT-1129 to have a marked impact in the treatment of cryptococcal
meningitis.
P024
Susceptibility testing of VT-1129, a novel fungal CYP51
inhibitor, against Cryptococcus neoformans and
Cryptococcus gattii
S. R. Lockhart,1 N. Iqbal,1 C. B. Bolden,1 N. T. Grossman,1
E. A. Ottinger,2 E. P. Garvey,3 S. R. Brand,3 W. J. Hoekstra,3
W. R. Moore3 and R. J. Schotzinger3
1
Centers for Disease Control and Prevention, Atlanta, GA, USA;
2
National Inistitutes of Health, Bethesda, MD, USA and 3Viamet
Pharmaceuticals, Inc, Durham, NC, USA
Background Therapeutic options to treat cryptococcal meningitis
(CM) in resource-limited countries are inadequate, leading to an estimated 600 000 deaths worldwide annually. Furthermore, fluconazole which is typically used as maintenance therapy following
meningitis must be taken daily and has limitations such as drugdrug interactions and a Class D pregnancy warning. VT-1129 is a
potent and highly selective novel inhibitor of fungal CYP51 (lanosterol demethylase). As an oral induction treatment, VT-1129 is superior to fluconazole in the murine model of CM caused by C.
neoformans (see adjoining abstract), and has a significantly longer
half-life than fluconazole which may decrease the dosing schedule
and improve patient compliance.
Aim To test the in vitro growth inhibition of VT-1129 against a global collection of C. gattii and C. neoformans isolates.
Methods The collection consisted of 100 isolates of Cryptococcus neoformans from patients in South Africa and 300 isolates of Cryptococcus gattii from Africa, Australia and North America. Cryptococcus
isolates were identified to the species level and isolates of C. gattii
were further characterized to molecular type. Isolates of all four C.
gattii molecular types were used in this analysis. Testing was performed as outlined in CLSI document M27-A3. MIC testing was performed using RPMI broth in 96-well microdilution plates with final
dilution concentrations of VT-1129 ranging from 0.015 to
8 g ml1 (with final in-assay DMSO concentration of 1%). Dilution
plates were stored at 70 C until used. All MIC values were determined visually as the lowest drug concentration at which there was
a 50% and a 100% decrease in growth after 72 h of incubation at
35 C. VT-1129 MIC values were compared to fluconazole MIC values for the same set of isolates.
Results C. neoformans MIC values against VT-1129 were very low
with a 50% inhibition range of 0.015 to 0.125 g ml1 and a
100% inhibition range of 0.015 to 2 g ml1. The VT-1129 MIC50
and MIC90 ranges were much lower (seven Log2 dilutions) than the
fluconazole MIC50 and MIC90 ranges against the same set of isolates
at 50% inhibition. The MIC values at 100% inhibition with VT-1129
were even lower than the MIC values with fluconazole at 50% inhibition. The C. gattii MIC values against VT-1129 were similarly low
with a 50% inhibition range of 0.015 to 1 g ml1 and a 100%
inhibition range of 0.125 to 8 g ml1), but the MIC50 and MIC90
values were two and three log2 dilutions higher, respectively, than
those of C. neoformans. The C. gattii VT-1129 MIC50 and MIC90 values at 50% inhibition were six and five log2 dilutions lower, respectively, than those of fluconazole.
Previous studies have shown that C. gattii antifungal susceptibility
patterns vary according to the molecular type of the isolate, with VGII
isolates generally having the highest MIC values. This was largely true
for VT-1129 as well. At 50% inhibition, the MIC50 and MIC90 values
for VGIV isolates were lowest (0.015 and 0.06 g ml1) followed by
VGI isolates (0.03 and 0.125 g ml1). VGII isolates and VGIII isolates
had the highest MIC50 and MIC90 values, but they were only 13 dilutions higher than those of the VGI isolates.
Conclusions MIC values for VT-1129 against C. neoformans and C.
gattii are low. VT-1129 has excellent activity against Cryptococcus
isolates from Africa where the burden of Cryptococcus remains very
high, and against isolates of C. gattii with high MIC values to fluconazole. Based on these data and the robust in vivo efficacy in a murine model of CM (see adjoining abstract), VT-1129 is currently
undergoing IND-enabling studies that will support the clinical investigation of its potential to reduce the high mortality rate of this devastating disease.
P025
In vitro antifungal susceptibilities of Ugandan clinical
isolates
K. Smith,1 B. Achan,2 T. McDonald,1 A. Akampurira,2
L. Okagaki,3 D. Meya,2 D. R. Boulware1 and K. Nielsen1
1
University of Minnesota, Minneapolis, MN, USA; 2Infectious
Disease Institute, Kampala, Uganda and 3University of North
Carolina, Chapel Hill, NC, USA
Cryptococcus neoformans infection results in over a half million deaths
in sub-Saharan Africa each year. A significant cause of this high
death rate in resource limited settings is due to inadequate treatment.
According to World Health Organization (WHO) guidelines, the optimal treatment for cryptococcal meningitis is amphotericin B supplemented with 5-flucytosine. In sub-Saharan Africa, 5-flucytosine is
expensive and not licensed for use in many countries, thus fluconazole is often used instead. The WHO also provides that fluconazole
may be administered singularly and is common in rural sub-Saharan
Africa due to limited resources and lack of intravenous treatment
capacity. Due to the widespread usage of fluconazole monotherapy in
sub-Saharan Africa, we examined the level of fluconazole and
amphotericin B antifungal drug resistance in Ugandan clinical isolates prior to treatment for cryptococcal meningitis. In addition,
while the CLSI photometric assay is considered the gold standard for
MIC determination, the CLSI assay requires equipment not commonly
available in resource limited settings. Thus, we also developed a macrodilution assay that could inexpensively and effectively be administered in a resource limited setting. Comparison of fluconazole
resistance results from the macrodilution assay versus the CLSI microdilution assay showed no difference between the two assays. Compared to previous studies, both the MIC50 and MIC90 appeared to
increase for fluconazole. While the amphotericin B MIC50 and MIC90
increased compared to previous studies, all isolates were still within
43
Poster Abstracts
P026
Evaluation of in vitro combination of antifungal drugs and
genotyping of clinical isolates of Cryptococcus spp. from
patients assisted at a University hospital in Brazil
F. Reichert-Lima,1 A. F. Busso-Lopes,1 L. Lyra,1 H. Taguchi,2
Y. Mikami,2 K. Kamei,2 M. L. Moretti1 and A. Z. Schreiber1
1
State University of Campinas, Campinas, Brazil and 2Chiba
University, Chiba, Japan
Background Despite all the advances in medicine, cryptococcosis
remains one of the most important systemic fungal diseases in Brazil.
The combination of amphotericin B (AMB) plus 5-fluocytosine (5-FC)
has been the best choice for the induction therapy. In Brazil, 5-FC is
not available and the treatment of cryptococcosis has been made
with AMB alone or in association with fluconazole (FCZ).
Aim the aim of this study was to evaluate the in vitro activity of the
combinations of antifungal drugs and to identify the genotypes of
Cryptococcus isolates from clinical samples in patients assisted at the
University Hospital of UNICAMP.
Methods 80 clinical isolates of Cryptococcus spp. collected from 57
patients with systemic diseases were studied. The antifungal susceptibility testing was performed according the CLSI M27-A3 (2008) for
AMB, 5-FC, FCZ, voriconazole (VCZ), itraconazole (ITZ) and terbinafine (TRB). Drug interaction was evaluated using checkerboard microdilution test design and four different combinations were
performed: AMB + 5-FC, AMB + FCZ, AMB + TRB and TRB + FCZ.
Cryptococcus species determination was accomplished using canavanine-glycine-bromothymol blue agar (CGB). Genotyping was accomplished by restriction fragment length polymorphism of the URA5
gene (URA5 RFLP).
Results 66 isolates were C.neoformans and 14 C.gattii. All C.gattii
belonged to VGII genotype and 62 (94%) isolates of C.neoformans
were in VNI genotype. Minimum inhibitory concentration (MIC)
ranges for C.neoformans were: AMB: 0.1251 lg ml1; 5-FC:
0.1252 lg ml1; FCZ: 0.258 lg ml1; TRB: 0.1252 lg ml1;
ITZ: 0.030.25 lg ml1 and VCZ: 0.0150.125 lg ml1. MICs for
C.gattii ranged from 0.25 to 1 lg ml1 for AMB; 0.54 lg ml1 for
5-FC; 216 lg ml1 for FCZ; 0.54 lg ml1 for TRB; 0.06
0.5 lg ml1 for ITZ and 0.060.25 lg ml1 for VCZ. C.neoformans
VNI genotype showed 75.80% synergistic interaction for AMB + 5FC; 79.03% for AMB + FCZ; 77.41% for AMB + TRB and 95.16%
for TRB + FCZ interaction. VNII genotype had 100% synergism effect
for all combinations. C.gattii VGII genotype showed 85.71% synergistic interaction for AMB + 5-FC; 85.71% for AMB + FCZ; 100% for
AMB + TRB and 85.71% for TRB + FCZ interaction.
Conclusion We found a good performance in all combinations, especially those involving TRB for both C.neoformans and C.gattii. C.gattii
showed more than 80% of synergism for all combinations and
44
P027
3-bromopyruvate as a potent anticryptococcal drug
M. Dylag,1 K. Niedzwiecka,1 P. Lis,1 Y. H. Ko,2 P.L. Pedersen,3
A. Goffeau4 and S. Ulaszewski1
1
Institute of Genetics and Microbiology, University of Wroclaw,
Wroclaw, Poland; 2KoDiscovery, LLC, UM BioPark, Baltimore,
MD, USA; 3Department of Biological Chemistry, John Hopkins
University School of Medicine, Baltimore, MD, USA and 4Institut
des Sciences de la Vie, Universite Catholique de Louvain,
Louvain-la-Neuve, Belgium
Background 3-bromopyruvate (3-BP), a small alkylating molecule,
is a potent anticancer drug. Its anticancer property was identified
in the laboratory of P.L. Pedersen in the year 2000. 3-BPs killing
efficacy toward liver cancer cells has been successfully demonstrated
by Y.H. Ko in animals and humans with no apparent side effects.In
our previous studies we have demonstrated that3-BPenters the
yeast Saccharomyces cerevisiae cells through the lactate /pyruvate H+
symporter Jen1 membrane protein. We also have demonstrated that
3-BP is not submitted to the PDR network of ABC efflux pumps in
yeast.
Aim The aim of our present study was to determine the spectrum
and molecular mechanism of antifungal activity of 3-BP toward different yeast-like and filamentous fungi.
Methods The killing effect of 3-BP on various strains was examined
by standard spot tests method and also by a microdilution bioassay
(CLSI protocol M27-A2). The influence of sub-MIC concentrations of
3-BP on the intracellular ATP level in Cryptococcus neoformans was
determined using the ATPliteTM system (PerkinElmer). The 3-BP
uptake assays were performed using [14C]-labeled 3-bromopyruvate
based on the method previously described for radioactive L-lactate.
The intracellular level of glutathione (GSH) was determined spectrophotometrically according to the published procedure.
Results Our studies demonstrate different susceptibilities toward 3BP even between strains from the same Cryptococcus like genus.
Among the tested fungi, all the C. neoformans strains were particularly sensitive to 3-BP (MIC 0.120.2 mmol l1). The best killing
activity of 3-BP toward this fungal pathogen correlates with its intracellular high accumulation and also with naturally low level of GSH.
A rapid and drastic decrease in intracellular ATP after 3-BP treatment leads to fungal cell death. The induction of DOPA-melanin synthesis in C. neoformans cells may offer some protection against the
toxic effects of 3-BP.
Conclusion Summarizing 3-BP could be a novel promising anticryptococcal drug.
Poster Abstracts
P028
Evaluation of three commercial methods for antifungal
susceptibility testing of non-grubii Cryptococcus
neoformans strains
M. F. Colom Valiente,1 C. Linares,1 F. Hagen,2 V. Rosa1 and
M. Torreblanca1
1
University Miguel Hernandez, Sant Joan dalacant, Spain and
2
Canisius-Wilhelmina Ziekenhuis, Nijmegen, The Netherlands
Background Among the non-Candida yeasts, members of the Cryptococcus neoformans-Cryptococcus gattii complex have been the most
common species recovered among clinical isolates, as well as the second most common cause of severe fungal infection due to pathogenic
yeasts. These cryptococcal infections are associated with high mortality rates. Some previous studies on the in vitro antifungal susceptibility of C. neoformans showed that species and genotype influenced the
susceptibility to antifungal drugs used to treat cryptococcosis. In our
geographical area (Spain and part of Europe) genotypes VNIII and
VNIV of C. neoformans are especially prevalent in contrast to the
world wide most prevalent C. neoformans var. grubii.
Aim The evaluation of three different commercial methods for the
antifungal sensitivity testing of Cryptococcus neoformans VNIII and
VNIV (C. n. var neoformans) isolates.
Methods Commercial methods tested were Sensititre Yeast One
-SYO- (Trek Diagnostic Systems), Etest (AB Biodisk) and SensiQuattro
Candida EU (Liofilchem). A total of 31 isolates of C. neoformans var.
neoformans serotype D(genotype VNIV) and C. neoformans hybrids
serotype AD (genotype VNIII) were tested. The isolates were previously tested by the reference microdilution standardized method for
yeasts (CLSI M27-A3). Amphotericin B, Posaconazole, Fluconazole
and Voriconazole were included in all test performed while Itraconazole was only tested by SYO. Flucytosine, Caspofungin, Micafungin
and Anidulafungin were only studied in Sensititre YeastOne (SYO)
and Sensiquattro panels.
Results As it was expected from previous published works, none of
the echinocandins tested showed antifungal activity against any of
the C. neoformans studied. All isolates produced clearly visible growth
only after 72 h of incubation in SYO and E-test and even longer periods (5 days) in SensiQuattro panel. We could not read the MIC endpoint clearly at 48 h in most of them because the growth in all
isolates and systems was insufficient.
Sensitivity
Specificity
PPV
NPV
P value
Average > 20 mm Hg
Average 28 mm Hg
Average > 35 mm Hg
Minimum 35 mm Hg
95%
70%
49%
40%
34%
66%
90%
95%
57%
65%
82%
88%
88%
71%
66%
64%
0.62
0.68
0.68
0.68
0.029
0.006
0.006
0.007
(0.52-0.77)
(0.56-0.80)
(0.56-0.80)
(0.56-0.80)
Sensitivity
Specificity
PPV
NPV
C-Statistic(95% CI)
P value
Average 5 mm
Average 6 mm
72%
58%
55%
58%
44%
79%
80%
34%
0.08 (0.28-0.57)
0.1 (0.103-0.56)
0.0006
0.0006
P029
Non-invasive assessment of intracranial pressure in
Cryptococcal meningitis using Tonometry and Ocular
Sonography
H. W. Nabeta,1 N. C. Bahr,2 J. Rhein,2 N. Fossland,2
A. N. Kiragga,1 D. B. Meya,1 S. Dunlop3 and D. R. Boulware4
1
Infectious Diseases Institute, Makerere University, Kampala,
Uganda; 2University of Minnesota, Minneapolis, MN, USA;
3
Hennepin County Medical Center, Minneapolis, MN, USA and
4
Center for Infectious Disease & Microbiology Translational
Research, Minneapolis, MN, USA
Background Cryptococcal meningitis (CM) is associated with
increased intracranial pressure (ICP). Daily therapeutic lumbar punctures (LP) are recommended when ICP is >250 mmH2O, yet manometers are unavailable in Africa and not always used where
available.
Aim We assessed whether measuring (i) intraocular pressure by
tonometry or (ii) optic nerve sheath diameter using ultrasound could
be used as non-invasive surrogates for predicting increased ICP in CM.
Methods Ninety eight HIV-infected Ugandans with suspected meningitis (86% CM) had intraocular pressure measured in both eyes using
a handheld tonometer (CareFusion, McGaw Park, IL, USA) prior to
LP (n = 77) and/or optic nerve sheath diameter (ONSD) measured by
ultrasound (Sonosite, Bothell, Washington) before and after LP
(n = 89). We determined the diagnostic performance of these noninvasive techniques for predicting increased ICP in comparison with
LP opening pressure using manometry.
Results The median ICP was 225 (IQR: 135, 405) mmH2O. The median intraocular pressure was 28 (IQR: 22, 37) mmHg. ICP and intraocular pressure moderately correlated (rho = 0.45; P < 0.001).
Above an average >28 mmHg intraocular pressure, there was 73%
sensitivity and 66% specificity for predicting ICP >250 mm H2O (odds
ratio = 4.6, 95% CI: 1.811.9, P = 0.002). As the intraocular pressure increased, the proportion with elevated ICP (i.e. positive predictive value) increased. The average intraocular pressure from both eyes
had better diagnostic performance than just one eye. Ultrasound correlated moderately with ICP (rho = 0.36, P = 0.0006). With an average of ONSD >5 mm, there was sensitivity of 72%, specificity of 55%
for predicting ICP >250mmH2O. (odds ratio = 1.60, 95% CI: 1.12
2.30, P = 0.0150). There was minimal difference between ONSD measurement before and immediately after lumbar puncture. There was
an observed difference between the measured ONSD in both eyes.
Conclusions Non-invasive intraocular pressure measurements by
ocular tonometry or ultrasound each correlate moderately with
intracranial measurement, but both are a suboptimal replacement for
actual ICP measurement using a manometer at time of LP.
45
Poster Abstracts
P030
Scorpion-venom derived antimicrobial peptides with
antifungal activity against Cryptococcus neoformans
F. Guilhelmelli, N. Vilela, L. S. Derengowski, M. R. Mortari,
E. F. Schwartz, P. Albuquerque and I. Silva-Pereira
Universidade de Brasilia, Brazil
Background All classes of organisms, including plants, vertebrates
and invertebrate animals and microorganisms produce antimicrobial
peptides (AMPs) as defense tools against infection. In mammalians,
AMPs are components of innate immune system that can act both as
antimicrobial agents and as important immune modulators enhancing its protective functions. Our group has been working in the identification of AMPs from arthropod venom glands with potential
antimicrobial or immunomodulatory activity, in special against pathogenic fungi. Considering the relatively low number of antifungal
drugs currently available and their high cost and toxicity, there is a
strong need for the development of therapeutic alternatives especially
with the increasing rise of resistant strains.
Aim The present work aimed to evaluate the antifungal activity of
scorpion-venom derived AMPs against the human pathogenic fungus
Cryptococcus neoformans. C. neoformans is a ubiquitous distributed
encapsulated yeast responsible for more than 600 thousand deaths
per year worldwide.
Methods Chemically synthetized AMPs derived from sequences
obtained from cDNA libraries of the venom gland of Brazilian Cerrado scorpions. The sequences were chosen based on structural similarities with previous isolated antimicrobial peptides from scorpions and
amphibians. Cells of different strains of C. neoformans were grown in
the presence or absence those peptides using the microdilution broth
susceptibility assay described in the M27-A2 guidelines with some
modifications and amphotericin B as a positive control. After 48 h of
incubation fungal growth was evaluated by optical density measurement at 630 nm. The MIC50 and MIC90 values for each peptide
were calculated using non linear regression analysis.
Results and discussion The MIC50 against C. neoformans strain
H99 ranged from 4.4 to 75.2 lmol l1, although none of the
46
P031
Erg6 is a potential drug target in Cryptococcus neoformans
F. F. M. Oliveira,1 H. C. P. Costa Paes,1 L. D. F. Peconick,1
P. Albuquerque,1 A. M. Nicola,1 M. H. Melo,1 F. L. Fonseca,2
M. L. Rodrigues,2 J. A. Alspaugh,3 M. S. Soares Felipe1
and L. F. Fernandes1
1
Universidade de Braslia, Brasilia, Brazil; 2Universidade Federal do
Rio de Janeiro, Rio de Janeiro, Brazil and 3Duke University
Center, Durham, NC, USA
Background Nowadays adverse effects of and cases of resistance to
the current drugs used against human fungal pathogens are causes
of great concern. Treatment failures lead to the urgent need of developing new antifungal drugs, but this requires the identification and
characterisation of new potential molecular targets. Erg6, a sterol C24 methyltransferase, acts on the ergosterol biosynthetic pathway in
the conversion of zymosterol to fecosterol and is of interest as a
potential antifungal target, as this reaction occurs exclusively in
fungi, whereas the mammalian hosts have specific enzymes for the
conversion of zymosterol to cholesterol.
Aim To characterize Erg6 as a potential antifungal target we have
investigated its role in Cryptococcus neoformans through deletion of
the ERG6 gene and phenotypic analyses of the mutant.
Methods A deletion cassette was constructed by DJ-PCRand inserted
on theH99 (serotype A) strain by biolistic transformation. The erg6
strain was confirmed by PCR and Southern blotting. A reconstituted
strain was generated by transforming the mutant strain with the
ERG6 gene alongside the pJAF1 plasmid containing the NEOR resistance marker. The mutant and reconstituted strain were tested in vitro for common phenotypes associated with virulence: capsule
induction, melanisation, growth at 37 C, urease and phospholipase
secretion and resistance to several stressor agents such as Congo
Red, SDS, caffeine, Calcofluor White (cell wall), NaCl and KCl (osmotic) and H2O2 (oxidative). The strains were tested for virulence in the
in vitro J774.A16murine macrophage model of phagocytosis followed
by CFU counts. The in vivo virulence was assessed by survival curves
in the Galleria mellonella alternative model of infection at 37 C. Drug
susceptibility tests were performed in solid medium with Fluconazole,
Itraconazole, Cetoconazole, Amphotericin B, Nystatin, FK506, Cerulenin, Brefeldin A and Terbinafin. The Minimal Inhibitory Concentration was determined for each drug according to NCCLS. The sterol
composition of the cell membranes of the mutant strain was evaluated by HPLC.
Poster Abstracts
P032
Clinical trial protocol and report on recruitment:
comparing flucytosine-fluconazole combination therapy to
standard of care in western Kenya
K. E. Feldman,1 M. A. Jacobson,1 C. C. Bii,2 A. Mohammed,2
J. K. Kwasa,3 E. A. Bukusi,2 C. R. Cohen1 and W. Meyer4
1
University of California, San Francisco, CA, USA; 2Kenya Medical
Research Institute, Nairobi, Kenya; 3University of Nairobi, Nairobi,
Kenya and 4Yale University, New Haven, CT, USA
Background Opportunistic Cryptococcus spp. infection is the second
leading cause of HIV-related deaths in resource-limited settings.
Screening asymptomatic HIV-infected individuals with advanced
immunosuppression for serum cryptococcal antigen (sCrAg) clearly
identifies a population at high risk of cryptococcal meningitis (CM)
and death in resource-limited settings. Currently there is wide variation in practice and little evidence to guide the treatment of early
cryptococcal infection, also known as asymptomatic cryptococcal
antigenemia, in HIV-infected individuals with advanced immunosuppression. The mainstay of anti-cryptococcal therapy in
resource-limited settings is oral fluconazole, though preliminary
evidence suggests this may not be an effective treatment for early
cryptococcal infection. Thus, there is a critical need for potent
therapies that (i) can be safely administered in resource-limited settings and (ii) are effective in a heterogeneous population of HIVinfected individuals with advanced immunosuppression and early
cryptococcal infection who are initiating anti-retroviral therapy
(ART).
Aim To compare the safety and preliminary efficacy of fluconazole
plus flucytosine in combination to the standard of care, fluconazole
treatment alone, in a population of HIV-infected patients with early
cryptococcal infection in Western Kenya.
Methods This is a Phase IIB randomized open label clinical trial
based at two sites supported by Family AIDS Care and Education Services (FACES) in Western Kenya. We will enroll a consecutive sample
of 100 HIV-infected adults with CD4 + T-cel count 100 cells ll1
and sCrAg titer 1 : 2 who have no signs or symptoms of severe,
systemic cryptococcal infection.
Individuals who meet inclusion and exclusion criteria and consent
to participate in the study will be randomized to combination therapy
with
oral
fluconazole
(1200 mg day1)
plus
flucytosine
(100 mg kg1 day1) or fluconazole alone for the fourteen days of
therapy. Subsequently both groups will receive anti-retroviral therapy
P033
Anticryptococcal activity of secondary metabolites
laevicarpin and polygodial isolated from Brazilian plants
S. U. Purisco,1 D. S. A. Maciel,2 J. H. G. Lago,2 A. G. Tempone1
and M. S. C. Melhem1
1
Adolfo Lutz Institute, Sa~o Paulo, Brazil and 2Sa~o Paulo
University, Sa~o Paulo, Brazil
Background Cryptococcosis is the second leading cause of death
among systemic mycoses in Brazil, and 23.9 out of every 1000 AIDS
related deaths are attributed to cryptococcal infection. Among C. gattii molecular types VGII is the most frequent type encountered in
Latin America. It is recognized that the clinical findings and therapeutic management of C. gattii cases present some distinct aspects
from those caused by C. neoformans. Cryptococcosis has a narrow
therapeutic arsenal and the antifungal agents used in clinical settings
are limited due to drug toxicity, high costs, low availability, and clinical resistance. So, there is an urgent need for the discovery of new
drugs to treat Cryptococcosis. Natural products are a rich source of
antimicrobial compounds and Brazil has a rich plant biodiversity
with a great potential for drug prototypes.
Aim In this study we investigated the efficacy of two metabolites,
laevicarpin and polygodial, isolated from Atlantic Forest plant species Piper laevicarpu (Piperaceae) and Drimys brasiliensis (Winteraceae), against C. gattii strain-type WM 178 designed molecular type
VGII.
Methods Hexane extract from steam bark of D. brasiliensis and
CH2Cl2 extract from leaves of P. laevicarpu were obtained using
accelerate solvent extractor ASE300. After solvent elimination, both
extracts were individually subjected to several chromatographic procedures, including column chromatography over SiO2 and/or
Sephadex LH-20 to afford, respectively, polygodial and laevicarpin.
The in vitro activity of polygodial and laevicarpin against VGII
strain-type was investigated according to the method (M27-A3,
2008) with modifications. We employed 96-well flat-botton plates
and range concentrations of 200 to 1.5625 lmol l1 of each compound. The dye Alamar blue was used to detect the cell viability.
The 100% inhibition was assessed by visual and spectrophotometer
(570 nm) reading after 48 h of incubation. All experiments were
performed in triplicate. C. krusei (ATCC 6258) and C. parapsilosis
47
Poster Abstracts
P034
Suceptibility profile of a Brazilian collection of clinical
Cryptococcus gattii isolates
L. X. Bonfietti,1 C. D. Pham,2 M. W. Szeszs,1 D. C. Silva,1
M. A. Martins,1 S. R. Lockhart2 and M. S. C. Melhem1
1
Instituto Adolfo Lutz, Arac_atuba, Brazil and 2Centers for
Disease Control and Prevention, Atlanta, GA, USA
Cryptococcosis is a life-threatening disease and remains a difficult
management issue in developing countries. Although cryptococcosis
has generally being associated with Cryptococcus neoformans species
worldwide, particularly in HIV-positive and transplant recipients,
Cryptococcus gattii is notable for causing disease in healthy persons.
Clinically, infections caused by C. gattii result in a higher incidence
of lung and brain granulomas and neurological complicationsin comparison with C.neoformans infections. Importantly, it is recognized
that C. gattii isolates usually show high minimum inhibitory concentrations (MIC) to fluconazole and other azole drugs used in the management of cryptococcosis cases. This study aimed to describe the
susceptibility of 54 clinical isolates of C. gattii molecular type VGII
and 4 of molecular type VGI from a Brazilian culture collection.
A total of 58 unique isolates of C. gattii were analyzed at the Centers for Disease Control and Prevention (CDC -Atlanta, GA, USA). Isolates were from human cryptococcosis cases collectedbetween 1994
and 2012 in hospitals located in the southeast part of Brazil. All isolates were previously identified by conventional and molecular methods to be C. gattii and the molecular types were confirmed by
multilocus sequence typing (MLST). The antifungal drugs tested were
amphotericin B (AmB), fluconazole (FZ), itraconazole (IZ), voriconazole (VZ), flucytosine (5-FC), andposaconazole (PZ). For all drugs
except AmB, MIC determinations were performed as recommended
by Clinical and Laboratory Standards Institute (CLSI) documents
M27-A3 (CLSI, 2008) using RPMI broth and frozen panels custom
manufactured by TREK Diagnostics (Cleveland, OH, USA). For AmB,
the MIC was determined by the E-Test (bioMerieux, Fr) method and
the result was defined as the point of 100% growth inhibition. Data
are reported as MIC ranges and MICs of each antifungal agent necessary to inhibit 50% (MIC50) and 90% (MIC90) of the isolates tested.
Quality control isolates C. parapsilosis ATCC 22019 and C. krusei
ATCC 6058 were included on each day of testing and were always
found to be within the recommended range. Because no breakpoints
are available, to interpret the susceptibility data we used the MIC values to classify the isolates into wild-type (WT) and non-wild-type
(non-WT) categories according to the epidemiological cutoff (ECV)
values. ECVsare the end point of the wild-type distribution of MIC
values and were outlined previously as the MIC value encompassing
at least 95% of the wild-type distribution. The distribution of MICs of
54 VGII and four VGI C. gattii molecular types are shown in Table 1.
For FZ we found two VGII and one VGI non-WT isolates (VGI, 8 mg/
l1 and VGII, 32 mg/ l1). None of the isolates showed MICs above
the ECV for PZ. The rate of itraconazole non-WT MICs were higher
(5/58, 8.6%) than those of the other three azoles. Although we
tested only four isolates of molecular type VGI,we didnt observed a
difference in antifungal susceptibility for FZ,IZ, PZ, 5-FC orAmB,
when compared to VGII strains. However, one strain ofmolecular
typeVGI (1/4, 25%) showed high FZ, IZ and PZ MICs (8, 0.25 and
0.25 mg/ l1). Our results show that, for the most part, C. gattii isolates from Brazil have similar antifungal MIC values to C. gattiiisolates from other parts of the world, and they confirm the previously
established ECVs.
Table 1 Distribution of MIC values of 54 VGII and fourVGI C. gattii molecular types.
MICs equal or higher than the ECVs proposed by Espinel-Ingroff and co-authors, 2012.
48
Poster Abstracts
Table 2a MIC range, mode, geometric mean (GM) MIC, MIC50 and MIC90 of all C. gattii isolates by molecular type of the isolate.
Table 2b
P035
P036
49
Poster Abstracts
(165 mg1x/day for 2 weeks). One patient received also 5FC (200 mg
daily) after the first week. Successful treatment was observed in all
patients in despite of occurrence of disseminate form in 2 patients.
Initial fungal burden for the six patients was: 2500; 10 000;
21 000; 57 000; 99 000 and 880 000 CFU ml1 of CSF.Sterile CSF
culture after day-7 of treatment was observed for two patients and
day-14 for one patient.Mycological cure was obtained for remain
three patients only after a month of therapy. Using isolates from four
patients, we found that the estimated RivDC for AmB was
0.5 mg l1, for the remaining 2 the RivDC was 1 mg. In all, but one
(CnVNII) case the etiological agent was CnVNI showing AmB-MIC of
0.5 mg l1, FLC-MIC > 8 mg l1. The AmB cidal-effect was similar
for all isolates (612 h).Checkerboard method showed indifferent
interaction (2 > FICI > 1.25) between AmB and 5FC in tests with all
isolates.
Discussion and conclusions All patients evolved to cure, even
with causative isolates showing high FCL-MIC values indicating low
susceptibility. Antifungal susceptibility testing with AmB suggested
good clinical-laboratory correlation (high cidal-effect by low TKvalues
and high inhibitory action by low MICs-AmB). Drug regimens in
50% of our patients did not render CSF sterile after 14 days of treatment suggesting therapy should be modified early in the course of
treatment to improve probability of survival to 10th week.Of note, all
cases presented significant fungal burden exceeding 2500 CFU ml1
in pretreatment CSFsample. This value represents the maximum
inoculum volume recommended in M27-A-CLSI method. In agreement with previous authors we found suitable additional studies to
evaluate an alternative parameter incorporating the measure of
severity of meningitis corresponding to number of organisms found
in the pretreatment CSFsample aiming predict response in individual
patients.
P037
Correlation of CLSI and EUCAST in-vitro antifungal
susceptibility with clinical outcome of patients with AIDS
associated cryptococcosis from India
A. Chowdhary,1 S. Kathuria,1 A. Prakash1 and J. F. Meis2
1
Vallabhbhai Patel Chest Institute, Delhi, India and 2CanisiusWilhelmina Hospital, Nijmegen, The Netherlands
Background Cryptococcus neoformans, is the most common etiologic
agent of cryptococcosis in immunosuppressed hosts and associated
with significant morbidity and mortality. India has world-wide the
second largest burden of cryptococcosis due to an estimated population of 3.1 million to 9.4 million persons living with HIV-AIDS. The
data on antifungal susceptibility profiles along with the outcome of
therapy in patients of cryptococcosis helps in the development of
effective treatment strategies.
Objective To investigate the statistical correlation of MICs obtained
by CLSI and EUCAST antifungal susceptibility testing of C. neoformans
var. grubii from 45 HIV-positive Indian patients with their therapeutic outcome.
Materials and methods A total of 45 patientswith culture proven
cryptococcosis were included in a prospective study during 2008
2012. Of 45 patients, 96 C. neoformans isolates comprising 75 from
CSF, 9 blood, 8 sputum, 2 urine and one from a bronchial aspirate
were analysed for AFST by CLSI and EUCAST methods. Twenty nine
patients received combination therapy of amphotericin B
(1 mg kg1) and fluconazole (400 mg day1) whereas 16 patients in
addition received flucytosine (100 mg kg1). Genotyping was done
using microsatellite typing. Susceptibility was determined for amphotericin B (AMB), flucytosine (FC), fluconazole (FLU), voriconazole
(VRC), posaconazole (POS), isavuconazole (ISA) and itraconazole
(ITC) by CLSI and EUCAST methods. Statistical differences between
mean MICs of CLSI and EUCAST were assessed using Students t-test.
Discrepancies of more than two dilutions among MIC results were
used to calculate the essential agreement (EA).
50
P038
Drug delivery in C. neoformans using nanoparticles
C. B. Nichols, C. Vazquez and J. A. Alspaugh
Duke University Medical Center, Durham, NC, USA
Background Many compounds with potential efficacy against
microbial pathogens are ineffective because of poor intrinsic ability to
enter the target cell. The cell surface of C. neoformans possesses two
structures that inhibit drug entry: the cell wall and polysaccharide
capsule. In prior studies we demonstrated that the farnesyl transferase inhibitors manumycin and tipifarnib have a higher efficiency
against capsule mutants of C. neoformans compared to wild type
strains, suggesting that the capsule offers protection against both of
these drugs. In recent years, the field of nanomedicine has developed
polysaccharide-based nanoparticles as drug delivery devices. Chitosan, a principal cell wall component of C. neoformans, is also one of
the polysaccharides capable of forming nanoparticles. Exogenous
chitosan also has antifungal properties against C. neoformans.
Aim We hypothesize that drugs packaged into chitosan-derived
nanoparticles may be able to traverse the C. neoformans capsule/cell
wall barrier into the cell resulting in increased activity against C.
neoformans.
Methods Purified chitosan was mixed with heparin to generate
chitosan-based nanoparticles. In control samples, chitosan nanoparticles were prepared in the absence of drug. In experimental samples,
various concentrations of compounds with varying antifungal activity were incorporated into the nanoparticles. These preparations were
tested against a wild type C. neoformans grubii strain (H99).
Results We observed minimal but measurable growth inhibition of
C. neoformans in samples containing chitosan/heparin nanoparticles.
In addition, there was increased growth inhibition in samples containing manumycin- and tipifarnib-containing chitosan/heparin
nanoparticles.
Discussion/conclusion As we hypothesized, chitosan-derived nanoparticles containing various antifungal agents inhibited growth of C.
neoformans. This demonstrated that chitosan nanoparticles were able
to incorporate these compounds. In addition, the chitosan/heparin
nanoparticles without drug also had a subtle inhibitory effect on C. neoformans. This demonstrates that the chitosan polysaccharide packaged
into a nanoparticle is able to inhibit growth and also that the combination of chitosan and drugs may have a synergistic effect against C. neoformans. In conclusion, our results indicate that chitosan-derived
nanoparticle drug delivery in C. neoformans is a viable strategy to
enhance cell entry for varied compounds, potentially bypassing the
barriers provided by the cell wall and polysaccharide capsule.
Poster Abstracts
P039
High-dose fluconazole for the treatment of Cryptococcal
meningitis in HIV-infected individuals
R. A. Larsen,1 K. Hollabaugh,2 U. G. Lalloo,3 M. M. Ssemmanda,4
L. M. Momanyi,5 D. K. Lagat,6 J. G. Hakim,7 T. T. Ly,8 E. Hogg,9
L. Komarow2 and J. A. Aberg10
1
USC Medical Center, Los Angeles, CA, USA; 2Harvard School of
Public Health, Boston, MA, USA; 3Nelson R Mandela School of
Medicine, Durban, South Africa; 4Joint Clinical Research Center,
Kampala, Uganda; 5Keyna Medical Research Institute, Kericho,
Kenya; 6MOI University Teaching Hospital, Eldoret, Kenya;
7
University of Zimbabwe, Harare, Zimbabwe; 8National Institutes
of Health, Bethesda, MD, USA; 9ATCT Operations Center, Silver
Springs, MD, USA and 10ICAHN School of Medicine at Mount
Sinai, New York, NY, USA
Background Amphotericin B (AmB) and fluconazole are often the
only antifungal agents available for the management of AIDS-associated cryptococcal meningitis. Fluconazole treatment at doses
>1200 mg day1 have only been tested in a small cohort of human
subjects which has limited its use at high doses due to potential concerns with both safety and efficacy.We report the pre-specified safety
analysis of stage 1 of the 2 stage ACTG A5225 phase I/II study of
oral fluconazole for the initial management of AIDS-associated cryptococcal meningitis.
Aim To address safetyand tolerability of high dose fluconazole and
obtain more robust estimates of 10 week efficacy.
Methods Anti-retroviral nave subjects with AIDS-associated cryptococcal meningitis were sequentially enrolled into three fluconazole
cohorts (1200, 1600 and 2000 mg day1) with a 3 : 1 randomization of fluconazole vs. AmB based treatment regimen. Quantitative
cerebrospinal fluid (CSF) cultures were obtained every 2 weeks.
Treatment with the assigned dose of fluconazole (adjusted for baseline weight) was continued for 2 weeks after the first negative quantitative CSFculture or to a maximum of 10 weeks. Fluconazole was
reduced to 400 mg daily if CSF cultures became negative prior to
10 weeks. Antiretroviral therapy was permitted after 4 weeks of
anti-fungal therapy with an efavirenz based anti-retroviral regimen.
The subsequent fluconazole cohort opened if 3 or fewer fluconazole
dose limiting toxicities (DLTs) were observed in the first 14 days of a
fluconazole dose cohort.
Results 96 participants enrolled -29 in cohort 1 (22fluconazole
1200 mg and 7 AmB), 35 in cohort 2 (26 fluconazole 1600 mg and
9 AmB) and 32 in cohort 3 (24 fluconazole 2000 mg and 8 AmB).
Participants enrolled in the fluconazole 1200 and 2000 mg day1
cohorts were more ill based upon observed baseline mental status
and quantitative cryptococcal CSF cultures (Table 1). There were no
changes in renal function, hemoglobin, or liver test abnormalities
attributed to fluconazole. QTc interval changes were identified in 10
P040
A modified loop-mediated isothermal amplification
method for diagnosis of Cryptococcal meningitis
M. Chen,1 L. Li,2 Z. Zhou,2 L. Li,2 Z. Zhang,1 Y. Meng,1
T. Boekhout,3 W. Liao1 and A. Pan2
1
Shanghai Key Laboratory of Molecular Medical Mycology,
Changzheng Hospital, Shanghai, China; 2Department of
Dermatology, Shanghai Changzheng Hospital, Shanghai, China
and 3CBS-KNAW Fungal Biodiversity Centre, Utrecht, The
Netherlands
Background Cryptococcal meningitis (CM) is a severe systemic
mycosis with high morbidity and mortality, particularly in immunocompromised patients in resource-limited countries. The Cryptococcus
neoformans/C. gattii species complex is the primary causative agent of
CM. However, the low sensitivity, accuracy, and time involved of
conventional diagnosis approaches hinder further reduction of the
mortality rate of CM. Loop-mediated isothermal DNA amplification
(LAMP) is a potential technology platform that could meet clinical
requirements in both the developed and developing world.
Aim We established a modified LAMP assay for direct identification
of all members of the C. neoformans/C. gattii species complex, evaluated its specificity and sensitivity and explored its suitability for clinical diagnostics of CM patients in two university hospitals in
Shanghai, China.
Amphotericin B
N=24
Fluconazole 1200
N=22
Fluconazole
1600
N=26
Fluconazole 2000
N=24
14 (4.2%)
12 (22.7%)
14 (11.5%)
12 (20.8%)
190 (120-300)
200 (105-300)
200 (150-290)
350 (180-550)
61 (13,144)
22,900 (2,130171,000)
24 (8-53)
101 (50-180)
152,000 (16.000780,000)
20 (9-55)
68 (30-145)
27,500 (96,000107,000)
59 (21-131)
64 (13-185)
144,000 (18,500443,000)
23 (9-64)
5.42 (5.17-5.74)
5.35 (5.09-5.95)
5.59 (5.09-5.82)
5.54 (5.16-5.93)
Maximum 15 points: Eye Opening 4 points, Verbal Response 5 points, and Motor Response 6 points
and % abnormal.
Figure 1
51
Poster Abstracts
P041
Chitin-like structures and cryptococcal physiology
jo,1 S. Frases,1
F. L. Fonseca,1 L. Kmetzsch,2 G. Arau
2
M. H. Vainstein and M. L. Rodrigues3
1
Federal University of Rio de Janeiro, Rio de Janeiro, Brazil;
2
Federal University of Rio Grande do Sul, Porto Alegre, Brazil and
3
Center for Technological Development in Health, Oswaldo Cruz
Foundation, Rio de Janeiro, Brazil
Figure 2. Sensitivity test of the modified LAMP assay using the genomic DNA of type strain of C.neoformans var. grubii.
52
P042
Detection of high CSF levels of (1-3)-Beta-D-glucan in
cryptococcal meningitis
J. R. Rhein,1 N. C. Bahr,1 Y. Zhang,2 M. Finkelman2 and
D. R. Boulware1
1
University of Minnesota, Minneapolis, MN, USA and 2Associates
of Cape Cod, East Falmouth, MA, USA
Background (1-3)-b-D-glucan (BDG) is a helpful tool in the diagnosis of many invasive fungal infections. It is not conventionally
thought to be useful in cryptococcal disease, however, and is specifically FDA-labeled as not being approved for detecting Cryptococcus.
This assumption is based on limited data from HIV-negative persons
with cryptococcal pulmonary disease.
Aim We evaluated the utility of BDG levels in CSF as an adjunct
diagnostic strategy for patients with HIV-associated cryptococcal
meningitis (CM).
Methods We determined the BDG levels in the CSF of 45 HIV-infected
Ugandan subjects with suspected CM using the Fungitell assay. We
also assessed whether BDG levels in CSF correlate with quantitative
cryptococcal cultures, cryptococcal antigen (CRAG) titers, the levels of
18 different cytokines in CSF, and clinical outcomes.
Results A cut-off value of 80 pg ml1 provided 97% sensitivity in
39 cases of confirmed CM, with one false negative result occurring in
an individual with very low CSF CRAG titer (1:2) and sterile CSF culture who later developed culture positive CM. BDG levels were
<80 pg ml1 in all 5 individuals without evidence of cryptococcal
Poster Abstracts
disease. One individual that was CRAG+ in serum but without evidence of meningitis (CSF culture and CRAG negative) had a CSF BDG
level of 130 pg ml1. BDG levels correlated with CSF fungal burden
by quantitative culture (r = 0.824, P < 0.001) as well as CRAG LFA
titers (r = 0.842, P < 0.001). CSF BDG levels also correlated with the
CSF immune response of: interferon-gamma (R = 0.362, P = 0.028),
MIP-1b (CCL4; R = 0.409, P = 0.012), and MCP-1 (CCL2; R = 0.309,
P = 0.063). In CM cases, high BDG levels were associated with death
within 1 week of diagnosis (MannWhitney U, P = 0.030).
Conclusions BDG can be detected in the CSF of HIV-infected
patients with CM and may provide useful diagnostic and prognostic
information. Further studies are needed to better define the role of
BDG in the immunology and management of cryptococcal disease.
P043
Cell wall-associated polyphosphates and acidocalcisomelike compartments in Cryptococcus neoformans
C. L. Ramos,1 F. M. Gomes,1 S. Frases,1 M. L. Rodrigues2 and
K. Miranda3
1
Federal University of Rio de Janeiro - UFRJ, Rio de Janeiro,
Brazil; 2CDTS/Fiocruz & UFRJ, Rio de Janeiro, Brazil and 3UFRJ &
INMETRO, Rio de Janeiro, Brazil
The most prominent morphological characteristic of Cryptococcus neoformans is the presence of a polysaccharide capsule, which coats the
cell wall and entraps divalent cations, including calcium and magnesium. These ions are required for capsular enlargement, which is
determinant for fungal pathogenesis. In this context, mechanisms
regulating the concentration of divalent cations at the cell surface
are likely essential for fungal pathogenesis. Acidocalcisomes are calcium storage acidic organelles that contain several polyphosphatebound cations. These organelles have been described in a variety of
organisms. Acidocalcisomes are acidified through the action of proton pumps such as the vacuolar proton ATPase and the vacuolar
proton pyrophosphatase. As described in other microorganisms, polyphosphates can participate in ion homeostasis, microbial growth
and in cellular responses to environmental stresses. In this study, we
analyzed the presence of cell wall-associated polyphosphates and acidocalcisome-like organelles in C. neoformans. Intracellular acidic
granules were observed after staining of fungal cells with acridine
orange. Cell wall-associated and intracellular polyphosphates were
observed after staining fungal cells with DAPI followed by analysis
by fluorescence microscopy. Transmission electron microscopy combined with X-ray microanalysis revealed the presence of phosphorus
in both the cell wall and in the intracellular space. Short- and longchain polyphosphates were extracted from yeast cells and the
amount of inorganic phosphate was determined spectrophotometrically after treatment of polyphosphate fractions with S. cerevisiae polyphosphatase (PPX1). The suggestion of intracellular and cell wall
polyphosphates in C. neoformans may lead to future studies involving
their participation in capsular architecture and ion homeostasis. Considering the essential role of the cryptococcal capsule in fungal pathogenesis, surface-associated polyphosphates might also participate in
the interaction of C. neoformans with host cells.
P044
Cryptococcus neoformans glucuronoxylomannan fractions
of different molecular masses are functionally distinct
P. C. Albuquerque,1 F. L. Fonseca,1 F. F. Dutra,1 M. T. Bozza,1
S. Frases,1 A. Casadevall2 and M. L. Rodrigues3
1
UFRJ (Universidade Federal do Rio de Janeiro), Rio de Janeiro,
Brazil; 2Albert Einstein College of Medicine - AECOM, New York,
NY, USA and 3CDTS/Fiocruz & UFRJ, Rio de Janeiro, Brazil
Aims Glucuronoxylomannan (GXM) is the major polysaccharide
component of Cryptococcus neoformans. We evaluated in this study
whether GXM fractions of different molecular masses were functionally distinct.
Materials and methods GXM samples isolated from C. neoformans
cultures were fractionated to generate polysaccharide preparations
differing in molecular mass. These fractions were used in experiments
focused on the association of GXM with cell wall components of C.
neoformans, as well as on the interaction of the polysaccharide with
host cells.
Results and conclusions GXM fractions of variable molecular
masses bound to a C. neoformans acapsular mutant forming punctate
patterns of surface distribution, in contrast to the usual annular pattern of surface coating observed when GXM samples containing the
full molecular mass range were used. The polysaccharide samples
were also significantly different in their ability to stimulate cytokine
production by host cells. Our findings indicate that GXM fractions
are functionally distinct depending on their mass.
P045
Association of CD44 polymorphism with Cryptococcal
meningitis in HIV-uninfected Chinese patients
X. Wang, R. Y. Wang and J. Q. Wu
Huashan Hospital, Fudan University, China
Background Hyaluronic acid (HA), one of the capsule components
of Cryptococcus neoformans, has been validated to play a role as an
adhesion molecule during infection by the yeast. CD44 is one of the
most common membrane HA receptors. In vitro and animal experiments have demonstrated that the interaction between C. neoformans
HA and CD44 on human brain endothelial cells plays an important
role in brain invasion.
Aim In this study, we identified the association between CD44 polymorphism and cryptococcal meningitis in HIV-uninfected Chinese
patients.
Methods A case-controlled genetic association study was conducted.
We genotyped 42 single nucleotide polymorphisms (SNPs) in the
CD44 gene in 150 patients with cryptococcal meningitis and 300
control subjects by multiplex SNaPshot technology. The distributions
of allele frequency, genotypes, and haplotypes were compared
between patients and control subjects.
Results Among the 150 patients with cryptococcal meningitis, 84
did not have predisposing factors. The genotype G/G of rs12419062
(OR = 1.74, 95% CI [1.042.93]; P = 0.034) and the genotype C/T
of rs353626 (OR = 1.81, 95% CI [1.152.85]; P = 0.009) were
under-presented and the genotype C/C of rs3751031 (OR = 0.36,
95% CI [0.130.99]; P = 0.048) was over-presented in the 150
patients with cryptococcal meningitis. In cryptococcal meningitis
patients without predisposing factors, the genotype C/T of rs353626
was also less frequently detected (OR = 0.55, 95% CI [0.310.96];
P = 0.035) than in controls.
Conclusions These findings suggested for the first time that CD44
rs353626 genetic variants have significant effect in susceptibility of
cryptococcal meningitis.
53
Poster Abstracts
P046
Phagocytosis and dissemination of Cryptococcus
neoformans spores
N. M. Walsh,1 M. R. Botts2 and C. M. Hull1
1
UW Madison, Madison, WI, USA and 2University of California San Deigo, San Deigo, CA, USA
Background Aerosolized Cryptococcus is inhaled into the lung and
then disseminates by an unknown mechanism to the brain, causing
meningoencephalitis that is uniformally fatal without treatment.
Cryptococcus exists in multiple cell types, including yeast, which
reproduce by budding, and spores, which are the products of sexual
development.
Aim In a mouse model of infection, we discovered that spores from
an avirulent yeast pair caused uniformly fatal disseminated disease.
This study examines the mechanisms by which spores cause cryptococcosis. We tested the hypothesis that spore-mediated disease is
facilitated by a Trojan Horse mechanism in which spores are phagocytosed, germinate into yeast, reproduce vegetatively, and escape the
lung within host phagocytes to infect the brain.
Methods For survival studies, C57Bl/6 mice were infected intranasally with a suspension of spores (purified from a cross of B3501 and
B3502) or yeast (B3501 + B3502), and survival was tracked. To
examine dissemination, mice were sacrificed at various time points
following infection, and organs were homogenized and plated to
determine CFUs. Phagocytosis of spores and yeast was observed by
co-incubation with immortalized murine macrophages (RAW 264.7)
or dendritic-like cells (JAWS II). Intracellular location of calcofluor
white labeled spores following phagocytosis was investigated by
staining of a phagolysosomal protein with a FITC-LAMP1 antibody.
P047
Pathogenic profiles in serotype C isolates of Cryptococcus
gattii are influenced by chitin-like structures
jo,1
J. Rodrigues,1 F. L. Fonseca,1 L. Kmetzsch,2 G. Arau
S. Frases,3 M. H. Vainstein2 and M. L. Rodrigues4
1
Universidade Federal do Rio de Janeiro - UFRJ, Rio de Janeiro,
Brazil; 2Universidade Federal do Rio Grande do Sul - UFRGS,
Porto Alegre, Brazil; 3UFRJ & INMETRO, Rio de Janeiro, Brazil and
4
UFRJ & Fiocruz/CDTS, Rio de Janeiro, Brazil
54
Poster Abstracts
P048
Identification of a role for virulence-related Sec14-1 of
Cryptococcus neoformans in export of cell wall enzymes
and cell separation using proteomics
J. T. Djordjevic,1 S. Lev,1 B. Crossett,2 C. F. Wilson,1
D. Desmarini,1 S. Y. Cha,1 C. Li,1 M. Chayakulkeeree,3
P. R. Williamson4 and T. Sorrell5
1
Westmead Millennium Institute, Sydney, NSW, Australia;
2
University of Sydney, Sydney, NSW, Australia; 3Siriraj Hospital,
Mahidol University, Bangkok, Thailand; 4NIH, Bethesda, MD, USA
and 5Marie Bashir Institute for Infectious Diseases and
Biosecurity, Sydney, NSW, Australia
Secreted proteins contribute to the pathogenesis of Cryptococcus neoformans (Cn), and are key mediators of host-pathogen interaction.
Secretion of the fungal invasin, phospholipase B1 (Plb1)1, is partially
regulated by the putative phosphatidylinositol transfer protein,
Cnsec14-11. However, the composition of the Sec14-1-dependent secretome is unknown. In addition to reduced secretion of Plb1, the
sec14-1delta mutant has a compromised cell wall and is less virulent
in mice, despite increased expression of its closely related homologue,
SEC14-21. Although deletion of SEC14-2 has no effect on the virulence composite it cannot be disrupted in combination with SEC1411.The aims of this study were to (i) identify the SEC14-1-dependent
and WT secretomes (ii) compare the efflux routes of selected Sec14dependent proteins and (iii) determine whether SEC14-1 and SEC142 are functionally redundant.
To address aim 1, the secretomes of WT (strain H99) and sec141delta were analyzed by mass spectrometry. 105 proteins were identified in WT secretions, 27 of which are canonically secreted (contain
signal-peptides). The abundance of 25 proteins was reduced in sec14-
1delta secretions. Of these, 7 are cell wall-associated and/or cell wallmodifying enzymes, including canonically-secreted Plb1, laccase
(Lac1) and a-1,3-glucan synthase (Ags1), which have proven roles
in pathogenesis, and acid phosphatase (Aph1). Using an APH1 deletion mutant, Aph1 was confirmed to be the major phosphate-repressible, extracellular acid phosphatase in Cn, and to contribute to the
virulence composite. Comparison of the subcellular localization of
SEC14-1-dependent Plb1 and Aph1, revealed that Plb1 was transported directly to the periphery, while Aph1 accumulated in endosome-like structures en route to the plasma membrane and vacuoles.
Both proteins were enriched in bud necks, implicating a role for them
in bud formation and/or septation. Microscopic examination of
sec14-1delta revealed that, in contrast to WT, mother and daughter
cells often remained connected via the septum following mitosis.
To address Aim 2, we placed SEC14-2 under the control of a galactose-inducible promoter in sec14-1delta. When SEC14-2 gene expression was suppressed by glucose, growth of sec14-1deltaGAL7SEC14-2
was inhibited, demonstrating that SEC14-1 and SEC14-2 are functionally redundant. We are now in a position to observe the effect of
full loss of sec14 function on Aph1 and Plb1 subcellular localization.
Taken together, our findings demonstrate that SEC14-1 regulates
export of cell wall enzymes via endosome-dependent and -independent secretion routes to promote cell wall integrity and bud formation, and ensure timely dissolution of the septum, and that loss of
the combined function of SEC14-1 and SEC14-2 is lethal.
Reference 1. Chayakulkeeree et al. Molecular Microbiology 2011;
80: 1088.
PW is supported by the Division of Intramural Research, NIAID,
NIH.
P049
Verification of Cryptococcus neoformans Gene deletion
mutants related to traverse the blood-brain barrier
H. K. Tseng,1 W. L. Cho,2 C. P. Liu,1 J. Y. Jong3 and
J. R. Perfect4
1
Mackay Memorial Hospital, Taipei, Taiwan; 2Mackay Medical
College, New Taipei City, Taiwan; 3University of Southern
California, Los Angeles, CA, USA and 4Duke University, Durham,
NC, USA
Pathogenic yeast C. neoformans is the most common central nervous
system (CNS) fungal infection these years. This pathogen has a predilection for the brain and causes devastating meningoencephalitis. In
order to cause the brain invasion, C. neoformans must cross the
blood-brain barrier (BBB). However, the brain invasion mechanism is
largely unknown. We utilized a mutant library of signature-tagged,
targeted gene deletion C. neoformans mutants to decipher how C. neoformans enter into brain. We identified two genes, FNX1 and PUB1,
related to BBB crossing (PLoS ONE 7:e45083) and the strategies to
identify mutants related to transmigration to CNS were described.
Briefly, screen strategy to find mutants with significant signature tag
loss mutants on mice brain transmigration assay (MBTA) model;
competition strategy to show statistically significant difference
mutants from parental strain CMO18 on MBTA. The project to
screen the whole mutant library with 1201 strains according
to above strategy is going and we plan to establish brain cell strategy
to confirm BBB transmigration related mutants. Currently twenty
mutants (Figure) were identified through MBTA in vivo. The in vitro
transcytosis assay by human brain microvascular endothelial cells
(HBMECs) for these mutants are in progress. This research helps to
understand the molecular level of Cryptococcal meningitis pathophysiology and may discover new treatment targets for this medical
important fungus.
55
Poster Abstracts
P051
Innate immune cell activation of a copper-dependent anticryptococcal agent
R. A. Festa,1 M. E. Helsel,2 K. J. Franz2 and D. J. Thiele1
1
Duke University Medical Center, Durham, NC, USA and 2Duke
University, Durham, NC, USA
P050
Consequences of SIR2 regulation on the pathogenesis of
Cryptococcus neoformans
T. Bouklas, N. Jain, X. Wang and B. C. Fries
Albert Einstein College of Medicine, New York, NY, USA
Background Replicative aging has been implicated in the pathogenesis of the debilitating fungus, Cryptococcus neoformans. However, the
underlying mechanisms for this unique contribution have not been
completely elucidated. Previous work has shown that age-related
resilience is acquired by C. neoformans cells as they undergo asymmetric divisions. The sum of these divisions, which is known as their
replicative life span (RLS) can impact the outcome of chronic infection when these cells are selected and become the dominant pathogen population in the host.
Aim This work investigated whether C. neoformans strains that demonstrate a variable RLS either from gene deletion or drug manipulation affect its virulence.
Methods A mutant (sir2D) and its complement were generated in a
serotype A (H99) and D (RC2) strain, and their phenotypes were
examined in vitro and in vivo.
Results and discussion/conclusion As expected loss of SIR2 attenuated doubling time and mating ability of C. neoformans. The mutant
had a 3368% shortened RLS compared to the wildtype (wt). Importantly, it was hypovirulent in Galleria mellonella (waxworm) and in
mice. Notably in mice, the mutant was only hypovirulent after intratracheal, not intravenous infection, as supported by histopathology
and fungal burden found in the lungs and brain. Since the mutant
crossed the blood-brain barrier equally well compared to the wt, we
hypothesized that this was due to the altered nutritional growth conditions in the infection sites. Indeed, RLS was dependent on levels of
glucose as calorie restriction (CR) either shortened (RC2) or extended
(H99) RLS in different strains; however, the extension was not seen
in the absence of SIR2. Also, RT-PCR analysis supported this observation as SIR2 was upregulated in H99 cells and was downregulated
56
Poster Abstracts
P053
P052
The role of tryptophan biosynthetic genes TRP3 and TRP5
on the survival of C. neoformans and their applicability as
drug targets
J. D. Fernandes,1 V. Tofik,2 J. Machado-Jr,2 M. A. Vallim2 and
R. C. Pascon2
1
Universidade de Sa~o Paulo, Sao Paulo, Brazil and 2Universidade
Federal de Sa~o Paulo, Diadema, Brazil
Defects in amino acid biosynthetic pathways generated by gene deletion in C. neoformans lead to auxotroph strains which often have no
virulence or attenuated virulence, in other cases the block in this
kind of metabolic route may create lethal phenotypes, impairing
growth and survival. Therefore, nutrient biosynthesis and/or acquisition pathways offer interesting opportunities as drug targets. Out of
twenty-three amino acids synthesized in plants, microbes and parasites, nine are essential in animal cells, among these there are the
aromatic amino acids, including tryptophan, that differently from
tyrosine and phenylalanine, has only one route of synthesis, increasing its interest as target for development of novel antifungal agents.
According to bioinformatics analysis, four genes (TRP2 to TRP5) are
expressed and necessary to convert chorismate into tryptophan in C.
neoformans. TRP3 encodes a triple function protein that acts on the
first, third and forth step of this pathway. Whereas, TRP2 protein
acts on the first, TRP4p on the second and TRP5p on the last step
of the pathway. In this work we attempted to delete TRP3 and
TRP5 genes to evaluate the impacts of lack of tryptophan biosynthesis on virulence factors and survival. An extensive search for
mutants bearing homologous integration leading to tryptophan auxotrophy was unsuccessful, therefore, we assumed the hypothesis that
the tryptophan biosynthetic genes are essential. To address this issue
we used RNAi to modulate TRP3 and TRP5 expression. All strains
grew well under RNAi repression (Glucose). However, during RNAi
induction (Galactose) Trp3i and Trp5i strains did not grow in rich
or poor medium with or without tryptophan supplementation. Synthetic medium supplemented with proline as the sole nitrogen source
and tryptophan at 25 C was the only condition we observed
mutant growth, even though growth rate was poor. qPCR was performed to confirm TRP3 and TRP5 mRNA suppression under RNAi
induction. This far, tryptophan is the second report of an essential
amino acid biosynthetic pathway in C. neoformans. One possible
explanation for this fact is that extracellular amino acids are inefficiently transported into the cell, due to, either fewer or lower affinity
permeases or both. To test this hypothesis we searched all amino
acid permeases in C. neoformans genome using 24 query related
sequences from S. cerevisiae and found only 8 putative permeases
(AAP1 to AAP8). By qPCR their gene expression was checked and
among these, only six had expression above the threshold. Comparative analysis under various growth conditions applied in these experiments showed a distinct expression pattern for AAP2, AAP4 and
AAP5 in response to preferred nitrogen source and the presence of a
pool of amino acids added to the medium. These results suggest that
C. neoformans, in fact, has fewer amino acid permeases, which could
explain low assimilation. The expression profile suggests permeases
are induced by a pool rather than single amino acids and also, they
may be controlled by global amino acid response, nitrogen catabolism repression and a signaling pathway that responds to the presence of the substrate, similar to SPS-sensing in S. cerevisiae. In
57
Poster Abstracts
P054
Leucine biosynthesis is required for iron homeostasis and
virulence in Cryptococcus neoformans
E. S. Do,1 G. Hu,2 L. Oliveira,2 M. Caza,2 W. Kronstad2 and
W. Jung1
1
Chung-Ang University, Anseong-Si, South Korea and 2University
of British Columbia, Vancouver, BC, Canada
Amino acid biosynthesis that is absent in mammals is considered an
attractive target of antifungal treatment. Leucine biosynthesis is such
a target pathway, consisting of a five-step conversion process starting
from the valine precursor 2-keto-isovalerate. Isopropylmalate dehydrogenase (Leu1) is an iron-sulfur cluster protein that is required for
leucine biosynthesis in Saccharomyces cerevisiae and is highly homologous to the iron regulatory protein Irp1 in mammalian cells. Moreover, our previous transcriptome data showed that the expression of
LEU1 is regulated by iron availability in Cryptococcus neoformans. In
this study, we aimed to characterize the role of leucine biosynthesis
in iron homeostasis and the virulence of the human pathogenic fungus C. neoformans. We found that deletion of LEU1 caused the cells
to become leucine auxotroph and that intracellular iron levels were
significantly distorted in the leu1 mutants. The leu1 mutants also displayed increased susceptibility to oxidative stress and cell wall/membrane disturbing agents. Furthermore, our results suggest that the
functions of superoxide dismutases, Sod1 and Sod2, are closely associated with the expression of LEU1 and that LEU1 is required for virulence in a mouse model of cryptococcosis. A mutant lacking the
beta-isopropylmalate dehydrogenase gene (LEU2), which encodes an
enzyme catalyzed in the subsequent step of leucine biosynthesis,
showed not only similar phenotypes to the leu1 mutant but attenuated virulence. Overall, our results suggest that leucine biosynthesis
is required for iron homeostasis and virulence in C. neoformans.
Aim To explore the 14-3-3 gene functions and its potential for virulence, we intended to generate a 14-3-3 mutant strain. We investigated the mutant characteristics by examining its roles in growth,
morphological alterations, MV biogenesis, and its adhesion ability to
human brain microvascular endothelial cells.
Methods C. neoformans 14-3-3 is apparently a single-copy, essential
gene. To explore the functions of 14-3-3, we replaced the promoter
region of the chromosomal 14-3-3 gene with the copper-controllable
promoter CTR4. The growth rate and its morphological changes were
examined to characterize the alterations of mutant strain. The roles
of 14-3-3 on the biogenesis of MVs were scrutinized by the yield of
isolated MVs, NanoSight display and 3-D plot, and proteomic analyses. In vitro BBB adhesion assay was also used to test its roles during
invasion.
Results The knockdown strain C1617 showed a reduction in growth
rate, slightly enlarged cell size, drastic change in morphology and
the reduction in the thickness of the capsule under copper repressed
conditions. The mutant cells became temperature-sensitive. Furthermore, the mutant cells produced lower amount of total proteins in its
extracellular MVs and reduced adhesion to the HBMEC in vitro. Proteomic analyses of the protein components under induction and suppression conditions reveals that the MV biogenesis may be tightened
with the 14-3-3 function(s).
Discussion/conclusion The 14-3-3 protein is highly conserved protein which plays pleitropic roles among organisms. Our studies of C.
neoformans 14-3-3 protein suggest that, as in other organisms, plays
several important roles relevant to its growth, morphology, cell division, and other functions. The 14-3-3 protein was first identified in
bovine adrenal chromaffin cells as a cytosolic protein Exo1, required
for exocytosis. In our 14-3-3 mutant studies, both of total MV proteins and activities of two enzymes associated with MVs, laccase and
acid phosphatase, were reduced. As secretion of MVs play a role in
the building block transport for its extracellular capsule biosynthesis,
it is possible that reduction of 14-3-3 protein level in C1617 results
in the decline of MV secretion, and subsequently drops the supplies
for capsule biosynthesis which results in the reduction of
capsule size.
The temperature-sensitive nature of 14-3-3 knockdown cells prevents from doing animal studies. However, the in vitro adhesion studies show that the reduction of 14-3-3 protein levels in the mutant
strain causes a reduction in its ability to bind to the HBMEC. This
reduction may be due to, a small part, of slow-growth rate at 37 C,
and also could be attributed by the reduction in the amount of MV
secreted in the mutant since MV can affect the binding of cryptococcal cells to HBMEC, or others. Given the fact that the 14-3-3 functions are relevant to its growth, morphology, possible cytokinesis,
capsule and MV biogenesis, it is perceivable that the C. neoformans
14-3-3 functions are closely associated with C. neoformans physiology
and pathogenesis.
P056
P055
Cryptococcus neoformans 14-3-3 is an essential gene for
virulence
Y. Jong,1 Y. C. Chang,2 K. J. Kwon-Chung,2 S. Huang,1
S. Shimada1 and X. Fu1
1
Childrens Hospital Los Angeles, USC Keck School of Medicine,
Los Angeles, CA, USA and 2NIAID, Bethesda, MD, USA
58
Poster Abstracts
budding as the growth phase progresses into late log to early stationary phase, resulting in a tendency to accumulate unbudded
cells with G2 rather than G1 DNA content. This was found to be
the organisms inherent response to stressful cultural conditions
such as changes in oxygen availability, pH and temperature and
was implicated as a possible additional virulence mechanism during
survival within host tissues.
We have reported the molecular characterization and physiological roles of the two main eukaryotic cell cycle genes, C. neoformans
cyclin dependent kinase 1 (CnCdk1) and cyclin homologues. Only a
single Cdk1-related G1 and G1/S cyclin homologue was found in
the genome sequence of C. neoformans and designated CnCln1. In
the light of the functional specialization of G1 and G1/S cyclins in
S. cerevisiae, it was surprising to find that C. neoformans was found
to have a single G1 and G1/S cyclin in the genome. Thus, it is
important to understand the mechanisms that govern this yeasts
unique cell cycle behavior during G1-S phase transition and the
role of this single cyclin in this unique stress response pathway.
We investigated the cell cycle control mechanism of CnCln1 by
comparing its activity with G1 and G1/S cyclins of S. cerevisiae
from a point of view of their structure-function relationship. CnCln1
was not only able to complement the function of the G1 cyclins of
S. cerevisiae, such as ScCln3, but also the G1/S cyclins of S. cerevisiae, such as ScCln1 and ScCln2. Our in silico analysis demonstrated
that the CnCln1/ScCdk1 complex was more stable than any of the
yeast cyclin and ScCdk1 complexes. These results are consistent
with in vitro analysis that has revealed the flexible functional
capacity of CnCln1 as a Cdk1-related G1 and G1/S cyclin of S.
cerevisiae.
In the obligate aerobic yeast C. neoformans, limited aeration has
been demonstrated to cause slowdown in proliferation and delayed
budding, resulting eventually in a unique unbudded G2-arrest. The
ability to adapt to decreased oxygen levels during pathogenesis has
been identified as a virulence factor in C. neoformans. We tried to
identify and characterize genes that are necessary for the proliferation slowdown and G2-arrest caused by limited aeration. Random
mutants were prepared and screened for lack of typical slowdown
of proliferation under limited aeration. The CNAG_00156.2 gene
coding for a zinc-finger transcription factor was identified in
mutants showing most distinctive phenotype. Targeted deletion
strain and reconstituted strain were prepared to characterize and
confirm the gene functions. This gene was also identified in parallel
studies as homologous both to calcineurin responsive (Crz1) and
PKC1-dependent (SP1-like) transcription factors. We have confirmed
the role of the cryptococcal homologue of CRZ1/SP1-like transcription factor in cell integrity, and newly demonstrated its role in
slowdown of proliferation and survival under reduced aeration, in
biofilm formation and in susceptibility to fluconazole. Our data demonstrate a tight molecular link between slowdown of proliferation
during hypoxic adaptation and maintenance of cell integrity in C.
neoformans and present a new role for the CRZ1 family of transcription factors in fungi.
P057
Genomic approaches to inferring recombination rates and
population structure of Cryptococcus neoformans var
grubii
J. Rhodes,1 M. A. Beale,1 C. Cuomo,2 S. Sakthikumar,2
T. Bicanic,3 T. S. Harrison3 and M. C. Fisher1
1
Imperial College London, London, UK; 2Broad Institute, Boston,
MA, USA and 3St. Georges, University of London, London, UK
Cryptococcus neoformans var grubii (Cng) can be broadly divided into
the phylogenetic lineages VNB, VNI and VNII. Population genetics
suggests that the Cng VNI lineage has emerged out of Africa to
cause the main burden of cryptococcal meningitis worldwide; however, the pathway and timing of global proliferation has yet to be
P058
Isolation of Cryptococcus gattii VGII from indoor dust in
the deep Amazon of the Rio Negro basin
F. Brito-Santos,1 G. B. Barbosa,2 L. Trilles,2 B. Wanke,2
W. M. Wieland,3 F. A. Carvalho-Costa4 and M. S. Lazera2
1
Fiocruz/IPEC, Rio de Janeiro, Brazil; 2Evandro Chagas Clinical
Research Institute, Fiocruz, Rio de Janeiro, Brazil; 3Centre for
Infectious Diseases and Microbiology, Sydney Medical SchoolWestmead, Sydney, NSW, Australia and 4Institute Oswaldo Cruz,
Fiocruz, Rio de Janeiro, Brazil
Cryptococcosis is a human fungal infection of significant mortality
and morbidity, especially in the meningoencephalitis form. Cryptococcosis is distributed worldwide and their agents, C. neoformans and
C. gattii, present eight major molecular types - VNI-VNIV and VGIVGIV respectively. The primary cryptococcosis caused by molecular
type VGII (serotype B, MAT alpha) prevails in immunocompetent
patients in the North and Northeast of Brazil, revealing an endemic
regional pattern to this molecular type. Since 1999, C. gattii VGII
has been involved in an outbreak in Canada, expanding to the
59
Poster Abstracts
P059
Functional analysis of puf3 mediated post-transcriptional
regulation in cryptococcus neoformans
J. Kaur and J. C. Panepinto
State University of New York at Buffalo, Buffalo, NY, USA
Background PUF proteins represent a conserved family of RNAbinding factors that are key regulators of mRNA translation, stability
and localization across the eukaryotic kingdoms. Investigation of the
C. neoformans genome has revealed that it encodes four PUF proteins
which are typically characterized by the presence of 8 consecutive
Puf repeats, however variations do occur. The PUF proteins characterized to date have been reported to bind to consensus sequence
characterized by a UGUR tetra nucleotide in their target RNA. Recent
phylogenetic studies have demonstrated that RNA binding domain of
Puf3 is conserved and there is significant enrichment of Puf3 binding
element in genes that are involved in mitochondrial translation
machinery in Saccharomycotina species, which is lost in higher
fungi. Our studies indicate that puf3-delta mutants exhibit no mitochondrial phenotype. Phenotypic characterization of a C. neoformans
puf3-delta strain revealed a defect in filamentation which led us to
hypothesize that Puf3 plays a role in C. neoformans morphogenesis.
Aim To characterize the role of PUF3 protein as post transcriptional
regulator of cryptococcal morphogenesis.
Methodology We performed bilateral mating assays for wild type
and puf3-delta mutants, in which equal numbers of opposite mating
cells were cocultured on V8 agar. To determine the ability of puf3D
mutants to produce pheromone, northern blot for MF alpha was
done. Puf3 was mCherry tagged using fusion PCR and fluorescence
microscopy was done to study its localization. Recombinant PUF3
protein was made using pLysS expression system and purified using
Ni-NTA columns. Protein binding activity of Puf3 to its cognate element was detected using UV-linked RNA EMSA. Site directed mutagenesis was done to mutate the RNA binding domain (RBD) of Puf3
as well as the Puf3 element present in the 50 UTR
Results Bilateral crosses of puf3-delta mutants are defective in the
production of dikaryotic hyphae as compared to the wild type, but
pheromone sensing and fusion were not affected. Using fluorescence
microscopy, we have shown that mCherry tagged Puf3 specifically
localizes to areas of hyphal growth, but is not visible in yeast cells.
Mating assay revealed that the RBD mutants are defective for filamentation as compared to the wild type. Mating spots of strains complemented with Puf3 in which the 50 UTR Puf3-binding element was
mutated were more filamentous as compared to the wild type
60
P060
Elongation of sexual filaments and spore morphogenesis
are coordinately regulated by Cryptococcus neoformans
Crk1 and Mub1
D. Liu and W. C. Shen
National Taiwan University, Taipei City, Taiwan
Cryptococcus neoformans is a heterothallic basidiomycete that grows
vegetatively as yeast and filamentous hyphae are produced in the
sexual state. The production of sexual filaments is inhibited by
Cwc1/Cwc2 complex upon light treatment. A genome wide genetic
screen has identified components such as CRK1 and MUB1 genes in
the pathway. C. neoformans CRK1 gene is a homologue of Ustilago
maydis crk1 and Saccharomyces cerevisiae IME2, and contains the
conserved Ser/Thr kinase domain and TXY dual phosphorylation
site. In C. neoformans, Crk1 negatively regulates mating differentiation and mating efficiency is increased in the crk1 mutant cross.
Compared to the wild type cross, production of dikaryotic filaments,
basidia, and basidiospores are earlier in the mutant cross; however,
elongation of dikaryotic filaments is perturbed in the crk1 mutant
cross. C. neoformans MUB1 gene, a homologue of Saccharomyces cerevisiae MUB1 (multi-budding) gene, is a MYND domain-containing
protein required for ubiquitination. Deletion of C. neoformans MUB1
gene caused compromised growth at 37 C. C. neoformans mub1
mutants similarly displayed a multiple-budding phenotype and
altered structures of bud scars were observed. Interestingly, elongation of dikaryotic sexual filaments was abnormal and formation of
basidiospore chain was defective in the mub1 bilateral cross. To further examine their epistatic relationship, the MATa crk1mub1 and
MATa crk1mub1 mutants were created and mating phenotypes were
characterized. Similar to the crk1 bilateral mutant cross, basidia and
basidiospores were also seen at 24 h in the crk1mub1 bilateral
mutant cross; however, spore morphogenesis was ceased at fourspore stage and production of basidiospore chains was impaired as
the mub1 mutants. These results indicated that both CRK1 and
MUB1 genes coordinately regulate dikaryotic filament elongation,
but they play different roles in the process of spore chain formation
in C. neoformans.
Poster Abstracts
P061
Molecular typing of environmental Cryptococcus
neoformans isolated from pigeon droppings in Tripoli,
Libya
M. S. Ellabib,1 M. A. Aboshkiwa,1 R. DAmicis2 and M. Cogliati2
1
Department of Medical Microbiology and Immunology, Faculty
of Medicine, Tripoli University, Tripoli, Libya and 2Lab. Micologia
Medica, Dipartimento di Scienze Biomediche per la Salute,
Universita degli Studi di Milano, Milano, Italy
Background Cryptococcus neoformans is the major cause of fungal
meningitis, a potentially lethal mycosis. Bird excreta can be considered a significant environmental reservoir of this species in urban
areas. At present, data concerning environmental distribution of C.
neoformans in Libya as well as in the city of Tripoli are lacking.
Aim This study was aimed to obtain an overview of the presence of
C. neoformans in pigeon droppings in Tripoli urban area as well as to
identify the molecular type and the mating type of the collected
isolates.
Materials and methods One hundred samples from pigeon excreta
were collected from three different sites in the city of Tripoli, Libya.
Samples were cultured on sunflower seed agar supplemented with
antibiotics and biphenyl. Identification of C. neoformans was performed on the basis of melanin pigment production on sunflower
seed agar, presence of a capsule observed in India ink preparation,
urease production on urea agar medium, and ability to grow at
37 C. Vitek2 compact system was used to confirm C. neoformans
identification. Molecular type and mating type allelic pattern were
determined performing two specific multiplex PCRs as described
elsewhere.
Results Thirty-two out of the 100 samples were positive for C. neoformans. Multiplex PCR amplifications reveal that all isolates belonged
to molecular type VNI (C. neoformans var. grubii) and mating type
alphaA.
Conclusion This study provides for the first time information concerning the ecology and genotypes of C. neoformans in the Tripoli
region of Libya.
var grubii divided into three molecular groups VNI, VNII and VNB;
the latter was originally described in Botswana, and demonstrates
greatest evidence of recombination. Using multi-locus sequence typing (MLST) of Cn isolates from a large clinical trial cohort, we
explored the genotypic diversity of Cn in South Africa, its geographical distribution within Cape Town, and its association with clinical
outcome.
Aims To understand the cryptococcal genetic diversity present
within the study population of South African clinical trials patients,
and to determine relationships between phylogenetic clade and burst
groups with clinical outcome.
Methods DNA extracted fromCn isolated from 222 South African
HIV-infected individuals with CM was amplified by PCR and
sequenced at seven loci to generate MLST profiles (ISHAM typing
scheme). Isolates were classified by phylogenetic relationship into
molecular types and further sub-divided into subclades using maximum likelihood analysis. Clinical outcome was analysed using Cox
proportional hazards survival analysis, adjusting for known adverse
prognostic indicators as well as CM induction drug treatment. Geographical location data was mapped using patient address to obtain
GPS coordinates via Google Maps, followed by plotting onto shape
files using ArcGIS.
Results Of 222 isolates,218 were Cn var grubii, with 4 C.gattii.
MLST profiling revealed great genetic diversity within South African
patients, with 48 different sequence types (STs), including at least 18
novel STs. Molecular types were VNI (n = 166), VNII (n = 42) and
VNB (n = 7). Patients infected with VNB molecular type had worse
survival compared to VNI (HR 2.8, 95% CI 1.26.5, P = 0.016),
even after adjustment for altered mental status, baseline fungal burden (CFU ml1 CSF) and treatment with amphotericin (HR 2.6, 95%
CI 1.16.3, P = 0.029). Survival comparisons using specific ST types
and Burst Groups were also explored and will be presented. Geographical mapping for 185 cases localised in Cape Town showed no
clustering by Molecular Type, Sequence Type, or Burst Group.
Discussion/conclusion South African clinical C.neoformans strains
are highly genetically diverse. CM caused by the VNB molecular type,
though rare, appears to be associated with poorer outcome. The lack
of geographical clustering of isolates argues against a common recent
environmental exposure, and lends support to the reactivation of
latent infection hypothesis. The underlying mechanisms of this association will now be explored using a combined genomic and transcriptomic approach, and further in vitro phenotypic analysis.
P062
P063
Cryptococcus neoformans molecular type VNB is associated
with mortality in HIV associated cryptococcal meningitis in
South Africa
M. A. Beale,1,2 E. Robertson,1,3 S. P. Simwami,2 K. Fuentes,1
J. N. Jarvis,1,4 A. Loyse,1 J. Bradley,5 G. A. Meintjes,6 D. Wilson,7
T. S. Harrison,1 M. C. Fisher2 and T. Bicanic1
1
Research Centre for Infection and Immunity, Division of Clinical
Sciences, St. Georges University of London, UK; 2Department of
Infectious Disease Epidemiology, School of Public Health, Imperial
College, UK; 3Department of Microbiology, University of
Minnesota, MN, USA; 4Department of Clinical Research, London
School of Hygiene and Tropical Medicine, UK; 5Department of
Infectious Disease Epidemiology, London School of Hygiene and
Tropical Medicine, UK; 6Institute of Infectious Disease and
Molecular Medicine, UCT Faculty of Health Sciences, University
of Cape Town, South Africa and 7Department of Medicine,
Edendale Hospital, Pietermaritzburg, South Africa
Background Cryptococcal meningitis (CM) is a major cause of mortality amongst HIV-infected individuals in Subsaharan Africa, from
where C.neoformans (Cn) is thought to have evolved. The organism is
divided into two subspecies (var grubii and var neoformans), with Cn
61
Poster Abstracts
P064
A chemical-genetic portrait of a human fungal pathogen
J. C. S. Brown,1 B. VanderSluis,2 R. Deshpande,2 J. Nelson,2
A. Butts,3 S. Kagan,4 I. Polacheck,4 D. J. Krysan,3 C. L. Myers2
and H. D. Madhani1
1
University of California, San Francisco, San Francisco, CA, USA;
2
University of Minnesota, Minneapolis, MN, USA; 3University of
Rochester Medical Center, Rochester, NY, USA and 4HadassahHebrew University Medical Center, Jerusalem, Israel
Background and aim Two major questions in microbial pathogenesis are how to systematically identify targets of anti-microbial drugs
and how annotate genes of unknown function involved in the virulence process. We sought to develop and adapt efficient, highthroughput methods to perform these functions and create datasets
of use to the Cryptococcus research community.
Methods We used chemical-genomic profiling to address the questions of drug target identification and functional annotation of virulence genes. This is the systematic measurement of the impact of
small molecules on the growth rate of a large number of deletion
mutants. We employed a plate-based colony array method to quantify the impact of >200 diverse growth-inhibitory compounds on
~1500 C. neoformans deletion strains.
Results (1) Identification of novel virulence factors: We performed hierarchical clustering on the resultant dataset, then focused our initial
studies on two clusters that each contained 12 genes known to be
62
P065
Molecular epidemiology of clinical Cryptococcus gattii
isolates from Colombia
C. Firacative,1 J. Lizarazo,2 P. Escandon,3 C. A. Agudelo,3
~eda3
W. Meyer1 and E. Castan
1
The University of Sydney, Westmead, Australia; 2Hospital
Universitario Erasmo Meoz, Cucuta, Colombia and 3Instituto
Nacional de Salud, Bogota, Colombia
Background Cryptococcosis caused by Cryptococcusgattii isendemic
in several countries and affects mostly immunocompetent patients,
both their pulmonary and central nervous systems. Worldwide the
number of cases of this mycosis is increasing mainly because C. gattii
is expanding its environmental niche, which leads to a wider geographic distribution. In Colombia, a national surveillance on cryptococcosis, including demographic, clinical and microbiologic data, is
being conducted since 1997, although molecular characterization of
the isolates using MLST has not been done.
Aim To characterize by molecular methods the clinical isolates of C.
gattii recovered in Colombia from 1997 until 2010, to provide
insights into the molecular epidemiology of this important pathogen
in the country and to contribute to the general knowledge of Cryptococcus and cryptococcosis in the world.
Methods From 1.207 surveys analyzed, 43 C. gattii cases from 15
departments were reported, with the majority of the cases (n = 15)
from Norte de Santander. Among all isolates, four were recovered
from HIV patients, one from a patient with arthritis and one with
diabetes. The remaining 37 patients did not have or report any risk
factor. Molecular type of the isolates was determined using PCR fingerprinting with the primer (GTG)5. Mating type a or alpha was
determined using specific primers. Multilocus Sequence Typing
(MLST) was carried out using the ISHAM consensus MLST typing
scheme for C. neoformans/C. gattii species complex, which includes
seven genetic loci, CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and the
IGS1 region.
Results The molecular type VGII was the most frequent among the
isolates (55.8%), followed by VGIII (27.9%), and VGI (16.3%).
Among the patients with risk factors associated with the development of cryptococcosis, the molecular type VGII and VGI were
Poster Abstracts
identified in two and one HIV+ patients, respectively. One VGII isolate was recovered from a patient with arthritis and one VGIII isolate
from a patient with diabetes. Mating type was determined as alpha
in 23 (53.5%) isolates and as a in 20 (46.5%). Among 42 isolates,
16 MLST sequence types (ST) were identified: two STs amongst VGI
isolates, nine amongst VGII isolates, with ST25 being the most common one (68%), and five amongst VGIII isolates. Eight STs of the
VGII isolates (ST8, ST12, ST31, ST46, ST321, ST322, ST323 and
ST324) and three of the VGIII isolates (ST59, ST64 and ST85) were
identified for the first time in this study. The obtained MLST data
was incorporated into the C. neoformans/C. gattii databases accessible
at http://mlst.mycologylab.org.
Discussion/conclusion The current study showed that the clinical
C. gattii isolates from Colombia are genetically diverse. Despite the
low number of isolates studied, several genotypes were identified
within each molecular type, opposite to the less diverse and rather
clonal C. gattii populations reported in other countries such as Canada, USA, Australia and Thailand, where few genotypes have been
identified in a much larger number of isolates. The identification of
the same ST in different departments, like the prevalent ST25 for
VGII isolates found in 7 different departments, the ST51 and ST58 in
VGI isolates, and the ST64, ST79 and ST146 in VGIII isolates, suggests the circulation of some genotypes in the country. The isolation
of C. gattii predominantly from otherwise healthy hosts rather than
from patients with impaired immune system supports the idea of C.
gattii as primary pathogen and as being clinically more pathogenic
than its sibling species C. neoformans. Our data is giving a more
detailed picture of the molecular epidemiology of cryptococcosis in
Colombia and includes the country as integral part of the global population genetics studies of the C. neoformans/C. gattii species complex.
P066
Purine biosynthetic enzymes as antifungal drug targets
R. B. Blundell, S. D. M. Arras, S. J. Williams and J. A. Fraser
University of Queensland, Brisbane, QLD, Australia
Background With increasingly large immunocompromised populations around the world, opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality;
C. neoformans is estimated to be responsible for over 600 000 deaths
every year. Fungal infections are difficult to treat owing to the similarities of their eukaryotic physiology to that of humans. The limited
antifungal agents available tend to target the few differences between
the two systems. To combat the current scarcity of antifungal therapeutic agents, research into fungal-specific drug targets is required.
Adenylosuccinate synthetase (AdSS) is a crucial enzyme in the ATP
biosynthetic pathway, catalyzing the formation of adenylosuccinate
from inosine monophosphate and aspartate. We have previously
characterised another enzyme in the GTP biosynthetic pathway, inosine monophosphate dehydrogenase and have developed a pipeline
with which to test the antifungal potential of these enzymes.
Aim To characterise AdSS from Cryptococcus neoformans and investigate its potential as an antifungal drug target.
Methods The AdSS-encoding gene ADE12 was deleted from typestrain H99, and the effects of this deletion on growth, adenine
requirements, the production of several virulence factors and virulence in both C. elegans and mice were investigated. Phenotypic
changes were confirmed using knock-out strains complemented with
wild-type ADE12 and the homologuous gene from Escherichia coli.
Cryptococcal AdSS was also expressed in E. coli and purified via
nickel-affinity and size-exclusion chromatography. Purified protein
was used to complete enzyme kinetic and multi-angle laser light scattering (MALLS) studies. Purified AdSS was crystallised both in its apo
form and bound to several substrates and inhibitors, and its structure
solved. This crystal structure was used to examine the structural differenced between human and AdSS to gain an insight unto potential
routes of antifungal development.
P067
Enhancing the complementation process in Cryptococcus
neoformans
S. D. M. Arras and J. A. Fraser
University of Queensland, Brisbane, QLD, Australia
Introduction Just as Kochs postulates formed the foundation of
infectious disease study, so do Stanley Falkows adapted molecular
postulates which define the approach to be taken in determining
whether a gene is involved in pathogenesis. Fundamentally, these
molecular postulates state that if a gene is involved in virulence, its
removal will compromise virulence. Likewise, its reintroduction
should restore virulence to the mutant. The deletion of genes in
Cryptococcus neoformans is a well-established technique, with the biolistic transformation of a dominant selectable marker and subsequent
deletion of gene of interest via homologous recombination. However,
the complementation of these mutants is less straightforward.
Currently one of two approaches tends to be taken: recreation of the
original locus and random integration.
Complementation of a deletion mutation by reinsertion of the gene
at the original locus is unlikely to interrupt any other genes and may
give a wild type level of expression, however these transformants can
be indistinguishable from a wild type contaminant if direct selection is
used, and if it is not it requires integration of a secondary marker
which may affect adjacent genes. Secondly and most importantly, one
may not be able tell if the original mutant phenotype was due to the
gene deletion or unintended affects from adjacent genes.
In contrast, random integration involves the complementation
construct being biolistically transformed into a random location in
the genome. While this strategy requires little experimental design
and results in an abundant number of transformants, as this species
has a gene rich genome it is probable that other genes are disturbed
during such events. Short of determining the precise insertion site it
is impossible to tell what genes are affected, and in turn what unintended phenotypic consequences have arisen. Importantly, these secondary phenotypes may only be obvious under certain conditions,
such as in a murine model. To counter the drawbacks of the current
approaches to complementation we have created a new tool to assist
in the to complementation mutant strains.
Aim To create a new resource that facilitates the complementation
of mutant strains in C. neoformans.
Methodology To avoid the disruption of random genes during
introduction of a complementation construct, it was decided to design a
plasmid that targets the transformed DNA to a preselected, silent location. A pBluescript-SK derivative plasmid was created containing
homology for the silent site, as well as a C. neoformans selectable marker.
In order to retain blue/white screening, these features were inserted into
the backbone of pBluescript, keeping the multicloning site intact.
Results We determined that the complementation plasmid integrates
at high frequency in the correct position, effectively complementing a
mutant strain without causing secondary mutations that complicate
analyses of our mutant strains. Transformants bearing the complementation plasmid at the correct genomic location were easily identified using a multiplex colony PCR assay. Importantly, once the
plasmid successfully integrates, qRT-PCR on the flanking genes on
either side of the silent region revealed no changes in their expression, and no secondary phenotypes were observed.
63
Poster Abstracts
P068
Deletion of PKC1 by biolistic transformation results in
aneuploidy in Cryptococcus neoformans
M. J. Donlin,1 R. Upadhya,2 W. Lam2 and J. K. Lodge2
1
Saint Louis University School of Medicine, St. Louis, MO, USA and
2
Washington University School of Medicine, St. Louis, MO, USA
Background Cryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell
wall is essential to viability and pathogenesis of C. neoformans, and
biosynthesis and repair of the wall is primarily controlled by the cell
wall integrity (CWI) signaling pathway. The protein kinase C,
encoded by the PKC1 gene is a DAG-activated kinase and the DAGresponsive C1 domain has been shown to be essential for production
of melanin. In a prior study, a pkc1D strain had severe cell wall phenotypes, sensitivity to a variety of cell wall stressors and requires an
osmotic stabilizer for growth in YPD. The pkc1D was confirmed by
multiple PCR screens and a Southern blot analysis for the correct
homologous recombination and single insertion. Complementation of
the pkc1D strain within the native locus resulted in wild-type growth
and loss of sensitivity to cell wall stressors. We conducted an RNA
sequencing experiment to compare pkc1D to wild-type KN99 and discovered that ~30% of the genes on chromosome 5 were up-regulated
compared to ~810% of the genes on any other chromosome. We
noted that passage of this deletion strain on YPD only eventually
resulted in a strain that grew normally even in the absence osmotic
stabilization. Taken together, these results led us to hypothesize that
deletion of PKC1 by biolistic transformation resulted in an aneuploid
chromosome and that this aneuploidy may be lost after passaging
without osmotic stabilization.
Aims Our first aim was to conduct a comparative genome-hybridization to determine if chromosome 5 in the pkc1D strain was aneuploid. Our second aim was to characterize the cell wall phenotypes of
any deletion strains that had lost the aneuploid chromosome. Our
third aim to repeat these experiments with additional isolates of the
PKC1 deletion.
Methods The pkc1D strain was generated by biolistic transformation
of wild-type KN99. A confirmed pkc1D strain was passaged for several generations on YPD only; this strain is subsequently referred to
as the pkc1D adapted. Genomic DNA was isolated from wild-type
KN99, pkc1D and a pkc1D-adapted strains and hybridized to H99
64
P069
Effect of histone deacetylases inhibitors on the virulence
phenotypes of Cryptococcus neoformans
F. Brandao, L. S. Derengowski, P. Albuquerque and
M. J. Pocas-Fonseca
University of Brasilia, Brasilia, Brazil
Cryptococcus neoformans infection is due to expression of various virulence factors among which we highlight the ability of the fungus to
grow at 37 C, presence of a polysaccharide capsule and melanin,
urease and phospholipases production. However, the mechanisms
that regulate the expression of these virulence factors are not well
known. Recent studies indicate that a key regulatory mechanism in
gene expression is the modulation of chromatin. Histone deacetylase
inhibitors (iHDAC), such as Sodium butyrate (NaBut) and Trichostatin A (TSA), have been employed in studies in different cell types
showing to be able to affect chromatin structure leading to changes
in gene expression.
Objective In this context, the aim of this work was to study the
effect of these HDAC inhibitors on expression of the major virulence
factors of C. neoformans, as well its effect onhost-pathogen
interaction.
Methods Different concentrations of the drugs were applied to C.
neoformans cultures grown on several conditions in order to analyze
the expression of virulence factors. Growth curves were performed by
measuring the cell density at 630 nm at 30 and 37 C. For assays of
capsule and melanin production, cells were incubated in minimal
medium with/without L- DOPA. Cell size was evaluated by flow
cytometry. Phospholipase activity was estimated in agar base containing egg yolk. Urease secretion was analyzed in Christiansens
urea agar medium. For mating assays, opposite mating types (K99a
and K99a) were inoculated on agar Filament at room temperature in
the dark and were observed daily in a microscope for hyphae and
other mating structures formation. Survival curve were performed
infecting caterpillars of the alternative host Galleria mellonella with C.
neoformans cells pretreated or not with iHDAC.
Results and discussion NaBut concentrations starting at
1 mmol l1 were able to interfere significantly in C. neoformans
growth at both 30 and 37 C. Flow cytometry revealed a fungal cell
size decrease in the presence of 10 mmol l1 of the NaBut. There
was also a significant reduction in capsule size at concentrations as
low as 1 mmol l1 of this drug. A decrease in the cell size in the
presence of 10 mmol l1 of NaBut was observed by flow cytometry.
Secreted phospholipase activity, melanin synthesis, and mating
hyphae formation were all affected at NatBut concentrations as low
as 5 mmol l1. TSA showed very subtle effects compared to NaBut
in all analyzes performed. It was able to reduce cell growth only at
Poster Abstracts
P070
The major cytoplasmic exonuclease Xrn1p affects virulence
and mating in Cryptococcus neoformans
C. Wollschlaeger,1 N. Trevijano,2 X. Wang,3 M. Legrand,1
O. Zaragoza2 and G. Janbon1
1
Institut Pasteur, Paris, France; 2National Center of Microbiology,
ISCIII, Madrid, Spain and 3Duke University, Durham, NC, USA
Background Extracellular vesicles (EVs) are membranous structures
used by a number of fungal species cells, including Cryptococcus neoformans, to deliver virulence factors to the extracellular milieu. C.
neoformans EVs are immunologically active and can regulate the antifungal activity of phagocytes. The interaction with environmental
predators is essential for the establishment of virulence mechanisms
used by C. neoformans. The ability of EVs to modulate the antifungal
activity of environmental phagocytes and the processes that mediate
C. neoformans uptake by Acanthamoeba castellanii are still unknown.
A. castellanii expresses a surface mannose-binding-receptor (MBR)
and this receptor is described as important for bacterial phagocytic
events with this predator.
Aim To characterize the effect of EVs produced by C. neoformans on
the antifungal activity of the environmental predator A. castellanii
and identify the association of MBR for amoeba phagocytosis and
killing of this yeast.
65
Poster Abstracts
P071
Genotypic variation of Cryptococcus neoformans variety
grubii from Asia and South Africa
K. Khayhan,1 J. Vreulink,2 B. Theelen,3 F. Hagen,4 A. Botha2 and
T. Boekhout3
1
Faculty of Medical Sciences, University of Phayao, Phayao,
Thailand; 2University of Stellenbosch, Stellenbosch, South Africa;
3
Fungal Biodiversity Centre (CBS-KNAW), Utrecht, The
Netherlands and 4Canisius-Wilhelmina Hospital, Nijmegen, The
Netherlands
Background Cryptococcus neoformans variety grubii is an opportunistic pathogen that causes diseases, particularly cryptococcal meningitis, pulmonary cryptococcosis and cutaneous cryptococcosis, in both
immunocompetent and immunocompromised individuals. In subSaharan Africa Asia, the number of cryptococcosis patients is estimated more than 720 000 new cases per year, followed by South
and Southeast Asia accounting for 140 000 new cases per year. In
this study, we used multilocus sequence typing (MLST) to study the
genetic diversity among cryptococcal isolates from different Asian
regions and from South Africa.
Objectives We determined genotypic variation of C. neoformans var.
grubii isolates collected from various geographical locations in Asia
and from Cape Town, South Africa using MLST. Furthermore, we
compared the genotypes obtained to those of C. neoformans var. grubii
isolates from the other continents.
Methods Four hundred and seventy-six C. neoformans var. grubii
were obtained from various countries in Asia, including China, Hong
Kong, India, Indonesia, Japan, Kuwait, Qatar and Thailand, and 31
isolates from Cape Town, South Africa. MLST was performed to
determine sequence types (STs). MLST data of C. neoformans var. grubii from previously publications were used for comparison at the global level.
Results MLST showed that 4 predominant STs (i.e. STs 4-6 and
ST93) occurred among 476 Asian C. neoformans var. grubii isolates
and 3 common STs (i.e. ST23, ST40 and ST69) occurred among 31
isolates from South Africa. Among the Asian population the distribution of STs was different in each country. The minimum spanning
tree of the global MLST C. neoformans var. grubii isolates showed that
most Asian isolates clustered together in one group and differed from
isolates from the other continents, whereas the South African isolates
had a scattered distribution.
Conclusion Multilocus sequence typing revealed a different distribution of genotypes of C. neformans var. grubii in different geographical
locations at the continental (Asia) and global (Cape Town) scale. The
66
predominate STs among the Asian isolates differed from those among
South African isolates. Asian isolates showed limited genetic variation, whereas isolates from South Africa showed more diversity and
occurred in all clusters present in the minimum spanning tree of the
set of global MLST data.
P072
Post-transcriptional regulation contributes to translational
prioritization during host-temperature adaptation in
C. neoformans
A. L. Bloom and J. C. Panepinto
SUNY University at Buffalo, Buffalo, NY, USA
Background In response to the hostile host environment, pathogens
undergo rapid reprogramming of gene expression to adapt to stress.
In C. neoformans, exposure to host-temperature results in rapid
repression of ribosomal protein (RP) transcripts and simultaneous
induction of ER stress response transcripts. These responses are transient as levels of both functionally related classes of mRNAs return to
basal-like quantities after 3 h. Ccr4-mediated degradation regulates
the intensity and duration of each of these responses through
enhanced destabilization of these mRNAs at the time at which each
class undergoes repression during host-temperature adaptation. In
the ccr4delta mutant, RP-mRNAs are insufficiently repressed, and the
ER stress response is constitutively active. The dissociable RNA polymerase II subunit, Rpb4, also contributes to these transient, fastrelaxing responses by coupling mRNA synthesis and degradation.
Following a shift to host-temperature, Rpb4 exits the nucleus, presumably bound to mRNAs, and mediates enhanced decay in the
cytoplasm.
Aim Our goal is to investigate the prioritization of protein translation
in response to host-temperature to understand the stress-induced cellular reprogramming required for host-temperature adaptation. We
specifically want to determine the contribution of post-transcriptional
regulation by Ccr4 and Rpb4 to this reprogramming.
Methods Stability of RP-mRNAs and ER stress mRNAs was determined by northern blot hybridization of transcript levels during inhibition of transcription. To investigate changes in translating pools of
mRNAs, polysome profiling was performed for whole cell lysates from
wt, ccr4delta, and rpb4delta strains during non-stressed conditions
and 1 and 3 h post-temperature shift from 30 to 37 C. RNA was
extracted from fractions collected during profiling, and distributions
of RPL2 (RP transcript) and KAR2 (ER stress transcript) were examined by northern blot analyses.
Results Polysome profiles do not change appreciably throughout
host-temperature adaptation in the wild type. However, polysomeassociation of RPL2 is reduced 1 hour after the temperature shift,
and increases to non-stressed levels by three hours. KAR2 exhibits
modestly more polysome-association at 1 h. Profiles are aberrant in
the ccr4delta and rpb4delta null mutants. Overall translation is
decreased following a shift to 37 C in the absence of Rpb4. While
fluctuation of RPL2 in and out of polysome fractions is maintained in
the rpb4delta mutant, the changes are not to the magnitude of the
wild type. In the ccr4delta mutant, RPL2 and KAR2 remain polysome-associated throughout the time course. Additionally, in contrast
to the wild type, as polysomes become heavier, less RNA is associated
when Ccr4 is absent.
Discussion/conclusion Our previous work demonstrates that
mRNA synthesis and decay are coupled processes. Our current investigations will address how uncoupling of mRNA synthesis and decay
impacts stress-responsive translational prioritization. Polysome analyses demonstrate that the association of RP-mRNAs with the translating pool is reduced 1 hour after a shift to 37 C. We previously
demonstrated that RP-mRNAs are maximally repressed by Rpb4- and
Ccr4-mediated enhanced decay at this time. The combinatorial effect
of these processes likely results in more available ribosomes to translate stress-specific proteins. Indeed, we observe higher levels of stressinduced ER stress transcripts, and these mRNAs become more
Poster Abstracts
P073
A whole population comparative genomics approach to
understanding the emergence of Cryptococcus gattii in the
American Pacific Northwest
D. M. Engelthaler,1 N. Hicks,1 J. Gillece,1 C. Roe,1 J. M. Schupp,1
R. C. May,2 K. Voelz,2 M. C. Fisher,3 C. Firacative,4 L. Trilles,4
G. R. Thompson,5 S. R. Lockhart,6 P. S. Keim1 and W. Meyer4
1
Translational Genomics Research Institute, AZ, USA; 2University
of Birmingham, Birmingham, UK; 3Imperial College, London, UK;
4
Unversity of Sydney, Sydney, NSW, Australia; 5University of
California Davis, Davis, CA, USA and 6Centers For Disease
Control and Prevention, Atlanta, GA, USA
Background The emergence of distinct populations of Cryptococcus
gattii (type VGII) in the temperate North American Pacific Northwest
(PNW) was unexpected, in that C. gattii was previously known to be
endemic only to tropical and semi-tropical regions. Beyond a new habitat niche, the dominant emerged populations displayed high virulence
and caused primary pulmonary disease, as opposed to neurologic disease often seen in other C. gattii infections. Since the emergence, there
has been speculation about the origin of the outbreak strains.
Aim In this study, whole genome sequence analysis was performed
on 118 Cryptococcus isolates, primarily C. gattii VGII, to better ascertain the genomic changes behind the PNW emergence.
Methods Using Illumina next generation sequencing technology, we
sequenced 113 C. gattii VGII isolates and one isolate each of VGI,
VGIII, and VGIV genotypes. Our analysis also used genomes from
previously sequenced isolates of C. neoformans var. neoformans and C.
n. var. grubii. We assessed all SNP mutations among isolates, and
compared the gene content of the PNW populations to all other
known global VGII subtypes and other Cryptococcus genomes.
Results While the overall VGII population was highly diverse, demonstrating large amounts of both mutation and recombination, the
three dominant subtypes in the PNW had low diversity and are completely clonal. Although, VGII was found on at least five continents,
all genetic sub-populations had representation, or closest relatives, in
South America. Numerous (>50) gene content differences were identifiable, including genes potentially related to habitat adaptation, virulence and clinical differentiation. Evidence was also found of a likely
introgression event, from C. n. var. grubii, of a gene complement
rarely seen in the global C. gattii population but found in all PNW
populations.
Discussion The phylogenetic data highlight multiple dispersal events
to North America and elsewhere, likely originating from South America. The identification of novel gene content among and between the
PNW populations, while not causal, provides evidence of genomic
evolution that allowed for the recent expansion of habitat and disease.
The findings here provide greater understanding of C. gattii adaptation in North America and its dispersal from South America.
P074
Multilocus sequence typing of clinical and environmental
isolates of Cryptococcus neoformans in Uberaba, Brazil
M. L. Silva Vergara, V. M. Moraes-Manzato, K. F. Ferreira Paim,
A. Vilas-Boas, T. B. Bragine Ferreira, L. O. Oliveira David,
D. J. Mora and L. A. Andrade Silva
Tria^ngulo Mineiro Federal University, Uberaba, Brazil
Background Cryptococcus neoformans is widely distributed and
accounts for most cases of cryptococcosis mainly in HIV infected
patients. According to WHO estimatives, yearly, one million cases of
cryptococcosis occur around the world, of whom, 620 000 die as
consequence of this infection. During the last decades, molecular
tools contributed to the development of Cryptococcus complex taxonomy, identifying several serotypes, genotypes and sub-genotypes
between the two pathogenic species. More recently, the multi locus
sequence typing (MLST) was defined a gold standard technique to
evaluate seven different loci among clinical and environmental isolates of Cryptococcus neoformans and Cryptococcus gattii.
Aim To characterize clinical and environmental isolates of Cryptococcus neoformans by MLST.
Methods A total of 100 C. neoformans isolates were analyzed. Of
these, 61 were recovered from 55 different AIDS patients with cryptococcosis, of which, 40 from the cerebrospinal fluid (CSF), 12 from
blood, five from urine, two from skin lesions and one of broncho
alveolar lavage (BAL). The remaining 39 isolateswere obtained from
environment surrounding areas of a teaching hospital. The sequencing experiments were done to the loci CAP59, LAC1, IGS1 and
URA5 according ISHAM MLST consensus scheme for Cryptococcus
neoformans and Cryptococcus gattii.
Results The CAP59 locus presented 99 single nucleotide polymorphisms (SNPs), and five ATs. The isolates identity compared to the
strain H99 ranged from 92.6 to 100% whereas the SNPs ranged
from 0-37. The locus IGS1 presented 41 SNPs and eight ATs. Identities and number of SNPs ranged from 85.7 to 100% and from 0 to
90, respectively. The locus LAC1 presented 48 SNPs and five ATs.
Identities and number of SNPs ranged from 90.1 to 99.8 and from 1
to 45, respectively. Finally, the locus URA5 presented 39 SNPs and
five ATs. Identities and number of SNPs ranging from 94.2 to 100%
and from 0 to 35, respectively. The phylogenetic analysis of the concatenated regions showed 13 sequence types (STs) and a total of 300
polymorphisms with identities ranging from 90.70 to 99.96%. The
IGS1 locus showed the highest number of polymorphic sites with
163 followed by LAC1, URA5 and CAP59 with 54, 42 and 41,
respectively. The highest efficiency of typing (0.122) was found in
the LAC1 locus and the greatest discriminatory power in the IGS1
ones (0.879). The ST with the largest number of isolates was ST13
with 57 isolates (87.7% clinical and 12.3% environmental). Both
ST1 and ST6 consisted of 100% and 96% of environmental isolates.
Of five patients with isolates obtained from different sites, two presented more than one ST. Both clinical and environmental isolates
were included in nine different STs.
Discussion/conclusion Through MLST it was possible to identify
nine STs among clinical and environmental isolates with a total of
13 different STs, which suggests high genetic variability of these isolates compared with other reports that evaluated a larger number of
loci. However, a whole genetic variability of the isolates herein evaluated, it will be obtained after typing the others loci suggested by
ISHAM MLST consensus. The finding of more than one ST in isolates
of the same patient seems to be an uncommon feature, since most
reports included only one isolate from each patient. A recently study
in Africa, found more than one ST in 16.7% of CSF samples genotyping more than one colony of the same patient, which can suggest
that mixed infections can be found in one or different samples. The
genetic characterization of clinical and environmental isolates of
Cryptococcus spp. by MLST contribute to understand the genetic variability and dynamic of these complex microorganisms around the
world.
67
Poster Abstracts
P075
Molecular epidemiology and ecology of environmental
Cryptococcus neoformans populations in Zambia
M. V. Vanhove,1 M. Beale,1,2 J. Rhodes,1 T. Bicanic2 and
M. Fisher2
1
Department of Infectious Diseases and Epidemiology, St Marys
Campus, Faculty of Medicine, Imperial College London, UK and
2
Saint George Hospital - University Medical Center, London, UK
Population genetics suggests that the Cryptococcus neoformans var.
grubii (Cng) VNI lineage has emerged out of Africa before spreading
worldwide. However, the population structure of the environmental
pathogen remains largely unknown in Africa. This project aims to
explore the population genetic structure of environmental populations of the basidiomycete yeast in southern Africa.
Multiple ecological niches have been screened to isolate Cng isolates from the environment. More than 400 sites have been investigated across four ecoregions throughout Zambia. Samples from tree
bark and soil were cultured on Niger-seed (NS) plates. The Cryptococcus neoformans/gattii species complex appears as dark-brown colonies
on NS agar. The fungal diversity present in these ecological niches
was also assessed by sequencing the internal transcribed spacer (ITS)
region of more than 500 individual colonies.
The sequencing of the capsular gene CAP59 allowed the differentiation between C. neoformans (n = 35) and C. gattii (n = 40) and
neighbour joining tree gave insights on the C. neoformans and C. gattii diversity present in Zambia. This preliminary work showed the
validity of the protocol to isolate Cryptococcus species and paved the
way to the sequencing of these Cng isolates using whole genome
sequencing (WGS) technologies.
Another question of interest is how this environmental yeast
acquired its pathogenicity and became an important human pathogen. Current knowledge suggests that environmental selection pressure from invertebrate hosts led to the pathogenicity of C. neoformans
observed in humans. Cryptococcus neoformans is facultative intracellular pathogen and is able to replicate inside macrophages. Previously,
an amoeba model, using Acanthamoeba castellanii was successful in
describing phenotypic variations between different strains. This model
was used using ImageStream imaging flow cytometry to quantify
cryptococcal cells uptake. Using different stains, cryptococcal cells
can be visualised and counted inside and outside amoeba. A cohort
of both clinical and environmental isolates has been selected depending of geographic and genetic characteristics. Phenotype differences
in Cryptococcus uptakes could lead to interesting observations regarding the development of hypervirulent lineages in humans. The functional analysis might reveal that local adaptation leads to variations
in Cryptococcus pathogenicity.
P078
P076
Phylogenetic analysis of cryptococcus laurentii isolates
reveals a high frequency of sibling species
M. L. Silva Vergara,1 T. B. Bragine Ferreira,1 L. A. Andrade Silva,1
D. J. Mora,1 F. Machado Fonseca,1 M. S. De Souza Carvalho
Melhem,2 D. Springer3 and K. F. Ferreira Paim1
1
Tria^ngulo Mineiro Federal University, Uberaba, Brazil; 2Adolfo
Lutz Institute, Sa~o Paulo, Brazil and 3Duke University Medical
Center, Durham, NC, USA
Background The Cryptococcus genus is comprised by more than
100 species, most of them considered saprophytic. At present, Cryptococcus neoformans and Cryptococcus gattii are considered pathogenic,
but some species such as Cryptococcus laurentii have recently been
described infecting immunocompromised hosts.
68
Poster Abstracts
distinct species. C. gattii VGII isolates are causing the Pacific Northwest outbreak, whereas VGIII isolates frequently infect HIV/AIDS
patients in Southern California and Africa. VGI, VGII, and VGIII have
been isolated from patients and animals in the Western US, suggesting these molecular types occur in the environment. However, only
two environmental isolates of C. gattii have ever been reported from
California: CBS7750 (VGII) and WM161 (VGIII). The incongruence
of frequent clinical presence and uncommon environmental isolation
suggest an unknown C. gattii reservoir in California.
Aim We sought to collect C. gattii from environmental samples from
areas that had confirmed reports of clinical and/or veterinary infections to identify the local environmental reservoir of C. gattii in
Southern California.
Methods Swab and soil samples were collected during the summers
of 2011 and 2012 utilizing BD BBL Single application CultureSwabs
with Liquid Amies. In 2011 samples were obtained from 9 locations,
64 trees (30 different species), and 25 soil samples in the greater Los
Angeles area. In 2012 the two sites that had previously yielded C. gattii were resampled and 15 additional sites were sampled. From these
trees 45 trees were swabbed and 33 soil samples were collected. Cryptococcus isolates were selected on Niger seed agar containing chloramphenicol (0.5 g l1). Yeast colonies producing brown pigmentation
were selected and colony purified. All clinical and environmental isolates were streaked onto canavanine-glycine bromothymol blue (CGB)
agar and incubated for 13 days to identify C. gattii isolates.
Clinical isolates were obtained from 48 patients who were treated
at the University of Southern California, Harbor-UCLA Medical Center, or Kaiser Permanente Downey Hospital between February 2008
and January 2013. The clinical isolates were de-identified and linked
to major cross streets near the patients homes.
Multilocus sequence typing was performed on 12 loci (SXI1a or
SXI2a, IGS, TEF1, GPD1, LAC1, CAP10, PLB1, MPD1, CAP59,
TOR1, SOD1, and URA5). Ploidy was determined by fluorescence
activated cell sorting. Whole Genome Sequencing was done by Illumina paired end reads. Fertility was assessed with standard mating
competent strains. Intracellular proliferation rate and tubular mitochondrial morphology were determined utilizing J774 macrophages.
Virulence was assessed in BALB/c and A/JCr mouse models.
Results Here we report frequent isolation of C. gattii VGIII MATa and
MATa isolates and infrequent isolation of VGI MATa from environmental sources in Southern California. VGIII isolates were obtained
from soil debris associated with tree species not previously reported as
hosts from sites near residences of infected patients. These isolates are
fertile under laboratory conditions, produce abundant spores, and are
part of both locally and more distantly recombining populations. MLST
and whole genome sequence analysis provide compelling evidence
that these environmental isolates are the source of human infections.
Isolates displayed wide-ranging virulence in macrophage and animal
models. When clinical and environmental isolates with indistinguishable MLST profiles were compared, environmental isolates were less
virulent. Taken together, our studies reveal an environmental source
and risk of C. gattii to HIV/AIDS patients with implications for the
>1 000 000 cryptococcal infections occurring annually for which the
causative isolate is rarely assigned species status. Thus, the C. gattii
global health burden could be more substantial than currently appreciated with diagnostic, prognostic, and treatment implications.
P079
Unisexual reproduction reverses Mullers Ratchet
K. C. Roach and J. Heitman
Duke University, Durham, NC, USA
Background Cryptococcus neoformans is a pathogenic basidiomycetous fungus that engages in outcrossing, inbreeding, and selfing
forms of unisexual reproduction as well as canonical sexual reproduction between opposite mating-types. Long thought to be clonal,
>99% of sampled environmental and clinical isolates of C. neoformans
are MATa limiting the frequency of opposite mating-type sexual
P080
The H99 family tree: variation in the common laboratory
reference strains of Cryptococcus neoformans var. grubii
characterised through whole-genome sequencing
K. L. Ormerod,1 E. J. Byrnes III,2 I. A. Wood,1 J. K. Lodge,3
J. Heitman4 and J. A. Fraser1
1
University of Queensland, Brisbane, QLD, Australia; 2National
Institutes of Health, Bethesda, MD, USA; 3Washington University,
St Louis, MO, USA and 4Duke University Medical Center,
Durham, NC, USA
Background Mutational analysis of a species inevitably relies on
choosing a laboratory reference or wild-type strain; this reference
strain provides the background against which phenotypic changes are
contrasted and consequently a better characterised reference aids both
interpretation and comparison of results. The most widely used reference for Cryptococcus neoformans var. grubii is H99, isolated in 1978
from a 30 year old male with Hodgkins lymphoma at Duke University
Medical Center. Since then, laboratory passage has led to the formation of two distinct lineages with different phenotypic characteristics:
the Stud lineage consisting of the Duke University stock H99F (the
genome sequence subculture), Stud (H99S), and the congenic pairs
KN99a/a and YL99a/a, and the Wimp lineage consisting of the Duke
University Eunuch (H99ED), the Washington University Eunuch
(H99E) and subsequent derivations CM018 (H99C) and Wimp
(H99W). Each lineage is distinguishable based on two key phenotypes:
while mating and melanisation is increased in the Stud lineage, it is
decreased in the Wimp lineage. Sequencing of strains within both lineages and comparison has already uncovered multiple small mutations
separating them however the picture currently remains incomplete.
Aim To fully characterise the genomic variation between laboratory
reference strains used by the Cryptococcus community.
Methods The genomes of each of the strains were sequenced to
approximately 30X coverage. Strains H99ED, H99W, H99S were
sequenced using Illumina 72 bp paired-end reads. All other strains
were sequenced at BGI using Illumina 90 bp paired-end reads. Reads
69
Poster Abstracts
were mapped against the H99 reference genome using BWA and
variation detected using a combination of the Genome Analysis Toolkit and BreakDancer. Mappings were visualised using IGV and CLC
Genomics Workbench (CLC bio). Further strain genotyping was conducted using Sanger sequencing performed at BGI and the Australian
Genome Research Facility.
Results Analysis of each of the genomes uncovered a small number
of SNPs and indels separating the key strains enabling us to establish
a detailed pedigree describing their history. Further analysis was conducted by tracing these mutations through a progeny set originating
from CM018 and KN99a. A clear association was identified between
two linked indels and decreased mating capacity and melanisation.
While one of these mutations was predicted to be silent, the other
occurred within a glutamine rich protein without functional annotation. A deletion mutant created in the Stud background revealed a
role in both mating and melanisation, with a slight reduction in melanisation and a significant reduction in mating proficiency observed.
For this reason we dubbed the gene LMP1 for low mating performance. The lmp1delta mutant was also avirulent in the mouse model
of infection.
Discussion/conclusion The process of characterising genes via the
creation of gene deletion mutants relies on understanding of the background against which the mutation is created. The full characterisation of the strains commonly used as references within the
Cryptococcus community will ensure accurate interpretation of results
and facilitate collaboration between laboratories. The small number of
SNPs and indels observed means that mutations behind other key
phenotypic variation between these strains can also now be identified.
P081
Evaluation of modified maldi-TOF-based approach to
identification and typing of Cryptococcus neoformans
clinical isolates
N. V. Vasilyeva,1,2 A. A. Atsapkina,1 I. A. Riabinin,1,2
T. S. Bogomolova1,2 and G. A. Chilina2
1
Mechnikov North-Western State Medical University, SaintPetersburg, Russia and 2Kaschkin Research Institute of Medical
Mycology, Saint-Petersburg, Russia
Background MALDI-TOF-mass-spectrometry is a promising method
in the laboratory diagnosis of cryptococcosis. Recent works in this
area are related not only with the species identification of Cryptococcus spp., but also with the recognition of molecular types established
by MLST. However, with other methods of typing (RFLP; AFLP, ITSgenotype, PCR-fingerprinting) such works are not carried out yet.
S. L. Cohen and B. T. Chait (Anal. Chem., 1996) indicated the possibility of using different solvent systems for the MALDI-matrix in the
mass-spectrometry of protein with molecular masses >6 kDa, but
since then there is no data on the use of these solvents for microbial
identification in MALDI Biotyper.
Aim The aim of this study is to compare results of Cryptococcus neoformans species identification, obtained by MALDI-TOF-MS with using
different solvents for MALDI-matrix, and to reveal the compliance
between molecular types of C. neoformans and mathematical classifications of proteome mass-spectra.
Methods Seventeen clinical isolates of C. neoformans from Russian
Collection of Pathogenic Rungi (RCPF) were previously analyzed
using M13-PCR-fingerprinting and URA5-restriction fragment length
polymorphism (RFLP) with HhaI and Sau96 restrictases in Molecular
Mycology Laboratory of Centre of Infectious Diseases and Microbiology, Westmead, Australia (W. Meyer et al., Problems in Medical
Mycology, 2003, in Russian). Used strains were attributed to three
types: VNI (11 strains), VNIII (3 stains) and VNIV (3 stains).
For MALDI-TOF-mass-spectrometrical investigation strains were
subcultivated in wort-agar plates during 24 h at 37 C. Protein
extraction from cell suspensions was performed according to Bruker
standard protocol. MALDI-TOF-mass spectrometry was made with
70
Poster Abstracts
P082
P083
71
Poster Abstracts
P084
Velvet proteins are mating regulators in Cryptococcus
neoformans
L. F. Fernandes Matos,1 T. C. Santos,1 A. L. N. Barros,1
L. D. F. Peconick,1 H. C. Paes,1 C. B. Nichols,2 J. A. Alspaugh,2
M. S. Felipe1 and L. F. Fernandes1
1
Universidade de Braslia, Braslia, Brazil and 2Duke University,
Durham, NC, USA
Background Velvet proteins comprise four highly conserved members among ascomycetes and basidiomycetes, all of which share the
common Velvet domain. They regulate different signaling pathways
in response to environmental stimuli and also coordinate secondary
metabolism and asexual and sexual differentiation in different fungal
species. Recently, Velvet proteins have been implicated in the virulence of some plant pathogens.
Aim To identify and elucidate the role of Velvet proteins in C. neoformans, the VEL1, VEL2 and VEL3 genes were deleted and the mutants
were analyzed for phenotype.
Methods Identification of Velvet genes: The Velvet homologues were
found by using the amino acid sequences of the Velvet proteins of
Aspergillus nidulans (VeA, VelB and VelC) as queries for the BLASTP
tool of the Broad Institute H99 C. neoformans genome database. The
retrieved best hits, CNAG_02387, CNAG_00564, CNAG_02697 were
named VEL1, VEL2 and VEL3, respectively. The fourth member of
this family, VOSA, was also found and pertaining data are presented
elsewhere (Peconick, et al).
Deletion and reconstitution: The knockout strains were generated by
DJ- PCR followed by biolistic transformation of the KN99a and
KN99a wild types using the selective markers NATR and HYGR,
respectively. The reconstituted strains were generated on the KN99a
background by transforming the mutant strains with the deleted gene
alongside the pJAF1 plasmid containing NEOR. Single insertion into
the appropriate locus was confirmed by Southern blotting or PCR.
Phenotypical analysis: The mutant and reconstituted strains were
tested in vitro for common phenotypes associated with virulence as
capsule, melanin, urease and phospholipase production, growth at
37 C and resistance to several cell wall, osmotic and oxidative stressors. Strains were mated on SLAD, Filament or MS agar in the presence and absence of light. The mating filaments were analysed
microscopically.
Virulence assays: The strains were tested for virulence in the in vitro
J774.A16 murine macrophage model of phagocytosis followed by
CFU counts.
Results and discussion C. neoformans Velvet homologues have the
Velvet domain. All Velvet mutants showed no defects related to virulence and stress conditions, but presented altered phenotypes on mating assays.
Deletion of VEL1 causes hypermating: the filaments from crosses
between vel1D mutants occurred two days earlier relative to the wild
type crosses; it also affects the sensing of the light, as mutants produce filaments even in the presence of white light, which is a known
mating repressor. Therefore, Vel1 is a negative regulator of C. neoformans mating. In contrast, deletion of VEL2 blocks the production of
hyphae on mating conditions. Furthermore, a defect in cell fusion
was observed on vel2D. The absence of fusion indicates imbalance in
the pheromone pathway in these mutants, which is essential for the
initial cell recognition stages and fusion. The vel2aD vs. KN99alpha
cross produced shorter filaments, but normal spore chains. Thus,
VEL2 is a positive modulator of mating, and a single gene copy is
sufficient to trigger the process. VEL3 plays a minor role on C. neoformans mating as the vel3D mutant crosses presented a slight
72
P085
Functional characterization of bZIP-domain containing AP-1
like transcription factor, Bap1 in human fungal pathogen
Cryptococcus neoformans
S. Maeng, H. Kim and Y. S. Bahn
Yonsei University, Seoul, South Korea
Background Cellular response and adaptation to oxidative stress are
essential for survival and proliferation of a human fungal pathogen
Cryptococcus neoformans during host infection. Although previous
studies reported that various enzymes are required for antioxidant
defense system against reactive oxygen species, transcription factors
involving in this mechanism still remain elusive in C. neoformans. In
Saccharomyces cerevisiae, Yap1 (Yeast AP-1 protein) is critical for the
oxidative-stress response and adaptation and induces a variety of oxidative stress-responsive genes.
Aim In this study, we aimed to identify and characterize the function
of Bap1 (bZIP-domain containing AP-1-like transcription factor 1) in
oxidative stress defense system and virulence of C. neoformans.
Methods BLAST search and protein domain analysis were performed
to identify a Yap1 ortholog in C. neoformans. Expression levels of
BAP1 during oxidative stress responsewere measured by Northern
blot analysis. The bap1D mutants were constructed by overlap PCR
followed by biolistic transformation method. Stress sensitivity was
assessed by treating wild-type and mutant cells with diverse stressinducing agents and antifungal drugs. Production of virulence factors, including capsule, melanin and urease, were measured and
mating ability was also monitored.
Results The absence of BAP1 increased cellular sensitivity to oxidizing agents, such as H2O2, tBOOH, diamide, and menadione. Supporting this, the expression of BAP1 was induced by oxidative stress. The
bap1D mutant also showed increased sensitivity to azole drugs and
resistance to a polyene drug, amphotericin B. Moreover, the deletion
of BAP1 reduced capsule biosynthesis and mating. In contrast, the
bap1D mutant exhibited enhanced urease and melanin production.
Conclusion/discussion Our data demonstrated that Bap1 played
important roles in environmental stress response and modulating virulence factors, proposing that Bap1 could be a potential target for
development of antifungal therapeutic approaches for the treatment
of cryptococcosis.
P086
Distinct and redundant roles of protein tyrosine
phosphatases, Ptp1 and Ptp2, in governing the
differentiation and pathogenicity of Cryptococcus
neoformans
K. T. Lee, B. Byun, J. Jung and Y. S. Bahn
Yonsei University, Seoul, South Korea
Background Mitogen-activated protein kinases (MAPKs) govern a
plethora of cellular processes in eukaryotes, such as proliferation, differentiation, programmed cell death, and stress responses. In
Poster Abstracts
response to a variety of chemical and physical stresses, MAPKs signaling is activated to sense environmental cues and eventually activate the expression of MAPK-target genes. During this process,
negative feedback regulation for controlling the duration and magnitude of signaling is as important as positive regulation because all
signaling cascades must be properly tuned to avoid deleterious overactivation or constitutive activation and subsequently desensitized to
recurrent external cues in a timely manner. However, the functions
of negative regulators or inactivating signaling components in MAPK
pathways are less well characterized than those of positive regulators. Protein tyrosine phosphatases (PTPs) serve as key negative feedback regulators of mitogen-activated protein kinase (MAPK)
signaling cascades. However, their roles and regulatory mechanisms
in human fungal pathogens remain elusive.
Aim In this study, we characterized the functions of two PTPs, Ptp1
and Ptp2, in Cryptococcus neoformans, which causes fatal
meningoencephalitis.
Methods Genes were elucidated by performing 5 and 3 rapid amplification of cDNA ends (RACE). Genes were deleted in C. neoformans
var. grubii strains H99, KN99a or other mutant backgrounds by biolistic transformation using overlap PCR or split marker/double joint
(DJ)-PCR strategies with a set of primers. Various stress response test
was performed by using solid YPD medium containing the indicated
concentration of stress-inducing agents and antifungal drugs.
Results PTP1 and PTP2 were stress-inducible genes, which were
controlled by the MAPK Hog1 and the transcription factor Atf1.
Ptp2 suppressed the hyperphosphorylation of Hog1 and was involved
in mediating vegetative growth, sexual differentiation, stress
responses, antifungal drug resistance, and virulence factor regulation
through the negative feedback loop of the HOG pathway. In contrast,
Ptp1 was not essential for Hog1 regulation, despite its Hog1-dependent induction. However, in the absence of Ptp2, Ptp1 served as a
complementary PTP to control some stress responses. In differentiation, Ptp1 acted as a negative regulator, but in a Hog1- and Cpk1independent manner. Additionally, Ptp1 and Ptp2 localized to the
cytosol, but were enriched in the nucleus during the stress response,
affecting the transient nuclear localization of Hog1. Taken together,
our data suggested that PTPs could be exploited as novel antifungal
targets.
Discussion Ptp1 and Ptp2 played minor and major roles, respectively, in the virulence of C. neoformans. While Hog1 was obviously a
major target of Ptp2, other signaling pathways appeared to also be
regulated by Ptp2 in C. neoformans. The PTP2 overexpression(oe)
strain often showed more severe or opposite phenotypes compared to
the hog1D mutant in response to stresses (e.g. CdSO4, H2O2, and flucytosine); however, these potential additional targets are not yet
clear. In our study, Ptp1 appeared to be largely dispensable for Hog1
signaling and the growth of C. neoformans, despite its Hog1-dependent induction. One notable role of Ptp1 is its involvement in sexual
differentiation. PTP1oe, but not PTP2oe, reduced normal mating efficiency and suppressed enhanced mating in the hog1D mutant. However, PTP1oe failed to suppress enhanced MFa1 expression, strongly
suggesting that Ptp1 could modulate mating without direct involvement in Cpk1 and pheromone production. In C. neoformans, Ptp1
and Ptp2 localized to both the cytosol and the nucleus, but were
enriched in the nucleus. Furthermore, the finding that deletion of
PTP1 or PTP2 reduced transient nuclear accumulation of Hog1
under stressed conditions implied that both Ptp1 and Ptp2 may have
Hog1-anchoring functions in the nucleus of C. neoformans. However,
it remains to be addressed how Ptp1 and Ptp2 modulate the cellular
localization of Hog1.
P087
Role of the inositol polyphosphate kinases, Ipk1 and Asp1,
in the pathogenesis of Cryptococcus neoformans
C. Z. J. Li,1 S. Lev,1 A. Saiardi,2 D. Desmarini,1 T. Sorrell3 and
J. T. Djordjevic1
1
Westmead Millennium Institute and the University of Sydney,
Sydney, NSW, Australia; 2University College London, London, UK
and 3Marie Bashir Institute, University of Sydney, Sydney, NSW,
Australia
Background Phospholipase C (Plc1) is essential for homeostasis and
virulence of Cryptococcus neoformans (Cn)(1). We recently identified a
Plc1 signalling pathway in Cn which involves phosphorylation of the
Plc1 hydrolysis product, inositol trisphosphate (IP3), by an inositol
polyphosphate kinase (IPK) called Arg1(2). Arg1 (an IP3 kinase), and
Kcs1, another IPK predicted to function downstream of Arg1 as an
IP6 kinase, convert IP3 to inositol polyphosphates (IP)4-5, and IP6 to
inositol pyrophosphates (PP-IP)7-8, respectively. IP3-6 have only a single phosphate at a given position on the inositol ring while PP-IP7-8
have one or more diphosphates at a single position. Both arg1(2)
and kcs1 showed a similar attenuation in their virulence composite,
and were avirulent and hypovirulent, respectively, in a mouse inhalation model of cryptococcosis (unpublished).
Aims Using gene deletion analysis, the aims of this study were to
determine the role of two other IPKs, Ipk1 (putative IP5 kinase) and
Asp1 (another putative IP6 kinase) in IP homeostasis and their contribution to the virulence composite of Cn.
Methods IPK1 and ASP1 gene deletion mutants (Dipk1 and Dasp1)
were created using biolistic transformation(3). Mutant IP profiles were
determined using inositol radiolabeling and HPLC as described(4).
Mutant phenotypes were assessed by spot dilution tests, enzymatic
assays, western blots and antifungal susceptibility testing. Virulence
was assessed using mouse inhalation and Galleria mellonella (30 C)
infection models. Mutant phagocytosis by the THP1 cell-line was
assessed by flow cytometry and monocyte activation using a custom
PCR cytokine array.
Results Similar toDarg1 and Dkcs1, Dipk1 showed a similar attenuated virulence composite: reduced growth at 37 C, increased susceptibility to antifungals, a cell wall defect, and reduced urease, laccase
and secreted acid phosphatase activity. Dipk1 was hypovirulent in
both animalmodels, with 20% of mice succumbing to infection
within 50 days. In the remaining 80%, Dipk1 lung infection persisted
for the duration of the infection period, even though mice maintained weight and vigour. Dissemination to the brain was also
observed. Dipk1 hypovirulence correlated with reduced Dipk1 uptake
and activation ofmacrophages, compared to WT. In contrast, virulence phenotypes were not compromised in CnDasp1.
Conclusion Ipk1 is required for the phenotypic expression of a similar set of virulence traits to Plc1, Arg1 and Kcs1. This suggests that
the PP-IP end products of the IPK pathway, which are commonly
absent in all the IPK mutants, are essential for production of these
virulence traits in Cn. Absence of an attenuated virulence phenotype
in asp1 suggests functional redundancy of Asp1 with Kcs1. Ipk1 is
therefore another potential antifungal drug target within the Plc1/
IPK pathway.
References 1. Chayakulkeeree M. et al. Molecular Microbiology
2008; 69: 80926.
2. Lev S. et al. Infection and Immunity 2013; 81: 124555.
3. Toffaletti DL. et al. Journal of Bacteriology 1993; 175: 140511.
4. Azevedo C, Saiardi A. Nature Protocols 2006; 1: 241622.
73
Poster Abstracts
P088
Role of a Ryp1 homologue in the virulence of
Cryptococcus neoformans
H. C. Paes,1 L. S. Derengowski,1 P. Albuquerque,1 A. M. Nicola,2
M. A. Vallim,3 J. A. Alspaugh,4 M. S. Felipe2 and
L. F. Fernandes1
1
Universidade de Braslia, Braslia, Brazil; 2Universidade Catolica
de Braslia, Braslia, Brazil; 3Federal University of Sa~o Paulo,
Braslia, Brazil and 4Duke University, Durham, NC, USA
Background Cyclic AMP-independent pathways pathways using
Gti1/Pac2 transcription factors have been shown to play a major role
in adaptation of ascomycetes to their environment and hosts. Among
other functions, these factors influence secondary metabolism, conidiation, mating, dimorphic transition and pathogenesis in fungi ranging from plant to human pathogens. However, nothing is known
about their function in basidiomycetes, despite the fact that their homologues have been annotated in genome projects.
Aim To fill a gap in our knowledge of regulatory processes in C. neoformans, we have deleted the PAC2 homologue and analysed the phenotype of the mutant.
Methods Identification of the target gene: the PAC2 homologue was
found by using the amino acid sequence of the Ryp1 protein of Histoplasma capsulatum (the only homologue characterised in a human
pathogen) as query for the BLASTP tool of the Broad Institute H99
C. neoformans genome database. The retrieved best hit,
CNAG_01983.7, was provisionally named RYP1.
Deletion and reconstitution: the ryp1::HYG strains were generated by
overlap PCR followed by biolistic transformation of the H99, KN99a
and KN99a wild types. The reconstituted strain ryp1::RYP1 was generated solely on the H99 background by transforming the mutant
strain with the RYP1 gene alongside the pJAF1 plasmid containing
the NEOr resistance marker. In both cases, transformants were
selected in YPD solid medium containing hygromycin or G418,
respectively. Single insertion into the appropriate locus was confirmed
by Southern blotting or PCR.
Phenotypical analysis: the mutant and reconstituted strains were
tested in vitro for common phenotypes associated with virulence,
namely capsule induction, melanisation, growth at 37 C, urease
and phospholipase secretion and resistance to several cell wall stressors. Strains generated on KN99 backgrounds were mated on SLAD
agar.
Virulence assays: first,the strains were tested for virulence in the in
vitro J774.A16 murine macrophage model of phagocytosis followed
by CFU counts. The in vivo virulence was assessed by survival curves
in the Galleria mellonella alternative model of infection at 37 C.
Results The ryp1::HYG strain has defects in several virulence factors. It fails to induce capsule growth, be it on DMEM/MOPS or on
Sabouraud/MOPS. In the latter, it also shows a remarkable flocculation phenotype (Fig. 1), not quite similar to the one described by
Wormley et al. (2005). Among cell wall stressors, only Congo Red
inhibited growth of the mutant, which had no difficulty growing at
37 C. Melanisation, as well as urease and phospholipase secretion,
were normal in the mutant. Of note, the mutant had a significant
growth deficit on DMEM/MOPS.
Surprisingly, given how important Pac2 homologues are to mating
and conidiation in other fungi, in C. neoformans Ryp1 seems to play
no role in mating, as mutants generated sexual filaments at the same
rate and frequency (on visual inspection) as their parental wild-type
strains.
The loss of Ryp1 impaired both survival of the fungus within macrophages in culture and its ability to colonise the invertebrate host
G. mellonella (Fig. 2), in which the mutant was completely avirulent.
Discussion The observed loss of virulence in the ryp1D::HYG strain
is not surprising given a similar finding in dimorphic and filamentous
fungi. In addition to confirming these observations on a murine
model of infection, we will follow up with an investigation of the role
of kinase signalling on Ryp1 regulation and with expression studies
74
P089
Clinical parameters of cryptococcosis are associated with
Cryptococcus strain genotype
T. McDonald,1 D. R. Boulware,1 K. Huppler Hullsiek,1
M. A. Rolfes,1 D. B. Meya,2 G. A. Meintjes,3 C. K. Muzoora4 and
K. Nielsen1
1
University of Minnesota, Minneapolis, MN, USA; 2Makerere
University, Kampala, Uganda; 3University of Cape Town, Cape
Town, South Africa and 4Mbarara University of Science and
Technology, Mbarara, Uganda
Cryptococcosis is a leading cause of AIDS-related mortality in subSaharan Africa. Previously, we showed a correlation between patient
mortality and strain genotype for 140 strains in a cohort of 111
patients with AIDS and cryptococcal meningitis. Here, we have
expanded these studies to include additional patients from multiple
regions in sub-Saharan Africa to determine the generalizability of the
association between fungal genotype and patient mortality. We analyzed a total of 660 clinically isolated strains, including 562 from
Kampala, Uganda, 22 from Mbarara, Uganda and 76 from Cape
Town, South Africa. We genotyped the strains using multi-locus
sequence typing (MLST) at eight standard loci. Genotypes were clustered using the BURST algorithm. We found 38 genotypes of Cryptococcus neoformans var. grubii (serotype A), including both VNI and
VNII strains. We found six hybrids between C. neoformans var. grubii
and C. neoformans var. neoformans (A/D hybrids) and one C. gattii
strain. Population structures differ between Uganda and South
Poster Abstracts
P090
Towards better understanding of the molecular
mechanism of fluconazole-induced aneuploidy in
C. neoformans var grubii
L. Kozubowski,1 S. Altamirano,1 S. Sridhar2 and K. Sanyal2
1
Clemson University, Clemson, SC, USA and 2JNCASR, Bangalore,
India
Background Heteroresistance is the intrinsic feature of Cryptococcus
neoformans that is characterized by the infrequent emergence of fluconazole (FLC) resistant subpopulations within a single colony of the
susceptible strain. Resistant cells are aneuploids and contain increased
copy number of genes that contribute to their survival in the presence
of FLC. While significant progress has been made to uncover multiplied genes that are responsible for the FLC resistance, the effects of
FLC on nuclear division and the exact molecular mechanism responsible for the formation of aneuploidy remain poorly characterized. Specifically, the magnitude of the effect of FLC on cell division and
change in ploidy in the overall population has not been characterized.
Moreover, it remains unclear whether the resistant aneuploid cells
are derived from initially formed diploids through subsequent chromosomal loss or are formed de-novo during FLC treatment.
Aim The purpose of this study was to examine the effects of FLC on
nuclear division of C. neoformans var. grubii
Methods Cells were grown in YPD liquid cultures supplemented
with FLC at concentrations ranging from 8 to 128 microgram/ml.
To estimate the effect of FLC on ploidy, samples were collected at various time-points, fixed, stained with propidium iodide, and analyzed
using fluorescence flow cytometry. To assess the effect of FLC on budding, unbudded cells were treated with FLC and budding index was
scored at various time-points. To assess the effects of FLC on mitosis,
localization of fluorescently tagged histone H4 and centromeric histone variant Cse4 was analyzed after FLC treatment.
Results FLC at 32 lg ml1 caused a significant inhibition of cell
division. However, FLC had no effect on bud initiation and the initial
bud growth. FLC treatment for 9 h at 8 lg ml1 resulted in a significant fraction of cells with various ploidy levels above 2n. At
64 lg ml1, the effect of FLC was significant already at 6 h and
longer incubation times increased the percentage of cells with higher
fluorescence indicative of further increase in DNA content. After 9 h
of treatment with 128 lg ml1 FLC, more than 20% of cells showed
signals corresponding to ploidy above 2n. Interestingly, no clear shift
to the diploid population was observed. At 6 h many cells with irregular shaped chromatin (GFP-H4) and cells with unequal amounts of
GFP-H4 were present, possible indicators of aneuploidy. At these later
time points mCherry-Cse4 intensity was higher in a fraction of the
population. Interestingly at time points 3 h and later, cells with two
buds began to emerge, possibly reflecting a post-anaphase and/or
cytokinesis defects. To test possible effects of FLC on the spindle
assembly checkpoint (SAC), two SAC mutant strains (mad2 and
bub1) were subject to FLC and ploidy was analyzed by flow cytometry. Strikingly, no difference was observed when compared to the wt.
Discussion/conclusions Our study revealed previously unanticipated significant dose and time-dependent effects of FLC on ploidy.
Our results suggest that aneuploidy is formed de-novo as a result of
aberrant chromosomal segregation rather than through chromosomal loss within FLC induced diploids. An epistatic-like relationship
between FLC treatment and the elimination of SAC (deletion of
BUB1 or MAD2) suggests that FLC inhibits SAC while imposing negative effects on nuclear division. We are presently testing this intriguing possibility.
P091
The Cryptococcus neoformans Velvet gene VOSA is a
positive regulator of mating
L. D. F. Peconick,1 H. C. Paes,1 C. B. Nichols,2 J. A. Alspaugh,2
M. S. Felipe1 and L. F. Fernandes1
1
Universidade de Braslia, Brasilia, Brazil and 2Duke University,
Durham, NC, USA
Background Cryptococcus neoformans is an opportunistic basidiomycetethat commonly infects HIV-positive and other immunocompromised patients. It is the causative agent of cryptococcosis, a
potentially fatal and cosmopolitan disease, the world incidence of
which is similar to that of tuberculosis. VosAbelongs to the Velvet
family, which is exclusive of fungi and was first described in Aspergillus, is involved in the regulation of sexual and asexual development
as well as in spore viability. It is also implicatedintrehalosebiosynthesis. Proteins of this family are highly conserved among ascomycetes
and basidiomycetesand regulate different processes in response to
environmental stimuli and in secondary metabolism.
Aim To elucidate the role of VosA in virulence and morphogenesis of
C. neoformans by gene deletion and mutant phenotypic analyses.
Methods The C. neoformans VOSAgene was found using the amino
acid sequences of the A.nidulansVosA protein as query for the
BLASTP tool of the Broad Institute H99 C. neoformans genome database. The retrieved best hit was CNAG_06580, re-labelled
CNAG_07989 in the latest assembly. A VOSA disruption cassette
containing the Hygromycin B resistance gene (HPH) was constructed
by DJ-PCR. The cassette was transformed by biolistics into KN99a/a
(seroA) wild type strains, thusgenerating the vosAaDandvosAaD
mutants. Likewise, the fragment containing the VOSAnative locus
was transformed into vosAaDto obtain a MATa reconstituted strain
(vosAaD::VOSAa). Disruption and reconstitution were confirmed by
Southern Blotting and PCR, respectively. The vosADmutants were
tested for various phenotypes: growth at 37 C, capsule, melanin,
urease and phospholipase production and disturbancesin cell wall
integrity.The strains were tested for virulence in the in vitro
J774.A16murine macrophage model of phagocytosis followed by CFU
counts. Ability-to-mate and fusion assays were also performed on
Murashige&Skoog (MS) medium to investigate the role, if any, of
VosA in the sexual reproduction ofC. neoformans.
Results/discussion The C. neoformans protein most similar to A.
nidulansVosAis 510aa long, with the Velvet domain in the N-terminal portion (1178), a PEST domain(187200), a bipartite nuclear
localization signal (NLS) beginning at position 180 and a nuclear
export signal between positions 132136. The vosAD mutants did
not show any phenotypic changes relative to the parental strains
with regard to any virulence factortested, including thein vitro macrophage assay; they also showed no cell wall defect. However, in the
mating assays, vosAD mutants showed reduced filamentation in unilateral crosses (WT vs. vosAD) and dramatic hypha loss in bilateral
crosses (vosAaD vs. vosAaD). Furthermore, a defect in cell fusion was
observed in the latter. Among the few filaments observed, the apical
basidia did not show any Basidiospore chains. The absence of fusion
in bilateral crosses indicates an altered pheromone pathway in these
mutants. Pheromone sensoringtriggers the mating process that
75
Poster Abstracts
P092
Mapping functional transcription factor networks to
identify novel capsule regulators
R. Gish,1 J. Maier,2 C. Haynes,2 M. Williams,1 Z. Wang,1
M. R. Brent2 and T. L. Doering1
1
Washington University St. Louis School of Medicine, St. Louis,
MO, USA and 2Washington University St. Louis, St. Louis, MO,
USA
Background The capsule of Cryptococcus neoformans is one of its key
virulence factors and changes dramatically in response to environmental conditions. A complete and integrated model of capsule regulation is fundamental for a comprehensive understanding of the
genetic networks involved in capsule synthesis.
We recently reported NetProphet, an algorithm we developed for
inferring transcriptional regulatory networks in S. cerevisiae (Haynes
et al, Genome Research, 2013). We have now applied NetProphet to
the regulation of capsule size in C. neoformans.
Aim Our goals were to develop and apply new computational methods to reconstruct the capsule regulatory network, to use the resulting network model to identify novel transcription factors involved in
capsule regulation, and to determine the role(s) of these novel transcription factors in cryptococcal biology and virulence.
Methods We used RNA-Seq to profile gene expression in a collection
of transcription factor deletion strains and analyzed the data with
NetProphet. We used the resulting network model to identify novel
transcription factors likely to act in capsule regulation, deleted the
corresponding genes, and assayed the resulting mutant strains for
capsule size. Mutants were also examined for additional virulence
factors and behavior in a mouse model of infection.
Results We used expression data from 30 cryptococcal mutants (3
biological replicates and wild type controls for each) to model the
capsule regulatory network. We identified 12 transcription factors as
likely to act in capsule regulation and successfully deleted 10 of the
corresponding genes; of this group 7 had phenotypes of altered capsule thickness and most of those were attenuated in virulence in a
short-term model of fungal growth in mice. One hypercapsular strain
was selected for further study and found to have defects in melanin
production and other traits relevant to infection; this mutant also
had severely attenuated virulence in mice. For several transcription
factors, direct targets were identified by ChIP-Seq. These studies confirmed NetProphets accuracy in mapping direct, functional interactions between transcription factors and their promoters.
Discussion/conclusion We have modeled the cryptococcal capsule
regulatory network and identified several novel transcription factors
that act in regulation of capsule synthesis. These will be the subjects
of further study. More generally, our experimental strategy combined
expression profiling and computational methods to (i) generate a
comprehensive network of upstream and downstream interactions of
genes involved in regulating a process of interest and (ii) predict
mutations that are likely to show a given phenotype of interest. This
76
method is applicable to other pathways in Cryptococcus and to important questions in other microbial systems.
P093
DNA repair, ubiquitination and virulence in Cryptococcus
neoformans
S. Verma and P. S. Shakya
School of Biological Sciences, University of Missouri-Kansas City,
Kansas City, MO, USA
Pathogenic organisms have evolved a number of ways of avoiding
the host defense systems to enable them to cause disease. Protection of its DNA from damage in response to environmental stresses,
and especially during the course of infection is one of the important
strategies adopted. An important DNA repair gene, RAD23 had
been implicated in the virulence of opportunistic pathogen Cryptococcus neoformans and knockout for this gene is shown to be hypovirulent (Liu et al, 2008). RAD23 has dual function: It is involved
in DNA repair as well as protein sorting ubiquitination pathway
(acts as ubiquitin receptor protein). We want to uncouple the function of the two roles of this gene in the virulence, to test which
role is more important for the virulence one or the other or both.
We want to figure out if Rad23 is required for nuclear DNA repair
or mitochondrial DNA repair? Another interesting aspect about
RAD23 is its proposed role in transcriptional regulation in yeast.
Apparently RAD23 is involved in regulation of almost two-third of
the UV regulated genes and almost one third of all the yeast genes
are mis-regulated in the RAD23 knockout (Wade et al, 2009 and
Wade and Auble, 2010). Rad23 has four domains, UBL, UBA1,
XPC-B, UBA2. The UBL, UBA1 and UBA2 have major implication
in protein sorting and ubiquitination pathway and XPC-B have role
in DNA repair pathway (Dantuma et al, 2009). We have performed
a full-length deletion of RAD23 and are also performing individual
domain knockouts to check their role in different DNA damage
stress and in virulence of the fungus. We are also checking these
domain deletion strains for their function in ubiquitination pathway. Gene knockouts for Rad23 are sensitive to UV stress and have
increased resistance to endoplasmic reticulum stress inducing antibiotic Tunicamycin. Rad23 -GFP fusion protein localizes to the
nucleus and cytoplasm and not to mitochondria, implicating its role
in nuclear DNA damage repair. We are in process of making different domain deletion strains of Rad23 and checking their survival
under different stress conditions and also their localization pattern.
Wax moth infection studies indicate RAD23 knockout to be hypovirulent in consistence with previous studies with mice model and
has to be checked for different strains. Completion of these studies
will shed more light on the virulence mechanisms of C. neoformans.
We will be able to uncouple the role of two different key pathways
in the virulence of the fungus.
References Liu OW, Chun CD, Chow ED, Chen C, Madhani HD,
Noble SM. Systematic genetic analysis of virulence in the human
fungal pathogen Cryptococcus neoformans. Cell 2008; 135: 174188.
Dantuma NP, Heinen C, Hoogstraten D. The ubiquitin receptor
Rad23: at the crossroads of nucleotide excision repair and proteasomal degradation. DNA Repair (Amst) 2009; 8: 449460.
Wade SL, Auble DT. The Rad23 ubiquitin receptor, the proteasome
and functional specificity in transcriptional control. Transcription
2010; 1: 2226.
Wade SL, Poorey K, Bekiranov S, Auble DT. The Snf1 kinase and
proteasome-associated Rad23 regulate UV-responsive gene expression. EMBO J 2009; 28: 291931.
Poster Abstracts
P094
P095
77
Poster Abstracts
P096
P097
Background Cryptococcus neoformans, an opportunistic fungal pathogen, produces a capsule that induces immunological unresponsiveness, interfering with normal phagocytosis, cytokine release, and
leukocyte migration. This definitive virulence factor is mainly
composed of the polysaccharides glucuronoxylomannan (GXM) and
glucuronoxylomannogalactan (GXMGal) with trace amounts of
mannoproteins. By molar ratio, mannose and glucuronic acid constitute the major sugar subunits, while xylose and galactose are incorporated into the structure to a lesser degree. Incorporation of these
monosaccharides into the capsule requires production of activated
donor molecules (e.g. GDP-mannose) in the cytosol followed by transport of these highly charged compounds into the Golgi where most
glycan biosynthesis occurs. Specific enzymes then catalyze the transfer of the monosaccharide from the nucleotide sugar donor to the
growing polysaccharide chain.
Although the biochemical pathways that are required to synthesize
the activated nucleotide sugar precursors have been elucidated, the
identity and regulation of the complete set of nucleotide sugar transporters (NSTs) responsible for import of these precursors into the
secretory pathway remain elusive. GDP-mannose transport has been
attributed to Gmt1 and Gmt2 (Cottrell et al, 2007), and UDP-galactose appears to be transported by Ugt1p (Moyrand et al, 2007). However, the transporters for the donors of other surface components
such as glucuronic acid, xylose, or potentially sialic acid have not
been identified. One possibility is that known NSTs translocate multiple nucleotide sugar substrates. Alternatively, these donors may be
transported by additional putative NSTs encoded in the cryptococcal
genome.
Aim Our long-term goal is to better understand the glycan synthetic
steps by which C. neoformans forms a polysaccharide capsule. Here,
our aim is to determine the biological role(s) of three nucleotide
sugar transporters: Gmt1, Gmt2, and NSTx.
Methods Mutants lacking GMT1, GMT2, and NSTx were generated,
and various strains were assayed for cell growth, capsule phenotype,
colony morphology, cell stress susceptibility, phagocytic uptake, and
protein as well as lipid glycosylation defects. Virulence in mice was
assayed by monitoring mouse survival following intranasal inoculation. In vitro transport assays were used to determine the specific
transporter substrate(s).
Results Gmt1 and Gmt2 demonstrated similar transport kinetics and
substrate specificities for GDP-mannose in vitro and showed partial
functional redundancy in vivo. Single gmt deletion mutants exhibited
defects in cell growth, capsule phenotype, colony morphology, and
protein glycosylation that were compounded in mutants lacking both
genes; the double mutant was also completely avirulent in mice.
Despite their apparently similar functions, however, Gmt1 and Gmt2
had distinct expression and localization patterns. Furthermore, swapping the coding sequences of the corresponding genes was insufficient to restore wild-type phenotypes. Mutants lacking NSTx
demonstrated capsule and growth defects that correlated with an
attenuation of virulence in mice, and heterologous complementation
studies suggested that the substrate of this transporter is an activated
acidic monosaccharide.
Conclusion Gmt1 and Gmt2 are GDP-mannose transporters that
play distinct roles in cryptococcal biology with only partial overlap in
function; this difference may reflect a non-identical requirement for
these transporters in various stress responses. NSTx appears to transport the donor of an acidic monosaccharide although further study is
required to define the exact substrate(s).
78
P098
Microsatellite typing show mixed infections with multiple
genotypes of Cryptococcus neoformans var. grubii in
Indian HIV positive patients with cryptococcosis
A. Chowdhary,1 F. Hagen,2 A. Prakash1 and J. F. Meis2
1
Vallabhbhai Patel Chest Institute, Delhi, India and 2CanisiusWilhelmina Hospital, Nijmegen, The Netherlands
Background Incidence of cryptococcal infection is high in developing countries such as India. Cryptococcal meningitis is the most common, life-threatening, opportunistic, fungal disease in HIV infected
individuals. So far, only few reports of cryptococcosis due to mixed
species or serotypes of Cryptococcus neoformans have been reported.
Aim To characterize the genotypes of C. neoformans var. grubii from
recurrent and non-recurrent cryptococcocal infections in HIV positive
patients.
Poster Abstracts
Materials and methods A total of 42 Cryptococcus neoformans isolates originating from 25 HIV positive patients and one from HIV
negative were studied from whom primary unpurified original cultures were stored. The isolates were recovered from CSF (n = 30,
71%), sputum (n = 9, 21%), blood (n = 2, 5%) and one from BAL. Of
these, 25 were repeat isolations from clinical specimens of 9 patients
(two or more isolates from a single patient at 16 months interval)
and the remaining 16 patients had single specimens while on AMB
or FLU therapy. Five single colonies from primary culture plates were
screened and were subjected to Amplified Fragment Length Polymorphism (AFLP) and were genotyped using multilocus microsatellite
typing (MLMT) based on nine markers specific for C. neoformans variety grubii (serotype A) or variety neoformans (serotype D). The antifungal susceptibility was determined for new azoles along with
standard antifungals using CLSI M27-A3 guidelines.
Results All isolates were AFLP1/VNI representing C. neoformans var.
grubii serotype A. MLMT revealed mixed infections with multiple genotypes of C. neoformans var. grubii in 9 (36%) of the 25 patients. This
included 7 (78%) patients with two and 2 (22%) with three different genotypes. Different strains were found at different anatomical sites (blood,
sputum and BAL) in two patients. Mycological failures after 2 weeks of
antifungal treatment were observed in 4 mixed-infection patients. All of
the isolates were susceptible to all the tested antifungal drugs.
Conclusions The prevalence of multiple strains of C. neoformans var.
grubii in the environment may likely result in acquisition of multiple
genotypes by human hosts leading to infection with multiple genotypes in cryptococcosis patients. Our data showed the presence of
multiple infecting genotypes of C. neoformans confirming previous
observations in other centers.
derived from the URA5 gene and restriction enzymes HhA1 and
Sau961. Reference strains were CBS10512 (serotype A, VNI,
AFLP1), CBS8710 (serotype A, VNII, AFLP1A), WM 628 (serotype
AD, VNIII, AFLP3), CBS10084 (serotype D, VNIV, AFLP2), WM 179
(serotype B, VGI, AFLP4), WM 178 (serotype B, VGII, AFLP6), WM
161 (serotype B, VGIII, AFLP5) and WM 779 (serotype C, VGIV,
AFLP7). The susceptibilty study was conducted using the disc diffusion method for amphotericine B, fluconazole, voriconazole and ketokonazole. Quality control was performed for every test using C.
krusei ATCC 6258 and C. parapsilosis ATCC 90018.
Results We studied 148 Cryptococcus isolates from spinal fluid of
122 HIV-infected patients with cryptococcal meningitis. The genotype identification shows the following results: sero-mating typing
revealed 21 Aa isolates, 115 Aa isolates, and 12 ADa isolates. Genotype characterization using URA5 - restriction fragment analysis
showed 117 VNI, 8 VN2 and 12 VNIII isolates. 133 from 147 isolates were susceptible to amphotericine B and 15 isolates were SDD,
133 were susceptible to fluconazole, six isolates were SDD, and seven
isolates were resistant. For Voriconazole 147 isolates were susceptible, while one strain was SDD. For ketoconazole we studied only 120
isolates and all were susceptible.
Discussion/conclusion Based on the molecular study the predominant cryptococcal species in HIV-patients in Jakarta is Cryptococcus
neoformans with serotype A and mating type a. The susceptibilty
study showed that most Indonesian Cryptococcus strains are susceptible to the antifungals available in Indonesia.
P100
P099
Genetic diversity and susceptibility study of single
cryptococcal isolates from HIV-infected patients in
Indonesia
R. Adawiyah,1 F. E. Siagian,2 D. Imran,3 R. Sjam,4 R. Ghaniem,5
N. Ariwati,6 T. Boekhout,7 B. Theelen7 and R. Wahyuningsih8
1
University of Indonesia; Programme of Biomedical Sciences,
Faculty of Medicine, Jakarta, Indonesia; 2Programme of
Biomedical Sciences, Faculty of Medicine, University of Indonesia,
Jakarta, Indonesia; 3University of Indonesia/Ciptomangunkusumo
Hospital, Jakarta, Indonesia; 4University of Indonesia, Jakarta,
Indonesia; 5Pajajaran University/Hasan Sadikin Hospital, Bandung,
Indonesia; 6Udayana University, School of Medicine, Denpasar,
Bali, Indonesia; 7Central Bureau of Schimmel Cultures, Utrecht,
Utrecht, the Netherlands and 8University of Indonesia, University
of Christian Indonesia, Jakarta, Indonesia
Background Cryptococcosis is an important opportunistic infection
in AIDS. Cryptococcus was divided into two species. i.e. Cryptococcus
neoformans and C. gattii. C. neoformans is composed of C. n var. grubii
(serotype A), C. n. Var. neoformans (serotype D) and the hybrid serotype AD. C. gattii consists of serotype B and C. Both species differ in
their virulence, geographical distribution, pathogenicity and clinical
appearance. Serotype A is known as the main cause of cryptococcosis
in HIV-infected patients and is distributed world wide. The mating
type is known as a virulence factor and plays a role in the epidemiology and evolution of the organisms.
Aim of the study To identify genetic variation, serotype, mating
type and susceptibility patterns of Cryptococcus isolates from HIVinfected patients in Jakarta.
Method We investigated 148spinal fluids derived from 122 HIVinfected patients in Jakarta (144 isolates), Bandung (one isolate), Bali
(one isolate), Pontianak (one isolate) and Papua (one isolate). Mating
and serotype was determined by a PCR method using four specific
primers. The genotype characterization was conducted using primers
79
Poster Abstracts
selected and the AIM-HII pipeline was used to identify sites of insertion. Of these, we have confirmed insertions in several previously
identified genes including ACA1 and SKN7, as well as a number of
unannotated genes. Through our analysis we have also identified
previously undescribed ATM insertion-induced events. These include
possible chromosomal rearrangements, large deletions, and extensions and truncations of the integrated T-DNA. Many of these
events would have been overlooked using the traditional PCR-based
methods.
Discussion/conclusion Our work introduces and utilizes a new tool
that will greatly facilitate insertional mutagenesis screens in the
future. Through this work we have identified a number of known
and novel genes involved in C. neoformans virulence attributes. Work
to further characterize the function of these genes is ongoing.
P101
D-amino acid oxidase genes in Cryptococcus gattii and
Cryptococcus neoformans
Y. C. Chang,1 A. Khanal Lamichhane,1 J. A. Bradley,2
L. H. Rodgers,3 P. Ngamskulrungroj4 and K. J. Kwon-Chung1
1
NIH, Bethesda, MD, USA; 2University of Louisville, Kentucky,
Louisville, KY, USA; 3University of Wisconsin, Madison, WI, USA
and 4Mahidol University, Bangkok, Thailand
Background Cryptococcosis is caused by two closely related sister
species, Cryptococcus neoformans and C. gattii, which differ considerably in their utilization of carbon and nitrogen sources. One of
the diagnostic characteristics that can distinguish between the two
species is the ability to utilize D-proline and a few other D-amino
acids by C. gattii but not by C. neoformans. The enzymatic mechanism of D-proline metabolism, however, has not been studied in
these species.
Aim In order to understand the enzymatic mechanism of D-amino
acid utilization by C. gattii but not by C. neoformans, we characterized
a gene required for D-proline utilization and studied the D-amino oxidase gene (DAO) in both species.
Methods We created an insertional library using Agrobacterium mediated transformation and analyze the clones that failed to utilize Dproline. Methods used include gene cloning, sequencing, targeted
gene disruptions, gene expression and animal infection model. Utilization of different D-amino acids was tested using yeast nitrogen base
without ammonia supplemented with various D-amino acids as the
sole nitrogen source.
Results Three homologs of the D-amino oxidase gene (DAO) were
identified in the genome of both the C. gattii strain R265 and the C.
neoformans strain H99. Expression profiles of the DAO gene in each
strain were examined using different D- amino acids as the sole nitrogen source. The contribution of each DAO gene in D-amino acid utilization was determined by deleting the DAO gene in both species.
Substrate specificity of the Dao proteins was determined by expressing the DAO genes in E. coli. We found the DAO2 gene to be important for growth of R265 in different D-amino acids as the sole
nitrogen source. Although deletion of each DAO gene individually
had no affect on fungal virulence, triple deletions of all DAO genes
significantly affected virulence in a murine model of R265 but not of
H99. This suggested that D-amino acid utilization is important for
the pathobiology of C. gattii. Interestingly, overexpression of RDAO2,
a DAO gene from C. gattii in H99 supported the growth of H99 in
D-amino acids. We are investigating further the molecular basis in
D-amino acid utilization in C. neoformans and C. gattii.
Conclusion Our study shows that D-amino oxidase genes are
responsible for D-amino acids utilization in cryptococci. Interestingly,
C. neoformans and C. gattii both possess homologs of three D-amino
oxidase genes which affect virulence in C. gattii but not in C. neoformans. A detailed investigation into the molecular differences of DAO
gene expression in the two species will shed light on their biochemical differences that impact their pathogenicity.
80
P102
Please see GW2.
P104
Characterization of granulomatous response in pulmonary
and cutaneous cryptococcosis in renal transplanted
patients
M. V. Solda, G. Ricci, S. Nishikaku, V. Ponzio, A. L. Colombo and
M. Franco
Federal University of Sa~o Paulo, Sa~o Paulo, Brazil
Cryptococcosis is the second major systemic mycosis associated to
kidney transplants, which is the most likely group to develop cryptococcosis among solid organ transplant recipients. There are few studies reporting the incidence of cryptococcosis in this risk group and
correlating the different granuloma patterns during infection and
pathogen/host interaction in those patients. Thus, the objective of
this work is to characterize the granuloma pattern in renal transplant recipients with cryptococcosis. The casuistic included a total of
14 paraffin blocks from 12 patients from the Hospital do Rim e Hipertens~
ao of S~
ao Paulo, who had cryptococcal infection post renal
transplantation, during the period of 20052012. The isolates of C.
neoformans/C. gattii complex collected from the lesions of the patients
were identified by biochemical and molecular methods. Paraffin HE
stain sections from eight skin biopsies and six of lung of these
patients were used for the histopathological analysis for characterization of tissue response, and Mayers Mucicarmin for visualization of
the fungal capsule. From a total of 14 cases analyzed, we observed
the pattern of compact epithelioid granulomas in 4 cases and loose
macrophagical granulomas in 10 cases (see Table 1). As observed in
Table 2, all cases showed the presence of inflammatory lymphomononuclear infiltrate cells rich in xanthomatous histiocytes, and of
multinucleated giant cells. Multifocal lesions were observed in all
patients accompanied by fibrosis and necrosis. Regarding the fungal
morphology, there was predominance of multiple budding yeast cells.
Overall, patients who developed compact granulomas had higher survival than those patients with loose granulomas. In conclusion, the
morphological findings of the present study show a diversity of granuloma patterns in kidney transplant patients with cryptococcosis. It
might occur possibly due to factors such as stage of infection, level of
immunosuppression and pathogen virulence. It is worth to emphasize, this is the first study describing the granulomatous response in
renal transplant patients with cryptococcosis.
Tissue
Granuloma
pattern
Yeast
proliferation
Clinical outcome
after treatment
1
2
3
4**
4
4
5
6
7
8
9
10
11
12
skin
lung
skin
skin 1
skin 2
skin 3
lung
skin
lung
lung
skin
lung
skin
lung
compact
loose
loose
loose
loose
compact
loose
loose
loose
loose
compact
loose
compact
loose
low
low
high
high
high
low
high
low
high
high
low
high
low
high
death*
death
death
discharged
discharged
discharged
death
discharged
discharged
death***
discharged
discharged
discharged
discharged
Poster Abstracts
Cell population
Other
lesions
Fungal
budding
Patient
Tissue
epithelioid
macrophagical
MGC=
LMN
fibrosis
necrosis
single
multiple
1
2
3
4***
4
4
5
6
7
8
9
10
11
12
Total
skin
lung
skin
skin 1
skin 2
skin 3
lung
skin
lung
lung
skin
lung
skin
lung
14
+*
+
+
4
-**
+
+
+
+
+
+
+
+
+
10
+
+
+
+
+
+
+
+
+
9
+
+
+
+
+
+
+
+
+
+
+
+
+
+
14
+
+
+
+
+
+
+
+
+
+
+
12
+
+
+
+
+
+
+
+
+
+
10
+
+
+
+
+
+
6
+
+
+
+
+
+
+
+
8
*Presence.
**Absence.
***Same patient with biopsies collected in different periods.
=
P105
Growth of Cryptococcus neoformans inside phagosomes: a
matter of resistance or a well orchestrated escape?
rgel,1 C. C. Coelho,2 A. Casadevall2
H. F. P. Tavares,1 P. H. M. Bu
1
and A. L. Bocca
1
UnB, Brasilia, Brazil and 2Albert Einstein College of Medicine,
New York, NY, USA
Phagolysosomes are vesicles located in the intracellular portion of
phagocytes, having the function of uptaking microbes and killing
them. The interior of phagolysosomes presents a harsh microenvironment for the microbes internalized. This is obtained by a number of oxidative, hydrolytic and acidifying enzymes present in
these vesicles. Phagolysosomes plays a vital role as part of the
maturation of the phagocytes, which in turn present high importance in suppressing the initial infection and recruiting other
leucocytes.
On the counterpart, microbes developed innumerous strategies for
escaping and/or impairing the phagocytes and their defenses, including the ability of survival inside the phagolysosome. In this matter,
Cryptococcus neoformans hasvarious virulence factors that allow its
survival and its escape from the interior of macrophages. Among
these factors, there are the enzymes of the phospholipase group
(PLB) which destabilize membranes by hydrolyzing ester linkages. It
is well known and described that the omission of the PLB in C. neoformans promotes a weaker infection with a diminished intracellular
growth inside macrophages.
Another strategy of the C. neoformans is the nonlytic extrusion,
which consists in the exocytosis of the fungal cell from the macrophage with both cells surviving. This interaction with the host is
unique and very peculiar, causing minimal host cell damage and
therefore not triggering a pro-inflammatory response. It is known
that the extrusion involves the damage of phagosome membrane,
therefore it has been shown that the omission of PLB causes less exocytosis of the fungal cell.
C. neoformans is described as a facultative intracellular pathogen
that doesnt interfere with the formation and maturation of phagolysosomes, being able to survive and multiply inside the acidified
vesicle. However, reports indicate that the constant damage caused
by the pathogen in the vesicle membrane impairs the acidification
of the media. It has also been demonstrated that the experimental
acidification of phagosomes altered the extrusion of the fungal cell.
P106
Differences between acapsular and encapsulated strain of
Cryptococcus neoformans in NLRP3 inflammasomedependent activation
rgel,1 P. H. Saavedra,1 K. G. Magalh~aes,1
P. H. M. Bu
R. J. Cordero,2 D. S. Zamboni,3 A. H. Tavares,1 P. Albuquerque,1
A. Casadevall4 and A. L. Bocca1
1
Universidade de Braslia UnB, Braslia, Brazil; 2Universidade
Federal do Rio de Janeiro UFRJ, Rio de Janeiro, Brazil; 3USP
Ribeira~o Preto, Ribeira~o Preto, Brazil and 4Albert Einstein College
of Medicine, New York, NY, USA
Cryptococcus neoformans is an encapsulated human pathogenic fungus
that affects primarily immunocompromised individuals. One of the
most important C. neoformans virulence factors in study is its capsule
which is involved in immune response evasion and fungal dissemination. Also C. neoformans capsule polysaccharides have been shown to
downregulate production of proinflammatory cytokines such as
tumor necrosis factor (TNF) and interleukin-1b (IL-1b). Recently it
has reported the existence of quorum sensing in C. neoformans, as virulence factor, regulating the cell growth of planktonic and biofilm
cells, glucuronoxylomannan (GXM) release, and melanin synthesis.
In this study we investigated whether C. neoformans molecules was
able of triggering inflammasome activation and evaluated the role of
its capsule in this event.
For the analysis, wild-type capsulated strain B3501 and wild-type
acapsulated strain were cultivated in minimum media for 5 days.
After that the media was filtrated in 0,22 m for the obtainment of
the conditioned medium. This medium was further processed using
ultracentrifugation for the obtainment of the conditioned medium
<1KDa. These mediums were utilized with murine BALB/C peritoneal
macrophages previously stimulated with LPS and later with nigericin
for inhibition assays. The results were obtained by the analysis of Il-
81
Poster Abstracts
P107
Searching invertebrate model hosts to study the
Cryptococosis agents
D. C. S. Santos,1 H. M. Soares,2 T. C. Roat,2 O. Malaspina,2
M. A. Martins,3 L. Oliveira,3 P. I. S. Junior,4 T. J. Oliveira4 and
M. S. C. Melhem3
1
Graduate Program of Coordination for Diseases Control,
Secretariat of Health, Brazil; 2University Estadual Paulista Julio de
Mesquita Neto, Brazil; 3Adolfo Lutz Institute, Public Health
Reference Center, Secretariat of Health, Sa~o Paulo, Brazil and
4
Center of Toxins, Immune-Response and Cell Signaling,
Butantan Institute, Brazil
Background Cryptococcus neoformans is a pathogenic yeast that is
the main causative agent of Cryptococcosis. Murine models for far
represents usefull strategy to study host- C. neoformans interactions.
Evaluation of C. neoformans virulence in a number of non-mammalian hosts suggests that this speciesis a non-specific pathogen.
Recently, non-vertebrate animals have been described as valuable for
studies on C. neoformans virulence factors and host innate immune
responses mimicking the natural scenario with environmental predators, as amoebae and nematodes.
Aim Identify appropriate invertebrate host for the study of aspects of
cryptococcal pathogenesis.
Methods We investigated in two proposed non-vertebrate models
the installation and progression of Cryptococcosis infection. The inoculation 1 9 105 cell ml1 was performed by three route of administration: topical application, oral delivery and injection in five groups
of 10 animals for Apis mellifera, and via injection in two groups of
10 animals for Zophobas morio.A control group inoculated with PBS
was included in all experiments. Additionally, we verify the identity
between the inoculated strain and the animal recovered strain. The
strain-type WM 148 C. neoformans molecular type VNI was used to
inoculate fourth instar larvae for both species. The follow-up of inoculated larvae was made up to adult phase (insect). A group composed by three animals was sacrificed weekly unless dead occurred.
The follow-up period depends on the species lifecycle. The dead
82
larvae was sliced for tissue microscopic examination and tissue culture was also performed in Sabouraud dextrose agar plate.
Results Larvae of Apis mellifera died due to injection route of administration. Oral delivery and topical application via cause death during
early follow-up with 100% mortality at the first week. Conversely,
larvae Zophobas morio were alive up to the end of experiments. Of
note, the injection route in this species stimulated the production of
heavy superficial slime. The multiple colonies obtained from the tissue larvae culture were submitted to PCR-fingerprinting using M13
single primer. All recovered colonies showed PCR fingerprinting patterns identical to those of the original inoculated strain-type.
Conclusion The Apis mellifera larvae seems to be a candidate for a
susceptible invertebrate model of infection, but both the inoculums
volume and administration via warranted additional studies to
improve the experiments. Our study suggested Zophobas morio as a
resistant non-vertebrate model of infection for Cryptococcosis via
injection. Future investigation are need to assess financial, space and
time commitment required for use the studied species, and importantly to determine virulence trait and host response before select the
most appropriated host.
P108
Macrophage autophagy in immunity to Cryptococcus
neoformans and Paracoccidioides spp.
F. C. K. Gustavo, A. Rossi Neto, K. T. Rangel and A. M. Nicola
Catholic University of Brasilia, Brasilia, Brazil
Background Autophagy is a conserved eukaryotic housekeeping
mechanism that allows cells to degrade and recycle bulky intracellular material such as protein aggregates and organelles. In addition to
these classical roles, a decade of research has shown that autophagy
play multiple central roles in immunity to pathogens as varied as
viruses, bacteria, protozoa and, more recently, fungi. Several groups
have found that when phagocytes internalize fungi the microorganisms end up in vacuoles with autophagosome characteristics. However, using different techniques different authors have found
disparate results as to what function this has on host-pathogen
immunity: some suggest autophagy to be host-protective, some concluded it makes no difference and some that it is beneficial to the
pathogen. Before autophagy modulation can be employed as immunotherapy for fungal infections, the precise mechanisms of its role in
antifungal immunity must be established.
Here we test the formation of autophagosomes surrounding C. neoformans or Paracoccidioides spp. cells that were phagocytosed by human
or murine macrophages. C. neoformans causes a life-threatening
meningoencephalitis that kills some 600 000 people each year. Fungi
from the genus Paracoccidioides are thermo-dimorphic and cause paracoccidioidomycosis (PCM), which affects lungs and reticuloendothelial
system and is the most prevalent systemic mycosis in Latin America.
Aim In order to further our knowledge onto the antifungal roles of
macrophage autophagy, this work focused on two specific aims:
1 Determine if human macrophages recruit the autophagosome
marker LC3 to vacuoles containing C. neoformans.
2 Test if internalized Paracoccidioides spp. cells induce LC3 recruitment in murine macrophages.
Methods We cultivated C. neoformans H99 strain in Sabouraud agar
media. P. lutzi Pb01 and P. brasiliensis Pb18 and Pb265 were maintained by weekly passages in Fava-Netto agar.
Murine macrophage (RAW264.7) and human monocytic (THP-1)
cell lines were grown respectively in DMEM and RPMI, both supplemented with 10% fetal calf serum and incubated at 37 C with 5%
CO2. THP-1 monocytes were differentiated onto macrophages by
treatment with phorbol 12-myristate 13-acetate (PMA). Macrophages
were plated in coverslips and infected with fungi (opsonized with
anti-capsule IgG1 in the case of C. neoformans) for 24 h. The cells
were then fixed, stained for LC3 immunofluorescence and observed
on an epifluorescence microscope.
Poster Abstracts
P109
Effects of Cryptococcus neoformans extracellular vesicles
on the phagocytosis and antifungal activity of the
environmental predator Acanthamoeba castellanii
A. Rizzo,1 V. Cabral,2 J.M. Wolf,2 J.D. Nosanchuk2 and
M.L. Rodrigues1
1
Federal University of Rio de Janeiro, Rio de Janeiro, Brazil and
2
Albert Einstein College of Medicine, New York, NY, USA
P110
CaMK4: a host kinase required for cryptococcal
engulfment and normal virulence
D. L. Srikanta, M. Williams and T. L. Doering
Washington University School of Medicine, Saint Louis, MO, USA
Background Cryptococcus neoformans, the causative agent of cryptococcosis,is an opportunistic fungal pathogen which kills over
600 000 individuals annually. Despite extensive research on cryptococcal pathogenesis, host gene products involved in the initial phagocytosis of C. neoformans and subsequent stages of infection need
further study. We have identified CaMK4 as playing an important
role in these events.
CaMK4 is a calcium/calmodulin-dependent protein kinase. It occupies a key position in a host signal transduction pathway that
responds to receptor signals and activates multiple processes, including cell differentiation, survival and cytokine release. This kinase
83
Poster Abstracts
P111
A role for protein palmitoylation in the interactions of
C. neoformans with host cells
F. H. Santiago-Tirado, M. Yang and T. L. Doering
Washington University School of Medicine, St Louis, MO, USA
Background Cryptococcus neoformans adherence to and uptake by
phagocytes are key events that are central in cryptococcal pathogenesis. Fungal engulfment by host cells and their subsequent intracellular proliferation have been implicated in latency, dissemination, and
virulence, but the full complement of C. neoformans gene products
that participate in these processes has not been defined. To address
this question, we used an automated high content method (Srikanta
et al., 2011) to assay the interactions between a human macrophage-like cell line and mutant fungi from a partial deletion collection (Liu et al., 2008).
We identified multiple genes whose deletion led to lower or higher
adherence and/or phagocytosis compared to wild-type. Several of
these have been previously reported to have perturbations of cell surface structures, validating our screening approach. The gene deleted
in one of these mutants encodes a probable protein fatty acyltransferase (Pfa), one of a family of DHHC domain-containing proteins that
catalyzes lipid modification of proteins.
84
Aim The goals of this project are to determine the defects associated
with specific loss of this putative Pfa and to uncover the mechanism
by which it alters the fungal-phagocyte interactions.
Methods To accomplish our goal, we generated mutant, complemented, and epitope-tagged strains; assayed them for phenotypes
including growth under stress conditions, phagocytosis by macrophages, intracellular survival, and virulence in mice; and examined
them by light microscopy. We also characterized proteins by gel electrophoresis, immunoprecipitation, and mass spectrometry.
Results We confirmed that deletion of the gene of interest, termed
PFA4 for its homology to that gene in S. cerevisiae, results in
enhanced adherence to and phagocytosis by human macrophages
compared to wild type. Mutant cells lacking the gene exhibit morphological defects that are exacerbated under host conditions. The
mutant is sensitive to a variety of cell wall stress conditions in vitro
and has a profound defect in intracellular growth; it is also avirulent
in a mouse model of cryptococcal infection. Interestingly, this mutant
has no obvious defect in capsule synthesis or induction. All of the
mutant phenotypes are reversed by genomic complementation and
are due to the lack of fatty acyltransferase enzymatic activity, as
point mutants in the enzyme active site phenocopy the gene deletion.
Proteomics studies and investigation of putative Pfa4 substrates are
currently under way to determine the mechanism of the defects
exhibited by the pfa4 mutant.
Discussion/conclusion Defects in lipid modification are known to
cause mislocalization or degradation of the substrate proteins, leading
to their dysfunction. Our results are consistent with aberrant palmitoylation of some cyptococcal protein(s) leading to alteration of the
cell surface; this could then cause increased recognition, phagocytosis, and killing by host macrophages and loss of virulence in mice.
Pfa4 is one of a family of proteins with the same biochemical activity. Our current efforts are directed at identifying the specific protein
substrate(s) responsible for the changes we observe in the pfa4
mutant, so that we can obtain a detailed picture of the molecular
mechanisms behind them.
P113
Automated imaging and analysis of C. neoformans for
high-throughput assays
A.L. Chang, D.L. Srikanta, M. Williams, M.R. Brent and
T.L. Doering
Washington University in St. Louis School of Medicine, St. Louis,
MO, USA
Background In the last decade, studies of C. neoformans have
expanded to the genomic scale, enabled by the genome sequence and
deletion collections. Initial forays have also been made into largescale analysis of host factors that influence fungal:host interactions
(Qin et al., 2011).
To capitalize on genome-wide experimental approaches, assays previously performed on a limited scale must be scaled up. One powerful
tool for this is high content screening (HCS), where high content refers
to processes defined spatially and temporally in the context of each
cell within an array of cells (Abraham et al., 2004). HCS refers to the
automation of imaging and analysis for screening purposes. It is a
powerful tool that can assist in analysis of libraries that range from
collections of deletion mutants to banks of chemical compounds. A
particularly exciting option is the application of HCS to probe the host
aspect of fungal:host interactions by using RNA interference (RNAi) to
target individual host genes on a large scale (Prudencio et al., 2009).
Host phagocyte engulfment of cryptococcal cells is central to cryptococcal pathogenesis and affects fungal survival, latency, growth,
and dissemination. However, details of their interactions remain
undefined, suggesting direct and unbiased assessment of key host
and pathogen features by screening as an appealing strategy to probe
these events. The current standard for assessing cryptococcal interactions with host cells is microscopic examination in multiple focal
Poster Abstracts
P115
P114
Viability of Cryptococcus neoformans/Cryptococcus gattii
in long term stored environmental samples from Colombia
P. Escandon and E. Castaneda
Instituto Nacional de Salud, Bogota, Colombia
Background Several studies carried out in Colombia have provided
valuable data about the ecology of C. neoformans and C. gattii. Extensive environmental samplings in different areas of the country have
provided us with both C. neoformans and C. gattii isolates recovered
from Eucalyptus spp, almond trees, Ficus spp, bird droppings, Eucalyptus ficifolia (Corymbia ficifolia) detritus, and some other species of
trees. Attempts have also been made in standardizing a molecular
detection technique in environmental samples of this fungus, being
able to extract and amplify Cryptococcus spp DNA from environmental
samples with adequate PCR specificity; therefore, the present availability of naturally colonized samples will be very useful in the standardization of a new specific C. neoformans/C. gattii detection
technique in environmental samples.
Aim To determine the viability of members from the C. neoformans/C.
gattii species complex in naturally colonized samples associated to
trees decaying wood and bird droppings stored since 2003.
Methods A total of 964 samples collected between 2003 and 2008
were processed. Of them, 274 were samples of filtrates from trees
decaying wood, 653 were samples from trees decaying wood, and
37 were samples of bird droppings. The molecular type of isolates
was established with PCR fingerprinting using the single primer
(GTG)5.
Results An overall positivity of 3.9% was obtained from the 964
samples processed; positive samples had been stored as filtrates from
trees decaying wood (36.9%), as decaying wood in bags at room
temperature (52.6%) or as bird droppings (10.5%); from these, 161
isolates were recovered and identified as members of the C. neoformans/C. gattii species complex. Eighty one isolates (50.3%) were
85
Poster Abstracts
0.020.94 mg l1. The MIC range for the 170 remained isolates
was: FCL-MIC 0.12 to 16 mg l1 and for AmB-MIC 0.121 mg l1.
High FCL- MIC (>8 mg l1) were observed in 2 (2/7; 28.6%) atypical-form isolates and in 5.3% of normal-cell isolates. Overall, data
from the time-kill curves showed fungicidal effect in 56% of cultures
in the initial 6 h-exposition to AmB. We observed designed re-growth
phenomenon in 2 (2/7; 28.6%) titan-cell isolates.
Discussion and conclusions Our findings suggested that morphologic changes are more frequent in C. neoformans isolates than C. gattii isolates, although we have studied larger number of C. neoformans
isolates since it is the main causative species. Previous work indicates
that being an accidental but successful pathogen with a broad host
range, C. neoformans has adapted to survive in multiple hosts that
could explain the morphologic changes.Furthermore, it was entertained that strains demonstrated a gradual increase in cell body size
with generational aging, and there is some evidence on in vivo selection of cells of advanced age in human cryptococcal meningoencephalitis. Moreover, older cells are likely accumulated during chronic
infection, and such cells appear to be resistant to macrophage,
H2O2, and AmB killing. We confirmed that almost all C. neoformans
strains that are recovered from patients are susceptible to FCL after
in vitro replication including those showing titan cells. The MIC had
not evaluated properly the cidal effect of AmB, according to low MIC
values obtained in this study. However, time-kill experiments suggested that titan cells seem to be more resistant to antifungal-mediated killing in vitro by AmB in comparison to our previous
experience (data not showed) in which 5.4% of 56 normal-cell C.
neoformans isolates re-growth in similar conditions. We hypothesized
that AMB would be less effective on atypical cells, conversely to FCL
that showed strong inhibitory activity against C. neoformans typical
and atypical isolates.
P116
Frequency of morphologic altered strains among
Cryptococcus neoformans - C. gattii complex isolates in a
Brazilian reference culture collection and antifungal
susceptibility pattern
L. Oliveira, D. C. S. Santos, D. M. Castro e Silva, M. W. Szeszs
and M. S. C. Melhem
Instituto Adolfo Lutz, Sa~o Paulo, Brazil
Background Typically the cryptococci budding cells are round to
elongate with 59 m diameter, but the occurrence of titan cells
(>10 lm), microforms (<5 lm) and hyphal forms are known. Environmental and host selection pressure could promote emerging of
morphological variants, and could accumulate in vivo promoting persistence. The extension and impact on the strain virulence and antifungal resistance or relevance to clinical outcome of atypical forms is
an enigmatic issue.
Aim We analyzed the frequency of atypical forms in 177 clinical isolates (112 Cryptococcus neoformans and 65 C. gattii) from a Reference
Culture Collection and correlated with culture melanization. All but
one (VN IV) isolates of C. neoformans were VNI and C. gattii isolates
were typed as VGII (61) and VGI (4).
Methods Cerebral spinal fluid and blood Cryptococcus cultures were
screening for melanine production onto Guizotia abyssynica agar and
for atypical forms. Atypical forms (Okagaki et al.,2010) was assured
at the medical Mycology Reference Laboratory of Spain, National
Centre for Microbiology of Instituto de Salud Carlos III. The atypical
isolates were submitted to PCR-RFLP method and antifungal susceptibility tests. Etest method was employed for amphotericin B (AmB)
and fluconazole (FCL). AmB fungicidal activity was determined by
time-kill curves method after 6, 12, 24, 48 and 72 h of exposition to
1 mg l1 of drug.
Results Among 177 clinical isolates tested 7 (7/177; 3.95%) isolates
showed titan cells including 3 (3/7; 41.9%) presenting hyphal forms.
No isolates containing micro cells were encountered among the studied isolates. All atypical forms were exclusively observed in C. neoformans molecular type VN I. The MIC range for the 7 isolates showing
titan cells were as follow: FCL-MIC 0.25 to 14 mg l1 and AmB-MIC
86
P117
First report on the environmental isolation of Cryptococcus
n, Colombia
neoformans molecular type VNI in Popaya
P. C. Castillo,1 C. A. Anacona,2 F. G. Gonzalez2 and
P. Escandon1
1
Instituto Nacional de Salud, Bogota, Colombia and 2Universidad
del Cauca, Popayan, Colombia
Background The importance of cryptococcosis has led to study the
ecology and the possible ecological niches of Cryptococcus neoformans/Cryptococcus gattii species complex. Reports on the isolation of
serotype A from bird droppings in Colombia started around 1968 in
Medellin, with a positivity of 18.8%; in the following years (1994),
Casta~
neda et al. reported a positivity of 53.8% for C. neoformans in
different cities and later a positivity of 49.6% was reported also from
avian droppings. Additionally, studies on the isolation of the complex
from Eucalyptus sp have been reported. These environmental studies,
together with a sampling that is being carried out in five regions in
Colombia, as well as the clinical data, are intended to be used to
delineate areas where the C. neoformans/C. gattii species complex is
established, and to predict the regions where the fungus may disperse in the future, thus generating an Ecological Niche Modeling
(ENM) for our country.
In Colombia the annual incidence rate for cryptococcosis is
3.3 9 103 for AIDS patients and in the general population
2.4 9 106. The annual incidence rate in the department of Cauca
(capital city Popay
an) was 1.3 9 106; no environmental reports
have been known on the isolation of members of the complex in this
city, being this is the first report on the recovery of C. neoformans
from bird dropping and trees in the rural and urban areas of
Popayan.
Aim To carry out an extensive sampling in the city of Popay
an and
characterize phenotypically and genotipically environmental isolates
of the C. neoformans/C. gattii species complex recovered, as a tool
heading for the creation of an ENM in Colombia.
Poster Abstracts
P118
Isolation of the Cryptococcus neoformans/Cryptococcus
gattii species complex in five cities of Colombia, and
association with ecological conditions
N. V. Velez,1 P. C. Castillo,1 M. A. Alvarez,2 B. C.De Bedout,3
F. G. Gonzalez4 and P. Escandon1
1
Instituto Nacional de Salud, Bogota, Colombia; 2Universidad del
Valle, CALI, Colombia; 3Corporacion para Investigaciones
Biologicas, Medellin, Colombia and 4Universidad del Cauca,
Popayan, Colombia
Background In Colombia several environmental studies have been
done describing the occurrence of the complex in diverse habitats;
isolates of serotypes A, B and C have been recovered from bird
droppings, Eucalyptus, Ficus sp and Terminalia catappa among others.
The characterization of the ecological conditions possibly related to
the habitat of the complex will allow us to get a closer knowledge
of the yeast.
Aim To carry out an extensive environmental sampling in five cities
in Colombia to recover isolates of the complex, characterize them
phenotypically and genotypically, and determine some ecological
conditions that may be related to its distribution in specific habitats.
Methods A total of 4501 samples, (avian droppings n = 609; tree
samples n = 3892) were collected in five cities: Bogota (n = 446),
located in the center of the country, the annual rainfall is 952
mm and average temperature of 14 C. C
ucuta (n = 495), located
Northwest, the annual rainfall is 806 mm and its average temperature is 30 C. Cali (n = 447), is located Southwest, the annual
rainfall is 900 mm and its average temperature is 23 C. Medelln
(n = 1515), located Northwest, the annual rainfall is 1656 mm
with an average temperature of 23 C. Popay
an (n = 1598),
P119
A new taxonomy for the Cryptococcus neoformans and
Cryptococcus gattii species complex
F. Hagen,1 K. Khayhan,2 B. Theelen,2 A. Kolecka,2 I. Polacheck,3
R. Falk,3 S. Parnmen,4 H. T. Lumbsch4 and T. Boekhout2
1
Canisius-Wilhelmina Hospital, Nijmegen, the Netherlands;
2
CBS-KNAW Fungal Biodiversity Centre, Utrecht, the
Netherlands; 3Hadassah-Hebrew University, Jerusalem, Israel and
4
The Field Museum, Chicago, IL, USA
Background During the past two decades, considerable genetic heterogeneity has been demonstrated to occur in the C. neoformans/C.
gattii species complex by a plethora of molecular methods, such as
amplified fragment length polymorphism (AFLP) PCR-fingerprinting
using M13, (GACA)4 and (GTG)5 primers, random amplification of
polymorphic DNA, restriction fragment length polymorphism fingerprinting based on the genes CAP10, CAP59, GEF1, PLB1 and URA5,
Fourier transform infrared-spectroscopy-based phenotyping, multilocus microsatellite typing and sequence analysis of a large number
of genes as well as whole genomes. The results of these studies
strongly question the currently used two species classification of the
complex.
Aim To settle the taxonomic status of the genotypic groups which
are recognized in the C. neoformans/C. gattii species complex.
Methods Phylogenetic analysis of 114 globally collected isolates of
the species complex was done using 11 nuclear loci CAP59, GPD1,
87
Poster Abstracts
IGS1, ITS, LAC1, PLB1, RPB1, RPB2, SOD1, TEF1 and URA5. These
molecular data were used to perform gene tree analyses in a maximum likelihood (ML) and Bayesian (B/MCMC) framework and coalescent-based species trees. We employed a combination of methods to
address the species delimitation, using gene tree estimation from single-locus and concatenated data sets, and species tree estimations,
including a genealogical species recognition method in which presence of clades in the majority of single-locus genealogies is taken as
evidence that these represent distinct lineages. We also used the coalescent-based general mixed Yule coalescent (GMYC) method, which
aims at locating the nodes that define the transitions between intraspecific (tokogenetic) and interspecific relationships using branch
lengths. Identification of the new species using a test set of 425 isolates was tested by MALDI-TOF MS.
Results Phylogenetic analysis of 11 loci and various genotyping
studies revealed significant genetic diversity with the pathogenic
Cryptococcus neoformans - C. gattii basidiomycetous yeast species complex. Genealogical concordance, cohesion-based, and species tree
approaches all supported the presence of distinct lineages within the
complex. Consequently, we propose to recognize the current C. neoformans var. grubii and C. neoformans var. neoformans as separate species, and within C. gattii five lineages occur. The type strain of C.
neoformans CBS 132 represents a serotype AD hybrid and needs to be
replaced. The newly recognized species differ in pathogenicity, prevalence for patient groups, as well as biochemical and physiological
aspects, such as susceptibility to flucytosine and fluconazole antifungals. MALDI-TOF MS proved to be a reliable and easy-to-use identification tool as no major errors were observed.
Discussion/conclusion Based on an extensive phylogenetic analysis
the currently known main genotypes in the C. neoformans/C. gattii
species complex are best interpreted as species. The type strain of C.
neoformans (and the genus Cryptococcus) CBS 132 has to be replaced
because of its hybrid nature. All species within the complex, and to a
large extent the hybrids as well, can be identified in the routine clinical laboratory by MALDI-TOF MS.
P120
Infective capacity of Cryptococcus neoformans and
C. gattii in a human astrocytoma cell line
~ eda and
M. C. Olave, J. C. Vargas-Zambrano, A. Celis, E. Castan
J. M. Gonzalez
Universidad de los Andes, Bogota, Colombia
Background Cryptococcal meningo-encephalitis is caused by the
members of the Cryptococcus neoformans/C. gattii species complex. C.
neoformans var. grubii is the most common etiological agent of cryptococcosis in immunocompromised individuals, and notably C. gattii
has emerged as the primary pathogen in immunocompetent populations. This fungal infection is the most important cause of death in
patients with acquired immunodeficiency syndrome (AIDS). The
pathogenesis of the disease in the central nervous system (CNS) is an
ongoing research topic; however the biology and mechanisms by
which C. neoformans and C. gattii invade and infect brain cells remain
unknown. Astrocytes, the main CNS cells population, play a fundamental role in the local immune responses. These cells may actively
participate during cryptococcosis either by direct infection or by
responding towards fungal antigens.
Aim This study evaluated the infection with C. neoformans and C.
gattii in a human astrocytoma cell line to determine their infectivity
and the induction of major histocompatibility complex (MHC) molecule expression.
Methods A glioblastoma cell line (ATTC: CRL-1718) with human
astrocytes characteristics was infected with C. neoformans and C. gattii yeasts labeled with FUN-1 fluorescent stain. The percentage of
infection and expression of HLA class I (HLA-ABC) and class II (HLADR) were determined by flow cytometry at day three post-infection.
Fluorescence microscopy was carried out in order to observe interactions between FUN-1-stained yeasts and astrocytes whose nuclei
88
P121
STAT1-induced classical macrophage activation is essential
for protection against Cryptococcus neoformans in mice
C. M. Leopold Wager,1 C. R. Hole,1 K. L. Wozniak,1
M. A. Olszewski2 and F. L. Wormley Jr1
1
University of Texas at San Antonio, San Antonio, TX, USA and
2
University of Michigan Health System, Ann Arbor, MI, USA
Background Protection against pulmonary inoculation with an
interferon-gamma producing strain of C. neoformans (H99 gamma) is
associated with classical activation of macrophages (M1) and
enhanced phosphorylation of the transcription factor STAT1 in these
cells. Studies utilizing general STAT1 KO mice inoculated with H99
gamma have revealed that STAT1 is essential for polarization of
macrophages toward an M1 activation phenotype and, coincidentally, for the induction of a protective immune response. However,
the necessity for M1 macrophage activation for combating cryptococcosis is unknown.
Aim The present studies were designed to determine the requirement
for STAT1-induced M1 macrophage activation in protection against
C. neoformans H99 gamma.
Methods Mice with macrophage-restricted STAT1 ablation (LysMCreSTAT1flfl mice) and control mice (STAT1flfl) were given an intranasal inoculation with H99 gamma and evaluated for survival and
pulmonary fungal burden. Pulmonary macrophages from the macrophage-restricted STAT1 KO mice and control mice were examined for
polarization phenotype by measuring gene expression of macrophage
activation markers. The anti-cryptococcal activity of macrophages
was determined by enumerating the intracellular cryptococci and,
following 24 h of culture, measuring nitrite levels in culture supernatants. We also determined the necessity of nitric oxide (NO) production by macrophages from control mice towards inhibiting the
intracellular growth of Cryptococcus.
Results Our data reveal that mice with macrophage-restricted
STAT1 ablation have a significant increase in pulmonary fungal burden compared to control mice. This increase correlates with a 10%
Poster Abstracts
P122
Dectin-2 polymorphism associated with Cryptococcosis in
HIV-uninfected Chinese patients
X. P. Hu,1 R. Y. Wang,2 X. Wang,2 Y. Q. Chen,2 Y. H. Cao,2
H. Z. Zhao,2 J. Q. Wu2 and L. P. Zhu2
1
Zhejiang Provincial Peoples Hospital, Hang Zhou, China and
2
Huashan Hospital, Fudan University, Shanghai, China
Background and aim Dectin-2 is a C-type lectin receptor which
can recognize critical structures of fungi, and previous studies have
indicated an important role of this receptor in antifungal immunity.
We did this research to analysis the distribution of Dectin-2 genetic
polymorphisms among Chinese Han patients with cryptococcosis and
healthy controls to investigate the association between Dectin-2 and
cryptococcosis.
Patients and methodology In this case control study, we genotyped 2 unknown functional change SNPs of Dectin-2 (rs11045418,
rs118059158). Only rs11045418 have polymorphisms in our population. A total of 252 patients with cryptococcosis and 464 healthy
controls were included in this study. The overall patients were
divided into three subgroups according to the different types of infection, including patients had cryptococcal meningitis and without pulmonary cryptococcosis (CM group), patients had pulmonary
cryptococcosis and had no central nervous system infection (PC
group), and patients had both these two types of infection (PC + CM
group).
Results Compared to the control group, there was a trend of
increasing of a heterozygote found in the overall 78 PC patients (48/
78 vs. 231/464, P = 0.055, OR = 1.61, 95% CI:0.992.64). And
the heterozygote was significantly increased in PC patients who had
no predisposed condition (36/53 vs. 231/464, P = 0.016,
OR = 2.08, 95% CI: 1.133.81). But no such difference was found
between controls and overall patients as well as patients with other
two types of infection. We also found that the heterozygote of
rs11045418 decreased in the 171 CM&PC + CM patients (patients
had cryptococcal meningitis), when compared with 78 PC patients
(who had no CNS involved), although without no significant difference (85/171 vs. 48/78, P = 0.083, OR = 0.62, 95% CI: 0.36
1.07). When excluded the cases which had predisposed conditions, a
notable decrease of this heterozygote was found in CM&PC+CM
P123
The role of macrophages in interleukin-4 receptor (IL-4R)dependent pathology in pulmonary cryptococcosis
ller,1 W. Stenzel,2 A. Grahnert,1
D. Piehler,1 U. Mu
hler,3 O. Frey,4 J. Held,2 T. Richter,1
M. Protschka,1 G. Ko
1
M. Eschke, T. Kamradt,5 F. Brombacher6 and G. Alber1
1
Insitute of Immunology, Leipzig, Germany; 2Department of
Neuropathology, Berlin, Germany; 3Institute of Pathology, Fulda,
Germany; 4Institute of Clinical Chemistry and Laboratory
Diagnostics, Jena, Germany; 5Institute of Immunology, Jena,
Germany and 6Division of Immunology, Cape Town, South Africa
Background In an experimental model of murine pulmonary cryptococcosis, susceptibility to infection is associated with alternatively
activated macrophages (aaMph). IL-4R plays a key role in the induction of aaMph as in IL-4R-deficient mice aaMph are almost absent in
several models studied. In murine pulmonary cryptococcosis alternative activation of macrophages is dependent on T helper (Th)2 cells,
especially polyfunctional Th2 cells, that produce more than one Th2
cytokine (IL-4, IL-5, IL-13) simultaneously (Mucosal Immunol 2012;
5(3): 299310).
Aim The aim of this work was to investigate the role of IL-4R-deficient macrophages that are insensitive to IL4 and IL-13 (while other
cells are unaffected) in pulmonary cryptococcosis.
Methods To study the function of aaMph, mice with genetically
ablated IL-4Ralpha expression on macrophages (Mph IL-4R/) were
infected with C. neoformans intranasally and compared to heterozygous littermates with heterozygous IL-4Ralpha expression (IL-4R+/).
The survival rate, lung burden, pulmonary histopathology, activation
state of macrophages, immunoglobulin levels, and cytokine production (measured by ELISA and intracellular flow cytometry) was
analyzed.
Results Mph IL-4R-/- mice are more resistant to pulmonary cryptococcosis, resulting in increased survival rates and lower lung burden
than non-deficient littermates. Interestingly, in Mph IL-4R/ mice
the number of alternatively macrophages is reduced but still detectable in comparison to IL-4R+/- mice. Moreover, macrophages from
Mph IL-4R/ and IL-4R+/ mice both show the potential to produce
the protective effector molecule NO; however, the capacity is higher
in macrophages from Mph IL-4R/ mice. Although Mph IL-4R/
mice are more resistant, the pulmonary Th2 response is comparable
in both groups. Pulmonary recruitment of eosinophils and mucus
production are not diminished in the more resistant Mph IL-4R/
mice.
Discussion/conclusion In a chronic Th 2-dependent inflammatory response mice can control pulmonary cryptococcosis by abrogation of IL-4Ralpha-dependent induction of aaMph. These findings
highlight the importance of IL4Ractivated macrophages in pathogenesis in pulmonary cryptococcosis (Int Immunol 2013; 25(8):
45970).
Funding: German Research Foundation grant DFG AL 371/5-4.
89
Poster Abstracts
P124
The role of interleukin-4 receptor (IL-4R)-dependent
polyfunctional T helper (Th) 2 cells in pulmonary
cryptococcosis
hler,3
ller,1 W. Stenzel,2 A. Grahnert,1 G. Ko
D. Piehler,1 U. Mu
O. Frey,4 J. Held,2 T. Kamradt,5 F. Brombacher,6 G. Alber,1
M. Eschke1 and T. Richter1
1
Insitute of Immunology, Leipzig, Germany; 2Department of
Neuropathology, Berlin, Germany; 3Institute of Pathology, Fulda,
Germany; 4Institute of Clinical Chemistry and Laboratory
Diagnostics, Jena, Germany; 5Institute of Immunology, Jena,
Germany and 6Division of Immunology, Cape Town, South Africa
Background In murine pulmonary cryptococcosis, T helper (Th)
cells and macrophages are important mediators of protection or pathogenesis depending on their polarization. A cellular immune
response, also called Th1 response, is protective, with classically activated macrophages and IFN-c-producing Th1 cells. On the other
hand, a Th2 response is detrimental in pulmonary cryptococcosis
and is associated with alternative activation of macrophages and differentiation of IL-4-producing Th2 cells. Susceptibility to pulmonary
cryptococcosis is associated with loss of fungal growth control and
subsequent spread of C. neoformans to other organs than the lung,
especially to the central nervous system.
Aim The aim of this work was to analyze in the murine pulmonary
cryptococcosis model the role of IL4Rdeficient Th cells that are insensitive to IL-4 (and IL-13) while other cells are unaffected.
Methods Mice with genetically ablated IL-4Ralpha expression on Th
cells were infected intranasally and compared to heterozygous littermates with heterozygous IL-4Ralpha expression (IL-4R+/-). The survival rate, lung burden, pulmonary histopathology, activation state of
macrophages, and Th cell cytokine production (measured by ELISA
and multiparameter intracellular flow cytometry) was analyzed.
Results In this study we show that IL-4R-dependent polyfunctional
Th2 cells simultaneously producing IL-4 and IL-5 or IL-13, are
essential in the pathogenesis of pulmonary cryptococcosis. A polyfunctional Th2 response enhances pulmonary eosinophil recruitment
and mucus production as well alternative activation of macrophages,
resulting in inhibition of fungal growth control. Th cell-specific IL4Ralpha ablation is able to confer resistance in pulmonary
cryptococcosis.
Discussion/conclusion Polyfunctional Th2 cells are the main producers of the pathology-associated Th2 cytokines leading to fatal
alternative activation of macrophages (Mucosal Immunol 2012; 5
(3): 299310).
Funding: German Research Foundation grant DFG AL 371/5-4.
P125
Interactions of murine phagocytes and Cryptococcus
neoformans strains in combination with voriconazole
L.V. Filippova,1 N. V. Vasilyeva,1 E. V. Frolova,1
A. E. Uchevatkina1 and E. P. Kiseleva2
1
North-Western State Medical University named after I.I.
Mechnikov, Saint-Petersburg, Russia and 2Institute of
Experimental Medicine Rams, Saint-Petersburg, Russia
Voriconazole is a triazole that offers extended activity against yeasts
that are not susceptible to earlier azole-type drugs. Clearance of
fungi depends on the ability of macrophages to destroy the yeast
cells, thereby preventing them from spreading. Phagocytosis of
fungi activates macrophages to production of various microbicidal
factors and cytokines, the balance of which determines the course
of infection.
90
The aim of this study was to investigate interactions between murine phagocytes and different virulence Cryptococcus neoformans strains
in combination with voriconazole.
Twelve clinical isolates of C. neoformans, received from the Russian
Collection of Pathogenic Fungi (RCPF), were investigated. All strains
were isolated from patients with AIDS or after renal transplantation.
Virulence of C. neoformans strains was study according to survival time
of Balb/c male mice 812 weeks old after intravenous inoculation
with 0.5 9 106 cells per mice. Highly virulent strains have caused
50% mortality of mice on 14th day after inoculation. Weakly virulent
strains have caused 50% mortality on 50th day and there was no
100% mortality after 65 days of observation. Phagocytosis of C. neoformans, production of nitric oxide and cytokines were studied in the
presence or absence of voriconazole in a concentration of 2 lg ml1.
Intact macrophages poorly uptake all C. neoformans strains.
Weakly virulent strains were better phagocytosed by macrophages in
comparison with results for highly virulent strains. Was established
that voriconazole increased the uptake by macrophages highly virulent strains of C. neoformans and did not affect their phagocytic activity against weakly virulent strains fungi. These data were obtained
confirmed the findings of other researchers about the absence of the
inhibitory effect of voriconazole on C. neoformans phagocytosis. In the
study of the macrophages ability to produce nitric oxide after uptake
C. neoformans, established that all strains of fungi in the interaction
with intact macrophages did not induce nitric oxide production, the
addition of voriconazole also did not affect its production. All investigated strains activated the synthesis of IL-10, IL-13, and ability of
fungi to cytokines induction was directly correlated with their virulence degree. Thus, we can suppose that the interaction with all
strains of Cryptococcus macrophages acquires alternatively activated
macrophages signs. Voriconazole did not affect the production of
IL-10 and IL-13 intact peritoneal macrophages, but significantly
inhibited the ability of highly virulent C. neoformans strains induce
macrophages to release IL-13. Perhaps, this is due to the modifying
effect of on chitin, a part of the fungi cell walls, which could interact
with dectin-1 macrophages. Thus, besides an antifungal action, voriconazole may influence on the production of cytokines responsible
for the antifungal activity of the macrophages. Furthermore, the
results suggest mechanisms by which voriconazole can cooperate
with immune defense systems in controlling of C. neoformans
infections.
P126
Role of an aspartyl protease (PEP1) and anti-PEP1
antibodies on the course of experimental cryptococcosis
F. Vernel-Pauillac and F. Dromer
Institut Pasteur, Paris, France
Background Cryptococcosis is a severe opportunistic infection still
associated with a 20% mortality rate despite adequate antifungal
therapy leaving room for improvement. Clinical and experimental
data suggest that both the host and the pathogen determine the variable outcome of infection. In a relevant murine model of disseminated cryptococcosis, we previously found that mice that survive the
inoculation of a usually lethal challenge with Cryptococcus neoformans
(Cn) were more likely to develop a delayed and monospecific antibody response against a 40 kDa protein that those which died from
cryptococcosis. The protein was identified as an aspartyl protease
PEP1.
Aim To assess whether vaccination with rPep1 and/or serotheray
with anti-PEP1 antibodies could alter the course of the infection.
Methods A recombinant protein (rPep1) and several monoclonal
antibodies specific for PEP1 (Mab) were produced. The effect of rPep1
or Mabs was tested prior to or after inoculation with Cn on survival
and fungal burden in target organs of BALB/c male mice.
Results Compared to 100% mortality rate in control mice, active
immunization with rPep1 prior to Cn inoculation was associated
with prolonged survival and decreased fungal burden both in H99
Poster Abstracts
and NIH52D-infected mice, with 25% and 60% survival rate at day
100 post inoculation, respectively and no residual infection in mice
surviving NIH52D inoculation. Therapeutic vaccine based on a single
injection of rPep1 in mice previously infected with Cn provided prolonged survival with partial control of the infection.
Passive serotherapy with 1 injection of anti-PEP1 Mabs the day
before, or 1 or 7 days after Cn inoculation led to a prolonged survival (dependent on the Cn strain, the Mab tested, the timing and
the Mab dose) compared to mice treated with an irrelevant Mab or
anti-capsular polysaccharide Mab E1. None of the protocol was associated with sterilisation of the target organs, but reduced fungal burden was obtained.
Discussion/conclusion These results suggest that immunomodulation with PEP1 or anti-PEP1 antibodies may be of benefit during disseminated cryptococcosis. Other experiments are ongoing to decipher
the role of the enzyme in the pathogenesis of the infection.
P127
Plasmacytoid dendritic cells and their role during the
protective immune response to experimental pulmonary
Cryptococcus neoformans infection
C. R. Hole, C. M. Leopold Wager, K. L. Wozniak and
F. L. Wormley Jr
University of Texas at San Antonio, San Antonio, TX, USA
Background Cryptococcus neoformans is a pathogenic basidiomycetous fungus that engages in outcrossing, inbreeding, and selfing
forms of unisexual reproduction as well as canonical sexual reproduction between opposite mating-types. Long thought to be clonal,
>99% of sampled environmental and clinical isolates of C. neoformans
are MATa limiting the frequency of opposite mating-type sexual
reproduction. Sexual reproduction allows eukaryotic organisms to
exchange genetic information and shuffle their genomes to avoid the
irreversible accumulation of deleterious changes that occur in asexual populations, known as Mullers Ratchet.
Aims To characterize whether unisexual reproduction, which dispenses with the requirement for an opposite mating type partner, is
able to purge the genome of deleterious mutations.
Methods Spores were dissected from unisexual matings of strains
carrying auxotrophic or temperature sensitive mutations. Parental
strains and F1 progeny were phenotypically characterized for
growth, competitive growth, and stress responses. Murine and Galleria models were infected by parental strains and F1 progeny.
Results The unisexual cycle can restore mutant strains of C. neoformans to wild-type genotype, phenotype, and growth rate. Furthermore, the unisexual cycle allows attenuated strains to purge
deleterious mutations and produce progeny that are returned to
wild-type virulence. Our results show that unisexual populations of
C. neoformans are able to avoid Mullers Ratchet and loss of fitness
through a unisexual reproduction cycle involving a-a cell fusion,
nuclear fusion, and meiosis. Similar types of unisexual reproduction
may operate in other pathogenic and saprobic eukaryotic taxa.
Discussion We show that unisexual reproduction between strains
that carry deleterious mutations generates progeny that have
returned to the wild-type genotype. Unisexual reproduction allows C.
neoformans to escape from Mullers Ratchet and produce phenotypically and genotypically fit offspring. In addition to restoring growth
rate, unisex is also able to produce virulent strains from avirulent
parents. There are clear implications of these findings for genetic
exchange and transmission of drug resistance and these processes
may occur in both the environment and in the host.
P128
Cytokine profile in human blood mononuclear cells
stimulated by GXM from AIDS patients with cryptococcal
meningitis
M. L. Silva Vergara, I. H. Rocha, L. A. Andrade Silva,
K. F. Ferreira Paim, R. R. Rocha Vasconcelos, A. Borges,
D. N. Silva-Teixera and D. J. Mora
Tria^ngulo Mineiro Federal University, Uberaba, Brazil
Background Globally, Cryptococcus neoformans is the main etiological agent of cryptococcal meningitis (CM) in AIDS patients. Yearly, it
accounts for one million cases of which 620,000 die particularly in
settings where access to anti-retroviral therapy is less available. Cryptococcus spp. are yeasts surrounded by a capsule primarily consisted
of glucuronoxylomannan (GXM). This capsular polysaccharide is an
important virulence factor able to inhibit opsonophagocytosis, T-cell
proliferation and downregulate proinflammatory cytokines production by peripheral blood mononuclear cells (PBMC).
Aim This study aimed to evaluate IL-2, IL-4, IL-8, IL-10, IL-12p40,
IL17-A, IFN-c and TNF-a production by GXM-stimulated peripheral
blood mononuclear cells from AIDS patients with CM.
Methods PBMC were obtained from 24 AIDS patients with CM (CM+
HIV+) at admission and during antifungal therapy. As controls, 32
HIV-positive individuals without CM (CM- HIV+) matched by CD4+ Tcells count and age and 47 non-HIV healthy donors (CM HIV)
paired by gender and age were recruited. Blood was anti-coagulated
with 20 U ml1 pyrogen-free heparin and diluted with equal volumes of RPMI 1640 medium. PBMC (1 9 106 cells ml1) were isolated by Ficoll-Hypaque density gradient centrifugation. One milliliter
was dispensed in each one of the 24-well plates and incubated at
37 C for 48 h in 5% CO2 with GXM (10 lg ml1). GXM was
obtained from culture supernatants of serotype A (ATCC 90112) by
CTAB-GXM precipitation and identified by Cryptolatex test and GXMspecific monoclonal antibody 18b7. Lipopolysaccharide (LPS)
(100 ng ml1) from Escherichia coli 026:B6 was used as a positive
control. Cultures were centrifuged (600 g for 15 min), and the supernatants stored at 70 C until be tested for cytokine concentration by ELISA.
Results Of 24 patients, 19 (79.1%) were male, median age of
34.2 years. Cryptococcal meningitis was the first AIDS-defining disease in 14 (58.3%) cases, while in 8 (57.1%) both diseases were
simultaneously diagnosed at admission. The CD4+ baseline values
were <100 cells mm3 in 18 (75%) of cases, whereas 15 (62.5%)
presented viral load levels >30 000 RNA copies per dl. At admission,
PBMC of CM- HIV- individuals produced higher levels of proinflammatory cytokines in response to LPS than those who were CM HIV+
and CM+ HIV+, except IL-17A to CM HIV+ and IFN-c to CM+ HIV+.
Kinetic studies showed significant increase of proinflammatory cytokines among CM+ HIV+ patients on week 16 when compared to
baseline value (P < 0.05). PBMC stimulated with GXM produced
baseline high levels of IL-4 and IL-10 in CM+ HIV+ patients which
progressively decreased during treatment (P < 0.05).
Discussion These features indicate that soluble GXM is able to
induce cytokine secretion by PBMC in AIDS patients with CM. Purified GXM suppressed the induction of proinflammatory cytokines
before and during antifungal therapy. In contrast, GXM stimulated
the induction of both IL-4 and IL-10 which are detrimental to the
cell-mediated immunity and consequent fungal clearance. This may
be clinically relevant, since high concentrations of this capsular antigen are frequently found in the body fluids of AIDS patients with CM
and are one of the most important negative prognostic factors associated to outcome.
Financial support: FAPEMIG grant BPD0050713.
91
Poster Abstracts
P129
P130
patients with CM who died before day 14 and those who survived.
92
Survivor
Non-survivor
P value
29 [11-85]
8.0 [6.9-9.0]
7272 [6154-8390]
33 [13-55]
5.6 [3.5-7.7]
4197 [2340-5653]
0.72
0.04
0.007
3.91 [3.82-4.00]
4.10 [3.86-4.32]
0.08
44% [39-50]
26% [15-37]
0.002
68.1 [60.0-76.3]
64.7 [53.1-76.4]
0.66
1033.5 (765.5-1480.5)
36.4 (13-68.3)
1410 [1196-1856]
84.9 (46.5-115)
0.02
0.01
Poster Abstracts
P131
Various expression patterns of cytokines and chemokines
can be observed after interaction with clinical isolates of
Cryptococcus neoformans
A. Sturny-Lecle`re, A. Alanio, F. Vernel-Pauillac, M. Gougeon and
F. Dromer
Institut Pasteur, France
Background Macrophages play a central role in cryptococcocis
pathogenesis, representing the first line of defense, a potential site for
yeast replication and dormancy, and also probably a vehicle for yeast
dissemination. We previously demonstrated that clinical isolates of
Cryptococcus neoformans showed variations in their interaction with
macrophages (phagocytosis and intracellular proliferation index of
the yeasts) that were associated with differences in clinical outcome
of the patients (1).
Objective and methods First, we investigated the diversity of macrophage cellular response following their interaction with 9 clinical
isolates previously selected for their phenotype, in comparison with
the reference strain H99 (1). The in vitro response of J774 macrophage cell line was assessed by quantifying the production of soluble
including pro- and anti-inflammatory
mediators (23-plex LuminexO)
cytokines (TNF-a, IL-1b IL-6, IL-10) and chemokines (MCP-1, MIP1a, MIP-1b, RANTES) in kinetics experiments. Second, we performed
in vivo experiments in OF1 outbred mice with 2 clinical isolates harboring low and high virulence compared to H99. We evaluated fungal load, tissue lesions and mediators production in individual sera,
brains and lungs of 7 mice/group.
Results and discussion Among the clinical isolates studied, isolates
SC5 and SC6 that differed in terms of virulence (median survival of
29 and 9 days post inoculation, respectively with a median CFU/g of
tissue in mice sacrificed on dpi 7 differing by more than 1 log respectively), and phagocytic index ratio compared to H99 (PI = 0.5 and
PI = 1.21, respectively) exhibited drastically different patterns of
cytokine/chemokine production in vitro and in vivo. In addition, in vitro analysis of the nine clinical isolates identified two major cytokine/
chemokine profiles that were associated with phagocytic index and
virulence. These data provide additional evidence on the influence of
fungal diversity on the host immune response.
Reference 1. Alanio A et al. mBio. 2011; 2(4): e0015811.
P133
TNF-alpha-induced stability of DC1 programming is
required for maintenance of protective Th1/Th17 immune
response against Cryptococcus neoformans
A. J. Eastman,1 J. Carolan,1 N. Potchen,1 M. J. Davis,1 Y. Qiu,1
A. Malachowski,2 I. Kryczek,1 K. Cavassani De Souza,1
S. Kunkel,1 J. Osterholzer2 and M. A. Olszewski1
1
University of Michigan, Ann Arbor, MI, USA and 2University of
Michigan/Ann Arbor VA Hospital, Ann Arbor, MI, USA
P132
Transient blockade of IL-10 signaling enhances fungal
clearance and Th1/Th17 effector responses in mice with
early and established Cryptococcal lung infection
M. A. Olszewski,1 B. J. Murdock,1 G. H. Chen,1 G. B. Huffnagle2
and J. Osterholzer1
1
VA Ann Arbor/University of Michigan, Ann Arbor, MI, USA and
2
University of Michigan, Ann Arbor, MI, USA
Background Pulmonary infection with Cryptococcus neoformans
(Cneo), a fungal pathogen acquired by the inhalational route, can
result in progressive infection and death or persistent infection associated with chronic lung inflammation and features of allergic airway
immunopathology. Infection of C57BL/6 mice with Cneo recapitulates
this persistently-infected phenotype. Studies performed in IL-10 deficient mice show that IL-10 is a critical determinant of this phenotype
by skewing the adaptive immune response away from the development of protective Th1/Th17 responses and towards ineffective Th2/
Treg responses.
Aim To determine whether transient antibody-mediated blockade of
IL-10 signaling during either the: (i) developing, or (ii) established
phase of infection enhances effector immune responses against Cneo
in C57BL/6 mice.
93
Poster Abstracts
Aim To address this question, we compared the effect of polyinosinicpolycytidylic acid condensed with poly-l-lysine and carboxymethylcellulose (Poly-ICLC), a stabilized version of Poly-IC, in murine cryptococcosis caused by the two species.
Methods Experimental models of pulmonary and systemic cryptococcosis were established using wild type (C57BL/6) and knock-out (Ifnabr/, TLR3/, MDA5/) mice to evaluate survival, tissue fungal
load and histopathology with and without Poly-ICLC treatment.
Quantitative RT-PCR, enzyme-linked immunosorbent assays were
used for measurements of type I IFN mRNA and cytokine detection,
respectively.
Results Poly-ICLC is a synthetic analogue of viral double-stranded
RNA (dsRNA) which is designed to stimulate prolonged, high-level
production of type I IFN. We observed a protective effect of Poly-ICLC
in both C. neoformans and C. gattii infection.As expected, the mice
lacking type I IFN-receptor 1 (Ifnabr KO mice) were significantly
more susceptible to both species than the wild type mice. Interestingly, however, while Poly-ICLC was not protective for Ifnabr-deficient mice from C. neoformans infection, it protected the mice from C.
gattii infection. Our results suggest that Poly-ICLC induced protection
against C. neoformans is type I interferon dependent but not against
C. gattii.
Conclusion Our study indicates the existence of major differences in
host defense against the two species. In addition, the study suggests
that Poly-ICLC can be an alternative anticryptococcal agent that can
be used for cryptococcosis maintenance therapy.
P134
94
P135
Capsule growth in Cryptococcus neoformans is
coordinated with cell cycle progression
R. Garca-Rodas,1 R. J. Cordero,2 G. Janbon,3 F. Moyrand,3
A. Casadevall4 and O. Zaragoza1
1
National Centre for Microbiology, ISCIII, Madrid, Spain; 2Federal
University Rio de Janeiro, Rio de Janeiro, Brazil; 3Institut Pasteur,
Paris, France and 4Albert Einstein College of Medicine, New
York, NY, USA
Poster Abstracts
P137
Golgi reassembly and stacking protein is involved in the
vesicular traffic of polysaccharides in Cryptococcus
neoformans
L. S. Sobrino Joffe,1 R. R. Pinheiro,2 J. Rizzo,3 L. Kmetzsch,4
C. C. Staats,4 C. L. Ramos,3 K. Miranda,3 S. Frases,3
M. H. Vainstein4 and M. L. Rodrigues5
1
Universidade Federal do Rio de Janeiro/Instituto de
Microbiologia Paulo de Goes, Rio de Janeiro, Brazil; 2Fundaca~o
Oswaldo Cruz - Fiocruz, Rio de Janeiro, Brazil; 3Universidade
Federal do Rio de Janeiro - UFRJ, Rio de Janeiro, Brazil;
4
Universidade Federal do Rio Grande do Sul - UFRGS, Porto
Alegre, Brazil and 5UFRJ & Fiocruz, Rio de Janeiro, Brazil
P136
The role of aspartyl aminopeptidase (APE4) in
Cryptococcus neoformans virulence and authophagy
M. A. Vallim,1 F. A. Gontijo,1 R. C. Pascon,1 J. Machado-Jr1 and
L. F. Fernandes2
1
Universidade Federal de Sa~o Paulo, Diadema, Brazil and
2
Universidade de Braslia, Brasilia, Brazil
In Saccharomyce cerevisiae the M18 metaloprotease Ape4 is activated
by nitrogen deprivation, its protease activity is against the aspartate
and glutamate aminoacids located at the protein amino-terminus.
This protein is engaged in autophagy and also is part of the cytoplasm to vacuole targeting pathway (Cvt), which is composed by the
proteins Ams1, Ape1, Ape4 e Atg19. The Ape4 protein under nitrogen starving growth condition is located in the autophagosome. Lack
of the Ape4 protein in S. cerevisae does not lead to high temperature
sensitivity whereas the ape4 Cryptococcus neoformans mutant is incapable to grow at 37 C. This information was gathered for this yeast
when a collection of mutants generated by insertional mutagenesis
was subject to a high temperature screening. To confirm that indeed
Ape4 was the responsible for this phenotype an independent gene
interruption was carried out generating a new mutant which was
unable to grow at 37 C. We demonstrated that the C. neoformans
ape4 mutant has defects for important virulence factors as melanin
and phospholipase production, capsule formation, and was unable to
survive inside the macrophage (linage J774A.1) compared to the
wild type. These date set suggested that the ape4 mutant have attenuated virulence. This hypothesis was confirmed by the animal experiment where the mutant was unable to kill the infected animals,
whereas the wild type and the reconstituted (ape4D+APE4) strains
where effective in causing the mice death within about 22 days postinfection. In S. cerevisae under nitrogen deprivation the Ape4 protein
migrates to the vacuole where it is active. We have fused C. neoformans Ape4 protein to GFP and we intend to demonstrate the cellular
localization for this protein. Also, we have cloned the APE4 gene in
a protein expression vector and we are going to isolate and purify
this protein to characterize its amino acid target in vitro employing a
polypeptide library.
Fapep: 2007/50536-3.
We have recently described that Golgi reassembly and stacking protein (GRASP) is required for polysaccharide secretion and virulence
in Cryptococcus neoformans. We have also observed in previous studies
that secretion of capsular polysaccharides in C. neoformans involves
extracellular vesicle (EV) formation. Since GRASP is potentially
involved in vesicular secretion in other eukaryotes, we hypothesized
that EV-mediated polysaccharide export and GRASP are functionally
connected in C. neoformans. In silico analysis of protein structure
revealed that C. neoformans GRASP contains unique regions, in comparison to its human homologues. Predictive studies suggested that
the fungal-specific GRASP regions have low hydrophobicity and high
antigenic potential. Wild type C. neoformans cells and a mutant lacking GRASP expression (Dgrasp) had comparable levels of EV formation. However, polysaccharide concentration in EVs produced by the
Dgrasp mutant was decreased, in comparison with WT cells. Vesicular polysaccharide fibers produced by the Dgrasp mutant also showed
reduced dimensions, as determined by dynamic light scattering. EVs
obtained from cultures of the Dgrasp mutant also differed from those
produced by WT cells in diameter distribution. Analysis of the C. neoformans surface architecture by scanning electron microscopy
revealed that, in comparison to WT cells, the mutant had capsular
polysaccharide fibers with reduced dimensions. These results were
suggestive of defects in polysaccharide traffic. We also evaluated
whether lack of GRASP would interfere with polysaccharide synthesis
in C. neoformans. Analysis of crude cellular extracts revealed that the
Dgrasp mutant was in fact less efficient than WT cells in synthesizing
capsular polysaccharides. These results demonstrated that GRASP is
required for both polysaccharide synthesis and EV-mediated traffic in
C. neoformans. Since polysaccharides are determinant for virulence in
this fungus, we conclude that EV formation, GRASP and pathogenesis are directly connected in the C. neoformans model.
P138
Galleria mellonella model identifies highly virulent strains
among all major Cryptococcus gattii molecular types
C. Firacative and W. Meyer
The University of Sydney, Westmead, NSW, Australia
Background Worldwide cryptococcosis is mainly caused by Cryptococcus neoformans. However, the number of cases due to C. gattii is
increasing, affecting mainly immunocompetent hosts. By different
molecular methods C. gattii has been divided into four major molecular types VGI to VGIV, which differ in their host range, epidemiology,
antifungal susceptibility and geographic distribution. Besides studies
on the Vancouver Island outbreak strains, which showed that the
sub-genotype VGIIa is highly virulent compared to the sub-genotype
VGIIb, little is known about the virulence of the other major molecular types.
95
Poster Abstracts
P139
Capsule synthesis occurs in a non-polarized manner and its
growth depends on mitochondrial activity
N. Trevijano-Contador, S. Landin, R. Garca-Rodas and
O. Zaragoza
National Center of Microbiology, ISCIII, Madrid, Spain
Background The dynamics of the capsule of Cryptococcus neoformans
is an important feature to understand the biology of the main virulence factor of this pathogen. The capsule is synthesized by addition
of new polysaccharide, but it is not known if the synthesis follows a
polarized or dispersed pattern. In addition, the size of the capsule is
not constant, and during infection it increases significantly. It is
believed that capsule growth is an energy-cost process, but this
aspect has never been addressed.
Aim We have investigated the dynamics of capsule formation and
the metabolic dependence of capsule growth, the dependence of capsule enlargement on mitochondrial activity. In particular, we wanted
to determine if capsule synthesis is polarized from a specific site of
the capsule. Concerning capsule growth, we have investigated the
importance of mitochondrial activity on this process.
Methods We used an acapsular strain (cap59) described by Kwonchung (1) in which the CAP59 gene has been reintroduced under
the control of the GAL7 promoter, so this strain is acapsular in glucose and encapsulated when transferred to galactose. In addition, we
have evaluated the role of mitochondrial activity on capsule growth.
For this purpose, we have studied the effect of specific inhibitors of
the electron respiratory chain on capsule enlargement. Furthermore,
we have estimated if during this process there are any changes in
96
mitochondrial activity by measuring cytochrome c oxidase and mitochondrial membrane potential with specific fluorescent probes
(mainly Rhodamin 123).
Results We have confirmed that the strain that expresses the GAL7::
CAP59 gene only presents the capsule when it is transferred to
galactose-containing media. When we examined the dynamics of
capsule appearance, we have observed that the capsule first appears
as spots randomly distributed throughout the cell, suggesting that
this process occurs in a non-polarized manner. We have also investigated the role of mitochondrial activity on capsule enlargement. For
this purpose, we used different metabolic inhibitors: rotenone, which
inhibits complex I, Antimycin A which inhibits complex III and Salicylhidroxamic acid that blocks the alternative cytochrome oxidase
pathway. In all the cases, we used concentrations that inhibited cell
division, but that did not affect the viability. We observed that addition of these inhibitors impairs capsule growth, confirming that this
process requires full mitochondrial activity. Current experiments are
ongoing to determine if during capsule growth there are changes in
mitochondrial activity.
Discusion/conclusion Our results highlight new aspects and elements involved in capsule dynamics in C. neoformans. We have demonstrated that capsule synthesis occurs randomly throughout the
cells, which is in agreement that capsule synthesis is linked with vesicle secretion. We have also confirmed that capsule enlargement is
an energy cost process that requires mitochondrial activity. This is
an important aspect, because capsule growth normally occurs in
conditions of nutrient limitation. So our results indicate that in these
conditions, an important amount of the limited energy supply is
invested in the growth of the capsule, which reflects the importance
of this process to survive in stress conditions and in the host
environment.
Reference 1. Chang and Kwon-Chung. 1994. MCB, 14: 4912
4919.
P140
Role of GTI1 homologue on Cryptococcus neoformans
virulence
L. S. Derengowski, H. C. Paes, L. F. Fernandes, M. S. Felipe and
I. Silva-Pereira
Universidade de Braslia, Brazil
Background Cyclic AMP independent pathways using Gti1/Pac2
transcription factors have been shown to play major role in adaptation of ascomycetes to the environment and hosts. Among other
Poster Abstracts
P141
The vacuolar protein Snf7 regulates export of virulence
determinants in Cryptococcus neoformans
R. M. C. Godinho,1 J. Crestani,2 L. Kmetzsch,2 C. C. Staats,2
A. Schrank,2 M. H. Vainstein2 and M. L. Rodrigues3
1
Universidade Federal do Rio Janeiro - UFRJ, Rio de Janeiro,
Brazil; 2Universidade Federal do Rio Grande do Sul, Porto Alegre,
Brazil and 3Universidade Federal do Rio de Janeiro & Fundaca~o
Oswaldo Cruz/CDTS, Rio de Janeiro, Brazil
Cryptococcus neoformans (CN) uses a wide range of pathogenic mechanisms to cause damage to host cells, including formation of a polysaccharide capsule, secretion of lytic enzymes and ability to melanize.
Several of the virulence determinants in CN are exported through
unconventional secretion mechanisms that require exosome-like,
extracellular vesicles. Despite their potential importance for pathogenicity, the origin of CN extracellular vesicles remains obscure. Exosome formation requires a number of functional proteins, including
those operating in the Endosomal Sorting Complex Required for
P142
Cryptococcus neoformans clinical isolates from Serbia genotypes, antifungal susceptibility and virulence
M. G. Pekmezovic,1 A. Barac,2 V. S. Arsic Arsenijevic,2 F. Hagen3
and J. F. Meis3
1
Faculty of Medicine, University of Belgrade, Belgrade, Serbia;
2
Institute of Microbiology and Immunology, Belgrade, Serbia and
3
Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands
Background Cryptococcus neoformans causes systemic fungal infection mainly in individuals with a suppressed immune system. It is
estimated that approximately 1 million cases of cryptococcosis occur
throughout the world that result in 650 000 associated deaths
annually. This species consists of varieties: C. neoformans var. grubii
(serotype A) and C. neoformans var. neoformans (serotype D), as well
as hybrids between both cryptococcal varieties (serotype AD). Amplified fragment length polymorphism (AFLP) fingerprinting has identified several genotypes among varieties: AFLP1, AFLP1A and AFLP1B
(serotype A), AFLP2 (serotype D) and AFLP3 (serotype AD). Also,
each C. neoformans strain can exist in form of haploid MAT-a or
MAT-alpha cell. MAT-alpha strains have been shown to be much
more prevalent and more virulent. Besides the presence of MATalpha locus, ability to grow at 37 C, capsule formation and the production of melanin, urease and phospholipase have been identified as
virulence factors of C. neoformans strains. Studies of cryptococcosis in
animal models infected with different strains of C. neoformans showed
that individual isolates differ in virulence, susceptibility and ecological characteristics.
Aim The purposes of this study were: (i) to identify the genotypes,
serotypes and mating types; (ii) to determine antifungal susceptibility
profile; (iii) to analyze virulence factors of C. neoformans clinical isolates from Serbia.
Methods This study investigated 34 clinical Serbian C. neoformans
isolates from cerebrospinal fluid and blood of 25 immunocompromised
patients in the 20-year period. AFLP fingerprinting was used for genotyping, while real-time PCR was used to determine the mating- and
serotype. The MICRONAUT plates were used to determine susceptibility to amphotericin B, 5-fluorocytosine, fluconazole, itraconazole,
posaconazole and voriconazole, according to the CLSI standardized
protocol. Each strain was examined for production of: (i) capsule by
India ink staining; (ii) melanin by culturing of minimal L-DOPA medium; (iii) urease by culturing on Christensens agar and (iv) extracellular phospholipase by culturing on Sabouraud-egg yolk agar.
Results Men predominated among 25 patients (male 92% vs. female
8%). HIV/AIDS (84%) was major predisposing factors for cryptococcosis, followed by non-Hodgkin lymphoma (12%) and kidney
97
Poster Abstracts
P143
Possibility of infection by Cryptococcus species for workers
in organic farming
M. A. Khiyami,1 A. H. Bahklia2 and H. EL-Zefzaf3
1
King Abdulaziz City for Science and Technology, Riyadh, Saudi
Arabia; 2King Saud Universities, Riyadh, Saudi Arabia and 3Plant
Pathology Research Institute, Agric. Res. Center, Giza, Egypt
Background Manure is organic matter used to prepare compost in
organic farming. Many factories in Saudi Arabia produce compost
however they do not follow the right procedure of preparation. A
pilot study illustrated that most of the composts contain pathogens.
The workers in the organic farms dealing with organic compost usually have limited education. Therefore, they might unknowingly
become infected with these different pathogens.
Aim of the study Assess health risks of microbial contamination of
industrial compost production.
Methods In this study a hundred samples of compost were collected
from five different factories. The compost samples were analysed for
ingredients and pathogens, including bacteria, fungi, yeast, and parasites. The microbial isolates were identified by microbiological and
molecular methods. Twenty five workers were selected for septic
screening. Sputum, blood and stool samples were collected to screen
for any pathogen.
Results Thirty-four out of one hundred samples of compost contained
several microbes includes fungi, yeast, bacteria and parasites. The
dominant bacteria in samples were E. coli and Enterobacter sp. Aspergillus sp, Penicillium sp, Fusarium sp, Mucor sp, and Cladosporium sp
were dominant fungi in all 30 samples. One strain of Cryptococcus was
found in two compost samples. Giardiasis, Isosporiasis and Entamoeba
histolytica were also isolated from all compost samples. The analysis of
stool samples collected from nineteen workers contained E. histolytica.
Work is still in progress and the rest of the analysis will elucidate
whether any of the workers was infected by immature composts or not.
Conclusion Compost was found to contain various microorganisms.
Cryptococcus sp. were very rare. Further work has to show the public health risks of compost production.
98
P144
Cryptococcus neoformans/Cryptococcus gattii species
complex in Cuban patients
M. T. Illnait-Zaragoz,1 Y. Rivera Gallego,1 E.X. Monroy Vaca,1
F. Hagen,2 C.M. Fernandez Andreu,1 G.F. Martnez Machn,1
M.R. Perurena Lancha1 and J.F. Meis2
1
Tropical Medicine Institute Pedro Kour, Havana, Cuba and
2
Canisius-Wihelmina Hospital, Nijmegen, The Netherlands
Background The incidence of cryptococcosis increased with the
beginning of the aids epidemic. Life expectancy and quality of life
among HIV-infected Cuban patients have increased due to the wide
HAART application during the past decades.
Aims To study clinical and microbiological aspects of patients suspected of having cryptococcosis who attended the Tropical Medicine
Institute Pedro Kour during 20112012.
Methods Twenty-five yeasts recovered form cerebro-spinal fluid and
blood were identified by conventional methods. Identification of Cryptococcus isolates were confirmed by URA5 amplification and ITS1/
5.8S/ITS2 sequencing. For every Cryptococcus sp. isolate the seroand mating-type was determined by PCR and the in vitro susceptibility was performed by using the ATB Fungus system. Clinical and epidemiological data of patients with confirmed cryptococcosis were
retrieved from their clinical records.
Results Conventional identification yielded 13 C. neoformans and
one C. gattii isolates. Amplification and sequencing confirmed the
identification. All the C. neoformans isolates were alphaA and no
resistance to the studied antifungal drugs were found. Clinical
records of patients revealed an average age of 38.5 years, the infection was twice more observed among male patients, nine patients
were HIV-infected, steroid treatment and renal transplant were found
in two patients, and main clinical findings were headache, fever and
neurological complications. Examination of cerebro-spinal fluid
showed at least one parameter altered in every patient and a positive
Indian ink in 90% of HIV-infected patients and 67% of non-HIV
patients; hematological studied indicated CD4+ less than
200 cells ml1 in 86% of patients; most patients received amphotericin B plus fluconazole during induction therapy followed by fluconazole for at least eight weeks; all isolates were susceptible to the used
drugs, 21.4% suffered from relapsed infections and 28.6% died.
Conclusion Cryptococcal infection is still a problem among HIVinfected Cuban patients. C. neoformans alphaA is the main causative
agent. More effective treatment for cryptococcal infections is required
since relapses and fatal outcome are frequent despite absence of in vitro resistance to recommended antifungal drugs.
P145
Pseudomonas aeruginosa as a potential source of agents
interfering with capsule formation in Cryptococcus
neoformans
V. Raclavsky,1 R. Novotny1 and V. Kolek2
1
Palacky University, Faculty of Medicine and Dentistry, Olomouc,
Czech Republic and 2University Hospital, Olomouc, Czech
Republic
Background Polysaccharide capsule represents a well-established
virulence factor in the pathogenic yeast Cryptococcus neoformans.
Although it is dispensable for its survival, it plays very important
role in its ability to evade the immune defense of host by interfering with its several aspects. Because C. neoformans is an opportunist
pathogen, possible imbalance between its virulence and the
immune status of host most probably decides whether a colonisation of airways will progress into tissue invasion and possibly lifethreatening systemic infection or not. Consistently, cases of
Poster Abstracts
P146
Cryptococcus neoformans and Cryptococcus gattii isolated
from woody debris samples collected from trees within a
public garden in Cape Town, South Africa
J. Vreulink,1 K. Khayhan,2 F. Hagen,3 A. Botes,1 L. Moller,1
T. Boekhout,4 H. Vismer5 and A. Botha1
1
University of Stellenbosch, Stellenbosch, South Africa;
2
University of Phayao, Phayao, Thailand; 3Canisius-Wilhelmina
Hospital, Nijmegen, The Netherlands; 4CBS-KNAW Fungal
Biodiversity Centre, Utrecht, The Netherlands and 5Formerly from
the Promec Unit, Cape Town, South Africa
Background Cryptococcosis is one of the leading causes of death
among HIV/AIDS patients, especially in sub-Saharan Africa. Infection
is acquired through the inhalation of basidiospores produced by Cryptococcus neoformans and Cryptococcus gattii in their natural habitat,
which seems to be woody debris. Globally, there have been numerous studies about the presence of these yeasts in the environment.
However, very little is known about the prevalence of these pathogens in the South African environment.
Aim The first aim was to determine if C. neoformans and C. gattii are
present in trees located in a public garden in Cape Town, South
Africa. If these yeasts were present, the second aim was to determine
the genotypes, mating and sequence types (STs) of the isolates.
Lastly, in order to determine if infection was acquired from the trees
located in the public garden, we compared the genotypes, mating
types and STs of these isolates to that of clinical isolates obtained
from the Medical Research Council (MRC).
Methods Woody debris samples were collected from trees located in
a public park in Cape Town, South Africa. Serial dilutions of the
woody debris samples were plated onto Niger seed agar plates. Representatives of C. neoformans and C. gattii were randomly isolated and
their genotypes were determined by restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PBL1) and
amplified fragment length polymorphism (AFLP) analysis. The mating types of the isolates were determined by PCR amplification of the
MATalpha/MATa pheromone genes or amplification of the STE12
locus. In addition, the sequence types (STs) of the isolates were determined using the ISHAM multi-locus sequence typing (MLST) scheme.
The genotypes, mating types and STs of clinical isolates obtained
from the former PROMEC Unit of the MRC were also determined and
compared to the environmental isolates.
Results During this study 65 woody debris samples were collected
from 17 trees located within a public garden in Cape Town, South
Africa. Thirteen (20%) of these samples produced pigmented colonies on Niger seed agar plates. A total of 40 pigmented colonies
were randomly selected and further characterized. The majority of
these isolates were C. gattii VGI/AFLP4 (80%), followed by C. neoformans VNI/AFLP1 (17%). Only one isolate was VNIII/AFLP3 (3%).
In contrast, the majority of the clinical isolates were VNI/AFLP1
(47%), while only one isolate was VGI/AFLP4 (5%). In addition five
clinical isolates were VNII/AFLP1B (26%) and one isolate was
VGIV/AFLP7 (5%). Interestingly, three clinical VNI isolates (16%)
were AFLP1A and are therefore VNB. The majority of the environmental isolates (85%) and all of the clinical isolates were MATalpha, while six environmental VGI/AFLP4 isolates (15%) were the
rare MATa mating type. The majority of the environmental isolates
were ST230 (56%), followed by ST23 (17%), while the rest of the
isolates were ST249 (7%), ST270 (5%), ST261 (2%), ST271 (2%),
ST330 (2%) and ST331 (2%). In comparison, the clinical isolates
were more diverse and belonged to the following STs: ST2 (5%),
ST5 (10%), ST23 (5%), ST40 (25%), ST69 (15%), ST93 (10%),
ST154 (5%), ST228 (5%), ST231 (5%), ST232 (5%), ST262 (5%)
and ST263 (5%).
Discussion/ conclusion Cryptococcus neoformans VNI/AFLP1 and C.
gattii VGI/AFLP4isolates were obtained from trees located within a
public garden in Cape Town, South Africa. These isolates belonged to
8 different STs. However, we only obtained one of these STs, i.e.
ST23, from a patient. Therefore, more environmental sampling needs
to be done in South Africa, in order to determine the location of the
other STs that were obtained from the other patients. Yet, our results
make a significant contribution to the knowledge about pathogenic
cryptococci present in the South African environment.
P147
Prevalence of CNS Cryptococcosis in a tertiary care
teaching hospital of Northern India
D. K. Chhina, P. Suri, V. Gupta and R. S. Chhina
Dayanand Medical College and Hospital, Ludhiana, India
Background The encapsulated yeast, Cryptococcus spp., is a major
cause of fungal meningitis and meningoencephalitis especially in
99
Poster Abstracts
P148
Synthetic peptides as promising antigens in the
immunodiagnosis of cryptococcosis
R. Brandao,1 L. Soares Martins,1 H. M. Andrade,2 A. R. Faria,2
A. Silva,1 B. Wanke,3 M. S. Lazera,3 M. H. Vainstein,4
R. P. Mendes,5 D. V. Moris,5 R. S. Cavalcante5 and S. Monte1
1
Federal University of Piau, Teresina, Brazil; 2Federal University of
Minas Gerais, Belo Horizonte, Brazil; 3Oswaldo Cruz Foundation,
Rio de Janeiro, Brazil; 4Federal University of Rio Grande do Sul,
Porto Alegre, Brazil and 5Medical School of Botucatu, Botucatu,
Brazil
Background Cryptococcosis is an important mycosis that affects
humans and animals all over the world. Globally, almost 1 million
cases of cryptococcal meningitis in HIV-infected subjects are estimated to occur every year and over 600 000 deaths. The diagnosis
of cryptococcosis is made through microscopy, culture followed by
biochemical tests, and detection of cryptococcal capsular antigen
(CrAg) by enzyme immunoassay or latex agglutination or lateral flow
assay. The application of synthetic peptides aiming to improve the
diagnosis for infectious diseases has been reported, but little has been
done as regards systemic mycoses. In leishmaniasis, Chagas disease,
paracoccidioidomycosis and tuberculosis have demonstrated that this
methodology has a potential for discovering antigenic targets.
Aim Test synthetic peptides as new antigenic targets for the development of a diagnostic test for cryptococcus.
Methods Sixty-three B cell epitopes from C. gattii immunoreactive
proteins previously identified were synthesized and evaluated as antigen in enzyme-linked immunosorbent assay (ELISA). Peptides were
first evaluated for their ability to react against sera from immunocompetent subjects carrying meningococcal meningitis. Peptides
whose first test yielded higher sensitivity and specificity were then
retested with sera from individuals with other fungal pathologies, for
cross reactivity determination.
Results Six synthetic peptides were recognized by antibodies in immunoassays, getting: (i) specificity of 100%, (ii) sensibility of 78%,
and (iii) minor cross-reactions. The recognition by antibodies of sera
coming from other pathologies was shown heterogeneous, and with
optical density (OD) values much lower than the ones observed for
cryptococcosis (P value ranged from P < 0.0001 to P = 0.0007),
except H18 (Fig. 1).
Discussion/conclusion From sixty-three synthetic peptides, six peptides were identified as good antigenic candidates for developing new
diagnostic tests for cryptococcosis. Out of these, three come from
100
Hsp70 (H18, H21, and H26). This chaperone shows both regions
conserved along the evolution and varied regions that allow a distinction between species, and it may present immunogenic epitopes.
In fact, recent works have shown that Hsps have been characterized
as key antigens inducing humoral responses to C. neoformans and C.
gattii and that Hsp70 is involved in the adhesion and phagocytosis
processes of C. neoformans. The remainder peptides derive from two
proteins whose information in the literature is still scarce: peptides
Hy49 and Hy50 (hypothetical protein CGNB 1302), and peptide S4
(protein Sks2). The peptides studied showed a high specificity, which
is essential in the clinical practice, as it excludes other morbidities
with similar clinical manifestations. In addition, the search for an
alternative to diagnose cryptococcosis in advance, together with the
lack of studies using synthetic peptides in developing serological tests
in mycoses, makes the validation of synthetic peptides for developing
a diagnostic test of cryptococcosis of utmost importance. In conclusion, by using immunoproteomics and bioinformatics techniques we
were able to identify new antigenic targets for developing diagnostic
tests for cryptococcosis and, in this study, six synthetic peptides
showed that they are promising antigens in the immunodiagnosis of
cryptococcosis.
Poster Abstracts
P149
Role of fungal burden in cerebrospinal fluid in hiv
management of cryptococcal meningitis
F. Concha Velasco and A. Bustamante Rufino
IMTAvH, Lima, Peru
Background Cryptococcal meningitis (CM) in HIV-infected patients
has high mortality despite the use of highly active antiretroviral therapy. Previous studies have shown that high fungal burden is an
important factor in mortality. Relation of fungal burden, assessed as
number of colony forming units (CFU), with colonies growing time
and cerebrospinal fluid (CSF) opening pressure (OP) could help in the
management of CM.
Aims 1. Quantify CFU in CSF culture and evaluate its association
with OP at start of treatment and after 2 weeks.
2. Determine the time to fungal clearance in CSF and evaluate its
association with OP.
3. Compare time-to-growth and number of CFU at baseline and
after 2 weeks of treatment
Methods We performed a retrospective study in HIV patients with
CM, hospitalized from January 2000 to September 2013, at Hospital
Nacional Cayetano Heredia in Lima, Peru. Patients with a positive
CSF culture for Cryptococcus at baseline and alumbar puncture at
2 weeks of treatment were included.
Results We included 113 patients, 62 (55%) of them had a positive
culture at week 2. We did not find a positive correlation between
baseline number of CFU (n = 51) and high OP ( 20 cmH2O)
(P = 0.06), but at week 2 of treatment there was a significant correlation (P = 0.0001). The median CFU was 4.23 0.93 log10 per ml
at baseline and 2.32 1.26 log10 per ml at week 2.
At week two, 44 (44%) did not have high OP (<20 cm H2O), 14
(14%) had mild OP (2024.9 cm H2O), 23(23%) moderate OP (25
34.9 cm H2O) and 19 (19%) had severe OP (35 cm H2O).
Also, there was a significant association between fungal clearance
(negative culture at 2 weeks) n = (51/113) with high OP
(P = 0.0001) and with severity degree (P = 0.01 for moderate OP
and P = 0.04 for severe OP Fig. 1).
Quantitative culture results were available for 51 patients at baseline and 85 at 2 weeks. There was a significant negative correlation
(P = 0.0154 and 0.000) between the number of CFU and time to
positivity of culture (Fig. 2). The median time for growth was
4 3.8 days at baseline and 5 6.2 days at 2 weeks.
At baseline 82.76% of C. neoformans cultures grew before 7 days,
14.94 % between 7-14 days and 2.3% over 14 days. At week 2 of
treatment, 62.71% grew in less than 7 days, 13.56% between 7 and
14 days and 6.78% in over 14 days.
P150
Prevalence of cryptococcosis in a tertiary care centre in
north India over 15 years
J. Chander,1 N. Singla,1 R. Singh,1 M. Kaur,1 U. Singhal1 and
F. Hagen2
1
Government Medical College Hospital, Chandigarh, India and
2
Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands
Background There is an increased prevalence of cryptococcosis
worldwide due to ongoing pandemic of AIDS and other circumstantial immunocompromised situations. Cryptococcus neoformans, the
classical etiologic agent of cryptococcosis, is currently recognized as a
species complex, comprising C. neoformans var. grubii, serotype A, C.
neoformans var. neoformans, serotype D, and C. gattii, serotypes B and
C. The vast majority of cryptococcal infections, particularly in immunocompromised patients, are caused by C. neoformans var. grubii
whereas C. gattii accounts for a smaller proportion of cases, which
frequently infects immunocompetent patients. Recent outbreak of C.
gattii in Canada and northwestern USA has posed a challenging task
before the medical community. In cryptococcal disease typing methods are useful in confirming phenotypic identification, resolving taxonomy, studying molecular epidemiology, improving the diagnosis of
cryptococcosis, identifying the genotype phenotype association and
investigating the evolution of pathogenic species of cryptococcus.
Aim The present study was undertaken to study profile of cryptococcosis and antifungal susceptibility testing pattern of isolates from
cases observed during 15 years i.e. 19992013 in the Government
Medical College Hospital, Chandigarh, India.
Methods The yeast isolates were identified based on cultural characteristics, biochemical reactions and serotyping. AFLP typing was
done at CanisiusWilhelmina Hospital, Nijmegen, The Netherlands.
Results Out of a total of 23 cases, twenty were diagnosed as cryptococcosis on culture basis. The CSF was clear with no turbidity
101
Poster Abstracts
Gender/Age
Co-morbidities
Localization
M/67
Meningitis
F/43
Meningitis
M/54
Disseminated
Strongyloides
stercoralis
Ancylostoma sp.
Disseminated
(lung,
meningeal,
gastrointestinal)
F/58
Meningitis
having two varieties i.e. var. neoformans and var. gattii. However,
based on the elucidation of the genomic sequences, C neoformans and
C. gattii are now considered two distinct species. These two species
have 5 serotypes based on antigenic specificity of the capsular polysaccharide; these include serotypes A, D and AD (C. neoformans) and serotypes B and C (C. gattii). Worldwide, C. neoformans serotype A causes
most of the cryptococcal infections in immunocompromised patients,
including patients infected with HIV. Cryptococcus gattii rarely infects
persons with HIV infection and other immunosuppressed patients and
usually involves immunocompetent individuals, respond slowly to
treatment and are at risk for developing intracerebral mass lesions like
cryptococcoma. C. neoformans is distributed worldwide.
Conclusion Identification of species complex can help not only in
recognizing the epidemiology of the strains prevalent in an area but
also in being vigilant about the changing antifungal patterns of those
isolates.
however Crypto-LA was positive. Microscopy of CSF revealed lymphocytic predominance and encapsulated budding yeasts in India ink.
On blood agar, Sabouraud dextrose agar and sunflower seed agar,
there were mucoid yeast-like colonies. Maximum isolates (13) were
Cryptococcus neoformans var. neoformans followed by Cryptococcus neoformans var. grubii (5) and two of the isolates were Cryptococcus gattii.
In three cases we could not isolate Cryptococcus species as the culture
turned out to be sterile and diagnosis was made on histopathology.
Most of the isolates (14) belonged to AFLP type 1 followed by one
isolates each of AFLP 2 and AFLP 8. As far as serotypes are concerned, maximum isolates (15) were serotype A followed by one isolate of serotype D and one of serotype BD. All the twenty isolates of
Cryptococcus species were found to be sensitive to amphotericin B and
fluconazole. Eighteen patients were adults and 2 were children (one
5 mol l1 and other 14 F). Among 18 patients, 12 were male and 6
were females. Maximum isolation has been from CSF samples 16, followed by skin 2, 1 bronchoalveolar lavage and one from gelatinous
cyst in abdomen after a blocked VP shunt.
Discussion Although the genus Cryptococcus contains more than fifty
species, only C neoformans and Cryptococcus gattii are considered principal pathogens in humans. Previously, C neoformans was defined as
102
P151
Cryptococcosis and HTLV 1: underdiagnosed cases
F. Concha Velasco and A. Bustamante Rufino
IMTAvH, Lima, Peru
Background Cryptococcosis is an opportunistic fungal infection
often described in HIV patients, who has a higher mortality rate than
in non-HIV patients. HTLV is a retrovirus, which in South America
affects mainly population from Brazil, Colombia and Peru. HTLV
infection is mostly associated with adult T cell leukemia/lymphoma
(ATL) and tropical spastic paraparesis. It has been reported 1232%
cases of cryptococcosis in co-Infected with HTLV-1 in Asia and Central America, while there is none report from Peru.
Aim Report clinical and epidemiological characteristics of cryptococcosis in peruvian patients co-infected with HTLV-1.
Methods Clinical records of patients with a definite diagnosis of
cryptococcosis (positive culture for Cryptococcus neoformans) and
HTLV-1 infection were reviewed.
Results All co-infected patients have lived in the highlands or in the
Amazonian regions. The clinical and epidemiological characteristics
of four patients are described in Table 1. HTLV-1 diagnosis was performed during hospitalization in two patients and in 1521 days
before in other two. All received amphotericin B deoxycholate
Poster Abstracts
(1 mg kg1 day1) plus fluconazole 800 mg day1 during the induction phase until negativization of cerebrospinal fluid (CSF).
Discussion and conclusion The 4 patients come from epidemic
areas in HTLV I, also they had present other additional diseases. Both
diagnoses, crytococcosis and HTLV 1 infection, were lately performed. Cause of death in two patients was not associated with the
fungal disease.
Search for HTLV infection should be conducted among HIV
patients diagnosed with cryptococcosis and resident in endemic areas
of this retrovirus. Participation of this entity as a risk factor for cryptococcosis should also be evaluated.
P152
Cryptococcus gattii infections in the Netherlands a
retrospective molecular diagnostic study using formalin
fixed paraffin embedded pulmonary coin lesions
F. Hagen, W. Vreuls, M. Wouters, M. J. Hendriks and J. F. Meis
Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands
Introduction Infections with members of the basidiomycetous yeast
genus Cryptococcus are in majority caused by C. neoformans and C.
gattii, while the sibling species C. adeliensis, C. albidus and C. laurentii
have rarely been reported as the culprit of disease. Cryptococcus gattii
has a limited geographic distribution pattern and was until recently
regarded as a pathogen related to tropical and subtropical climate
zones. However, the distribution pattern has largely been changed
due the occurrence of C. gattii outbreaks in temperate climate zones
in Europe, North America and Western Australia. Cryptococcus gattii
infections are exceptionally rare in the Netherlands as observed during a 30-year retrospective nationwide epidemiological survey. Surprisingly, this tropical pathogen was recently isolated from the
environment in the Netherlands, hence suggesting that C. gattii can
be autochthonous acquired.
Aim Retrospective investigation of the presence of C. gattii in lung
samples of coin lesions pathology archives among Dutch residents.
Material and methods Via the nationwide pathology database
(PALGA) a search was initiated for formalin-fixed paraffin embedded
(FFPE) tissue samples of coin lesions that were previously scored for
the presence of cryptococcal cells. Archived FFPE-tissue samples were
obtained from pathology departments in the Netherlands. Genomic
DNA was extracted from two 10 lm FFPE-tissue slices, showing morphological presence of yeasts, using the blackPREP FFPE DNA extraction kit (Analytic Jena, Germany). To detect DNA from Cryptococcus
species, a published real-time PCR assay was applied, while a novel
real-time PCR assay was generated and applied to detect C. gattii DNA.
Results The PALGA-database revealed that during the period 1981
2011 a total of 88 patients were presumely diagnosed with a pulmonary cryptococcoma and that 97 samples from these patients were
available. Finally, 18 pathology units participated in the study by providing 62 FFPE-tissue samples from 33 patients. From the 62 FFPEsamples, 34 were found to be positive for Cryptococcus species. DNA
corresponding to 22 patients (66%). From these positive samples, three
(9.1%) were positive for C. gattii by real-time PCR, corresponding to
three patients. Two patient were asymptomatic after removal of the
coin lesion, from one patient no follow up was available.
Conclusion Although molecular diagnostic investigation of formalin-fixed paraffin embedded samples remains a challenge when it
comes to the detection of fungal pathogens we were able confirm a
cryptococcal infection in 66% (22 out of 33) of the patients with a
cryptococcoma resembling coin lesion. From these 22 patients,
nearly ten percent had a C. gattii cryptococcoma, suggesting that this
tropical pathogenic fungus is causing more infections than was
recently observed during a retrospective study that made use cultured cryptococcal isolates from the same period. Unfortunately, it
remains a question where these patients acquired the infection.
P153
First isolation of Cryptococcus neoformans genotype VNI
alpha mating-type from hollow trunk of Hymenaea
courbaril
M. S. C. Melhem, D. M. Castro e Silva, D. C. S. Santos,
M. A. Martins, M. W. Szeszs, L. Oliveira and M. L. Moura
Adolfo Lutz Institute, Sao Paulo, Brazil
Background Cryptococcal fungal infection is transmitted by the
inhalation of contaminated air. Cryptococcus members can be dispersed in several sources, including fruits, vegetables, soil, insects,
honey bees, and decaying wood. Information on Cryptococcus species
inhabiting plants may be clinically relevant due to the epidemiological role of these habitats as possible sources of human infection with
C. neoformans and C. gattii.Recent data have shown the presence of
C. neoformans var. neoformans in the hollows of different tree species,
suggesting that trees could be a natural habitat.
Aim We describe a new vegetal specimen harboring C.neoformans
genotype VNI alpha mating-type.
Methods During a 12-month period we collected samples from nine
trees in one public park in S~
ao Paulo city. All trees were located in
the biggest city Sao Paulo of Brazil. The park offers visitors direct
contact with nature, fauna and flora. Hollow specimens of the following species were sampled quarterly: Casuarina equisetifolia, Alchornea
sidifolia, Tibouchina granulosa, Eriobotryajaponica (yellow plum), Rapaneaumbellata, Machoeniumnictitans, Ocoteapuberula (gooey cinnamon),
Ficusmicrocarpa, andHymenaeacourbaril. The inner part of the hollow
trunk was scraped (~15 g) with a sterilized spatula and transported
in sterile plastic bags to the Reference Mycology Center, Adolfo Lutz
Institute, to be processed. Approximately 10 g of each sample was
suspended in 40 ml of sterile saline. After vortexing (2 min), the suspension sat for 10 min. The supernatant was aspirated (8 ml) and
mixed with 2 ml of penicillin solution containing 1.2 million IU
(4.5 mg ml1) streptomycin (10 mg ml1) solution. After 15 min,
10 ll of the resultant mixture was transferred to 10 Petri dishes containing bird seed agar (BSA). The plates were incubated at 30 C
and observed daily for up to 7 days. All brown, creamy and mucoid
colonies were subcultured on BSA and stored for future phenotypic
identification. The presence of capsule, urease production, positive
nitrate reduction test, absent sugar fermentation and typical profile
assimilation tests confirmed Cryptococcus neoformans identification.
Species and molecular typing was determined using URA5-RFLP
analysis after amplification of the URA5 gene from high-molecularweight DNA. The obtained URA5-RFLP patterns were visually
assigned by comparison with the patterns of the following reference
strains: WM 148 (serotype A, VNI), WM 626 (serotype A, VNII),
WM 628 (serotype AD, VNIII), and WM 629 (serotype D, VNIV).
Results The typical Brazilian flora Hymenaea courbaril yielded
cryptococcal colonies during summer, autumn and winter seasons
with a high (102 CFU g1) fungal burden. Using PCR fingerprinting
analysis with the minisatellite-specific core sequence of the wild-type
phage M13 we could observed 14 distinct molecular patterns
among 60 colonies obtained during sampling. All colonies have
been identified as alpha mating-type. Additionally, we observed
through the European reference antifungal susceptibility testing
AFST-EUCAST, different minimum inhibition concentration values
of fluconazole (MIC range 4 to >64 mg l1) along with different
molecular profiles.
Discussion and conclusions These observations extend the geographic distribution and substantiated new urban environmental
niche of C. neoformans, the principal Cryptococcosis agent. Furthermore, our study reinforces the natural genetic complexity of the C.
neoformans VNI molecular type, the most implicated in human infections in our region. To our knowledge, this is the first study describing the occurrence of C. neoformans var. grubii VNI in the wood of
hollow trunks of Hymenaea courbaril. The importance of studies
reveng an increase in isolation from native trees around the world
could contribute to a better knowledge of the lifecycle of C. neoformans var. grubii in association with plant material. Further
103
Poster Abstracts
P154
Role of cerebrospinal fluid interleukin-6 as a prognostic
biomarker of Cryptococcal meningoencephalitis
M. Chayakulkeeree, J. Kobkijcharoen, D. Waywa and
R. Sirijatuphat
Faculty of Medicine Siriraj Hospital, Thailand
Background Interleukin 6 (IL-6) is a pro-inflammatory and antiinflammatory cytokine secreted by T cells and macrophages to stimulate the immune response. IL-6 assay has been commercially available and used as a biomarker in several inflammatory diseases.
Previous studies showed that IL-6 secretion from peripheral blood
mononuclear cells was stimulated by cryptococcal components and it
may play roles in cryptococcal pathogenesis. However, the usefulness
and clinical relevance of IL-6 in patients with cryptococcal meningitis
were limited.
Aim To investigate the role of interleukin-6 in predicting outcomes
of HIV-infected patients with cryptococcal maningoencephalitis.
Method This is a sub-group analysis of another study. We analyze
11 HIV-infected patients with cryptococcal meningitis who had been
tested for cerebrospinal fluid (CSF) IL-6. Clinical response, mycological response and mortality were analyzed in association with CSF IL6 level.
Result Of 11 patients, 8 (73%) were male with mean age of 37 (range
2359) years. Mean CD4 was 44 (range 3160) cells mm3. Fungemia
was found in 9 patients (82%). Clinical response was 64% and microbiological response was 73%. Overall mortality was 18%. All 5 patients
(100%) with CSF IL-6 less than 100 pg ml1 had a favorable clinical
and mycological response, whereas those with CSF IL-6 more than
100 pg ml1 exhibited a clinical response of 33% (2/6) and a mycological response of 50% (3/6). Early mycological response (negative
culture within 14 days) was demonstrated in 60% and 17% of patients
who had CSF IL-6 less than 100 pg ml1 and more than 100 pg ml1,
respectively. All patients with CSF IL-6 less than 100 pg ml1 were
survived more than 6 months but those with CSF IL-6 more than
100 pg ml1 had a 6-month mortality of 2/6 (33%). In fact, 2 patients
who did not survive had CSF IL-6 higher than 500 pg ml1 (7661 and
736 pg ml1). According to the low number of patients, the differences
were not statistically significant.
Conclusion High level of CSF IL-6 (more than 100 pg ml1) showed
a trend toward a poor clinical response, poor mycological response
and mortality in HIV-infected patients with cryptococcal meningoencephalitis. CSF IL-6 may potentially be used as a prognostic biomarker to predict outcomes in patients with cryptococcal meningoencephalitis. Further study with a larger number of patients is
warranted.
P155
Study of multiple single-colony isolates of the
Cryptococcus neoformans/Cryptococcus gattii species
complex in Colombia
P. Escandon and E. Castaneda
Instituto Nacional de Salud, Bogota, Colombia
Background The assumption that a determined infection in a host
is caused by a single strain is considered to be valid for some diseases, however, some of them have been possibly caused by mixed
infections with multiple strains of a pathogen. For Cryptococcosis,
some reports have been made on experimental infection of mice with
multiple strains of the fungus resulting in mixed infections in the
104
P156
Comparison between latex agglutination test and lateral
flow assay for the detection of Cryptococcus antigen in
serum
R. Wahyuningsih,1 H. Herqutanto,1 D. Imran,2 R. Estiasari,2
T. Natriana,3 N. Komari,2 S. Sucipto2 and E. Yunihastuti2
1
University of Indonesia, Jakarta, Indonesia; 2University of
Indonesia/Ciptomangunkusumo Hospital, Jakarta, Indonesia and
3
Drug Dependence Hospital, Jakarta, Indonesia
Background The classic method for the diagnosis of cryptococcal
meningitis the india ink test and culturing from spinal fluid. But,
lumbar puncture, to provide spinal fluid is an invasive procedure that
often in convenient for the patient. Furthermore, the sensitivity and
specificity of both methods are not as high as we expected. Antigen
detection can be conducted for spinal fluid and serum. By doing antigen detection in serum we have a presumptive diagnosis. Two methods for antigen detection are recommended by WHO i.e. latex
agglutination test (LA) and lateral flow assay (LFA). The first was
used for >10 years, while the second was just launched in Indonesia
Poster Abstracts
last year. Are both methods equally suitable for the detection of cryptococcal antigen in serum?
Aim of the study Comparing lateral flow assay (LFA) and latex
agglutination test (LAT) for the detection of Cryptococcus antigen in
serum.
Method Serum tested originated of HIV infected and non-HIV
patients. Clinical samples were sent to our laboratory for the diagnosis of cryptococcosis using LFA test (IMMY, Lab USA) and LA test
(Pastorex - Crypto plus BioRad France) to detect Cryptococcus antigen. Both methods were done according to procedures described by
the manufacturer. Fisher exact test was done to test any association
between the two methods, with confidence interval (CI) <5%. In
addition, the clinical condition was also noted.
Result We investigated 80 sera, of which 79 were from HIV-infected
and one from non-HIV infected patients for Cryptococcus antigen.
Using both methods (LFA and LA methods), 20 out of 80 sera tested
were positive for Cryptococcus antigen. Two samples were positive of
Cryptococcus antigen by LFA test but negative by LA test, but two
samples were negative by LFA test but positive by LA test. The result
of Fisher exact test, revealed that both methods have a very strong
association (P < 0.0001). Thus, there was a an equal capabilityLFA
and LA methods to detect cryptococcus antigen in sera. Patients with
positive results were also diagnosed for other opportunistic infection
such as toxoplasma encephalitis, tuberculous meningitis and other
brain infection. Most of the patients were not diagnosed as cryptococcal meningitis, only three patients whos the sample tested were suspected as cryptococcal meningitis.
Conclusion/discussion Both methods are equally well in detecting
Cryptococcus antigen in serum. A positive result in serum is a presumptive diagnosis for cryptococcosis which guide us to search for
cryptococcal infection in other organs particularly the central nervous system.
P157
Synchronous fungal infections in an immunocompromised
host sampling of multiple suspect sites is a requisite for
discrimination and management
Y. A. Chai, K. Lim and J. E. Seet
National University Health System, Singapore
Background In immunocompromised patients at risk of opportunistic infections, obtaining clinical specimens for microbiology and histopathology can be challenging for several reasons. The general ill
status of many such patients, the presence of coagulopathies or the
poor accessibility of the site may deem an invasive sampling procedure risky. Weighing these considerations, the clinician is oftenatimes faced with the difficult decision on choosing the best accessible
and representative lesion to biopsy, particularly in the setting of suspected disseminated fungal disease.
Aim In most cases, sampling a representative lesion serves well in
clinching the diagnosis. However there are circumstances whereby
such a pragmatic approach may fail.
Methods We describe here, a case of patient status-post liver transplant who presented with synchronous fungal infections.
Results This is a 62 years old diabetic male with Childs B liver cirrhosis from Hepatitis C and newly diagnosed 1.3 cm hepatocellular
carcinoma. He underwent a living donor orthotopic liver transplant in
April 2013. In October, he presented with a 2.5 cm nodular and fleshy lesion over the right forearm after sustaining an abrasion against
a wooden door frame. He also complained of weight loss, lethargy and
low grade fever. Routine chest X-ray had demonstrated a prominent
left hilum. A CT scan that followed revealed mediastinal and left hilar
lymphadenopathy as well as left pulmonary nodule. There were no
chest symptoms of note. His immunosuppressive regimen then consisted of tacrolimus 1 mg daily and myfortic 360 mg twice daily
A skin biopsy of the right forearm nodule was performed. Histopathology showed scattered broad filamentous fungi. Requesting early
discharge from hospital, the patient was started on empiric
posaconazole 200 mg four times daily for disseminated invasive fungal infection. The fungal culture subsequently returned as Lichtheimia
corymbifera. The patient was re-admitted and underwent a wide margin excision of the forearm fungal lesion which confirmed the earlier
finding. The excised margins were clear of fungi. Decision was made
to also perform a transbronchial unltrasound-guided lung biopsy during this hospitalization. Interestingly cytology of the biopsy revealed
intracellular yeasts and the culture grew Cryptococcus neoformans.
The serum Cryptococcus antigen test was >1 : 1024. There was no
growth of moulds.
The patient was started on an initial therapy of conventional
amphotericin B and flucytosine targeted for at least 8 weeks. However, this treatment regimen had to be switched after 6 weeks due to
progressive renal impairment. Theorizing that the forearm Lichtheimia
lesion had been adequately dealt with the above treatment modalities, the patient was switched to oral fluconazole. Four months after,
he remains to be on fluconazole maintenance therapy and is doing
well. His latest serum Cryptococcus antigen level was 1:256 and
there was no recurrence of the Lichtheimia infection.
Discussions/conclusions Sampling an accessible, representative
lesion remains a practical approach for the diagnosis of IFI. However,
clinicians need to be mindful that immunocompromised patients can
present with multiple co-infections in the same setting and should
weigh the benefits versus risks of adopting an aggressive sampling of
multiple suspect sites for discrimination and effective management of
such synchronous fungal infections.
P158
Cryptococcal antigen in the serum of anti-retro viral nave
HIV-infected patients
R. Wahyuningsih,1 D. Imran,2 H. Herqutanto1 and C. K. Muda2
1
University of Indonesia, Jakarta, Indonesia and 2University of
Indonesia/Ciptomangunkusumo Hospital, Jakarta, Indonesia
Background Host immunity determines the faith of Cryptococcus in
human body. It can be dormant in immune-competent host, but able
causes infection in immune-compromised host. The most prevalent
clinical manifestation is meningitis, but the existence of Cryptococcus
in the human body can cause an inconspicuous clinical signs, and at
the same time releases antigen into body fluid that can be detected.
Aim of the study To detect Cryptococcus antigen in serum of ARVnave HIV infected patient and their clinical profile and outcome.
Method Serum tested was from HIV infected who anti retro viral
(ARV) nave. The method used was lateral flow assay (LFA) method.
The clinical profile was also noted.
Result A total of 78sera from ARV nave HIV infected subjects were
examined. Cryptococcus antigen was found in five out of 78 sera
tested. They consisted of three male and two female and their age
range from 18, 39 years, and only one patient is 43 years. Four subjects have CD4 count less than 50 cells ll1 and they belong to AIDS
105
Poster Abstracts
group, and one subject has CD4 > 200 cells ll1 who belong to none
AIDS group. All positive patients were suggested underwent lumbar
puncture (LP), but two patients refused and they died within a month
after antigen was detected. Another three patients were underwent
LP, with india ink, culture and antigen detection were negative and
fluconazole was given, and they are survived. Two patients, who
refused doing LP, have mental alteration i.e. cognitive disturbances.
Conclusion/discussion Cryptococcemia isassociated with mortality,
which normally detected by culture. Culture even though definite
diagnosis is time consuming, while antigen detection is easy to be
conducted and fast and cheap. We suggest to do antigen detection
before ARV treatment, since it can prevent mortality which causes
by the disease itself or IRISs associated mortality.
P159
Phenotypic switching of Cryptococcus neoformans var.
grubii VNI in a case of retroperitoneal diffuse large B-cell
lymphoma following modified Rituximab-CHOP
chemotherapy
H. K. Tseng,1 C. P. Liu,1 Y. C. Chen2 and M. W. Cheng1
1
Mackay Memorial Hospital, Taipei, Taiwan and 2National
Taiwan University Hospital, Taipei, Taiwan
An 88 year-old woman with past history of breast cancer post mastectomy had retroperitoneal diffuse large B-cell lymphoma, germinal
center type treated by modified rituximab-CHOP chemotherapy (rituximab, cyclophosphamide, vincristine, and prednisolone). She had
neutropenic fever and final blood culture and urine culture yield two
morphological phenotypes (smooth and mucoid) of Cryptococcus neoformans which VNI was confirmed. Central nervous system infection
was favored but her family member declined lumbar puncture for
CSF study due to personal issue. After completion of induction therapy with amphotericin-B and flucytosine two weeks, we switched to
maintenance oral fluconazole 400 mg daily. Due to the controlled of
her infection and improvement of her clinical condition, she was
discharged.
Figure 2. Colonies of smooth and mucoid capsule.
P160
Relationship of susceptibility testing of Cryptococcus
neoformans to survival and mycological clearance in HIV
associated cryptococcal meningitis
J. N. Day, V. A. Duong, T. T. H. Chau, T. N. Hoang and
M. Wolbers
Oxford University Clinical Research Unit, Ho Chi Minh City,
Vietnam
106
Background Antifungal susceptibility testing of Cryptococcus neoformans is cumbersome and its value is uncertain in patients presenting with cryptococcal meningitis. Susceptibility measured at the
point of diagnosis has not reliably been linked to clinical outcome.
Most studies have been small and have not accounted for differences in disease severity at abseline. The Sensititre Yeastone is a
relatively simple and easy to interpret broth microdilution susceptibility assay.
Aims To determine the effect of antifungal susceptibility as measured
by the YeastOne Sensititre on (i) clinical outcome at 70 days, and (ii)
clearance of yeast from cerebrospinal fluid.
Methods 299 HIV infected patients with a first episode of cryptococcal meningitis were enrolled into a 3 arm trial of combination antifungal therapy in Vietnam. Patients received induction therapy with
either amphotericin 1 mg kg1 day1 monotherapy for 4 weeks,
amphotericin combined with flucytosine 100 mg kg1 day1 for
Poster Abstracts
P161
Understanding synergy between iron chelators and
antifungals using gene co-expression networks
A. Carter,1 Y. W. Lai,1 C. N. I. Pang,2 L. T. Campbell,1
M. Truong,3 S. Chen4 and M. Wilkins2
1
University of Sydney, Darlington, NSW, Australia; 2University of
New South Wales, Kensington, NSW, Australia; 3University of
Technology, Sydney, Broadway, NSW, Australia and 4Westmead
Hospital, Westmead, NSW, Austria
Background Cryptococcosis remains an extremely difficult infection
to resolve. Current recommendations for induction therapy are
amphotericin B (AMB) combined with 5-flucytosine (5FC), but AMB
is highly toxic, requiring vigilant monitoring, and 5FC is expensive,
putting it out of the reach of the low income countries that need it
most. Finding and developing new antifungals is very difficult, therefore a promising avenue of antifungal research is to enhance existing
therapies with synergistic agents. Iron chelators administered with
certain antifungals have been found to improve the clearance of
some fungal infections. However, mechanistic data on exactly how
these work, and why they sometimes do not, are lacking. In addition,
iron depletion can be damaging to the host. In this study, we explore
various chelator-drug combinations and use gene co-expression networks to identify pathways and processes that are differentially regulated during a synergistic response. The goal is to identify targets for
new synergistic therapies without needing to administer chelators.
Aims 1. To find antifungals + iron chelator combinations that result
in synergy when used to treat Cryptococcus;
2. To use RNA-Seq and co-expression networks to analyse the synergistic response at the level of transcription and identify important
mediators of synergy.
Methods Checkerboard assays were used to determine synergy for
combinations of the following antifungals and iron chelators: Antifungals: AMB, fluconazole (FLC), itraconazole (ITZ), voriconazole (VRZ),
P162
Clinical features of pulmonary cryptococcosis in non-HIV
patients in Japan
S. Kohno,1 H. Kakeya,2 K. Izumikawa,1 T. Miyazaki,1
Y. Yamamoto,3 K. Yanaghihara,1 K. Mitsutake,4 Y. Miyazaki,5
S. Maesaki,4 A. Yasuoka,6 T. Tashiro,1 M. Mine,1 M. Uetani1 and
K. Ashizawa1
1
Nagasaki University Graduate School of Biomedical Sciences,
Nagasaki, Japan; 2Graduate School of Medicine, Osaka City
University, Osaka, Japan; 3Graduate School of Medicine and
Pharmaceutical Sciences for Research, University, Toyama, Japan;
4
Saitama International Medical Center, Saitama Medical
University, Saitama, Japan; 5National Institute of Infectious
Diseases, Tokyo, Japan and 6Omura Municipal Hospital,
Nagasaki, Japan
Objective To clarify the clinical features of pulmonary cryptococcosis in Japanese non-HIV population.
Methods Retrospective investigation of 151 pulmonary cryptococcosis cases between 1977 and 2012 was executed. The underlying disease (UDs), aggravating factors, radiological characteristics, and
treatment were examined.
107
Poster Abstracts
P163
Antifungal Activities of T-2307 an arylamidine compound
against Cryptococcus gattii: an emerging fungal pathogen
H. Nishikawa,1 K. Hirano,2 T. Takazono,2 S. Nakamura,2
Y. Imamura,2 K. Izumikawa,2 K. Yanagihara,2 T. Tashiro2 and
S. Kohno2
1
Toyama Chemical Co., Ltd, Toyoma, Japan and 2Nagasaki Univ.
Graduate Sch. of Biomedical Sci., Nagasaki, Japan
Background Cryptococcus gattii causes life-threatening disease in
otherwise healthy hosts and to a lesser extent in immunocompromised hosts. T-2307, an arylamidine compound, exhibits broad-spectrum and potent antifungal activity and is in clinical trials. In this
study, we evaluated in vitro and in vivo antifungal activities of
T-2307 against C. gattii in comparison with other antifungal agents.
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MIC50 (lg/ml)
MIC90 (lg/ml)
Range (lg/ml)
T-2307
5FC
FLC
ITC
VRC
MFG
AMB
0.0313
8
4
0.5
0.125
>128
1
0.0625
32
16
0.5
0.25
>128
1
0.0078 0.0625
0.5 >64
1 16
0.125 1
0.0625 0.25
>128
1
The MIC range of T-2307 against 16 isolates was lower than that
of any other antifungal agents tested. T-2307 and AMB showed concentration-dependent fungicidal activity with a reduction of >3
Log10 CFU/ml at 4 MIC or higher within 72 hours though FLC
showed no fungicidal activity even at 64 MIC. When the administration was performed once daily for 21 days from 2 hours postinfection
in the murine model, T-2307 at 0.5 mg/kg/day significantly reduced
the viable counts in both lung and brain, which was comparable to
FLC at 40 mg/kg/day and AMB at 4 mg/kg/day. In an advanced
infection model in which the administration was performed from
14 days postinfection once daily for 7 days, T-2307 significantly
reduced the viable counts in both lung and brain at 1 mg/kg/day.
AMB did not reduce the viable counts in lung and brain at 2 mg/kg/
day. FLC did not reduce the viable counts in lung at 320 mg/kg/day,
however significantly reduced the viable counts in brain at 160 mg/
kg/day.
Conclusion T-2307 showed excellent in vitro fungicidal and in vivo
antifungal activity against C. gattii in comparison with other antifungal agents. It is a promising candidate for the treatment of cryptococcosis caused by C. gattii.