mycoses

Diagnosis,Therapy and Prophylaxis of Fungal Diseases

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
2010–2013. 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 2.6–60), male/female
ratio 1/1, adults were 62.5%. Main underlying conditions were:
acute myeloid leukemia – 25%, acute lymphoblastic leukemia – 25%,
non-Hodgkin’s 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 СД4+ 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 72–360 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 Nigeria’s 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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Second prospective survey on cryptococcosis in Italy after
10 years
M. Cogliati,1 A. M. Tortorano1 and FiMUA Cryptococcosis
Network
1
Universita degli Studi di Milano, Milano, Italy
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

doi:10.1111/myc.12196

Poster Abstracts

December 2013. Clinical information were collected using the same
form adopted ten 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. neoformans var. grubii (VNI), 6 by C. neoformans var. neoformans (VNIV),
and 16 by AD- hybrid strains (VNIII). No C. gattii was isolated.
Discussion The results of this cryptococcosis survey show that the
number of recorded cases is significantly reduced compared to the
previous survey (156 cases) although the period and geographical
coverage were different. The percentage of HIV negative patients is
changed rising from 2.8% to 41%. This result confirms that, after
the introduction of HAART, HIV infected patients are less susceptible
to Cryptococcus infection. The ratio male/female is changed from
5.5 : 1 to 2.9 : 1 as a consequence of the higher percentage of nonHIV infected patients. The molecular analysis of Cryptococcus isolates
showed that VNI was the molecular type predominantly isolated
(50%) followed by VNIII (36%) and VNIV (14%), in contrast with
the percentages observed in the previous survey (35%, 35%, 30%
respectively). In conclusion our results highlight the need of a constant monitoring of cryptococcosis in Italy since the epidemiology of
this pathogen is changing.
The FIMUA cryptococcosis network
Ancona: E. Manso; Bergamo: M. Passera; Genova: M. Mikulska; Milano: A. Grancini, R. Grande, C. Ossi, M. Cainarco; Bolzano: P. Innocenti; Monza: S. Bramati; Novara: S. Andreoni; Pavia: C. Cavanna;
Pisa: A. Leonildi; Roma: A. Vella; Torino: A. Barbui; Udine: A. Sartor;
Varese: A. Colombo; Verona: G. Lo Cascio.

cases was of 1–3 weeks. At admission altered consciousness and
intracranial hypertension were present in 23 (52.3%) cases whereas
CSF assessment showed hypocellularity in 17/31 (54.9%). CSF cultures yielded Cryptococcus neoformans in 29/29 (100%) cases. The
CD4 cell baseline count ≤100 cells mm3 was present in 13/15
(86.6%) cases. Of 29 (64.4%) patients who started antifungal therapy, 23 (79.3%) died over the second week on therapy. Most patients
were antiretroviral naive and in the remaining 16 (35.5%), the cryptococcosis diagnostic was performed at necropsy. Disseminated infection involving mainly CNS 38 (84.4%), lungs 31 (68.9%), lymphnodes 25 (55.6%), kidney 22 (48.9%), liver and spleen 21 (46.7%),
adrenal gland 10 (22.2%), pancreas 10 (22.2%), prostate (20%) and
bone marrow (20%) was evidenced in 31 (68.9%) cases. Similar proportion of disseminated cryptococcosis was found among necropsied
patients who had received or not antifungal therapy. The remaining
15 cryptococcosis cases were localized in the CNS alone. Besides,
toxoplasmosis 13 (28.9%), candidiasis 7 (15.6%), cytomegalovirus 5
(11.1%) and 5 cases (11.1%) with mycobacterial infections were
concomitantly observed. Demographic, epidemiologic and most clinical features observed in these patients were similar to those noticed
in survivors. However, the presence of altered consciousness, intracranial hypertension and severe malnutrition at admission were statistically associated with a poor outcome.
Discussion/conclusion This cryptococcosis case series in AIDS
patients confirmed or diagnosed at necropsy, evidenced the epidemiological and clinical profile already described by others in poorresource settings. The severity of fungal infection showed by multiple
organ involvement in most cases and the presence of clinical poor
prognostic factors are directly associated to late AIDS diagnosis
together with the advanced immunodeficiency observed at admission.
Commonly, this infection is the first AIDS defining condition and
most patients are not receiving ART. Nowadays and despite specific
diagnosis and antifungal therapy, cryptococcosis mortality rate of
55% is still observed in Brazil and other Latin American countries.
The necropsy performance in teaching hospitals is a valuable tool to
know the outcome of AIDS patients and to improve the accuracy of
clinical diagnosis.

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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Poster Abstracts

discharged. During the follow up, she remained asymptomatic, her
CD4 T count increased to 250 cells mm3 and the viral load was
persistently undetectable. At admission she presented slow thinking
and mild ataxia. The skull CT scan evidenced moderate non communicant hydrocephalus and signs of intracranial hypertension. The
MRI imaging confirmed these features. The CSF assessment: cells:
25 mm3, neutrophils 7%, lymphocytes 90%, monocytes 3%, glucose
17 mg dl1 and proteins 90 mg dl1. CSF smear was negative to
bacteria, mycobacteria and fungal structures. India ink and Cryptolatex tests were negative. CSF culture was negative to bacteria, mycobacteria and fungus. VDRL and FTA-abs were non-reactive,
Toxoplasma gondii IgG antibodies 1 : 16, ELISA test to cysticercus
antibodies was non-reactive. Patient performed ventriculostomy and
was discharged. One week later, she was readmitted with worsening
of headache, vomits, photo and phonofobia and right side paresthesia. A new CT scan showed the same findings and mild hemorrhagic
foci in frontal lobe on the craniotomy site. A external shunt was performed and CSF obtained showed: Cells 21 mm3, glucose
17.9 mg dl1 and proteins 102.5 mg dl1. The Cryptolatex and
India ink tests were negative. CSF culture yielded Cryptococcus neoformans on day five. Amphotericin B + fluconazole were prescribed and
a ventriculoperitoneal shunt was performed due to the obstruction of
external derivation. Her clinical picture progressively improved and
after 27 days, she was discharged to continue the antifungal therapy
at day hospital which was kept for 10 weeks. During the follow up,
patient remains asymptomatic and the CT brain scan performed five
months later showed a significant reduction of hydrocephalus and
ventricular asymmetry.
Discussion Hydrocephalus is a complication of cryptococcal meningitis as a consequence of intracranial increased pressure which
occurs due to CSF outflow obstruction caused by the sum of several
factors such as: aggregation of capsular polysaccharide, inflammation
immune-mediated and osmotic effect of mannitol derived of fungus.
Most cases of hydrocephalus are associated to other signs and symptoms of meningitis or meningoencephalitis, different from the present
report. This patient presented a chronic headache which led to evidence moderate ventricular dilatation and CSF findings similar to
those seen in fungal and tuberculosis infection. Probably, she presented a very low fungal burden which explains the insidious clinical
picture, and diagnosis difficulty and could be the consequence of her
partial immune reconstitution after three years on regular antiretroviral therapy. To our knowledge it is an uncommon first clinical presentation of cryptococcal meningitis in AIDS patients.

hospitalized in her town, received blood transfusion and then was
transferred because she had worsened and the ELISA anti-HIV test
was positive. At admission, her clinical status was critic, presenting
severe malnutrition, dyspnea and acrocyanosis. Axillary temperature
was 37.5 °C and the body weight of 30 kg. White lesions in the oral
cavity suggestive of thrush were seen. Cardiac and pulmonary auscultations were normal, and no evidence of lymph-nodes, hepatomegaly
and/or splenomegaly was found. No meningeal signs were present.
Ophthalmological exam showed multiple white oval patches around
optical nerve and mild retinal hemorrhagic points. The clinical diagnosis was retinochoroiditis by Pneumocystis jirovecii. Complete blood
count showed pancytopenia. At Chest X-ray, bilateral interstitial infiltrate and pneumonic consolidation in both lung basal regions were
evidenced, and confirmed at chest CT. Arterial blood gases measurement evidenced severe hypoxemia, the CD4 cell count was
30 cells mm3, and no viral load was available at the time. At the
emergency room intravenous trimetoprim-sulfamethoxazole plus
hydrocortisone were prescribed to treat a presumptive P. jirovecii
pneumonia. A lumbar puncture was also performed and the CSF
analysis showed: 5 cells mm3, of which 98% were lymphocytes and
2% monocytes, glucose 27.7 mg dl1 and protein 43.0 mg dl1;
India ink and Crypto-latex tests were positive and CSF culture yielded
Cryptococcus neoformans. Amphotericin B deoxycholate plus Fluconazole were then prescribed. During the following days, her clinical picture worsened, presenting unresponsive cardiorespiratory arrest at
day 8. Necropsy was performed, and showed severe and disseminated
cryptococcosis involving meninges and brain, eyes, bone marrow,
lungs, stomach, colon, liver, spleen, pancreas, hypophysis, adrenal
gland and kidney. At histopathology exam of both eyes, a mild inflammatory infiltrate formed by lymphocytes and macrophages was evidenced through the choroids which presented multiple yeasts stained
with Mayer’ Mucicarmin. The retina, optical nerve and sclera were
not involved.
Discussion This case illustrates how difficult it is to establish the etiology of choroiditis in AIDS patients. Despite adequate diagnosis and
antifungal therapy, patient died with disseminated cryptococcosis evidenced at necropsy. Due to the presumptive clinical diagnosis of ocular involvement by P. jirovecii, it was asked to exam the eyes, which
presented fungal infection limited to choroids. This finding is in line
with previous reports showing that this structure is commonly
affected by Cryptococcus neoformans, different from CMV, Herpes simplex and Toxoplasma gondii infections which more frequently involve
retina and optical nerve. Of ocular cryptococcosis cases already
described, few, including the present, were confirmed by histopathological exam.

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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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 d’Alacant, Spain;
2
Noah’s Path, Elche, Spain; 3Clınica Veterinaria Alcabidechevet,
Alcabideche, Portugal; 4Clınica 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

adequate identification and environmental search to improve our
knowledge of the epidemiology of cryptococcosis.
Aim Review of the occurrence of cryptococcosis in ferrets in Spain
and Portugal and their possible role as sentinels of the presence of
the yeast in the environment.
Methods We describe three cases of cryptococcal infections in ferrets
from Spain and Portugal. One of them was identified as the first case
of cryptococcosis by C. gattii in Spain. All of them were exhaustively
studied with a complete clinical evaluation. The three cases were
detected on the basis of the anatomopathologic findings. Needle aspirates from different tissues (nasal mass, thoracic mass and lymph
node) were studied at the mycology laboratory. Cultures onto Sabouraud agar allowed the development of characteristic yeast colonies
for all three cases. Asymptomatic carriers detection and environmental search of the yeast in relation with these cases was performed.
Nasal swabs from the owners and other animals living in the same
household of two of the cases were tested for asymptomatic carriage
of Cryptococcus spp. Sampling of trees and soils in the vicinity of the
animals were also performed. Olive trees (Olea europaea), carob trees
(Ceratonia siliqua), pinus trees (Pinus halepensis) and false pepper trees
(Schinus molle) were sampled by the swabbing method. Samples were
processed for the search of Cryptococcus spp. Yeast colonies from clinical and environmental samples were phenotypically typed by carbohydrate compounds auxonograme (Auxonograme2-Biorad), capsule
production, urea hydrolysis, growth at 37 °C, melanin production
and growth on canavanine-glycine-bromothymol Blue medium
(CGB). Isolates identified as C. neoformans/C. gattii species complex
were subsequently molecularly characterized by Amplified Fragment
Length Polymorphism fingerprinting (AFLP). For the C. gattii isolates,
also Multi-locus sequence typing (MLST) was carried out.
Results The study of clinical isolates showed C. neoformans variety
grubii AFLP1/VNI from the nasal mass of the ferret in Balearic Islands;
C. gattii AFLP6/VGII from the thoracic mass of the Portuguese ferret
and C. gattii AFLP4/VGI from the lymph node of the ferret in Barcelona. In environmental samples five isolates of C. neoformans var. grubii genotype AFLP1/VNI were obtained in the vicinity of the ferret
from Majorca (Balearic islands) and 11 isolates of C. gattii AFLP4/VGI
from environmental samples related to the animal in Barcelona. In
Portugal all environmental samples and carriers detection were negative. Nasal swabs from two persons and two healthy ferrets related
with the case from Barcelona, were positive and the same species and
genotype of the infected ferret were obtained.
Conclusions The molecular characterization of the isolates and their
genetic profiles evidenced a very close relationship between clinical
strains and the environmental isolates of the same area, suggesting
the same origin and the acquisition of the disease from natural
sources. Adequate investigation of the genotype helps determining
the virulence of the yeast and the potential danger for the people
who live with the infected animal. Veterinarians should be aware of
this and carry out the proper tests to identify cryptococcal infections.

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 (5–60). 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
01°430 04″S 49°320 27″W 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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Poster Abstracts

colonies were isolated and identified phenotypically. From six positive
samples, three harbored C. gattii and C. neoformans, one only C. gattii
and one only C. neoformans. High positivity was observed in dust of
two houses of patients (>10 000 CFU g1) and the house of the
8 years old patient was negative, but a jambo tree in the cemetery, in
which the patient used to play, was positive. Eighty colonies were
identified as C. gattii and five as C. neoformans. The molecular analysis
(multilocus sequence typing – MLST) for subtyping is still ongoing.
The results reinforce evidence of indoor and outdoor exposure risks
for cryptococcal infection, and of the underdiagnosed cryptococcal
respiratory manifestations.

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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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 Doencßas Tropicais Natan Portella, Teresina/Piaui,
Brazil and 2Universidade Federal do Piauı, 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 Piauı, 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 Piauı and Maranh~
ao.

P012
Lethality of Cryptococcal meningitis in a Reference
Hospital in Teresine, Piauı, 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 Doencßas Tropicais Natan Portella, Teresina/Piaui,
Brazil and 2Universidade Federal do Piauı, 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 Piauı, Brazil.
Methods This study analysed data from 92 patients hospitalized for
cryptococcal meningitis in a referral centre for infectious diseases in
Teresina, Piauı, 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

Genotypes of Cryptococcus neoformans and Cryptococcus
gattii as agents of endemic cryptococcosis in Teresina,
Piauı (Northeastern Brazil)
L. Soares Martins,1 B. Wanke,2 M. Santos Lazera,2 L. Trilles,2
G. Goncßalves Barbosa,2 R. C. Lima Macedo,2 M. A. Salmito
Cavalcanti,3 K. Dantas Eulalio,3 H. Alves da Silva Machado,3
R. Melo Santos Serpa Brandao,1 J. A. Fonseca Castro,1
A. Socorro Silva,1 F. Ferraz Nascimento,1 V. Alves Gouveia4 and
S. Jamil Hadad Monte1
1
Universidade Federal do Piauı, Teresina-Piauı/Brasil, Brazil;
2
Fundacßa~o Oswaldo Cruz, Rio de Janeiro, Brazil; 3Instituto de
Doencßas Tropicais Natan Portella, Teresina, Brazil and
4
Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

Azole susceptibility of Cryptococcus and other melanized
yeast isolates from environment exposed to fungicides
J. P. Takahashi,1 L. M. Feliciano,2 D. M. Castro e Silva,2
S. D. P. Ramos,2 R. A. Oliveira,2 D. Attili-Angelis,3
N. R. Rodrigues,3 J. L. M. Sampaio4 and M. S. C. Melhem2
1
Graduate Program of Coordination for Diseases Control,
Secretariat of Health, Sao Paulo State, Brazil; 2Adolfo Lutz
Institute, Public Health Reference Center, Secretariat of Health,
Sa~o Paulo State, Brazil; 3Brazilian Collection of Microorganisms
from Environmental and Industry Cpqba/Unicamp, Paulinia, Sa~o
Paulo, Brazil and 4Grupo Fleury, Sa~o Paulo, Brazil

In Brazil, cryptococcosis caused by C. neoformans occurs in all regions
and it often causes systemic mycosis in AIDS patients and is the third
leading cause of opportunistic CNS infection in these patients. However, C.gattii acts predominantly as a primary pathogen, attacking
immunocompetent hosts, including children and young adults in
North and Northeast, and is therefore considered endemic to the
states of Amazonas (AM), Par
a (PA), Roraima (RR), Piauı (PI), Pernambuco (PE) and Bahia (BA). The southern and southeastern
regions show sporadic infections by C. gattii. In PI, previous studies
have shown that cryptococcosis caused by C. gattii is strongly associated with human immunodeficiency virus (HIV)-negative patients,
afflicting a significant portion of children and young adults. Here, we
report on the identification of the molecular pattern of the Cryptococcus species that caused meningitis in patients in Teresina, which is
the capital of the northeastern state of PI, Brazil, providing a quick
tool for diagnosis. In this study, the molecular types of 63 cryptococcal isolates recovered from the cerebrospinal fluid of meningitis
patients diagnosed between 2008 and 2010 in Teresina, Piauı, Brazil, were analysed. Out of the 63 patients, 37 (58.7%) were human
immunodeficiency virus (HIV)-positive and 26 (41.3%) were HIVnegative. URA5-restriction fragment length polymorphism analysis
identified 37/63 (58.7%) isolates as the C. neoformans VNI genotype,
predominantly in HIV-positive patients (32/37, 86.5%), and 24/63
(38.1%) as the C.gattii VGII genotype, mostly in HIV-negative
patients (21/26, 80.8%). The occurrence of C. gattii VGII in six
apparently healthy children and in seven adolescents/young adults
in this region reaffirms the endemic occurrence of C.gattii VGIIinduced primary cryptococcosis and early cryptococcal infection. In
our study, the occurrence of cryptococcal meningitis caused by C.
gattii was high among children and Young adults (not older than
20 years) (13/63, 19.9%). This result was similar to studies from
1995 to 2003 in this same region, as well as in AM and PA. In all
of these studies, it is worth noting that none of the children showed
signs of pulmonar compromise at the time of their diagnosis with
cryptococcal meningitis. Therefore, with pulmonary infections overlooked by physicians, the diagnosis of the mycosis usually occurs
when the infection has already spread to the CNS, which certainly
aggravates the prognosis. Lethality occurred in 18/37 (48.6%) of the
HIV-positive subjects and in 13/26 (50%) of the HIV-negative
patients. The growing number of recent cryptococcosis cases is irrefutable. In this current study, we showed that the C.gattii VGII genotype is possibly spreading in several cities of PI and MA (Brazilian
middle-north). More eco-epidemiology studies are necessary for a better understanding of its risk factors, virulence and geoclimatic distribution, as well as its correlation with the outbreak in Vancouver
Island and the extent of the expansion of this pathogen in northeast
Brazil. It is likely that the geographical distributionis much wider
than currently documented. Our results provide new information on
the molecular epidemiology of C. neoformans and C. gattii in Brazilian
endemic areas.

38

Background In fungi some mechanisms of resistance to azole drugs
are known. It has been observed that these phenotypes develop in yeast
populations either due to mutations or selection processes. In clinical
setting the long azole exposition is observed in Cryptococcosis associated to AIDS-cases. Azoles agents extensible employed for agriculture
purpose could result in azole resistance in environmental isolates. Taking account data from 2008, the amounts of agrotoxic compounds sold
for use in agriculture indicated Brazil ranked first world-wide.
Aim Evaluate the in vitro azole-susceptibility of Cryptococcus and
other melanized yeast isolates recovered from environmental Brazilian rural area exposed to azole fungicides.
Methods Samples of soil and water (irrigation and washing water)
were collected from two vegetal plantation area located in S~
ao Paulo
State, Brazil. The two studied areas differ regarding the usage of
azole-fungicides. We will measure the occurrence of fungicide in collected soil and water samples through gas chromatography methodology. Melanized yeast were recovered and identified according to
macroscopic and biochemical characteristics. MALDI-TOF was
employed for speciation of unconcluded identification. Additionally,
we performed molecular identification by rDNA sequence analysis
using LSU D1/D2 sequences amplified with the NL1 and NL4 primers
and to C.neoformans we used a technique the RFLP and mating-type.
The obtained sequences were compared with those existing in
GenBank and CBS databases. In vitro antifungal susceptibility was
measured according to AFST-EUCAST E.Def 7.2 guidelines for voriconazole, fluconazole and itraconazole. The activity of pure powder of
azole-fungicides obtained from the manufacturer (Syngenta, USA)
were also assayed against all isolates.
Results Among 79 yeast colonies we found seven Cryptococcus isolates. The sequences results presented 100% similarity to strains
deposited in GenBank and CBS databases as: C.laurentii (n =
2;28,57%), C.albidus (n = 3;42,85%), C.terrestris (n = 1;14,28%),
and C.neoformans VNI (n = 1;14,28%) and mating-type alfa. We
found minimum inhibition concentration (MIC) values of azoles used
in clinics up to 1 mg/l1, among Cryptococcus isolates from both
exposed and not exposed to azole-fungicide areas. The MIC range
were: fluconazole 0,12 to 1 lg/ml1; itraconazole 0.015 lg/ml1
and voriconazole >8 to 0.015 lg/ml1. The difenocolazole-MIC were
8 lg/ml1 for all isolates and for ciproconazole the MIC ranged from
0.25 to 8 lg/m l1.
Discussion and Conclusion It’s a hard task the recovering of melanized yeasts from water and soil due to fast growing-moulds habiting
from soil that overgrown on inoculated agar medium. Furthermore,
Candida and Rhodotorula colonies develop faster than Cryptococcus isolates on primary culture. Even under such contamination we could
recover 10% of Cryptococcus species isolates. The fluconazole-MICs
were the highest. The fungicide-MICs were higher than clinical azoleMICs being. The less susceptible isolates showed high fungicide-MIC
and clinical azole-MIC values. Of note, the isolates showing the highest MICs were obtained from fungicide exposed soil and water. Our
findings suggest that exposition to azole-fungicide compounds could
explain highest azole-MICs encountered in this study. We emphasized
the relevance of monitoring the azole-resistance of environmental
Cryptococcus isolates for routine in fungicide exposed area, in particular, in agricultural countries with large usage of those compounds.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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. Castan˜eda2
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 (1993–2010) 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 Piauı. However, Colombian
prevalence is similar to those reported in Uruguay (1.3%), Venezuela
(0.91%) and USA (0.85–1.4%). 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

Figure 2. The minimum spanning tree (MST) was constructed with
BioNumerics software.

-Cryptococcus gattii species complex. The climate of Taiwan, located at
tropical and subtropical regions, is optimal for the growth ofthem.
Recently, we published a nationwide survey to explore the molecular
epidemiology [1]. However, phylodynamic analyses of clinical Cryptococcus and the genetic correlation with global isolates were not clear.
Methods and findings Forty eight C. neoformans var. grubii isolates
(48 VNI and one VNII) and 9 C. gattii isolates (3 VGI isolates and 6
VGII isolates) were chosen for multilocus sequence typing (MLST)
analysis according to individual M13 fingerprinting pattern. Our
study identified seven sequence types (STs) of clinical C. neoformans
var. grubii, which ST5 (86%, 42/49) as the major genotype. Moreover, two strains (T107, T169) identical to Vancouver Island minor
strains VGIIb (R272) were identified which was ST7. Two STs
(ST328 and ST329) of C. gattii were novel in this study. Contrast to
the C. neoformans var. grubii, the population of clinical C. gattii isolates was diverse in Taiwan.
Conclusions The population of clinical C. neoformans var. grubii was
highly clonal, and the evolutionary change with time was small
before 2009 in Taiwan.

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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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 bird’s 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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

neoformans var. grubii (VNI), 6 by C. neoformans var. neoformans
(VNIV), and 16 by AD- hybrid strains (VNIII). No C. gattii was
isolated.
Discussion The results of this cryptococcosis survey show that the
number of recorded cases is significantly reduced compared to the
previous survey (156 cases) although the period and geographical
coverage were different. The percentage of HIV negative patients is
changed rising from 2.8% to 41%. This result confirms that, after
the introduction of HAART, HIV infected patients are less susceptible
to Cryptococcus infection. The ratio male/female is changed from
5.5 : 1 to 2.9 : 1 as a consequence of the higher percentage of nonHIV infected patients. The molecular analysis of Cryptococcus isolates
showed that VNI was the molecular type predominantly isolated
(50%) followed by VNIII (36%) and VNIV (14%), in contrast with
the percentages observed in the previous survey (35%, 35%, 30%
respectively).
In conclusion our results highlight the need of a constant monitoring of cryptococcosis in Italy since the epidemiology of this pathogen
is changing.
The FIMUA cryptococcosis network
Ancona: E. Manso; Bergamo: M. Passera; Genova: M. Mikulska;
Milano: A. Grancini, R. Grande, C. Ossi, M. Cainarco; Bolzano: P. Innocenti; Monza: S. Bramati; Novara: S. Andreoni; Pavia: C. Cavanna;
Pisa: A. Leonildi; Roma: A. Vella; Torino: A. Barbui; Udine: A. Sartor;
Varese: A. Colombo; Verona: G. Lo Cascio.

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

Y. Liu, H. Ye, C. Zhong, K. Liu and X. Lu
West China Hospital, Sichuan University, Chengdu, China
Background Cryptococcal meningitis is a global invasive mycosis
associated with significant morbidity and mortality. Amphotericin B
(AmB) combined with flucytosine were recommended as initial treatment for cryptococcosis management in 2010 IDSA guideline. Since
CNS remains a pharmacologic barrier to AmB regardless of formulation, the concentration of AmB in CNS was only 2–4% vs. plasma
concentration. Furthermore, AmB need to be gradually increased
from lower dosage because of its significant adverse reactions. So it’s
very difficult of AmB reaching effective fungicidal concentration in a
limited time. While fluconazole has more safety and efficacy with
high permeability of the blood-cerebrospinal fluid, it is worthy to
evaluate fluconazole added to induction therapy of Cryptococcal
meningitis.
Aim To investigate the therapeutic effects of non-HIV infected
patients with Cryptococcal meningitis treated with not only amphotericin B and flucytosine but also fluconazole from beginning.
Methods A retrospective study was conducted including 45 cases of
non-HIV-infected Cryptococcal meningitis in West China Hospital
during the past 8 years. The patients were divided into two groups:
the duplex therapy (initialized with AmB and flucytosine, n = 18)
and the triple therapy (initialized with AmB, flucytosine and fluconazole, n = 27). The dosage of AmB was 0.5 mg–1.0 mg
kg1 day1, that of flucytosine was 100 mg kg1 day1 and fluconazole was 400 mg kg1 day1. Treatment was considered successful if the patient had three consecutive negative CSF cultures. Then
they were continued with fluconazole till recover. The comparison
between two groups was done by t test and Chi-squared test.
Results Cure rate of triple therapy group and duplex therapy group
were 81.48% (22/27) and 66.67% (12/18), respectively. But no significant statistical differences were found between patients in two
groups with successful treatment (P = 0.257). Nonetheless, there
were significant statistical differences of the mean length of time until
treatment was successful between the triple therapy group and
duplex therapy group (71.9  37.32 days vs. 116  59.08 days;

41

Poster Abstracts

P = 0.021). The total dosage of amphotericin B of the triple therapy
group was less than the duplex one (3090.6  1540.21 mg vs.
4889.89  2787.48 mg, P = 0.035). Furthermore, the adverse
effects of triple treatment group were less than the duplex group
(48.15%, 13/27 vs. 77.78%, 14/18; P = 0.047).
Conclusions Triple therapy with amphotericin B, flucytosine plus
fluconazole in induction therapy phase could more effective for cryptococcal meningitis. Although we could not improve the cure rate
when we treat cryptococcal meningitis with AmB, flucytosine and
fluconazole at the outset, but it maybe cost patients’ shorter length
of stay and less adverse effects. Further prospective clinical trials with
large sample size in multi-center will be worthwhile to confirm the
conclusions.

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 Tukey’s 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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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 0.79–0.92).
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
40–60% 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 µg 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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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 µg ml1 and a
100% inhibition range of ≤0.015 to 2 µg 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 µg ml1 and a 100%
inhibition range of 0.125 to 8 µg 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 µg ml1) followed by
VGI isolates (0.03 and 0.125 µg ml1). VGII isolates and VGIII isolates
had the highest MIC50 and MIC90 values, but they were only 1–3 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

the susceptible concentrations. This re-affirms that amphotericin B
should be administered as a primary treatment for cryptococcal meningitis. The high rate of fluconazole resistance observed in our study
suggests fluconazole monotherapy is unlikely to be effective at controlling cryptococcal infections and could be a causative factor in the
high mortality observed in sub-Saharan Africa. We developed and
validated a low cost/infrastructure drug resistance screen assessable
in resource limited settings to routinely test the level of susceptibility
of C. neoformans isolates to fluconazole. When compared to previous
studies, the MIC50 and MIC90 in current Ugandan clinical isolates
was higher for both fluconazole and amphotericin B. Human-tohuman transmission of cryptococcal meningitis is extremely rare,
thus primary resistance, as seen in our study, is likely acquired in
the environment. Aspergillus fumigatus has acquired drug resistance
due to tri-azole fungicides used in farming. Widespread tri-azole fungicide use in Uganda to control the banana pathogen, Mycosphaerella
fijiensis, could be causing the increased resistance to fluconazole in C.
neoformans.

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: ≤0.125–1 lg ml1; 5-FC:
≤0.125–2 lg ml1; FCZ: 0.25–8 lg ml1; TRB: 0.125–2 lg ml1;
ITZ: 0.03–0.25 lg ml1 and VCZ: ≤0.015–0.125 lg ml1. MICs for
C.gattii ranged from ≤0.25 to 1 lg ml1 for AMB; 0.5–4 lg ml1 for
5-FC; 2–16 lg ml1 for FCZ; 0.5–4 lg ml1 for TRB; 0.06–
0.5 lg ml1 for ITZ and 0.06–0.25 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

C.neoformans VNI isolatesshowed better synergistic effect for FCZ +
TRB combination (95.16%) and VNII genotype showed 100% of synergism for all combinations, suggesting the relevance of C.neoformans
genotyping to guide treatment. C.neoformans VNI was also the predominant genotype affecting patients with cryptococcosis in Campinas region. In difficult-to-treat or unresponsive cryptococcosis the
combination of antifungal drugs such as AMB+TRB or FCZ + TRB
might be an alternative therapeutic approach in countries where 5FC is unavailable. More studies are necessary to elucidate the role of
Cryptococcus genotypes, clinical presentations and drug resistance.
Additional studies using antifungal combination in vitro and in vivo
should be performed in order to identify alternative therapeutic
approaches in the treatment of Cryptococcus infections.

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-BP’s 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 0.12–0.2 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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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 d’alacant, 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.

Table 1 Summary of diagnostic intraocular pressure thresholds to
predict elevated intracranial pressure.
Performance to detect Intracranial Pressure (> 250 mm H2O)
Intraocular Pressure from
both eyes

Sensitivity

Specificity

PPV

NPV

C-Statistic (95% CI)

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)

PPV = Positive predictive value, NPV = negative predictive value.
C-Statistic is the area under the curve (AUC) of the receiver-operator characteristic (ROC) curve for sensitivity and specificity.

Table 2 Summary of diagnostic ONSD thresholds to predict elevated
intracranial pressure.
Performance to detect Intracranial Pressure (> 250 mm H2O)
ONSD from both eyes

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

PPV = Positive predictive value. NPV = negative predictive value.
C-Statistic is the area under the curve (AUC) of the receiver-operator characteristic (ROC) curve for sensitivity and specificity.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

The three commercial methods showed a good correlation with
CLSI for Amphotericin B and azoles which are the main drugs for
the treatment of cryptococcosis. Nevertheless, for Fluconazole some
remarkable differences were detected: Etest showed higher MICs than
CLSI (GM: 33 lg ml1 for Etest and 8 lg ml1 for CLSI) and SYO
and SensiQuattro lower values than CLSI (4 and 1.4 lg ml1 respectively). Results for Flucytosine are more difficult to analyze because
SensiQuattro panel can only discriminate from a minimum value of
4 lg ml1. The geometrical mean (GM) for this drug by Sensititre
YeastOne was 4.4 lg ml1 and by SensiQuattro panel 79% of strains
showed values under 4 lg ml1.
Conclusion Results should be read after 72 h incubation for SYO
and Etest and more than 96 for SensiQuattro panels. Sensititre YeastOne showed the best global correlation with the reference method.
For the three commercial systems, results were coincident for
Amphotericin B and most azole drugs. Nevertheless, for Fluconazole
MICs values obtained with colorimetric microdilution panels (SYO
and SensiQuattro) tend to be lower than the ones from the reference
method and the opposite situation occurs with Etest.

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: 1.8–11.9, 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

Figure 1 Morphological changes in C. neoformans cells induced by
AMP6.

46

peptides was more potent than amphotericin B. C. neoformans B3501
strain showed lower inhibitory values when compared to the H99
strain. Capsule induction assays in the presence of AMP6, the most
potent peptide in our analysis, revealed a significant decrease in capsule size in the presence of this molecule. Additionally, scanning electron microscopy of C. neoformans cells incubated with 13 lmol l1 of
AMP6 for 1 h demonstrated severe deleterious defects in fungal
structure (Fig. 1 right panels) in comparison with untreated cells
(Fig. 1 left panels). Preliminary colony forming unit assays suggested
that AMP6 activity is fungistatic, rather than fungicidal. Peptide
sequence modification to enhance alpha-helix structures abolished
AMP6 antifungal activity suggesting that membrane insertion might
not be the main mechanism of its antimicrobial activity. Presently,
we are evaluating the role of melanin and capsule size in C. neoformans susceptibility to this peptide and the possible molecular mechanisms of how it acts. We also intend to analyze the effects of this
peptide against C. neoformans biofilms and evaluate its effects in combination with currently available antifungals.
Conclusion Our results demonstrated that AMPs are promising antifungal alternatives. Further characterization of the molecular mechanisms involved in their antifungal activity as well as in vivo studies
are essential to establish their true potential in our search for better
antifungal therapy options.

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 Brasılia, 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 erg6Δ
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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Poster Abstracts

Results The ERG6 deletion causes several alterations in cellular
homeostasis, such as increased sensitivity to osmotic and oxidative
stress and to cell wall stressors. The erg6Δ strain is also more susceptible to Azoles and more resistant to Polyene compounds. The
mutant was unable to grow at 37 °C on solid medium and a growth
curve in liquid medium showed a marked delay at this temperature.
Melanin and capsule production were not affected by the deletion,
but still the mutant was unable to survive within macrophages and
to colonise G. mellonella caterpillars. Finally, HPLC analysis showed a
change in membrane sterol composition with a dramatic reduction
in ergosterol content.
Discussion/conclusion The protein transport and anchoring
depends on membrane integrity and can be affected by the loss of
Erg6. Our results confirm yet again the importance of membrane
ergosterol to the viability and adaptability of the fungus to diverse
conditions. Therefore, Erg6 being an enzyme found exclusively in
fungi that plays an important role on ergosterol biosynthesis, it is a
potential molecular target for new antifungal drugs that can be used
alone to impair the fungus survival in the host or in combination
with established drugs to reduce their dosage, and thus the side
effects, through synergism. With the present work, Erg6 is confirmed
on C. neoformans as a very promising molecular target for the development of new antifungal drugs.

as well as fluconazole 800 mg day1 for 8 weeks followed by
200 mg day1. The primary safety endpoint will be the incidence of
treatment-related adverse events and serious adverse events. The primary efficacy endpoint will be survival at 12 weeks. Several secondary
outcomes including, but not limited to, survival at 24 weeks, number
of individuals who develop cryptococcal meningitis, number of individuals who develop immune reconstitution inflammatory syndrome due
to Cryptococcus, and cryptococcal meningitis-free survival at 24 weeks
will also be assessed (ClinicalTrials.gov Identifier: NCT01562132).
Results and discussion Patient screening and enrollment began in
October 2013 and at the time of this report, 297 individuals had
CD4+ T-cel count ≤100 cells ll1. Among these, 21 individuals had
a sCrAg titer ≥1 : 2, and 5 met inclusion criteria and consented to
participate. The median age of participants was 31 years (ranging
from 26 to 48) and 40% were female. The median CD4+ T-cell count
was 18 (ranging from 3 to 56), and the lowest sCrAg titer was
1 : 640 using the lateral flow assay (LFA). All 5 subjects consented
to an optional lumbar puncture, and all had a positive CSF CrAg titer
with the lowest titer being 1 : 64 by latex agglutination (LA). Thus
far, blood or CSF cultures were positive for Cryptococcus spp. in all
participants. Importantly, one participant was found to have C. albidus in the blood, rarely reported as a human pathogen. While the
study is ongoing, the preliminary culture results may indicate that
despite their lack of symptoms, individuals with low CD4+ T-cell
counts and cryptococcal antigen in the serum have central nervous
system infection.

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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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

(ATCC 22019) were included as quality control in all tests. The
minimal fungicidal concentration (MFC), minimal inhibitory concentration (MIC) of 50% and 50% inhibitory concentration (IC50) were
determined.
Results Structures of polygodial and laevicarpin were elucidated
based in analysis of NMR and MS spectra and compared to the literature. Polygodial showed an IC50 of 0.51 lg ml1 (95% confidence
interval 0.30–0.84 lg ml1), MIC of 1.46 lg ml1 and MFC of
0.73 lg ml1. Laevicarpin showed an IC50 of 2.35 lg ml1 (95%
confidence interval 1.67–3.31 lg ml1), MIC of 7.5 lg ml1 and
MFC of 60 lg ml1.
Discussion and conclusion In the course of selection of novel
drugs candidates we verified for the first time in literature, a high
antifungal activity of polygodial and laevicarpin against C. gattii. Further in vivo experiments using a cryptococcal model could confirm
the potential of these candidates. Our results suggested that polygodial and laevicarpin could be used as scaffolds for future drug design
studies against Cryptococcosis agents.

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 didn’t 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.

a

MICs equal or higher than the ECVs proposed by Espinel-Ingroff and co-authors, 2012.

48

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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

Heterorresistance to fluconazole in Brazilian clinical strains
of Cryptococcus gattii
L. M. Feliciano, L. X. Bonfietti, M. A. Martins, D. C. S. Santos,
T. N. Roberto, S. D. P. Ramos, M. W. Szeszs, A. Kubo and
M. S. C. Melhem
Adolfo Lutz Institute, Sao Paulo, Brazil

Antifungal susceptibility profiles assessed by a modified
broth macrodilution method using patient-specific
inoculum method of clinical isolates obtained from
cryptococcal meningitis cases attended at a tertiary public
~ o Paulo, Brazil
hospital Sa
L. Oliveira,1 D. C. S. Santos,1 M. W. Szeszs,1
M. C. S. M. Pappalardo,2 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 C. gattii strains have high potential to develop in vitro
resistance to fluconazole (FLC), which could explain the constant
therapeutic failures and relapses in patients affected by Cryptococcosis under FLC long-term therapy. Heteroresistance is the phenomenon described as the emergence of a minor subpopulation of
resistant cells within a single colony of the susceptible strain that
can tolerate FLC concentrations higher than the strain’s MIC levels.
Aim We aimed in this study investigate the level of FLC-heteroresistance exhibited by 46 C. gattii VGII and 4 C. gattii VGI clinical strains
(one per patient) from the culture collection (1994–2004) of Institute
Adolfo Lutz (SP, Br).
Methods Firstly, the MIC of FLC was determined by Etest method
for each strain, and then its level of heteroresistance to FLC (LHF)
was assessed. Cell suspensions of all strains were inoculated onto
YPD plates containing various concentrations of FLC (4–
128 mg l1). The lowest FLC concentration at which minor resistant
subpopulations emerged was identified as each isolate’s LHF. To
obtain the highly resistant subclones, the heteroresistant colonies
were isolated and inoculated on YPD agar containing stepwise (twofold) increases in the concentrations of FLC (up to 256 mg l1). The
culture plates of each passage were incubated at 30 °C for 72–96 h.
Finally, we have analyzed if the transferring of the highly resistant
subclones on drug-free media caused reversion to the original level of
heteroresistance. The H99 strain, and VGI-VGIV strain-types (kindly
sent by Prof. W. Meyer) with designed LHF were used as control test.
Results The MICs for these 50 strains ranged between 2 to
32 mg l1. Similarly to the unique published study we have demonstrated that all C. gattii tested strains manifested FLC-heteroresistance
with LHFs that ranged between 32 mg l1 and 128 mg l1. The
highly resistant subclones have been raised in a stepwise manner to
256 mg l1. All strains reverted to the original LHF upon daily
transfers in drug-free medium in distinct time-period.
Discussion and conclusion The study on heteroresistance may
reveal a novel adaptive mechanism for survival under the azole stress
and can offer helpful insights into the management of long-term
therapy. We observed that in C. gattii the level of FLC-heteroresistance was high (LHF ≥32 mg l1) and varies between strains. Moreover, all 50 tested clinical strains (100%) showed highly resistant
subclones (growth at 256 mg l1). We confirmed it is a reversible
adaptive response to the presence of the drug.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Background Antifungal susceptibility tests are largely employed as
tool for therapy for an individual patient, but lacking of interpretative
clinical breakpoints for Cryptococcus isolates against practice used
drugs cause controversial issue in Cryptococcosis.Up to date, the
drug-MIC values are not likely to predict the clinical outcome in
Cryptococcosis.In fact, patient’s pretreatment severity of meningitis is
considered the major risk factor associated with outcome.Due to drug
toxicity, particularly, AmB and/or 5FC, it is warranted to avoid
unnecessary drug administration. In previous studies it was proposed
a modified macrobroth dilution assay(MMD) for predicting the
response of individual patient to treatment with AmB, FCL, or the
combination of AmB plus 5FC.
Aim We used the MMD test for assessing susceptibility of clinical isolates obtained from AIDS-associated cryptococcal meningitis. Additionally we reviewed the medical records to assess the clinical
decision and outcome for each patient and correlated with antifungal
susceptibility testing results.
Material and methods Data and isolates were from five Brazilian
patients infected by C.neoformans (Cn) VNI and one infected by CnVNII.Severity of meningitis before antifungal-treatment and the quantitative responses at 0, 7 and 14 days were measured by CFU
counting per milliliter of cerebrospinal fluid. The inoculum employed
for MMD was matched to number of CFU ml1 present in pretreatment CSF for each individual patient. The range of AmB concentrations tested was from 0 to 3.5 mg l1, FCL from 0 to 500 mg l1,
and 5FC from 0 to 1000 mg l1. The end-point was based on serial
dilutions taken directly from each test tube after 48 h-incubation.
The drug-concentration that produced the quantitative in vitro
response that corresponded to patient’s quantitative biologic response
in the CSF at day-14 of treatment was determine as previously
described (relevant in vitro drug concentration = RivDC).Additionally,
AmB-MIC, 5FC-MIC and FCL-MIC were determined by Etestmethod, and time kill-curves were performed to assess cidal
endpopint of 1 mg l1 of Amb. Checkerboard method was employed
for analysis of drug interactions through FICIvalues.
Result All patients (21–45 years-old) showed CD4 < 150 cells
mm3 and were treated with AmB desoxycolate(1 mg kg1 day1)
plus FCL(400 mg 12/12 h) for 4 weeks. In one patient the desoxycolate formulation was changed at Day 5th to liposomal AmB

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 (6–12 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 Student’s t-test.
Discrepancies of more than two dilutions among MIC results were
used to calculate the essential agreement (EA).

50

Results All 96 isolates were found to be C. neoformans var. grubii
AFLP1/VNI and the majority belonged to microsatellite cluster (MC)
MC1 (n = 64; 66%), followed by MC3 (n = 21; 22%) and MC2
(n = 8; 8.3%). Three (3.1%) isolates could not be linked to known
MC type. All isolates showed good in vitro activities for all antifungals
except two which had high MICs of FC. The mean MICs were significantly higher by CLSI for AMB, (P = 0.0001), ITC (P = 0.035) and
POS (P = 0.003) whereas by EUCAST for FLU (P = 0.022) and VRC
(P = 0.001). EA between the two methods was highest for VRC
(94.5%) followed by FLU (89%), ITC (89%), AMB (87.3%), POS
(82%), and FC (70%). The mortality rates were equal in both treatment groups with higher fatalities among patients with disseminated
cryptococcosis involving two or more sites.
Conclusions The present study reports on the comparison of in vitro
antifungal susceptibility profiles of C. neoformans var grubii by using
CLSI and EUCAST methods revealed good concordance (97% EA)
between the two methods. Two microsatellite types dominate in India
and exhibit low MICs of FLU and AMB. The low MICs were associated with successful treatment in 67% of cases.

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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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 na€ıve 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

participants receiving these doses fluconazole. In four participants
there was QTc interval prolongation within the normal range while
in six participants (three on 1200 mg and three on 2000 mg day1)
the QTc internal was prolonged beyond the normal range. None with
a prolonged QTc had significant arrhythmias and all resolved with
dose reduction of fluconazole. Three participants assigned amphotericin B had a QTc interval change >70 ms but none had a prolonged
QTc. Concomitant medications (azithromycin, quinolone antibiotics,
and the antiemeticsgranisetron and ondansetron) known to prolong
the QTc interval were prescribed in four participants and appeared to
exacerbate the QTc prolongation associated with fluconazole in two.
Discussion All three dose levels of fluconazole met safety criteria of
three or fewer DLTs. After review by an ACTG Safety Monitoring
Committee, Stage 2 of the clinical trial will randomize an additional
72 participants in a 1 : 1 : 1 treatment ratio among fluconazole at
1600 mg day1, fluconazole 2000 mg day1 and amphotericin B.

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.

Table 1 Median Baseline Quantitative Values of Study Participants.
Treatment
Assignment
Glasgow Coma
Scale†
Intracranial Pressure
(mm CSF)
CSF Protein (mg/dL)
Quant. CSF Culture
(#/mL)
CD4 Cell Count
(#/mm3)
HIV Viral Load
(log10/mL)

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.

Median and interinterauartile rane.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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.

Methods The LAMP primers were generated by Primer Explorer V4
software (http://primerexplorer.jp/e/) based on the rRNA intergenic
spacer 1 (IGS1) sequence (GenBank accession number: EF211264) of
the dominant C. neoformans type strain H99. Cryptococcus DNA was
extracted from clinical CSF using the ZR Fungal/Bacterial DNA MiniPrepTM Kit (ZYMO Research Co., Ltd, Los Angeles, USA) according to
the instructions. The LAMP reactions were carried out twice in the special reaction tube (Eiken Chemical Co., Ltd, Tochigi, Japan) at 63 °C
for 90 min in Loopamp real-time turbidity meter (LA-320C). The
LAMP protocol was tested using 116 isolates of the C. neoformans/C.
gattii complex and related species altogether representing 38 species.
Results The modified LAMP assay yielded positive results for all 63
tested isolates of the C. neoformans/C. gattii species complex. Results
with DNA of related species was negative. Thus, the analytical specificity of our method was 100% in our study. The detection limit of
DNA of all eight genotypes of the C. neoformans/C. gattii species complex was approximately 20 fg per PCR. Furthermore, our modified
LAMP assay detected cryptococcal DNA of 95.7% (90/94) of clinical
CSF in 90 min.
Discussion/conclusion Compared to previously published studies of
LAMP assays on the C. neoformans/C. gattii species complex, our
modified LAMP method can directly identify all members of the C.
neoformans/C. gattii species complex with high specificity (100%). The
detection limit of 20 fg DNA per PCR revealed a sensitivity of approximately 30 Cryptococcus genomic DNA copies per PCR. Thus, our
results are comparable to a real-time PCR assay (5 cells per run), a
nested-PCR assay (10 cells per run), and the Luminex xMAP assay
on Cryptococcus (101–103 cells per run). Furthermore, our modified
LAMP assay detected cryptococcal DNA from 95.7% (90/94) clinical
CSF. Thus our method meets the ASSURED criteria [Affordable, Sensitive, Specific, User-friendly, i.e. simple to perform in a few steps with
minimal training, Robust and Rapid, i.e. results available in 30 min,
Equipment free, and Deliverable to the end user] for clinical diagnostics and surveillance of CM at the point of care in resource limited
countries.

52

Chitin and chitooligomers are glycans composed of beta-1,4-linked
N-acetylglucosamine. These glycans interact with glucuroxylomannan (GXM), the major capsular polysaccharide of Cryptococcus species
forming immunologically active hybrids. We have recently demonstrated that chitin-related structures participate in the pathogenesis
of C. neoformans, but the general role of these structures in the physiology of cryptococci remains obscure. In addition, the functions of
chitin-like structures in C. gattii cells have not been explored. In this
work, we compared a number of general properties of chitin-like
structures in C. neoformans and C. gattii. ELISA revealed that chitooligomers bind C. neoformans and C. gattii GXM with similar affinity.
Supplementation of fungal cultures with soluble chitooligomers followed by analysis by scanning electron microscopy resulted in profound alterations in the capsular architecture of C. neoformans, but
not of C. gattii. These alterations included increased dimensions and
formation of intracapsular areas that were apparently unfilled with
capsular components. In C. neoformans, blocking of chito-oligomers
with the wheat germ lectin (WGA) affected capsule formation and
GXM release to extracellular space. The lectin had no effect on capsule formation or GXM secretion in C. gattii. Lectin treatment downregulated the expression of eight capsule-related genes in C. neoformans, but stimulated expression of PKA1 in C. gattii. This observation
was validated in experimental models using inhibitors of protein
kinase A. These results support the supposition that chitin-related
structures have key roles in the physiology of cryptococcal cells. In
addition, they reveal an important functional divergence in C. neoformans and C. gatti that might impact pathogenic mechanisms.

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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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 (Mann–Whitney 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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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 [1.04–2.93]; P = 0.034) and the genotype C/T
of rs353626 (OR = 1.81, 95% CI [1.15–2.85]; P = 0.009) were
under-presented and the genotype C/C of rs3751031 (OR = 0.36,
95% CI [0.13–0.99]; 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 [0.31–0.96];
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.

Figure 2. Early dissemination to the mediastinal lymph node (MLN)
in spore mediated infection.
Results In a mouse intranasal model of infection, spores isolated
from a cross of the type strains B3501 and B3502 caused uniformly
fatal disease by 70 days post-infection, while the yeast parents were
avirulent. Through tracking fungal burdens over time in mouse tissues, we discovered that spores (but not yeast) are able to disseminate to the draining lymph node of the lung shortly after infection,
and spore-derived cells continue to colonize other organs, eventually
leading to a fatal CNS infection. Both immortalized and primary
phagocytes robustly phagocytose spores, while their yeast counterparts are largely resistant to phagocytosis. Fluorescence microscopy
revealed that spores are trafficked to the harsh environment of the
phagolysosome following phagocytosis. Surprisingly, the vast majority of phagocytosed cells are able to survive in this environment even
when the macrophages were activated.
Discussion/conclusion Following uptake, spores enter the phagolysosome and are able to survive, germinate into yeast, and replicate.
The ability of spores to germinate and disseminate in the host is
essential for disease progression as cryptococcal disease culminates in
an overwhelming fungal burden composed of yeast. These data support the hypothesis that phagocytes are providing a protected environment and vehicle for spores to grow and spread within the
mammalian host. Future studies will focus on further mapping the
relationship between spores and phagocytes in the host; including
identifying phagocytic receptors and how specific phagocytes may
influence dissemination.

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

Figure 1. Trojan horse model of C. neoformans spore mediated
infection.

54

b-1,4-N-Acetylglucosamine oligomers (chito-oligomers) participate in
surface architecture and pathogenesis in the Cryptococcus neoformans
model. In this work we aimed to establish connections between

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Poster Abstracts

surface architecture and pathogenesis in the C. gattii serotype C
model, with focus on the participation of chito-oligomers in fungal
dissemination. The serotype C cells studied here included strains
106.97, ATCC24066, HEC40143 and WM779. Scanning electron
microscopy (SEM) revealed typical capsular morphologies for all
strains, but immunofluorescence analysis demonstrated that strains
106.97 and WM779 had low reactivity with monoclonal antibodies
against GXM. Analysis of other virulence factors indicated that urease activity was similar in all strains. Pigmentation tests, however,
revealed that strains ATCC24066 and HEC40143 failed to produce
melanin in the presence of L-DOPA. In vivo studies in mice models of
infection confirmed two different pathogenic profiles. Strains 106.97
and WM779 efficiently colonized the brain of infected animals. The
ATCC24066 and HEC40143 isolates, however, were contained in
the lung and took significantly longer than 106.97 and WM779
strains to kill mice. Since chito-oligomers have been recently associated with fungal dissemination in the cryptococcosis model, we asked
whether these structures would influence the different pathogenic
profiles observed in our study. The higher-virulence strains (106.97
and WM779) were significantly more efficient than the low pathogenicity isolates (ATCC24066 and HEC40143) in inducing chitinase
activity in the lung of infected animals. This observation was associated to an increased detection of fungal chito-oligomers in lung tissues infected with strains 106.97 and WM779. Blocking of chitooligomers using the wheat germ lectin (WGA) prior to infection
delayed dissemination of both strains to the brain and resulted in
increased association of these fungal cells with macrophages. WGA
treatment, however, had no effect on phagocytosis profiles or animal
pathogenesis of strains ATCC24066 and HEC40143. These results
demonstrate different pathogenic profiles in serotype C isolates of C.
gattii and suggest a correlation between chitinase-induced chito-oligomer production and fungal dissemination. The diversity in the
pathogenic potential of serotype C strains of C. gattii reveals the need
for a detailed exploration of the relationship between virulence and
surface architecture in the Cryptococcus genus.

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-

ª 2014 The Authors
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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

in RC2 cells under CR. RNA sequencing of the mutant showed
downregulation of genes involved in galactose metabolism
(CNM00600,
CNM00620),
oxidation-reduction
(CNF03470,
CNN00260, CNI01580), and transmembrane transport (CNJ01360,
CND05980). Additionally, we studied the RLS modifying drug, isonicotinamide, which has been shown to act on SIR2 in Saccharomyces cerevisiae to extend RLS. We found that when it extended RLS in
C. neoformans (H99), it rendered it hypovirulent, and when it shortened RLS (I65), it rendered it hypervirulent. Thus, we provide further
support that a shortened RLS may be beneficial to the pathogen during chronic infection, especially if it helps it attain age-related resilience faster. In conclusion, SIR2 could serve as a potential target for
therapeutic inventions against cryptococcosis.

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

Figure 1. Cryptococcus mutants related to transmigration in MBTA
model.

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 33–68% 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

Background The increasing number of cryptococcal infections
worldwide demands new approaches for treating Cryptococcus neoformans. The host immune system uses a variety of weapons in an
attempt to eliminate infectious agents, such as production of reactive
oxygen and nitrogen species as well as iron sequestration. In addition, evidence is mounting to support a role for elevated copper (Cu)
during infection. In fact, under control of the Cu responsive transcription factor, Cuf1, C. neoformans mounts a strong defensive
response to elevated Cu in the lung through expression of two
Cu-specific metallothioneins. Without these metallothioneins, C. neoformans is unable to establish a successful infection. This response
highlights Cu as an important player in the lung, and presents a possible target for anti-Cryptococcal therapies.
Aim We seek to perturb Cu homeostasis of C. neoformans both in vitro and in vivo to determine the effects on cell viability as well as the
outcome of infection. To do this we use a protected form of the Cubinding molecule, 8-hydroxyquinoline (8HQ), to selectively harness
its antifungal activity in the context of either activated macrophages
in vitro or in the murine lung infection model. The nontoxic protected form of 8HQ, called QBP, contains a pinanediol boronic ester
that blocks metal coordination and is deprotected via reactive oxygen
species that are produced by activated immune cells to elicit Cudependent killing of the fungal pathogen C. neoformans. This strategy
is aimed at minimizing off-target effects, by focusing the antimicrobial properties of 8HQ to activated macrophages or sites of
inflammation.
Methods In this work, we utilize in vitro culture based assays to
assess the effects of 8HQ and QBP in the presence or absence of Cu
on both C. neoformans and macrophage-like cells. We assess the
impact of these molecules on cell viability, gene expression, and
metal content. LC-MS analysis was utilized to observe the conversion
of QBP to 8HQ by activated, but not na€ıve macrophages. Furthermore, we used in vitro co-culture experiments to determine the
impact of QBP conversion by macrophages on the survivability of C.
neoformans. Finally, we administered QBP to mice infected with C.
neoformans to establish its effects on fungal load in the lungs.
Results We determine that the Cu-dependent antifungal effects of
8HQ bound to copper are fungicidal rather than fungistatic, and the
mechanism of 8HQ-fungal killing is through its action as a Cu ionophore, leading to a large increase in cell-associated Cu, highly elevated expression of the fungal metallothioneins, and ultimately cell
death. While 8HQ is cytotoxic to mammalian cells, QBP is tolerated
at much higher concentrations. However, when activated, macrophages are able to convert QBP to 8HQ, and this process is able to
stimulate killing of C. neoformans when co-cultured in vitro. Furthermore, we show that QBP treatment is able to reduce fungal burden

ª 2014 The Authors
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Poster Abstracts

during lung infections, suggesting that QBP is converted to active
8HQ in infected lungs where it can exert its antifungal activity.
Discussion/conclusion There is a delicate balance of Cu between
the host and C. neoformans and we begin to address how this aspect
of infection can be targeted and perturbed. As Cu is a critical component of innate immunity, the activation of Cu ionophores or other
anti-fungal compounds, in concert with immune cell activation, may
provide first in class antimicrobial therapeutics.

summary, our results showed that tryptophan biosynthetic pathway
is an excellent drug target, since TRP genes are essential. Also our
results on amino acid permeases shed some light on why C. neoformans survival is highly dependent upon biosynthesis rather than
amino acid assimilation.

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

ª 2014 The Authors
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Characterisation of Cryptococcus strains isolated from
mammals living in an environmental site with high
cryptococcal presence in South-Western Australia
P. D. Danesi,1 R. M. Malik,2 M. K. Krockenberger2 and
W. Meyer3
1
Istituo Zooprofilattico Sperimentale delle Venezie, Legnaro
(Padova), Italy; 2The University of Sydney, Sydney, NSW, Australia
and 3Center for Infectious Diseases and Microbiology, Sydney
Medical School-Westmead, Sydney, NSW, Australia
Background Cryptococcosis is one of the most important systemic
fungal infections of mammals in Australia. Investigations of veterinary isolates and surveillance studies of captive koalas identified a
wildlife park in Perth, WA with an exceedingly high environmental
occurrence of Cryptococcus gattii. Furthermore, there is a high rate of
nasal colonisation of animals in this location, particularly in koalas,
with both subclinical and symptomatic disease.
Aim To further monitor the extent of colonization and the associated
genetic variability within the Cryptococcus neoformans/gattii species
complex in animals living in this park. Specifically, we were interested in whether colonisation of individual animals or focal environmental sites is clonal, or involved more than one cryptococcal strain/
molecular type.
Methods In October 2012, 58 samples, comprising swabs from captive koalas (n = 27), dingoes (n = 2), a wombat (n = 1) and the environment (n = 18) were collected at Caversham Wildlife Park. After
transport to the laboratory (at 5 °C; for 5 days), swabs were inoculated onto birdseed agar plates and incubated at room temperature
(approx. 21 °C). The extent of cryptococcal colonisation or environmental presence was codified as being of low, intermediate and high
grade according to the number of colonies showing a brown-colouring per plate (low < 50; intermediate 50–100; high grade >100 colonies). Following DNA extraction, restriction fragment length
polymorphism (RFLP) analysis of the URA5 gene and a mating type
specific PCR were performed to determine the molecular type and the
mating type, respectively, for one colony from each plate, representing isolates from an individual animal or a single environmental site.
Genetic variation among Cryptococcus isolates was studied using the
ISHAM consensus multi-locus sequence type (MLST) scheme for the
C. neoformans/C. gattii species complex. Additionally, to determine
whether different Cryptococcus clones were present amongst brown
colonies grown from a single plate from an individual animal or
environmental site, 74 colonies isolated from four plates (n = 18/
plate from one koala; n = 19/plate from another koala; n = 20/plate
from an environment sample; n = 17/plate from a different environmental sample) were analysed by M13 PCR fingerprinting.
Results Positive cultures were obtained from 14 of the 27 koalas
(52%) and from 10 of the 18 environment samples (56%). The extent
of colonisation or environmental presence was low (8 koalas and 3
environmental samples), intermediate (5 environmental samples) and
high grade (6 koalas and 2 environmental samples). Among the positive cultures, C. gattii VGII (n = 16; 67%), C gattii VGI (n = 5; 21%)
and C. neoformans VNI (n = 3; 12%) were identified. VNI was identified only from koalas. All isolates were of the a-mating type. MLST
analysis revealed six distinct sequence types (ST) among the 24 isolates: ST7 (14), ST23 (2), ST48 (2), ST51 (1), ST58 (1) and ST 154
(4). PCR fingerprinting profiles showed identical patterns among colonies from the same plate for the four plates so analysed.
Discussion/conclusions C. neoformans and C. gattii were both isolated from nasal swabs of koalas, whereas only C. gattii was found in

57

Poster Abstracts

the environment. The wide variety of MLST types of C. gattii isolates
supported the possibility of genetic recombination at this location,
consistent with earlier work and the previous finding of rare a mating types in this environment. On the other hand, PCR fingerprinting
profiles suggest that within an individual animal or environmental
site, clonal replication prevails, based on the number of plates studied. The persistent high environmental presence of VGII in Caversham Park, with corresponding high prevalence of asymptomatic
nasal colonisation, subclinical infection and clinical disease in animals, especially koalas and wombats, emphasizes the need for ongoing surveillance at this location. Clonal outbreaks might develop in
this location after genetic recombination, possibly giving rise to a
Vancouver Island-like outbreak in the vicinity.

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
Children’s Hospital Los Angeles, USC Keck School of Medicine,
Los Angeles, CA, USA and 2NIAID, Bethesda, MD, USA

Towards understanding cell cycle control and hypoxic
adaptation in Cryptococcus neoformans
S. Kawamoto,1 E. Virtudazo,1 Z. Moranova,2 M. Ohkusu,1
K. Shimizu,1 A. Suganami,3 Y. Tamura3 and V. Raclavsky2
1
Chiba University Medical Mycology Research Center, Chiba,
Japan; 2Faculty of Medicine and Dentistry, Palacky University,
Olomouc, Czech Republic and 3Chiba University, Graduate
School of Medicine, Chiba, Japan

Background Cryptococcus neoformans invades into brain and causes
severe meningoencephalitis. The mechanism of cryptococcal brain
invasion is largely unknown, and recent studies suggest that its
extracellular microvesicles (MV) may be involved in the invasion process. The 14-3-3 protein is abundant in the extracellular MVs of C.
neoformans, but the physiological role of 14-3-3 has not been
explored.

Cryptococcus neoformans is an opportunistic pathogen of worldwide
distribution and responsible for life-threatening infections among
immunocompromised persons. We have been studying the molecular mechanisms of the cell cycle control in C. neoformans and have
reported that the cell cycle behavior of this yeast is different from
the cell cycle control model exhibited by the common budding
yeast, Saccharomyces cerevisiae. C. neoformans exhibits a delay in

58

ª 2014 The Authors
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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 organism’s 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 yeast’s
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. George’s, 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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

identified, and this hypothesis needs further testing. We are using
whole genome sequencing (WGS) of isolates sampled worldwide to
infer the population structure and recombination rates of Cng. These
genome sequences will also be used to build a scenario of the dispersal of Cng across the globe, and assess the likely geographical
origin.
We are sequencing clinical and environmental isolates have been
collected from sites around the world, with a focus on Africa and
Asia. Illumina sequencing has been performed on these isolates to
generate sequences with >1009 coverage across the genome,
allowing high confidence variant calling following alignment to the
Cng H99 reference genome. These genome-wide SNP data were
used by the coalescent-based method LDhat ‘interval’ to estimate
population-scaled recombination rates within the VNB, VNI and
VNII Cng populations. A 4-gamete test (4GT) was also completed to
confirm the existence of recombination in these populations. Since
linkage disequilibrium is influenced by a number of factors, such as
recombination rate, the rate of mutation and selection, various statistical analyses were used to measure linkage disequilibrium. The
level of population differentiation at individual loci was assessed
using pairwise FST statistics amongst pairs of populations. The standardised index of association, rBarD, was also calculated. Software
such as fineSTRUCTURE and ARGweaver, which were initially
developed for inferring the ancestral recombination graphs of
humans, have also been used to determine the ancestry of Cng
populations.
Results from resequencing an initial panel of 39 Cng isolates collected worldwide confirm the expansion of the VNI lineage. rBardD
analysis confirms that this population is extremely clonal. SNP density plots of individual chromosomes also show the VNI population
as a clonal population, with low genetic diversity, compared to the
VNII and VNB populations.
Recombination analyses using LDhat ‘interval’ show the VNB population to be highly recombinogenic; this result is confirmed by the
4GT, which shows there is three times more recombination occurring
within the genomes of the VNB population, compared to the VNI
population. The calculation of FST statistics also highlighted that
regions of chromosomes 5 and 6 have a significantly lower FST score
when comparing VNB and VNI populations.
A significantly lower FST score for specific regions of chromosomes
5 and 6 when comparing VNB and VNI populations suggest that
open reading frames (ORFs) in these regions may be exhibiting purifying or diversifying selection. Since the VNB population displays
extensive recombination, it can be hypothesised that this population
is the ancestor of the low diversity VNI population.

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

Northwest of the United States, two temperate regions. Exposure to
propagules dispersed in the environment initiates the infection process, related to various organic substrates, mainly decomposing wood
in and around dwellings. The present study investigated the presence
of the agents of cryptococcosis in dust from dwellings in the upper
Rio Negro, municipality of Santa Isabel do Rio Negro in Amazonas
state. Indoor dust was collected from 51 houses, diluted and plated
on bird seed agar. Dark brow colonies were identified phenotypically
and genotipycally by restriction fragment length polymorphism of
the gene URA5 and multilocus sequence typing (MLST). The mating
type was identified using specific primers for pheromone. Three
houses were positive for C. gattii molecular type VGII, MAT alpha
and MAT a, showing significant density of this agent. MLST studies
identified eight subtypes, VGIIb (ST7), VGIIa (ST20), (ST5) and 5
new unique subtypes of the region. For the first time in the state of
Amazonas, C. gattii VGII MAT alpha and MATa were isolated from
the environment and correlates with endemic cryptococcosis in this
state. This is the first description of MLST subtypes on environmental
isolates in the Brazilian Amazon, indicating domiciliary dust as a
potential source for human infection with different subtypes C. gattii
VGII MAT alpha and MAT a.
[Correction added on 9 May 2014, after print publication:
Erroneous abstract has been replaced with the correct abstract.]

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

mating. UV cross-linked RNA EMSA has shown that Puf3 protein
specifically binds to its consensus TGTACATA cis element present in
50 UTR.
Discussion We have demonstrated a role for Puf3 in the regulation
of morphogenesis during C. neoformans sexual development. As there
is no defect in pheromone induction and fusant formation, it suggests
that bilateral mating of puf3D mutants is defective for post-fusion
hyphal extension. The intact RBD is essential for the functional role
of Puf3. Visualization of mCherry tagged PUF3 has depicted that
PUF3 localizes to the areas of hyphal growth or septation, but is
absent in vegetative yeast cells. This morphotype-specific production
of Puf3 may be mediated by auto-regulation through a Puf3-cognate
cis element in the PUF3 50 UTR. Future studies will determine the
mechanism of Puf3 regulation on potential target transcripts which
would enable us to establish a link between the physiology and the
Puf3 regulon of C. neoformans.

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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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. D’Amicis2 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 1.2–6.5, 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 1.1–6.3, 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. George’s 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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Two evolutionarily conserved post-transcriptional
regulatory pathways converge to regulate the ER stress
response of Cryptococcus neoformans
V. E. Glazier and J. C. Panepinto
SUNY University at Buffalo, Buffalo, NY, USA
Background Cryptococcus neoformans is one of a small number of
fungi able to transition from ambient environmental temperatures to
human core body temperature. We previously reported a role for the
ER stress response in host temperature adaptation of C. neoformans,
with post-transcriptional gene regulation controlling the intensity
and duration of this response. Activation of the ER stress response
results in the unconventional splicing of the pre-spliced HXL1u
mRNA to the active HXL1s mRNA at the ER surface by Ire1. Once
spliced, the HXL1s transcript is translated into Hxl1, a transcription
factor that promotes the expression of ER stress transcripts. Studies
in S. cerevisiae have identified a consensus sequence in HAC1 (an
HXL1 homolog) that affects the localization of HAC1 mRNA to the
ER surface where it is spliced by Ire1. The HAC1 consensus sequence
is conserved in several fungal species however is notably absent in
the C. neoformans counterpart, HXL1, suggesting an alternate method
of regulating HXL1 splicing in C. neoformans.
Aim Characterize the role of Puf4 in the post-transcriptional regulation of the ER stress response

61

Poster Abstracts

Methods We assessed tunicamycin and temperature sensitivity
through spot plate assays of H99, puf4-delta and puf4-delta: PUF4.
Northern blot analysis was used to measure steady state levels of ER
transcripts during tunicamycin treatment and growth at 37 °C. To
measure mRNA stability 1,10 phenanthroline was added prior to
harvesting cells to halt transcription then RNA isolation and northern blot analysis performed. To determine the rate of HXL1 splicing,
we used semi-quantitative PCR amplification of cDNA utilizing primers that span the HXL1 splice site. The electrophoretic mobility shift
assay contained TYE-705 labeled oligonucleotides and recombinant
Puf4 electrophoresed on a DNA retardation gel. Virulence studies
included tail vein injection, intranasal inoculation and a competition
assay.
Results Spot plate assays reveal an increase in temperature sensitivity of puf4-delta, combined with increased resistance to the ER stress
inducing drug tunicamycin, suggesting the ER stress response is
altered in puf4-delta. puf4delta cells exhibit misregulation of ER
transcripts when compared to wt cells during tunicamycin treatment
and growth at 37 °C. The mRNA encoding the ER stress response
transcription factor, HXL1, has two potential Puf4 consensus
sequences. EMSA results show the 50 UTR PUF element of HXL1 is
able to bind Puf4, suggesting a direct regulation of HXL1 by Puf4.
Puf4 appears to regulate both the rate of splicing and mRNA stability
of HXL1 as the puf4delta mutant has a defect in the rate of HXL1
splicing during ER stress, and an increase in HXL1 transcript stability
when compared to wt. Although Puf4 is involved in the regulation
of a pivotal component of the ER stress response, HXL1, deletion of
Puf4 has no effect on virulence in a mouse model, but exhibits
reduced fitness in an in vivo competition assay.
Conclusions Our results suggest a novel convergence of the PUF
post-transcriptional regulatory module and the ER stress response
signaling pathway in Cryptococcus. Puf4 appears to regulate the rate
of HXL1 splicing, possibly through localization of HXL1 mRNA to
Ire1 foci. This delay in HXL1 splicing has a downstream effect on ER
transcripts that rely on HXL1 splicing and production of Hxl1. In
addition to the effects of Puf4 on HXL1 splicing Puf4 also appears to
modulate HXL1 transcript stability. The interaction between Puf4
and HXL1 mRNA occurs through binding of Puf4 to a consensus
sequence in the 50 UTR of HXL1. Future studies will be required to
identify other mRNA targets that make up the Puf4 RNA regulon.

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 1–2 genes known to be

62

required for biosynthesis of polysaccharide capsule, along with a half
dozen genes of unknown function. We assessed all of the mutants
within each cluster for capsule polysaccharide production and found
that the majority impact capsule production and/or attachment.
Mutants attenuated for capsule production are also attenuated for
growth in a mouse lung.
(2) Development of drug synergy biomarkers: We hypothesized that
genes whose knockouts show altered growth to compound pairs
known to be synergistic could be used to predict new drug synergies.
We tested this hypothesis experimentally for two drugs and found
this novel approach to be highly successful at identifying synergistic
drug combinations compared to a randomly generated control group
of drugs.
(3) Identification of the target of a drug-like molecule: We discovered
that deletion of the gene coding for the Wee1 kinase produced dramatic resistance to an uncharacterized growth-inhibitory drug-like
compound called TDZ8. Wee1 blocks G2/M transition by phosphorylating Cdk1; the phosphatase Cdc25 opposes this. Our data suggested
that TZD8 acts by blocking Cdc25. Indeed, TZD8, but not a highly
related compound, produces a G2/M cell cycle arrest. We also
expressed and purified C. neoformans Cdc25, measured its phosphatase activity in vitro, and found that TZD8 directly inhibits Cdc25
phosphatase activity.
Discussion/conclusions Chemical-genomic profiling is a versatile
method that can efficiently address several rate-limiting questions in
microbial pathogenesis research, including identification of drug
targets and functional annotation of virulence genes.

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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Results We have characterised the affect of ade12Δ deletion on
growth and virulence factor production, and documented multiple
phenotypic changes as well as the resulting changes in virulence in
an animal model. The differences between human and Cryptococcal
AdSS in both crystal structure and enzyme kinetics suggest that it
may be possible to develop a fungal-specific inhibitor.
Discussion/conclusion Overall our results indicate that AdSS is a
promising antifungal drug target, and pave the way for further investigation into possible inhibitory compounds and species specificity.

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 Koch’s postulates formed the foundation of
infectious disease study, so do Stanley Falkow’s 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

Discussion and conclusions Based on the problems faced during
complementation of mutants in C. neoformans, we have successfully
created a new molecular resource for the Cryptococcus community
ensuring that unrelated genes and by extension virulence are not
affected during the complementation process. We believe that this
new strategy helps overcome problems with the current approaches,
and while it will be more difficult to integrate into the genome as
opposed to random integration, it eliminates any affects on other
genes, ultimately fulfilling Koch’s postulates.

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 ~8–10% 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

Figure 1. Relative genomic expression levels between the deletion
strains and wild-type KN99 for selected chromosomes.

64

transcript array. The DNA expression levels were compared between
wild-type and the two pkc1D strains, relative expression levels were
averaged across seven genes and plotted by chromosome.
Results When the levels of DNA hybridized to the transcripts are
compared between wild-type and the pkc1Dand pkc1D-adapted, chromosome 5 in the pkc1D is, on average, almost twice that of the wildtype strain (Fig. 1) whereas none of the other chromosomes in the
pkc1Dstrain nor any of the chromosomes in the pkc1D-adapted strain
show any differences from wild-type (Fig. 1). The pkc1D-adapted
strain remains very sensitive to high temperature, SDS and caffeine
but has lost sensitivity to high salt, calcofluor white and Congo red.
We are continuing to analyze this strain as well as additional isolates
and that data will be reported.
Conclusions This is the first reported case of chromosomal duplication found in a C. neoformans gene deletion strain that is obtained
after biolistic transformation. We found that passaging on YPD
media resulted in loss of the aneuploidy and loss of some, but not all
cell wall sensitivity phenotypes. It suggests that aneuploidy may represent one potential source of differential phenotypes observed with
multiple isolates of single gene deletion strain.

P069
Effect of histone deacetylases inhibitors on the virulence
phenotypes of Cryptococcus neoformans
F. Branda˜o, L. S. Derengowski, P. Albuquerque and
M. J. Pocßas-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 Christiansen’s
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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Poster Abstracts

A

E

B

F

C

G

H

I

Figure 1. C. neoformans capsule size is reduced by HDAC inhibitors.
Capsule was assessed by the zone of exclusion after India ink staining
(magnification, 935; bar, 10 lm). Pictures are representative of the
cells observed in each condition. A: Control, B: NaBut 1 mM, C:
NaBut 5 mM, D: NaBut 10 mM, E: DMSO, F: TSA 1.5 lM, G: TSA
3 lm, H: TSA 6 lm and I: TSA 10 lm. Boxes represent 75 percent
of the data distribution, the median divides the quadrants. Bars represent the minimum and maximum values. Statistical test applied
was Kruskal-Wallis and post test Dunn. * indicate P < 0.05,
***P < 0.001, **** P < 0.0001. Scale bars represent 10 lm. Three
different experiments were performed with similar results.

37 °C at concentrations above 1.5 lmol l1. Capsule formation was
affected at concentrations from 1.5 lmol l1, being more pronounced at higher concentrations. There was a decrease in cell size
in the presence of 10 lmol l1 of TSA and in mating hyphae formation in concentrations starting at 6 lmol l1. TSA did not interfere
visibly on phospholipase activity. Neither drug affected urease production in the in any of the tested concentrations. Finally, no difference was observed in survival curves of the invertebrate host G.
mellonella infected with pre-treated yeasts with NaBut or TSA compared with the untreated control. This fact agrees with the recovery
of virulence phenotypes in vitro since iHDAC are removed. The transient effects observed in cell growth curve of the fungus treated with
TSA, as well as the less pronounced effects of drug in longer duration
experiments, such as the mating assay, may be explained by the lack
of the stability followed by the short duration of TSA action described
by other studies.
Conclusion So far, these results indicate that iHDAC induce changes
in the expression of several virulence phenotypes of C. neoformans.
Further molecular approaches will be employed to reveal the possible
mechanisms involved in these observations.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Figure 2. HDAC inhibitors dose-dependent effect on the formation of
C. neoformans mating hyphae. Strains of opposite mating types were
mixed and cultured under mating-inducing conditions for 15 days.
Pictures are representative of two different assays with similar
results.

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

Methods Our approach included EV purification, analysis of amoeba
viability after exposure to EVs and methyl-alpha-D-mannopyranoside
(mannose), stimulation of A. castellanii with EVs, and mannose and
determination of the phagocytosis index and antifungal activity for
H99 strain, an encapsulated serotype A of C. neoformans.
Results Our initial results demonstrate that C. neoformans EVs with
a diameter range between 50 and 550 nm do not impact the viability of amoebae, but exposure to EVs increases fungal uptake and survival inside the environmental predator, compared to untreated
amoebae. Mannose treated amoebae also exhibited an increase in
fungal uptake and survival and were more susceptible to death during 24 h co-cultures with C. neoformans.
Discussion Our results to date demonstrate that fungal EVs impact
C. neoformans interactions with A. castellanii, indicating that C. neoformans vesicle release to the extracellular space may have developed in
response to interactions with environmental predators. Additionally,
our findings suggest that MBR can mediate fungal phagocytosis by
amoebae, opening a new field of investigation about evolutionary
steps that could culminate in intracellular pathogenic strategies for
C. neoformans to infect and proliferate inside mammalian
macrophages.

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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Poster Abstracts

polysome-associated in the wild type. In the rpb4delta mutant, we
observe reduced translation following the temperature shift indicating
a role for Rpb4 in maintaining translation during host-temperature
stress. In our ccr4D mutant, many classes of mRNAs are stabilized,
likely resulting in greater cellular RNA and a larger RNA::ribosome
ratio. This may explain the increase in low molecular weight polysome peaks and loss of heavy polysome peaks. In the ccr4delta and
rpb4delta mutants RP- and ER stress transcripts are aberrantly distributed over the polysome profile, suggesting that mRNA degradation contributes to reprogramming of the actively translating mRNA
pools required for host-temperature adaptation.

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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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 Mary’s
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.

Aim This study aimed to evaluate biological and molecular aspects
of C. laurentii strains from Brazil, United States, Canada and Africa
through the sequencing of three ribossomal regions.
Methods A total of 100 environmental isolates phenotipically identified as C. laurentii were evaluated by sequencing of the 50 end of
small subunit of 18S gene, D1/D2 region of the large subunit of 28S
gene and Internal Transcribed Spacer (ITS) region. The applicability
of these markers as DNA Barcodes was also evaluated.
Results BLAST search of the sequenced 550, 650 and 550 bp
amplicons obtained from the 18S, D1/D2 region of 28S and ITS
region allowed the identification of 75 C. laurentii strains which
showed 100% of identity with the C. laurentii NRRL Y-2536 strain
from Taiwan and C. laurentii RY1 strain from India. Nine strains
presented 99% of identity with the Bullera sp. VY-68 strain from
Japan and C. laurentii RY1 through the 18S sequencing. One of
them, the isolate P482A from S~
ao Paulo, showed 99% of identity
with the recently described Cryptococcus rajasthanensis (CBS 10406)
strain from India, while the remaining eight isolates presented 100%
of identity with the Cryptococcus sp. APSS 862 strain from India by
the D1/D2 region of LSU-rDNA 28S and ITS region analysis. Sixteen
isolates presented 99% of identity with the Cryptococcus flavescens
(CBS 142) reference strain by the 18S gene analysis. From these,
only six were confirmed by the D1/D2 region of 28S and ITS region
sequencing, while the remaining 10 presented 99% of identity with
the Cryptococcus terrestris (CBS 10810) reference strain recently
described in Brazil and United States. Among the three ribosomal
DNA markers evaluated, the ITS region presented the highest variability and the most defined Barcode gap, followed by the 28S and
18S. Despite the high variability observed for the ITS region, two
strains of C. flavescens clustered near the C. terrestris group. The concatenated sequence analyses allowed the identification of seven
Sequence Types in C. laurentii, three in C. flavescens, one in Cryptococcus sp., one in C. rajasthanensis and one in C. terrestris isolates
through Neighborhood-joining, goeBURST and Median-joining network analyses.
Discussion/conclusion Since the first description of C. laurentii by
Kufferath in 1920, its taxonomy have undergone several changes,
especially in the last decade with the introduction of the molecular
techniques. The high intraspecific variability of C. laurentii isolates
have already been described by others, despite few strains have been
included. Before, the results herein presented,, the variability of this
species was unknown. The intraspecific variability of 2.4% found in
this study represents an original result. In addition, other C. laurentii
sibling species including one unknown species were described, and
reinforce the relevance of the sequencing technique to species identification. Of the three ribosomal regions herein evaluated, the ITS was
the most suitable for DNA Barcode, which is in accordance with previous studies.

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

Cryptococcus gattii VGIII isolates causing infections in HIV/
AIDS patients in Southern California: identification of the
local environmental source as arboreal
D. Springer,1 R. B. Billmyre,1 E. Filler,2 K. Voelz,3 R. Pursall,3
P. A. Mieczkowski,4 R. A. Larsen,5 R. C. May,3 S. G. Filler,2
J. Heitman1 and F. S. Dietrich1
1
Duke University Medical Center, Durham, NC, USA; 2UCLA, Los
Angeles, CA, USA; 3University of Birmingham, Birmingham, UK;
4
University of North Carolina, Chapel Hill, NC, USA and
5
University of Southern California, Los Angeles, CA, USA
Background Ongoing Cryptococcus gattii outbreaks in the Western
United States and Canada illustrate the impact of environmental reservoirs and both clonal and recombining propagation in driving
emergence and expansion of microbial pathogens. C. gattii comprises
four distinct molecular types: VGI, VGII, VGIII, and VGIV, with no
evidence of nuclear genetic exchange, suggesting these represent

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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 1–3 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 Muller’s 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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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 Muller’s 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 Muller’s 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 Muller’s 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.

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 Hodgkin’s 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

Figure 1. MSP-dendrogram and molecular types of investigated
strains of C. neoformans.

using Autoflex speed TOF/TOF (Bruker Daltonics). Mass-spectrometry
was performed twice with two different alpha-cyano-4-hydroxycinnamic acid solutions, one on the basis of a standard solvent (TFA/
acetonitrile/water), and another on mixture of formic acid/water/isopropanol (0.2 : 3 : 2) for two points of the target. Thus each strain
was passed eight times during 2 months. Collected spectra were identified with using Biotyper 3.1 database.
Series of 50 top rates of identification obtained with different
matrix-solvents were compared by Wilcoxon T-test. Best identified
spectra were converted into MSP and compared by MSP-dendrogram
and cluster of principal component analysis (PCA), which were constructed by different algorithms implemented in MALDI Biotyper OC.
Results Using a standard solvent for the MALDI-matrix is significantly better to obtain the highest results of species identification (Tcriterion = 291, critical range = 397, P = 0.01), however for the
one strain, C. neoformans var. grubii RCPF Y-1180 the best rate of
identification (2,358) was derived using formic acid/isopropanolic
mixture. In total 14 strains (82%) were identified with rates above
2.018 and 3 strains (18%) with rates in range 1.785–1.990.
None of the methods of constructing MSP-dendrograms and PCAclusters allowed to group the mass-spectra in accordance with the
molecular types of C. neoformans (Fig. 1). In PCA-cluster points
belong to single strains, but derived from mass-spectra obtained in
different time periods often are not grouped closely.
Conclusion This study showed that MALDI-matrix on basis TFA/
acetonitrile/water mix is more proper for Cryptococcus identification
than matrix in modified solvent. Wide variation of proteome profiles
detected on PCA-cluster indicates a high phenotypic variability of C.
neoformans, that must be considered when identifying and typing of
Cryptococcus isolates.
T. Mihara et al. (Med. Mycol., 2013) using isolates of C. neoformans
from the environment and non-HIV-infected patients showed that
results of molecular typing obtained by MLST and RFLP/URA5-algorithm were not closely correlated. This fact is one of probable reasons
causing the mismatch of molecular types and distribution of proteome mass-spectra on MSP-dendrogram.
Further studies are required to clarify the correspondence between
the distribution of the proteome mass-spectra and molecular types of
Cryptococcus neoformans obtained by various genetic methods.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Poster Abstracts

P082

P083

When could loss be a gain: the two faces of genome
instability in RNAi deficient Cryptococcus lineages
E. W. L. Chow, S. Clancey, R. B. Billmyre and J. Heitman
Duke university, Durham, NC, USA

Structure-function analysis of the centromere-kinetochore
and spindle assembly checkpoint machinery in
Cryptococcus neoformans var grubii
K. Sanyal,1 S. Sridhar,1 V. Yadav,1 J. Heitman2 and
L. Kozubowski3
1
JNCASR, Bangalore, India; 2Duke University Medical Center,
Durham, NC, USA and 3Clemson University, Clemson, SC, USA

Background The RNAi pathway has been shown to play a role in
sex-induced silencing in Cryptococcus neoformans, and deletion of
RDP1, a key component in the RNAi pathway results in a significant
increase in transposon movement during meiotic reproduction2. Of
the pathogenic Cryptococcus species, Cryptococcus gattii VGII is missing
key RNAi pathway components. Because this subspecies is responsible
for the Vancouver Island and Pacific Northwest outbreaks over the
past decade, it is possible that the loss of the RNAi pathway could be
beneficial, allowing for more rapid genetic evolution. Similarly, loss of
RNAi in C. neoformans mutants might also allow higher rates of genomic evolution, such as aneuploidy, chromosomal rearrangements and
transposition, during mitotic or meiotic reproduction.
In fungi, aneuploidy has been shown to be an advantageous adaptative mechanism, conferring antifungal drug resistance and rapid
genetic evolution in response to hostile environments. Earlier studies
in the lab have shown that meiotic reproduction, both unisexual and
bisexual, generates aneuploidy in C. neoformans1. Loss of RNAi may
impact the rate of aneuploidy development during meiotic reproduction, derepress transposon activity, resulting in increased genome
instability.
Aim Given that meiotic reproduction generates aneuploidy and that
the RNAi pathway is required for centromere function (in Schizosaccharmoyces pombe) and suppresses transposon activity during Cryptococcus meiotic reproduction, we are investigating if the deletion of
RDP1 leads to increased rates of aneuploidy generation and chromosomal rearrangements in C. neoformans serotype D.
Methods Wild-types, unilateral wild-type x rdp1, and bilateral rdp1 x
rdp1 mating crosses were conducted and random spore dissection
performed to obtain ~100 progeny from each mating cross. Unisexual mating assays were also performed for the wild-type and rdp1
mutants. Mating assays were performed on MS medium and dissected spores were germinated on YPD medium. Mating progeny
strains were spotted in 10-fold serial dilutions and grown under the
following conditions for 3 days: (1) YPD at 30 °C, (2) YPD plus
8 lg ml1 fluconazole, (3) YPD plus 100 ng ml1 rapamycin, (4)
YPD plus 1 lg ml1 FK506, and (4) YPD at 37 °C.
Multiplex PCR, as previously described1, was performed as a diagnostic screen for aneuploid strains. PCR of serotype D STE20a and
STE20alpha MAT genes were used as markers to determine the mating type of the progeny. Whole chromosomal plugs will be made for
strains that display phenotypic variation in comparison to wild-type,
and whole chromosomal PFGE separation will be performed to detect
any karyotypic changes.
As described previously2, FPR1, the gene encoding FKBP12, can
be used as a transposon trap. A serotype D JEC43-derived rdp1 deletion mutant was used in comparison with a wild-type JEC43 isolate
to determine the molecular mechanisms resulting in inactivation of
FPR1 with and without the RNAi pathway. Mutants were selected
on FK506 media and the FPR1 locus of mutants cross-resistant to
rapamycin was sequenced. Similar studies were conducted with the
naturally RNAi-deficient strain VGII C. gattii strain R265.
Results Wild-type RNAi proficient isolates developed spontaneous
FK506/rapamycin resistance primarily as a result of mutations in
FPR1, while in the rdp1 deficient mutants FK506/rapamycin resistance was largely the result of transposon insertions into the FPR1
locus. In contrast, with R265 we did not observe transposon insertion in the locus, and instead the FPR1 locus was inactivated via
mutation, suggesting possible long-term adaptative changes to loss of
RNAi and that the consequences of short-term vs. long-term loss of
RNAi may differ. Phenotypic and karyotypic analyses of the progeny
from the rdp1 bilateral and unilateral bisexual and unisexual mating
crosses are currently in progress.
References 1. Ni M. et al. PLoS biology 11, e1001653.
2. Wang X, Darwiche S, Heitman J. Genetics 193, 1163–1174.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Background High fidelity chromosome segregation ensures faithful
inheritance of genetic material. Equal chromosome segregation
between daughter cells requires the linking of the chromosome to
the dynamic pulling and pushing force of the spindle microtubules.
Any error in this process, if not detected/corrected, leads to aneuploidy - a hallmark of many cancers. Several proteins assemble to
form the kinetochore on centromeric chromatin linking the chromosome to the dynamic plus ends of spindle microtubules. The spindle
assembly checkpoint (SAC) detects unattached kinetochores and halts
the cell cycle at the metaphase-anaphase transition until the errors
are corrected. Intriguingly, the centromere DNA sequence in various
eukaryotes is highly divergent but the basic architecture and function of the kinetochore remains conserved. However, the process of
kinetochore assembly including localization patterns and localization
dependency varies significantly.
Results Centromere location, structure and organization: CENP-C is an
evolutionarily conserved kinetochore protein found to be associated
with all functional centromeres studied to date. We identified centromeres as genome-wide binding sites of CENP-C in Cryptoccous neoformans var grubii (H99) by ChIP-seq analysis. A significantly enriched
binding region (<5 kb) was found to be present on each of the 14
chromosomes. These sites are present in 20–65 kb long ORF-free
repetitive regions that are rich in transposons (Tcn1–Tcn6). Kinetochore architecture and assembly: Towards understanding the kinetochore structure and the process of its assembly in various stages of
the cell cycle, we tagged two proteins each from inner (CENP-A,
CENP-C), middle (Mtw1, Nuf2) and outer (Dad1, Dad2) layers of the
kinetochore with mCherry or GFP. The localization patterns obtained
by time-lapse confocal microscopy reveal that unlike the pathogenic
yeast Candida albicans, the kinetochore assembles in a hierarchical
manner. In contrast to asomycetous yeasts, centromeres were found
to be unclustered until the onset of mitosis in C. neoformans. In addition, while most yeasts undergo closed mitosis, strikingly, we observe
a partial opening of the nuclear envelope during mitosis in C. neoformans suggesting a semi-open mitosis. Kinetochore localization dependency: Utilizing a stringent controllable promoter (the GAL7
promoter) to drive expression of fluorescently tagged kinetochore proteins, we sought to address the essentiality, localization dependency
and regulation of assembly of various proteins that belong to different layers of the proposed tri-laminar kinetochore network. Our
results suggest a putative kinetochore architecture wherein depleting
the protein pools of an outer layer (farther from the centromere DNA
but nearer to the microtubule ends) does not appreciably affect the
localization of underlying layers, while disrupting the inner layers (closer to the centromere DNA) perturbs the entire multi-subunit assembly
of the kinetochore. Spindle assembly checkpoint response: To our surprise
it was observed that depletion of inner or middle kinetochore proteins,
barring the fungal specific outer kinetochore Dam1 protein complex,
did not result in checkpoint arrest. Thereby these cells depleted of a
specific kinetochore protein proceeded through the cell cycle with
defective segregation machinery resulting in cellular destruction due to
faulty chromosome segregation. We speculate that this event is a consequence of disrupting the platform required for SAC signalling at the
kinetochore. We are currently testing this hypothesis.
Conclusions This study provides the first insights of the centromerekinetochore structure-function in a basidiomycete. Long repetitive
transposon-rich centromere organization, ordered kinetochore assembly, hierarchical dependency of kinetochore localization, and partial
opening of the nuclear envelope exhibited by this organism suggest
an evolutionary transition from ascomycetes to metazoan species.

71

Poster Abstracts

More work is needed for a definitive understanding of the interplay
of kinetochore architecture and the mitotic checkpoint which will
provide us clues as to the origins of aneuploidy conferring azole drug
resistance observed in this organism.

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 Brasılia, Brasılia, 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

acceleration of filament production. VEL3 is possibly the A. nidulans
VelC homologue, the least studied member of this family, and its role
is not yet well understood.
Conclusion The Velvet regulators in C. neoformans play no role in
pathogenicity and virulence, but are directly involved in the sexual
stage of the fungus. Although some of the findings are in agreement
with the literature for Ascomycetes, in C. neoformans Velvets proteins
may have other functions, given the structural differences in their
sequences. Two-hybrid assays are in progress to clarify how those
proteins interact.

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

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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.

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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 Δarg1(2)
and Δkcs1 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 Δasp1 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: 809–26.
2. Lev S. et al. Infection and Immunity 2013; 81: 1245–55.
3. Toffaletti DL. et al. Journal of Bacteriology 1993; 175: 1405–11.
4. Azevedo C, Saiardi A. Nature Protocols 2006; 1: 2416–22.

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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 Brasılia, Brasılia, Brazil; 2Universidade Catolica
de Brasılia, Brasılia, Brazil; 3Federal University of Sa~o Paulo,
Brasılia, 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

Figure 1. Ryp1 is necessary for virulence in the Galleria model. The
mutant strain was avirulent relative to wild-type and reconstituted
strains (P < 0.001).

Figure 2. Loss of Ryp1 causes flocculation on MOPS/Sabouraud.
Notice the pseudohyphal cells in the mutant. 209 objective, 7 days,
37°.

to identify genes regulated by it. Work with the Gti1 homologue is
algo in progress.

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

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Poster Abstracts

Africa. The population in Uganda is composed of one major clonal
genotype and several minor genotypes, some of which are singletons
in this analysis and some of which are minor clonal clusters. The
population in South Africa has more genotypes, including both
minor clonal clusters and singleton genotypes in this analysis, but no
major clonal clusters. In addition, clinical parameters of disease, such
as age/sex of the patient, CD4 counts, HIV viral load, cryptococcal
colony-forming units in the cerebral spinal fluid (CSF), cryptococcal
capsule titers in the CSF, opening pressure (via lumbar puncture, a
measure of fluid in the brain), mortality, time to death, and others,
were collected for each patient. Cytokine profiles in the CSF were
determined for a subset of patients upon entry into treatment. Of
these clinical parameters of disease, we found an association between
fungal genotype and mortality. Ultimately the genotype and mortality data can be used to select fungal strains for more in-depth
sequencing to identify novel virulence factors that differ between
high-mortality and low-mortality fungal genotypes.

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

ª 2014 The Authors
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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 Brasılia, 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 (1–178), a PEST domain(187–200), a bipartite nuclear
localization signal (NLS) beginning at position 180 and a nuclear
export signal between positions 132–136. 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

culminates in cell fusion and sexual spore production. A fluorescence
microscopy assay was performed to evaluate the defects related toloss
of VosA further:the inspection of unilateral crosses revealeddefective
clamp connections that do not seem to attach to adjacent hyphal
cells. Clamp connections are formed along the hyphae to ensure the
transfer of nuclei between cells during septation. Finally, no ramification was observed along the hyphae.
Conclusion This study connectsVosA, a member of Velvet family,
with the regulation of sexual reproduction in C. neoformansandcontributes to a better understanding of its role in the biology of this
pathogen. From the observations it can be concluded that VosA is a
positive modulator of mating controlling the formation of sexual
structures and sporulation, and a single gene copy is sufficient to
trigger mating, even if it is defective.

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 NetProphet’s 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: 174–188.
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: 449–460.
Wade SL, Auble DT. The Rad23 ubiquitin receptor, the proteasome
and functional specificity in transcriptional control. Transcription
2010; 1: 22–26.
Wade SL, Poorey K, Bekiranov S, Auble DT. The Snf1 kinase and
proteasome-associated Rad23 regulate UV-responsive gene expression. EMBO J 2009; 28: 2919–31.

ª 2014 The Authors
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Poster Abstracts

P094

P095

DNA repair and ubiquitination in virulence of Cryptococcus
neoformans
S. Verma, P. S. Shakya and A. Idnurm
School of Biological Sciences, University of Missouri- Kansas City,
Kansas City, MO, USA

Mitochondrial morphology and virulence in Cryptococcus
gattii
V. P. Shakya and A. Idnurm
University of Missouri Kansas City, Kansas City, MO, USA

Background Pathogenic organisms have evolved a number of ways
of avoiding the host defense systems to enable them to cause disease.
Protection of pathogen DNA from damage in response to environmental stresses, and especially during the course of infection, is one
of the important strategies adopted. The DNA repair gene RAD23 is
implicated in the virulence of Cryptococcus neoformans as knockout
for this gene is shown to be hypo-virulent (Liu et al, 2008). RAD23
has dual functions: it is involved in DNA repair and the protein sorting ubiquitination pathway (acting as ubiquitin receptor protein).
Another interesting aspect about RAD23 is its proposed role in transcriptional regulation in yeast. RAD23 is involved in regulation of
almost two-third of the UV regulated genes and almost one third of
all the Saccharomyces cerevisiae genes are mis-regulated in the RAD23
knockout strain (Wade et al, 2009 and Wade and Auble, 2010).
Aim 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.
Methods Rad23 has four domains, UBL, UBA1, XPC-B, UBA2. The
UBL, UBA1 and UBA2 domains are involved in protein sorting and
the ubiquitination pathway, and XPC-B has a role in DNA repair
Dantuma et al, 2009). We have generated strains with a complete
deletion of RAD23, and also complemented this mutANT by expressing RAD23 under its native promoter. We are performing individual
domain knockouts to test their role in different DNA damage stress
and in virulence. We are also checking these domain deletion strains
for their function in ubiquitination pathway.
Results and discussion 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, consistent with previous studies in a mice model.
Completion of these studies will shed 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: 174–88.
Dantuma NP, Heinen C, Hoogstraten D. The ubiquitin receptor
Rad23: at the crossroads of nucleotide excision repair and proteasomal degradation. DNA Repair 2009; 8: 449–60.
Wade SL, Auble DT. The Rad23 ubiquitin receptor, the proteasome
and functional specificity in transcriptional control. Transcription
2010; 1: 22–26.
Wade SL, Poorey K, Bekiranov S, Auble DT. The Snf1 kinase and
proteasome-associated Rad23 regulate UV-responsive gene expression. EMBO J 2009; 28: 2919–31.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Background Cryptococcus gattii has the potential to infect healthy
individuals along with the immunocompromised hosts [1]. The Vancouver Island outbreak starting in 1999 was caused by C. gattii, and
infection is prevalent mainly in Washington, Oregon and British
Columbia. In C. gattii pathobiology, the mitochondria likely play an
important role. Mitochondria are very dynamic organelles of eukaryotic cells that undergo fission and fusion to maintain function during
normal and stress conditions. Fission is required for the production of
new mitochondria while fusion of mitochondria helps the cell to cope
with stresses. In Saccharomyces cerevisiae the fission and fusion of
mitochondria is mediated through DNM1 and FZO1, respectively,
belonging to the GTPase family of proteins and are conserved in flies
and mammals [2]. The virulence of C. gattii is attributed to the
induction of tubular mitochondrial morphology upon engulfment in
host macrophages [3]. The individual punctate mitochondria may
fuse to form tubular mitochondria as a result of stress. The tubular
morphology correlated with higher intracellular proliferation in macrophages cell line.
Aim Our hypothesis is if we can impede the mitochondrial dynamics
of fission and fusion, it should affect the formation of tubular mitochondria and in turn virulence of C. gattii.
Methods To investigate the role of tubular morphology of mitochondria as a virulence trait of C. gattii, we have selected for study two
genes that regulate fission and fusion of mitochondria in eukaryotes
- DNM1 and FZO1. To test our hypothesis we are examining the
phenotypes of the two gene knockout strains. We are also investigating the role of the two genes in mitochondrial genome recombination
to address the issue of generation of recombinant mitochondria and
possible transmission of virulence traits. The generation of new hypervirulent stains through recombination events may also lead to
increased incidences of infection.
Results and discussion The knockout of DNM1 in C. gattii has
abnormal fused mitochondrial morphology. Moreover DNM1 knockout strains have functional defects in ER (endoplasmic reticulum)
protein sorting pathway as inferred from Tunicamycin stress studies.
Crosses of DNM1 knockout strains in CBS1930 and R265 strain
backgrounds was performed, and analysis of progeny is in progress
to see its role in recombination if any. The role of FZO1 is yet to be
verified and studies are in progress. We plan to perform the proliferation assays within macrophage cell lines and animal studies of the
knockout strains to investigate the effect of the two genes on the survival and virulence of C. gattii. Our studies will help to understand
the role of mitochondrial morphology in virulence and genome
recombination of C gattii.
References 1. Kronstad JW, Attarian R, Cadieux B, Choi J, D’Souza
CA, et al. (2011) Expanding fungal pathogenesis: Cryptococcus breaks
out of the opportunistic box. Nat Rev Microbiol 9: 193–203.
2. Hoppins S, Lackner L, Nunnari J (2007) The machines that
divide and fuse mitochondria. Annu Rev Biochem 76: 751–80.
3. Ma H, Hagen F, Stekel DJ, Johnston SA, Sionov E, et al. (2009)
The fatal fungal outbreak on Vancouver Island is characterized by
enhanced intracellular parasitism driven by mitochondrial regulation.
Proc Natl Acad Sci USA 106: 12980–85.

77

Poster Abstracts

P096

P097

Nucleotide sugar transporters: key players in capsule
glycan biosynthesis
L. X. Li, Z. Wang and T. L. Doering
Washington University St. Louis School of Medicine, St Louis,
MO, USA

Genotypic diversity within clinical and environmental
Cryptococcus neoformans var grubii population in India
A. Prakash,1 F. Hagen,2 S. Kathuria,1 J. F. Meis3 and
A. Chowdhary1
1
Vallabhbhai Patel Chest Institute, Delhi, India; 2Radboud
University Medical Centre, Nijmegen, The Netherlands and
3
Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands

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

Background Cryptococcus neoformans is an encapsulated, ubiquitous
environmental yeast that causes cryptococcosis, a potentially serious
disease that affects immunocompromised individuals, especially
patients with AIDS. The studies determining the genotypic diversity
in C. neoformans population is important to gain insightful knowledge
of this important pathogen. Microsatellite typing has become a popular molecular tool to study genotypic diversity in fungal pathogens
due to its high discriminatory power and reproducibility.
Aim To investigate genetic relatedness and antifungal susceptibilities
of a large collection of C. neoformans isolates from clinical and environmental sources in India collected during 2001–2010.
Materials and methods Four hundred and fifty three (224 environmental and 229 clinical) Indian Cryptococcus neoformans isolates were
subjected to Amplified Fragment Length Polymorphism (AFLP) and
genotyped by microsatellite typing based on nine markers specific for
C. neoformans variety grubii (serotype A) or variety neoformans (serotype D). In vitro antifungal susceptibility was determined for standard
antifungals using CLSI M27-A3 guidelines.
Results All 453 isolates were AFLP1/VNI genotype, representing C.
neoformans variety grubii serotype A. Microsatellite typing revealed
that the majority of isolates belonged to microsatellite cluster (MC)
MC3 (n = 183; 40.4%), followed by MC1 (n = 160; 35.2%), MC2
(n = 24; 5.2%), MC13 (n = 19; 4.1%), MC22 (n = 7; 1.5%), and others (8 MCs, n = 20; 4.4%). Forty (9.2%) isolates could not be linked
to a known MC from previous studies. MICs90 of AMB, FC, FLU, ITC,
VRC, POS and ISA were 1, 16, 8, 0.25, 0.125, 0.25, and
0.06 mg l1, respectively. Geometric mean MICs revealed that isolates in MC1 were significantly less (P < 0.0001) susceptible to ITC,
ISA, FC and AMB (P = 0.002) than isolates in MC3.
Conclusions The present study is the largest series of C. neoformans
var. grubii reported so far from a single country. Environmental isolates were genetically more diverse than clinical isolates comprising a
larger number of MC types than clinical MCs. Two microsatellite
types of C. neoformans var. grubii dominate in India and were uniformly distributed over clinical and environmental isolates. Fluconazole and flucytosine had high MICs (>8 mg l1) in 2% and 7% of the
isolates respectively, whereas the new azole isavuconazole exhibited
the lowest GM MIC of 0.06 mg l1.

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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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 1–6 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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

AIM-HII: a new method to rapidly identify agrobacteriummediated insertion sites in C. neoformans
S. K. Esher, J. A. Granek and J. A. Alspaugh
Duke University, Durham, NC, USA
Background Agrobacterium-mediated transformation (AMT) is a commonly used tool to generate mutations in a variety of plants and fungi,
including Cryptococcus neoformans. A. tumefaciens is a soil bacterium
which has the ability to deliver a portion of its own DNA to plants and
fungi. The transfer or T-DNA is carried on a plasmid and flanked by
border repeats. The border repeats define the insert DNA and the portion between them can be manipulated, commonly to include a selectable marker. The T-DNA integrates into the host genome, generally
yielding a single, random insertion. Insertional mutagenesis libraries
can therefore be generated and screened for phenotypes of interest, followed by identification of responsible mutation events. Traditionally,
the locations of T-DNA insertion in C. neoformans have been identified
by tedious PCR-based methods such as inverse PCR and TAIL-PCR,
creating a significant bottleneck, limiting the efficiency of the system.
Aim Develop a high-throughput method of insert identification in C.
neoformans AMT.
Method We have developed a high-throughput genomic sequencing
and analysis pipeline to facilitate the identification of insertions generated by AMT. Agrobacterium-mediated Insertional Mutagenesis
High-throughput Insert Identification (AIM-HII) combines batch sampling, whole genome sequencing, and bioinformatics analysis tools to
rapidly identify the locations of insertion. This method allows for the
identification of of 20–30 pooled mutants in the time it would take
to identify 1–2 mutants using traditional methods. In brief, a library
of C. neoformans AMT mutants was screened for phenotypes of interest. The genomic DNA of 28 mutants was harvested, pooled, and
sequenced. Whole genome sequencing data was generated, and reads
including the T-DNA insert sequence were extracted from the raw
data files. Next, the T-DNA sequence was clipped off of these reads,
yielding only C. neoformans genomic DNA sequence. Finally, this
resulting genomic sequence was aligned to the C. neoformans genome, and locations of insertion were identified.
Results With this approach in mind, we screened a library of
1500 ATM mutants for defects in growth on high pH, high salt,
and resistance to the detergent SDS. 28 mutants of interest were

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 2005–2012. 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 Mayer’s 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.

Table 1 Clinical outcome, granuloma pattern and yeast cell prolifera-

tion in 12 renal transplant patients with cryptococcosis.
Patient

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

*Death due to tuberculosis.
**Same patient with biopsies collected in different periods.
***Death due to bacterial sepsis.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Poster Abstracts

Table 2 Description of the histopathological findings in 12 renal
transplant patients with cryptococcosis.
Granuloma

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.
=
§

MGC = multinucleated giant cell.

LMN = linfomononuclear cell.

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 doesn´t 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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Therefore, it seems that the C. neoformans is, by some extent,
affected by the intracellular acidification, justifying the necessity to
analyze more deeply the connections between the acidification of
the phagosome, the increased extrusion and possible danger to the
fungus viability.
For the analysis, wild-type strain H99, mutant strain Dplb1 and
reconstituted strain Dplb1/rec were used in an interaction assay
with murine peritoneal macrophages previously stimulated with S.
cerevisiae cell wallb-glucan (Zymosan). The viability of the fungal
cells was accessed after 3 and 24 hours by CFU evaluation. The
acidification of the phagosome previously stimulated with Zymosan
was measured using confocal microscopy using the Lysotracker
probe.
Our preliminary results show that the stimulus of macrophages
with Zymosan was able to promote an acidification of the phagosome
and that this acidification was more constant and prominent in
phagosomes containing fungal cells. The interaction assay also demonstrated that the wild-type strain of C. neoformans has a diminished
intracellular growth when phagocytosed by an already activated
macrophage. Ultimately, the interaction assay utilizing the mutant
strain showed that further than inhibiting the growth, the activated
macrophage was able to kill the internalized fungus.
The capacity of the previously activated macrophage to inhibit the
intracellular growth and the acidification of the phagosome by this
activation lead to a hypothesis that the C. neoformans doesn´t absolutely thrive in an acidified vesicle environment. The results obtained
with the mutant strain corroborate with this hypothesis indicating
that the fungal cell that remains for a longer period inside the acidified phagosome has trouble in proliferate and survive, leading to his
death. Therefore, it may be possible that the decrease in the vesicular
phagosome pH is sensed somehow by the C. neoformans, triggering
the phenomenon of the extrusion.

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 Brasılia UnB, Brasılia, 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 µm 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

1b secretion and caspase-1 activation, by ELISA assay and cytometry
analysis of FAM-FLICA capase-1 probe, respectively.
The acapsular mutant of C. neoformans, but not the wild type,
induced high levels of IL-1b secretion. In parallel, caspase-1 was activated upon infection with acapsular but not with WT C. neoformans.
IL-1b secretion induced by C. neoformans was assessed in WT, Asc/,
Casp1/, Nlrp3/ and Nlrc4/ bone marrow-derived macrophages, showing IL-1b secretion to be dependent on NLRP3 inflammasome. In addition, inflammasome components were dispensable for C.
neoformans uptake or killing. The mechanisms underlying IL-1b processing and release were dependent on reactive oxygen species
(ROS), lysosomal destabilization, potassium efflux and Syk tirosine
kinase signaling. In this model IL-1b signaling is not required to
restrict intracellular yeast, but limited the rate of infection and yeast
growth. Moreover, the data show that acapsular C. neoformans activates the NLRP3 inflammasome and IL-1b secretion in a caspase-1
dependent manner and the mechanisms involved in this event rely
on ROS, lysosomal damage, potassium efflux and Syk signaling, leading to restriction of infection rate.
Furthermore, our results show that the addition of conditioned
medium of encapsulated C. neoformans is able to decrease inflammasome activation and IL-1b secretion through small molecules
(<1 kDa) secreted during infection rather than by the major capsule
component, GXM. Unfortunately, we were not able to identify the
specific molecule responsible for this process during the present study
but this activity was resistant to acid, alkali and the action of proteases and is sustained by the polar fraction.

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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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

Figure 1. LC3 immunofluorescence in macrophages infected with C.
neoformans or Paracoccidioides spp.

Results As shown in the figure, human THP-1 monocyte-derived
macrophages recruited LC3 to the vacuole containing C. neoformans
in a similar way to that previously described for murine macrophages. RAW264.7 cells infected with Paracoccidioides spp. yeast cells also
contained the fungi in autophagosomes regardless of the species (P.
lutzi or P. brasiliensis) or virulence in animal models (Pb18 – high
virulence; Pb265 – low virulence). Important controls that are not
shown in the figure confirm the results: (i) LC3 was recruited as
expected around C. neoformans in RAW264.7 cells and (ii) no similar
structures were found in experiments in which the primary LC3 antibody was omitted.
Discussion Recent publications have shown that LC3 is present on
the phagosomes containing C. neoformans in Drosophila S2 cells, as
well as in the murine cell lines RAW264.7 and J774.16. Our results
show that the same phenomenon occurs in human macrophages,
which can now be used to shed light in a more clinically relevant
fashion on why autophagy seems to protect the host in some cases
(Nicola et al., Infect Immun 2012) or benefit C. neoformans in others
(Qin et al., PLoS Pathog 2011).
Additionally, the observation that an autophagosome also forms
around phagocytosed Paracoccidioides spp. yeast confirms how widespread this phenomenon is, as it has already been observed with Saccharomyces cerevisiae, C. neoformans, Candida albicans and Aspergillus
fumigatus. This suggests that autophagy may be a universal response
to fungal infection.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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.
Methods Our approach included EV purification, analysis of amoeba
viability after exposure to EVs and methyl-alpha-D-mannopyranoside
(mannose), stimulation of A. castellanii with EVs, and mannose and
determination of the phagocytosis index and antifungal activity for
H99 strain, an encapsulated serotype A of C. neoformans.
Results Our initial results demonstrate that C. neoformans EVs with
a diameter range between 50 and 550 nm do not impact the viability of amoebae, but exposure to EVs increases fungal uptake and survival inside the environmental predator, compared to untreated
amoebae. Mannose treated amoebae also exhibited an increase in
fungal uptake and survival and were more susceptible to death during 24 h co-cultures with C. neoformans.
Discussion Our results to date demonstrate that fungal EVs impact
C. neoformans interactions with A. castellanii, indicating that C. neoformans vesicle release to the extracellular space may have developed in
response to interactions with environmental predators. Additionally,
our findings suggest that MBR can mediate fungal phagocytosis by
amoebae, opening a new field of investigation about evolutionary
steps that could culminate in intracellular pathogenic strategies for
C. neoformans to infect and proliferate inside mammalian
macrophages.

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

regulates transcription factors with key roles in immune response
and inflammation, and is predominantly found in cells of the nervous
and immune systems.
Aim Our overall goal is to identify host genes that influence engulfment of C. neoformans by mammalian cells and thereby alter the
course of infection. Our specific focus for this project is one of these
genes, which encodes CamK4. We aim to determine the specific
mechanism by which CaMK4 influences cryptococcal pathogenesis.
Methods To identify host genes involved in phagocytosis of C. neoformans, we used a previously established high-throughput imagebased assay (Srikanta et al., 2011) to screen a human macrophagelike cell line (THP-1) subjected to RNAi. We used the same assay to
perform secondary screens for specificity, and also performed followup studies using primary phagocytes, enzyme inhibitors, and animal
infections.
Results In preliminary studies we treated THP-1 cells with siRNA
targeting genes whose products are known to act in phagocytosis. As
expected, this resulted in decreased uptake of Cryptococcus. Based on
these results, we performed a primary RNAi screen targeting 901
human kinase and phosphatase genes in THP-1 cells; this identified
77 host factors that influenced fungal adherence and internalization.
To rule out non-specific alteration of host processes, we re-screened
these genes in parallel with both inert particles (latex beads) and C.
neoformans. For 25 of them, silencing consistently and significantly
altered uptake and/or adherence of cryptococcal cells while causing
minimal to no perturbation of inert particle (latex bead) phagocytosis. For a further subset of 8 genes, silencing specifically affected
cryptococcal uptake, but not uptake of the model yeast S. cerevisiae.
We focused on one gene, encoding CamK4, whose silencing significantly and specifically altered cryptococcal uptake. We found that
this result held in primary human and mouse phagocytes as well as
THP-1 cells. Excitingly, we also found that mice lacking this gene are
significantly less susceptible to C. neoformans in an inhalational
model of infection.
Discussion/conclusion CaMK4 plays an important role in cryptococcal pathogenesis and its absence significantly influences the outcome of infection. We are now investigating the precise mechanism
by which CaMK4 influences fungal:host interactions and thereby
cryptococcal disease. Our RNAi screen also yielded multiple other
host gene products in cryptococcal uptake by phagocytes, several of
which have never been implicated in fungal pathogenesis; these will
be the focus of future studies. Our exciting findings will shed light on
host aspects of cryptococcal pathogenesis and potentially other host:
microbe interactions as well.

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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Poster Abstracts

planes, but this is time-intensive and laborious. Several groups have
developed flow cytometry-based analysis methods (Voelz et al., 2010;
Alanio et al.; Nicola et al., 2011), but these are difficult to scale up to
the level required for screening. We have applied high content analysis to several aspects of cryptococcal biology relevant to pathogenesis,
including adherence to and engulfment by host phagocytes and capsule thickness.
Aim Our broad goal is to facilitate the investigation of cryptococcal
biology and pathogenesis by developing high-throughput imaging
and analysis protocols. The specific focus of this project was to optimize HCS assays for cryptococcal adherence, engulfment, and capsule
growth.
Methods We compared GE IN Cell, ImageXpress, and BioTek Cytation3 automated imagers with manual light microscopy, followed by
analysis through open-source, collaborator-developed, and proprietary image processing software. Cryptococcal phagocytosis, adherence, and capsule growth were assessed.
Results We examined multiple imaging and analysis tools, assessing
image quality, analysis quality, cost, and speed, and have developed
optimized HCS assays for cryptococcal adherence, uptake, and capsule growth. We identified several combinations that are particularly
powerful for high-throughput cell interaction assays. The IN Cell and
Cytation3 are excellent imaging options, and the GE Developer software is most powerful for complex analysis of captured images.
Discussion/conclusion Image and analysis quality must be balanced with efficiency and cost, and the best HCS option depends on
the requirements of the investigator and the assay. However,
through this examination of high-throughput screening methods for
C. neoformans, we have developed criteria that may be used to assist
the decision-making process. Host genome screens with RNAi have
proven informative, and the availability of cost-efficient, rapid, and
accurate systems will accelerate discovery of host and cryptococcal
factors important in pathogenesis.

identified as C. gattii; the remaining 80 (49.7%) were C. neoformans.
C. gattii isolates were recovered from samples of Eucalyptus spp,
Corymbia ficifolia, Terminalia catappa (almond tree) and Ficus spp
decaying wood. Sixteen (19.7%) isolates, recovered mostly from Ficus
spp., were typed as VGI; 43 isolates (53.1%) recovered from Eucalyptus trees, as VGII, and 22 (27.2%) recovered from C. ficifolia, almond
trees and Ficus spp., as VGIII isolates. C. neoformans isolates were
recovered from Ficus spp., Eucalyptus spp. and bird droppings. Molecular type VNI was observed in 78 (97.5%) isolates recovered from all
the above-mentioned sources; the remaining two isolates recovered
from Ficus spp. were VNII. In two samples recovered from Ficus spp.
both C. neoformans and C. gattii were found. CFU were estimated
between 0.5 9 102 and 11.4v104 CFU g1 of sample.
Discussion/conclusions In this brief observation, it is clear that C.
neoformans/C. gattii are able to survive in environmental samples
stored at room temperature either as soil/plant material or as filtrates
from the samples themselves. When re-processing the samples previously collected during our study, we established that both species
can be isolated from the same sample and tree species; this shows
the importance of isolating and characterizing as many colonies as
possible from those suspected to be C. neoformans/C. gattii in a sample. Specific environmental conditions are necessary for species to
exist in a particular environment, obtain nutrients, elude predators
and reproduce; therefore, their survival and abundance in a niche
depend on a series of factors that are essential for their viability. In
this study we found that agents of the C. neoformans/C. gattii species
complex can survive in stored samples for as long as nine years. Further studies are needed to determine how virulence factors express in
these isolates and compare their expression levels with those of the
strains initially recovered.

P115
P114
Viability of Cryptococcus neoformans/Cryptococcus gattii
in long term stored environmental samples from Colombia
P. Escandon and E. Castan˜eda
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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Anatomy of an outbreak: recombining clonal clusters
comprise the VGII C. gattii population
R. B. Billmyre,1 D. Croll,2 W. Li,1 P. A. Mieczkowski,3
J. Kronstad2 and J. Heitman1
1
Duke University, Durham, NC, USA; 2University of British
Columbia, Vancouver, BC, Canada and 3University of North
Carolina, Chapel Hill, NC, USA
Background Over the past 15 years, an outbreak of the typically
tropical and subtropical pathogen Cryptococcus gattii has developed in
the temperate Pacific Northwest of the United States and neighboring
British Columbia in Canada. This outbreak consists of three clonal
populations, designated VGIIa, VGIIb, and VGIIc. Robust populations
of C. gattii with high similarity to the VGIIb clonal cluster exist in
Australia and related isolates have been reported from South America, while VGIIa and VGIIc appear to be more unique to the Pacific
Northwest. Recent studies implicate South America as the ancestral
origin of C. gattii, however it is unclear where the more proximal origin of the PNW outbreak is located.
Aim We aimed to utilize whole genome sequencing in place of multilocus sequence typing to gain increased resolution to both (i) distinguish members of clonal clusters of the C. gattii population from each
other and (ii) determine the relationships and origins of these isolated
clonal populations.
Methods We utilized whole genome sequencing with the Illumina
platform, in conjunction with both reference-based assembly to the
R265 VGII reference genome and de novo assembly using velvet. To
distinguish the origin of the outbreak, we have sequenced VGIIb and
related isolates previously identified from both Australia and the
Caribbean. In addition, we have sequenced a number of VGII isolates
of all three clonal lineages from the PNW and incorporated previously published genomes from the CDC.
Results Whole genome sequencing has allowed dramatically
increased resolution of the VGIIa, b, and c groups from the Pacific
Northwest. Interestingly, these isolates are primarily differentiated

85

Poster Abstracts

by private polymorphisms and show evidence of a strong founder
effect, followed by radiation. Additionally, the VGIIb group shares
substantially more similarity with VGIIb isolates from Australia than
with a Caribbean Islands isolate, and the Australian isolates cluster
within the same group as those from the PNW. Furthermore, by
sequencing the entire genome, we have discovered that the majority
of VGII isolates share various identity islands throughout their genomes. These shared genomic islands are indicative of a robustly
recombining population, with common ancestry. Whole genome
allele compatibility tests provide robust evidence for recombination
in the population.
Discussion/conclusion The population structure of C. gattii
revealed by whole genome analysis is consistent with intermittent
mating, followed by periods of clonal expansion. This is most obvious
in the Pacific Northwest where three clonal groups have undergone
rapid expansion and are causing an outbreak. However, these three
isolates share regions of identity with each other, suggesting that
sexual exchange and recombination were important factors in the
origins of each outbreak lineage. Each of these isolates is MATalpha,
and MATa isolates are absent in the outbreak populations and
underrepresented globally. This suggests that unisexual reproduction
may be contributing to the recombination observed in the population, or isolates are undergoing sexual reproduction with MATa isolates in an under-sampled environmental niche and disseminating
into sampled locations. These are not mutually exclusive processes,
and both a-alpha bisexual and alpha-alpha unisexual reproduction
may contribute to both diversify and amplify genotype within the
population.

0.02–0.94 mg l1. The MIC range for the 170 remained isolates
was: FCL-MIC 0.12 to 16 mg l1 and for AmB-MIC 0.12–1 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 5–9 µm 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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Poster Abstracts

Methods Samples were collected between September 2012 and
August 2013 from trees (leaf, bark, flowers, soil and fruits) and
bird droppings in areas with a high density of trees and birds. Five
hundred and seventy seven avian droppings samples were collected,
454 of Columba livia (pigeons), 118 Egretta thula (Snowy Egret )
and 5 Bulbucus ibis (Cattle Egret) from churches, roofs, dovecotes
and parks of the historical area in Popay
an and its rural area, and
454 samples of trees. Phenotypic identification of the C. neoformans/C. gattii species complex was based on growth of brown colonies on Niger Seed Agar (NSA) and biochemical characteristics;
genotypic characterization was done using PCR Fingerprinting
(GTG)5 and Restriction Fragment Length Polymorphism (RFLP) of
the gen URA5.
Results Out of 1,031 samples of bird droppings and trees, 180 were
positive, 162/577 (28%) of pigeon droppings and 18/454 (4%) from
trees samples, recovering 515 isolates, 441 (85.6%) from pigeon
droppings and 74 (14.4%) from tree samples. C. neoformans var. grubii molecular type VNI was found in 99.3% and VNII in 0.7%. C. gattii was not recovered. Most of the isolates recovered from pigeon
dropping were sampled in churches, roofs and dovecotes.
Discussion C. neoformans was recovered, confirming the same
results obtained in others studies in Colombia, and reinforcing the
predominance of this species in environmental samples. Popay
an is
considered a region with a lower incidence of cryptococcosis than
other cities in Colombia, although a high environmental prevalence
of the fungus was found in droppings from places with high density of pigeons, being this a risk for immunosuppressed and immunocompetent patients, reinforcing that pigeon excreta is a favorable
environment for C. neoformans. Despite these significant findings,
there is still a lack of registration for cryptococcosis, not only in
Popay
an but in the whole country, where the disease is not of
compulsory notification, therefore, more strategies have to been
undertaken to improve the passive surveillance of cryptococcosis in
Colombia.
(Colciencias Project code: 2011-3600115683).

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. Medellın
(n = 1515), located Northwest, the annual rainfall is 1656 mm
with an average temperature of 23 °C. Popay
an (n = 1598),

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

located Southwest, the annual rainfall is 1991 mm with an average temperature of 18 °C.
The samples were collected between September 2012 and 2013
and processed with conventional techniques, fungus identification
and the variety were determined using biochemical testing, molecular type was determined by RFLP-URA5 and PCR fingerprint.
Climatic data (relative humidity, sun light, temperature and rainfall) was supplied by the National Institute of Hydrology, Meteorology
and Environmental Studies IDEAM.
Results From a total of 4501 samples processed, 238 (5.2%) were
positive, of which 232 (97.4%) were positive for C. neoformans and 2
(0.8%) for C gattii, 4 (1.6%) samples were positive for both C. neoformans and C. gattii: 3.8% (17/446) were positive in Bogot
a, 2% (10/
495) in C
ucuta, 2.8% (14/495) in Cali, 0.8% (13/1515) in Medellın
and 12.3% (198/1598) in Popay
an.
An 82% of positive samples were recovered from bird droppings (C.
neoformans) and 18% correspond to tree samples (C. neoformans/ C gattii). C. neoformans was recovered more frequently in dry bird droppings,
while C. gattii was found mainly in samples of bark, soil and leaves of
trees such as Eucalyptus spp., Ficus spp., T. catappa and Acacia spp.
As for now, molecular type VNI (n = 84) has been associated to C.
neoformans, C. gattii has been typed as VGII (n = 2) and VGIII
(n = 4); overall, the majority of positive samples 63.4% (n = 151)
were recovered in the dry season for the cities of Popay
an, Cali and
C
ucuta, while 8.4% (n = 20) were recovered in the rainy season in
Bogot
a and Medellin.
Further analyses are in progress to determine the association on
the isolation of the fungus with climatic conditions for each city.
Discussion The yeast was isolated mostly from avian droppings,
confirming this habitat as an important reservoir for this opportunistic pathogen; at the same time, VNI is identified as the most frequent
molecular type in C. neoformans.
Our data show that the highest positivity is obtained in the dry
season, and that the fungus was obtained more frequently from bird
droppings than from trees.
Ecological studies are relevant in the epidemiology study of this
pathogen; therefore the continuity of the research is important for
ecological studies which may allow us to determinate areas where
the fungus can find favorable conditions to develop in Colombia.
(Colciencias Project code: 2011-3600115683).

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

were stained with DAPI. To explore the viability of both Cryptococci
after infection, astrocytes were lysed and intracellular yeasts were
recovered and cultured in Sabouraud dextrose agar. The yeast
growth was assessed by colony forming units (CFU) after 48 hours of
culture. Statistical analysis was performed using non-parametric
tests.
Results The percentage of astrocyte infection with C. gattii was
35.3% (SD  19.5%) and 30.1% (SD  21.9%) with C. neoformans,
demonstrating no statistical difference (N = 13, P = 0.394, Mann–
Whitney U). There was similarly no difference in the percentage of
HLA-ABC expression (nearly 100%) or mean fluoresce intensity
(MFI) comparing fungi-infected cells with non-infected astrocytes.
The HLA-DR expression was higher in cells infected with C. neoformans 39.4% (SD  17.7%) than C. gattii 20.1% (SD  9.3%). Both
these populations had different expression than control cells 0.44%
(SD  0.1%) (P = 0.004, Kruskal–Wallis). Fluorescence microscopy
analysis demonstrated more live yeasts in C. neoformans-infected astrocytes than cells infected with C. gattii. This trend was confirmed
with the count of intracellular yeasts recovered after 48 h of culture,
C. neoformans 1943 CFU ml1 (SD  133.2) and C. gattii 1340 CFU
ml1 (SD  175.2).
Discussion/conclusion There was no difference in the percentage
of astrocyte infection by both species, but C. neoformans induced a
higher expression of HLA-DR than C. gattii. Since astrocytes can be
involved in antigen presentation to T-cells, these results suggest
that infection with C. neoformans could activate T-cells locally. As
more live yeasts in C. neoformans-infected astrocytes were recovered
than cells infected with C. gattii, C. neoformans could have different
virulence mechanisms that allow its survival in human glia-derived
cells.

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%

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Poster Abstracts

survival rate in the macrophage-restricted STAT1 KO mice compared
to a 100% survival rate in the control mice, indicating that STAT1
signaling in macrophages is required for protection. Macrophages
from macrophage-restricted STAT1 KO mice have an increase in
gene expression for alternative macrophage activation (M2) marker
Arg1, but no change in gene expression for M1 macrophage marker
iNOS compared to macrophages from control mice. This indicates an
iNOS/Arg1 ratio in STAT1 deficient macrophages that favors an M2
phenotype which is associated with a non-protective immune
response. Furthermore, compared to STAT1 sufficient macrophages,
STAT1 deficient macrophages have an increase in intracellular cryptococci. This coincided with a decrease in production of NO, a
byproduct of M1 macrophages that has been shown to have antimicrobial activity. The inverse relationship of intracellular yeasts to
NO production suggests that the macrophages are phagocytizing but
not killing the yeast. Additionally, inhibition of NO production in pulmonary macrophages cultured from inoculated control mice is
accompanied by a diminished capacity for control of cryptococcal
growth, indicating that NO is required for anti-cryptococcal activity
of macrophages.
Conclusions Overall, data indicate that STAT1-mediated M1 macrophage activation is essential for the induction of protective immune
responses to C. neoformans. Importantly, production of NO by M1
macrophages is critical for fungal growth suppression. These studies
provide the first evidence of a specific effector cell population and
mechanism for protection against pulmonary cryptococcosis.

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 People’s 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:0.99–2.64). 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: 1.13–3.81). 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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

group (51/103 vs. 36/53, P = 0.040, OR = 0.49, 95% CI: 0.24–
0.97).
Conclusion Our study firstly shows that the heterozygote for
rs11045418 is associated with the susceptibility and disease procedure of cryptococcosis, which suggests that Dectin-2 might play an
important role in this infection.

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): 299–310).
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):
459–70).
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): 299–310).
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 8–12 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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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 Muller’s 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 Muller’s 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 Muller’s 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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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

Immunoproteomics and immunoinformatics analysis of
Cryptococcus gattii: novel candidate antigens for diagnosis
L. Soares Martins,1 H. Monteiro Andrade,2 M. Henning
Vainstein,3 B. Wanke,4 A. Schrank,3 C. B. B. Bemfica Balaguez,3
P. R. S. Ribeiro Santos,3 L. S. Santi,3 S. F. P. Fonseca Pires,2
A. Socorro Silva,1 J. A. Fonseca Castro,1 H. Alves da Silva
Machado,5 R. Melo Santos Serpa Brandao1 and S. Jamil Hadad
Monte1
1
Universidade Federal do Piauı, Teresina-Piauı/Brasil, Brazil;
2
Universidade Federal de Minas Gerais, Belo Horizonte, Brazil;
3
Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil;
4
Fundacßa~o Oswaldo Cruz, Rio de Janeiro, Brazil and 5Instituto de
Doencßas Tropicais Natan Portella, Teresina, Brazil

Impaired monocyte production of TNF-alpha and reduced
HLADR expression on CD14+ CD16+ monocytes are
associated with mortality in cryptococcal meningitis
J. E. Scriven,1 L. Graham,2 C. Schutz,2 R. J. Wilkinson,3
D. R. Boulware,4 B. Urban,1 D. G. Lalloo1 and G. A. Meintjes2
1
Liverpool School of Tropical Medicine, Liverpool, UK; 2University
of Cape Town, Cape Town, South Africa; 3Imperial College,
London, UK and 4University of Minnesota, Minneapolis, MN,
USA

The potential for C. gattii to cause illness in immunocompetent
patients and its rapid spread worldwide justify the implementation of
a public health effort to increase the awareness of both the public
and healthcare professional. Information from the recently published
C. gattii genome, combined with data obtained via proteomic
approaches, has created opportunities for the development of diagnostic tools and therapeutic targets in the context of cryptococcosis
caused by C. gattii. The development of such tools has been improved
by the production of antigens, which increases the range of alternative tests for immunoassay-based pathogen detection. In this study,
we identify both immunoreactive proteins of C. gattii and predicted
B-cell epitopes for their potential use as antigens in new serologic
tests. We combined 2D gel electrophoresis, immunoblotting and mass
spectrometry to identify immunoreactive proteins from four strains of
C. gattii genotype VGII (CG01, CG02, CG03 and R265). Next, we
screened the identified proteins to map B-cell epitopes. We identified
68 immunoreactive proteins and highlight that only six of them
were reactive in three isolates simultaneously without a reaction
for negative sera. Due to their antigenicity in different strains, we
believe that these proteins would be most promising for testing as
antigens. Taken together, assessing the specificity of these antigens
will be the next step to validate them for the diagnosis of cryptococcosis. The major finding of this work was the identification of C. gattii
proteins recognized as molecular targets by antibodies produced by
patients with cryptococcal meningitis. In addition, we mapped 374
peptides potentially targeted by B cells. Considering the evolutionary
relevance of the identified proteins, we may speculate that they could
be used as the initial targets for recombinant protein and peptide
synthesis aimed at the development of immunodiagnostic tools for
cryptococcosis. In conclusion, the applied combination of immunoproteomics and immunoinformatics methods was demonstrated to
be a specific and powerful tool for identifying novel antigens from
mycological pathogens. In addition, we identified potential antigens
that may be used for the development of more accurate serological
diagnoses in human cryptococcal meningitis. The immunogenic proteins and Bcell epitopes identified herein need to be investigated as
antigens in serological tests to diagnose cryptococcal infection. These
proteins could present cross-reaction with cryptococcal infection
caused by other species. Although these proteins can react with multiple fungal species, some of these fungi may not be present in the
patient. However, this is the first important step to selecting new target antigens to facilitate the immunodiagnosis of cryptococcosis.

Introduction Cryptococcal meningitis (CM) remains a significant
cause of death amongst individuals with HIV-1 infection. Immune
correlates of protection are lacking. Animal and in vitro studies suggest macrophage and monocyte activation plays a key role in determining outcome from infection.
Aims To determine whether abnormal monocyte activation is associated with poor outcome in individuals with HIV-associated cryptococcal meningitis.
Methods Patients with HIV-1-associated CM were recruited from
hospitals in Cape Town. Initial anti-fungal therapy was IV amphotericin 1 mg kg1 and oral fluconazole 800 mg daily for 14 days.
Monocyte sub-populations and their activation phenotype were characterized in whole blood at presentation using flow cytometry. Intracellular cytokine responses were assessed following 6-hour
stimulation with lipopolysaccharide (TLR4 agonist) and R848
(TLR7/8 agonist). Comparisons in baseline monocyte activation were
made between subjects who survived to 14 days and those who died.
Student’s T-test was used for normally distributed variables; non-normally distributed variables were either log transformed prior to parametric testing or a Mann–Whitney U test was used.
Results (see table) Fifty nine subjects were enrolled; median CD4
count was 34 9 106/l and 14-day mortality was 24%. At presentation, non-survivors had a significantly lower proportion of circulating
monocytes, significantly reduced expression of HLA-DR on inflammatory monocytes (CD14+CD16+), and a non-significant trend towards
increased surface expression of CD163 on inflammatory monocytes
(CD14+CD16+). Non-survivors were also found to have significantly
higher baseline serum concentrations of soluble CD163 and C-reactive protein (see table). In addition, monocytes from non-survivors
had impaired responses to lipopolysaccharide, with significantly fewer
cells producing TNFa. No differences in monocyte responses were
seen following TLR7/8 stimulation (see Table).
Conclusions This study suggests impaired monocyte function may
be an important factor determining poor outcome in cryptococcal
disease. Despite serum markers suggesting significant baseline
immune activation, circulating monocytes from patients who died
from cryptococcal meningitis had a less pro-inflammatory phenotype
and appeared to be functionally unreactive. Reversal of this defect
may be a potential immune-based therapeutic strategy in HIV-associated cryptococcosis.

Table 1 Differences in immune parameters at presentation between

patients with CM who died before day 14 and those who survived.

6

CD4 count (x10 /L) (median, IQR)
Monocytes, (%WBC) (mean, 95%CI)
HLADR MFI (CD14+CD16+ Mo)
(mean, 95%CI)
Log10 CD163 MFI (CD14+CD16+ Mo)
(mean, 95%CI)
%TNFa +ve Mo (after LPS stimulation)
(mean, 95%CI)
%TNFa +ve Mo (after R848 stimulation)
(mean, 95%CI)
sCD163 (ng/ml) (median, IQR)
CRP (mg/L) (median, IQR)

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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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): e00158–11.

Methods C57BL/6 mice were infected by the intratracheal route
with Cneo (strain 52D). Two cohorts of mice received either: (i) antiIL-10 receptor antibody or (ii) an isotype control (0.5 mg per 200 ml
PBS by the intraperitoneal route) at 3, 7, and 10 days post-infection
(dpi) to assess the effect of IL-10 blockade during the developing
phase of infection. Two separate cohorts of mice received the same
antibodies at 22, 25, and 28 dpi to evaluate the effect of IL-10 blockade during the established phase of infection. At 35 dpi, all four
cohorts of mice were euthanized and evaluated for the following
immune parameters: (i) lung and brain fungal burden (by CFU
assay), (ii) the percentage and total numbers of numerous lung leukocyte subsets (by specific antibody staining and 8–10 parameter
flow cytometric analysis), and (iii) CD4+ T cell subsets (by intracellular cytokine staining and flow cytometric analysis).
Results Blockade of IL-10 signaling during either the developing
phase or during the established phase of persistent cryptococcal lung
infection significantly improved fungal clearance at 35 dpi. Improved
clearance was associated with enhanced accumulation of CD11b+
dendritic cells and increases in the number of Th1 and Th17 cells.
IL-10 blockade also enhanced the total number of exudate (but not
alveolar) macrophages in the lungs.
Conclusion Blockade of IL-10 signaling promotes the clearance of
cryptococcal lung infection, likely through mechanisms involving the
DC-mediated enhancement of Th1 and Th17 immune responses and
the subsequent accumulation of fungicidal exudate macrophages.
Thus, IL-10 signaling blockade represents an attractive therapeutic
target in patients with newly-acquired or established fungal lung
infection.
Funding: Biomedical Research & Development Service, Dept. of
Veterans Affairs, Career Development Award (JJO) and Merit Review
Award (MAO). NIH T32 Training Grant (trainee BJM).

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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Background The cytokine tumor necrosis factor alpha (TNF-a) is
recognized as being critical to mount an effective immune response
to C. neoformans (C. neo): C. neo strains that induce less host-derived
TNF-a are more pathogenic, AIDS patients that do not produce substantial TNF-a have poor prognosis against C. neo infection, and
patients undergoing TNF-a monoclonal antibody therapy are more
susceptible to C. neo infections. Th1/Th17-polarized T cell responses
support clearance/containment of C. neo, while Th2 polarization is
non-protective. Dendritic cells (DC) that prime a Th1/Th17 response
display DC1/classical activation phenotype characterized by production of Th1/Th17-driving cytokines and high levels of iNOS expression. DC2 that prime a Th2 response are reported to display features
of an alternative activation phenotype including increased expression
of Arginase1, Fizz, and YM-2 and increased production of Th2/regulatory cytokines. While TNF-a has been demonstrated to promote DC
maturation, it is unknown how TNF-a signaling affects the balance
between these DC1/DC2-activation phenotypes and how TNF-a
affects DC-phenotype stability during C. neo infection.
Aim To determine the mechanisms by which early TNF-a signaling
promotes generation and stability of protective Th1/Th17 response to
C. neo, we tested whether TNF-a signaling is necessary to generate
stable DC1, thereby supporting the development and long-term stability of a protective Th1/Th17 response.
Methods CBA/J mice (resistant) received TNF-a antibody or isotype
control antibody injected into the peritoneum at the time of

93

Poster Abstracts

intratracheal infection with C. neo 24067. This treatment neutralizes
TNF-a for the initial 7 days of infection with TNF-a levels recovering
by d14. However, the immune response remains non-protective past
d28. We harvested mice at d7, d14 and d28 post-infection and characterized DC and T cell activation profiles by qPCR and flow cytometry and assessed the fungal lung burden and brain and spleen
dissemination. We then investigated how TNF-a signaling affects DC
activation and stability of DC1 or DC2 phenotypes. We generated
bone marrow-derived DC (BMDC) and subjected them to 24 hours of
interferon-gamma (IFN-g) stimulation with or without TNF-a, then
switched the cytokine stimulation to interleukin-4 (IL4) for 24 h and
assessed DC activation and phenotype markers by qPCR and flow
cytometry. Lastly, we investigated the link between DC epigenetic signatures and phenotype stability by measuring expression of histone
modification enzymes and immunoassays for activating and repressing histone methylations.
Results In vivo we found that the robust Th1/Th17 response in control mice was replaced by a non-protective response with many Th2
features and fluctuating cytokine levels in the TNF-a antibody-treated
infected group. The DC1 signature in CD11c+ cells was suppressed
throughout infection (d28), and similar suppression could be detected
in myeloid DC precursors (CD11b+) early in the TNF-a antibody-treated mice (d7). Furthermore, the activating histone modification
H3K4 trimethylation was diminished in myeloid precursor cells (d7)
and lung DC (d7 and 28) of TNF-a antibody-treated mice, suggesting
decreased activation of certain genes at the epigenetic level. Levels of
the H3K27 methyltransferase EZH2 were also decreased in the DC
of TNF-a antibody-treated mice, suggesting less epigenetic silencing
of certain genes. In vitro, the combination of TNF-a and IFN-g reduce
the induction of the DC2 marker Fizz and DC2 cytokine Interleukin10 and increase DC1 cytokines Interleukin-12a and 12b when the
DC are switched to IL4.
Discussion We conclude that TNF-a contributes to programing DC
to become stable DC1, which promote and sustain protective Th1/
Th17 responses to C. neoformans. Our results suggest that TNF-ainduced chromatin modification in DC precursors and lung DC support DC1 stability. Studies on the effect of TNF-a on the epigenetic
signatures profiles, the specific genes epigenetically silenced or activated, and how this relates to pulmonary DC1 stability are currently
underway.

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

Background The most important virulence factor of the fungal
pathogen Cryptococcus neoformans is a polysaccharide capsule that
surrounds the cell. The size of the capsule is variable and it increases
during infection. However, the pathways and genes involved in this
process are unknown.
Aim In this work, we investigated the relationship between capsule
enlargement and cell cycle progression in C. neoformans. We hypothesized that capsule enlargement was regulated by the cell cycle for the
following reasons: (i) During capsule growth, there is a correlation
between the size of the capsule and the size of the cell body, so we
believe that the factors that regulate the cell body growth (which
occurs in G1) also regulate the capsule size; and (ii) We argue that
capsule enlargement stops during G2-M because in these phases, the
accumulation of new polysaccharide at the capsule might interfere
with the emergence and separation of the bud from the mother cells.
For these reasons, we hypothesized that capsule growth occurs
mainly in the G1 phase of the cell cycle.
Methods We used flow cytometry, time-lapse microscopy and cell
cycle inhibitors to study if cell cycle changed during capsule enlargement. Furthermore, we characterized a mutant strain lacking a gene
that encodes a G1-S cyclin (cln1). We studied capsular phenotypes of
this mutant and its reconstituted strain both in vitro and in vivo
using the invertebrate model Galleria mellonella.
Results Capsule growth occurred primarily during the G1 phase.
Real-time visualization of capsule growth demonstrated that this process occurred before the appearance of the bud and that capsule
growth arrested during budding. Besides, benomyl, a cell cycle inhibitor that arrests the cells in G2/M, impaired capsule growth. However,

Differences in host response toward C. neoformans and
C. gattii
E. Sionov, D. L. Barber, K. D. Mayer-Barber, Y. C. Chang and
K. J. Kwon-Chung
National Institutes of Health, Bethesda, MD, USA
Background Cryptococcosis is one of the most important AIDS-associated opportunistic infections with an estimated global burden of
one million cases with 600 000 deaths annually. Although both
Cryptococcus neoformans and C. gattii are the etiologic agents of cryptococcosis, the AIDS associated infection is mostly caused by C. neoformans and only occasionally by C. gattii even in the C. gattii
endemic regions. C. gattii apparently affects immunocompetent hosts
more often than immunocompromised hosts, hence it is considered
as a primary pathogen. To understand the differences in the hostpathogen interactions of the two species, we compared the pathogenicity of the two species in mice with defects in the immune system
critical for defense against infectious agents. During acute infection
with HIV, several antiviral and immunoregulatory cytokines, particularly type I interferon (IFN), are known to be up-regulated and type I
IFN has been used in therapy for several viral infections and malignancies. We investigated the effect of exogenous induction of type I
IFN during infection by the two cryptococcosis agents.

94

P135
Capsule growth in Cryptococcus neoformans is
coordinated with cell cycle progression
R. García-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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Poster Abstracts

rapamycin, which causes G1 arrest, increased capsule size. The cln1
mutant showed a delay in S entry, and also an increased capacity to
enlarge the capsule, both in vivo (using Galleria mellonella as host
model) and in vitro. The cln1 mutant displayed morphological alterations, such as a different WGA binding pattern and aberrant forms
during budding. Besides, it was avirulent at 37 °C, which correlated
with growth defects at this temperature.
Discusion/conclusion Our results support a model in which capsule enlargement is a differentiation process that causes G1 arrest.
Our model also suggests that cell cycle progression in these conditions does not occur until the capsule has reached a certain size. In
addition, the reduced virulence of cln1 mutant opens the possibility
to design antifungal strategies using cell cycle regulatory elements as
targets.

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; 2Fundacßa~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 Brasılia, 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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

95

Poster Abstracts

Aim To investigate the virulence potential of C. gattii strains of all
major molecular types and different MLST genotypes, by comparing
their pathogenesis and assessing the cryptococcal virulence factors in
an invertebrate model of infection.
Methods Galleria mellonella larvae were inoculated with ten globally
selected strains per molecular type, which were previously studied by
MLST. Larvae were checked daily, deaths were recorded and survival
plots were constructed. Cells of all C. gattii strains were isolated from
the larvae after inoculation and cell and capsule sizes were measured. Cell and capsule sizes of the strains before inoculation were
also measured. Melanin production and growth at 37 °C was determined in all strains.
Results Among the studied strains, one VGII, one VGIII and one
VGIV strains were more virulent (P < 0.05) than the highly virulent
Vancouver Island outbreak strain VGIIa (CDCR265); eleven strains
(four VGI, two VGII, four VGIII and one VGIV) had similar virulence
(P > 0.05) and twenty-five (six VGI, six VGII, five VGIII and eight
VGIV) were less virulent (P < 0.05). Overall, strains of the molecular
type VGIV showed to be the leats virulent. Cell and capsule size of all
strains increased drastically during larvae infection (P < 0.001). Melanin production was directly associated with the level of virulence of
the strains: highly virulent strains produced more melanin that low
virulent strains (P < 0.05). All strains had the ability to grow at
37 °C and there was no difference among the strains regarding their
growth rate (P > 0.05).
Discussion/conclusion The results indicate that all major C. gattii
molecular types exhibit a range of virulence, with the potential of
some strains amongst them being highly virulent. As reported in C.
neoformans, the present study supports the fact that fungal virulence
factors like capsule, melanin and growth at physiological temperature, which are involved in mammalian pathogenesis, are also
needed for G. mellonella infection and killing. This study highlights
the importance to determine the major molecular type/genotype of
the isolates as part of the epidemiology of Cryptococcus and cryptococcosis and warrants further studies to identify specific genotypic
traits of highly virulent C. gattii strains that are associated with their
virulence and which are relevant for the development and progress
of cryptococcosis.

P139
Capsule synthesis occurs in a non-polarized manner and its
growth depends on mitochondrial activity
N. Trevijano-Contador, S. Landin, R. Garcıa-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 Brasılia, 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

Figure 1. Morphology of gti1D::HYG colony grown on Sabouraud
medium at 30 °C for 48 h. The mutant strain grows as donutshaped colony.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Poster Abstracts

functions, these factors influence secondary metabolism, conidiation,
matting, dimorphic transition and pathogenesis in fungi ranging
from plants to humans pathogens. However, nothing is known about
their function in basidiomycetes, despite the fact that their homologues have been annotated in genome projects.
Aim In order to fill a gap in our knowledge of regulatory processes
in C. neoformans, we have deleted the GTI1 homologue and analyzed
the phenotype of the mutant.
Methods Identification of the target gene: The Gti1 homologue was
found by using the amino acid sequence of Gti1 from Schizosaccharomyces pombe as query for the BLASTP2 of the Broad Institute H99
C. neoformans genome database. The retrieved best hit,
CNAG_05835.7, was provisionally named Gti1. Deletion and reconstitution: The gti1::HYG strain was generated by double-joint PCR
followed by biolistic transformation of the H99 wild type as described
by Kim et al., 2009. The reconstituted strain was generated by transforming the gti1::HYG strain with the Gti1 gene a long side pJAF1
plasmid containing the Neor resistance mark. In both cases transformants were selected in YPD solid medium containing hygromicin or
G418, respectively. Single insertion into to appropriated locus was
confirmed by Southern Blot or PCR. Phenotypic analysis: The mutant
and reconstituted strains were tested in vitro for common phenotypes
associated with virulence, namely capsule induction, melanization,
growth at 37 °C, urease and phospholipase secretion and resistance
to several cell wall stressors. Virulence assays: First, the strains were
tested for virulence in the in vitro J774 murine macrophage model of
phagocytosis followed by CFU counts. The in vivo virulence of the
strains was assessed by survival curves in the Galleria mellonella alternative model of infection at 37 °C.
Results The gti1::HYG strain is able to grow at 37 °C similarly to
the wild type strain. In addition, the mutant has no defect on capsule induction, melanin production and urease and phospholipase
secretion. Curiously, the gti1::HYG strain grows as donut-shaped
colony on Sabouraud (Fig. 1) and YPD agar (not shown). Interestingly, it is not capable of growing on DMEM/MOPS medium.
Finally, the knockout of GTI1 gene impaired survival of the fungus
within macrophages in vitro and the mutant also seems to be hypovirulent in the invertebrate host G. mellonella comparing to the
wild type.
Discussion The present results suggest a partial loss of virulence of
the mutant strain in G. mollonella model which we will be follow up
by assessment of virulence in the murine model. The donut-shaped
colony is a striking observation, giving that single cells showed no difference in morphology relative to wild type cells. Whether this is due
to different growth patterns of cells in different areas of the colony, or
to an altered secretion pattern by the mutant, remains to be identified. Work with the Pac2 homologue is also in progress in our group.

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 & Fundacßa~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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Transport (ESCRT)-III. In this study, the role of the ESCRT-III vacuolar
protein Snf7 in the export of CN virulence factors was evaluated
through the phenotypic characterization of snf7D and snf7D::SNF7
(reconstituted) strains generated on the H99 background. The snf7D
mutant showed increased sensitivity to alkaline pH and high LiCl2
concentrations, in comparison to wild type (WT) and snf7D::SNF7
strains. The snf7D mutant also displayed reduced efficacy to produce
melanin when cultivated in both Niger seed agar and L-DOPA at
37 °C. In comparison to WT and reconstituted cells, the snf7D strain
manifested severely reduced glucuronoxylomannan (GXM) secretion.
This phenotype was linked to a drastic reduction in capsule size, as
concluded from counterstaining of fungal cells with India ink, immunofluorescence using an anti-GXM monoclonal antibody (18B7) and
scanning electron microscopy. Lack of Snf7p was also associated with
a significantly reduced ability of CN to kill mice in an intranasal infection model of cryptococcosis. These results demonstrated that Snf7p
regulates unconventional secretion mechanisms involved in the
release of several CN virulence factors, suggesting that the cryptococcal ESCRT machinery plays a central role during infection by C.
neoformans.

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 Christensen’s 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

transplantation (4%). Strains were isolated from cerebrospinal fluid
(82%) and blood (18%). AFLP analysis revealed that the majority of
isolates belonged to genotype AFLP1 (58.8%), followed by AFLP2
(29.4%), AFLP3 (8.8%) and AFLP1B (2.9%). Out of 25 patients, 21
(84%) were infected with MAT-alpha isolates. All isolates were found
to be susceptible to amphotericin B, posaconazole and voriconazole,
while low resistance level was observed for fluconazole (2.9%) and 5fluorocytosine (5.9%). A high percentage of isolates was found to be
susceptible dose-dependent to itraconazole (47.1%). All clinical isolates produced phospholipase, capsule and melanin, but these activities varied with individual isolates. Statistical analysis showed that
isolates obtained from HIV-negative individuals are significantly less
susceptible to 5-fluorocytosine (t test, P < 0.01). In relation to production of virulence factors, our results showed that AFLP2 isolates
produced more melanin, comparing to AFLP1 and AFLP3 (Chisquared test, P < 0.05). No correlation between expression of virulence factors and susceptibility profile was observed.
Discussion This genotype and mating type distributions are consistent with previous European studies. All isolates were found to be
susceptible to amphotericin B, posaconazole and voriconazole, as previously shown. Comparing to similar studies, the percentage of isolates resistant to fluconazole (2.9%) was lower, while the percentage
of 5-fluorocytosine resistant isolates in this study (5.9%) was higher.
Statistical analysis showed no correlation between expression of virulence factors, as well as between virulence and susceptibility profile.
Further studies are needed to investigate host factor contribution in
pathogenesis of C. neoformans.

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-Zaragozı´,1 Y. Rivera Gallego,1 E.X. Monroy Vaca,1
F. Hagen,2 C.M. Fernandez Andreu,1 G.F. Martınez Machın,1
M.R. Perurena Lancha1 and J.F. Meis2
1
Tropical Medicine Institute “Pedro Kourı´”, 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 Kourı’ during 2011–2012.
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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Poster Abstracts

reactivation of infection being dormant for several years have been
described following alteration of the immune status. In a study at
Mayo Clinic, C. neoformans was isolated from lungs of 43 patients
without signs of systemic cryptococcal disease; 26 of the 43
patients suffered various bronchopulmonary disorders with chronic
obstructive pulmonary diseases (COPD) and asthma being the most
common [1]. Colonisation of airways by diverse microbes that can
worsen the disease during exacerbations is typical in COPD, as well
as in cystic fibrosis (CF), another progressive chronic pulmonary
disease. Chronic colonisation by Pseudomonas aeruginosa strains that
evolve toward an increased mutation rate and increased antibiotic
resistance plays important role in progression of disease. Concurrently, the diversity of the microbial community is gradually
reduced, with mucoid P. aeruginosa strains prevailing at the end
stage of disease. This course of disease may also be atributed to
interspecies competition where hypermutational P. aeruginosa is able
to fight down its competitors through production of antimicrobial
agents. In a broader search for pseudomonadal toxic action we
have also explored its effect on C. neoformans.
Aim Check for influence of P. aeruginosa isolates from sputum of
patients suffering chronic pulmoary disease (CF and COPD) on C.
neoformans.
Methods Strains were inoculated on plates and checked for visible
inhibition of growth at crossing of inoculation lanes. Micromorphology was also observed on nutrient-poor media cultured under CO2
atmosphere for induction of capsule formation.
Results No apparent growth interference was observed, however,
some strains of P. aeruginosa were able to reduce the formation of
capsule measured by capsule thickness under inducing conditions.
Discussion So far, antimicrobial peptides [2,3], anti-beta-glucan
antibody [4] and viral protease inhibitors [5] have been described to
interfere with capsule formation in C. neoformans. Our observation
suggest that P. aeruginosa may also be a source of agents potentially
useful for blocking the production of this important virulence factor.
Such agents may possibly shift the balance towards favorable side in
the host-pathogen interaction.
Acknowledgements The Ministry of Health (NT/13560-4) and the
Internal Grant Agency of Palacky University Olomouc
(LF_2013_012) supported this work. The infrastructural part of this
project (Institute of Molecular and Translational Medicine) was supported by the Operational Programme Research and Development for
Innovations (project CZ.1.05/2.1.00/01.0030).
References 1. Duperval R, Hermans PE, Brewer NS. et al. (1977)
Chest 72: 13–19.
2. Barbosa FM, Daffre S, Maldonado RA et al. (2007) FEMS Microbiol Lett 274: 279–86.
3. Silva FD, Rossi DC, Martinez LR et al. (2011) FEMS Microbiol
Lett 324: 64–72.
4. Rachini A, Pietrella D, Lupo P et al. (2007) Infect Immun 75:
5085–94.
5. Sidrim JJ, Perdigao-Neto LV, Cordeiro RA et al. (2012) Can J
Microbiol 58: 932–6.

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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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

immunocompromised patients. Its incidence has increased in recent
years. Worldwide, approximately 1 million new cases of cryptococcal
meningitis occur each year, resulting in 625 000 deaths.
Aim To study the prevalence of CNS cryptococcosis in our hospital.
Material and methods The presentstudy was conducted in Dayanand Medical College and Hospital, Ludhiana, Punjab (India). All
patients with the clinical diagnosis of chronic meningitis from January 2013 to December 2013 were included in the study.CSF sample
obtained from the patients was processed according to standard laboratory procedures.Cryptococcal meningitis was confirmedon the basis
of India ink examination, culture and cryptococcal antigen latex
agglutination test (CALAS by Meridian Biosciences).
Results A total of 747 samples were received during the study period out of which 14 (1.9%) were positive by any one of the methods.
Maximum positivity was observed with antigen detection (100%).
Majority of the casesof CNS cryptococcosis weremales.
Conclusion Prevalence of CNS cryptococcosis in our study was
1.9%. A high index of suspicion and routine mycological surveillance is required to help in an early diagnosis and appropriate therapy as clinical picture may be confusing with viral or tubercular
meningitis.

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 Piauı, 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

Figure 1 ELISA reactivity of subjects’ sera against peptides H21,
Hy49, S4, H18, H26 and Hy50.

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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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 (20–24.9 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.

Figure 2. Baseline CFU vs time-to-growth.

Discussion and conclusions These results support the value of
cryptococcal fungal burden as a tool in the management and evaluation of CM disease. They also suggest that the baseline fungal burden
probably is not the only contributor to high OP, but seems to be one
of the most important factors at week 2. In addition, they highlight
the importance of incubation of fungal CSF cultures for over two
weeks before being considered negative at baseline and week 2 of
treatment.

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. 1999–2013 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 Canisius–Wilhelmina 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

Figure 1. Fungal clearance vs opening pressure.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

101

Poster Abstracts

Table 1 Characteristics of cases.
Case

Gender/Age

Co-morbidities

Localization

1

M/67

Diabetes, high blood
pressure,
corticosteroid
therapy

Meningitis

2

F/43

Fungoid mycoses Tcell lymphoma
Scabies

Meningitis

3

M/54

Disseminated
Strongyloides
stercoralis
Ancylostoma sp.

Disseminated
(lung,
meningeal,
gastrointestinal)

4

F/58

Diabetes, high blood
pressure

Meningitis

Figure 1. Ulcerative lesions over the arm of a cryptococcosis patient.

Clinical characteristics and
outcome
3 months: fever, headache,
nauseas, vomiting,
neck stiffness. Glasgow: 11,
CrAg* titer: 1/256.
Death due to Acinetobacter sp.
Bacteremia.
2 weeks: headache, nauseas,
vomiting, neck
stiffness and weight loss of 5
Kg. CrAg titer: 1/8.
Cure.
5 years: diarrhea, fever, cough,
weight loss of 10 Kg.
1 week: headache. CrAg titer:
1/4000. Thorax
X-rays with alveolar
infiltrates in bases.
Death due to Acinetobacter sp.
bacteremia.
7 months: headache, tremor,
weigh loss of 10 Kg,
bladder
incontinency, hyperreflexia and
meningeal signs.
CrAg: 1/8.
Cure

CrAg* Cryptococcal latex agglutination test.

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.

Figure 2. Cryptococcus neoformans var. grubii seen in the pyogenic
lesion (H&E 1009).

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 12–32%
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 15–21 days
before in other two. All received amphotericin B deoxycholate

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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

environmental studies are likely to show a wider spectrum of host
trees and molecular variety among environmental isolates.

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
23–59) years. Mean CD4 was 44 (range 3–160) 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. Castan˜eda
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

brain or the lungs. This issue has been demonstrated by Dromer et
al., who reported that mixed infections in a single patient are composed of different mating types, serotypes and/or genotypes. In
Colombia, the passive surveillance on Cryptococcosis has provided us
with an important collection of strains in which this type of analysis
could be done.
Aim To analyze clinical isolates of the C. neoformans/C. gattii species
complex to determine if mixed infections are present in our patients.
Methods Clinical cultures were collected during the passive surveillance on Cryptococcosis that is being held in Colombia since 1997.
Original strains correspond to cultures obtained from the biological
specimen on agar. The selection was made randomly, from isolates
received between 2006–2013, from different regions in Colombia,
and not based on the patient’s characteristics. Sixteen C. neoformans
isolates and 7 C. gattii isolates were included. A suspension of
approximately 9x102 CFU ml1 of the original culture was plated
onto Sabouraud agar and Niger seed agar plates. Five colonies per
plate were selected based on colony morphology differences (if any),
otherwise randomly when the phenotypes were similar. For each isolated colony, conventional biochemical test were done, and molecular typing by PCR fingerprinting with (GTG)5.
Results All colonies studied for each isolate were confirmed as C.
neoformans or C. gattii as they were initially characterized. Molecular
typing has been done in all of the (16 9 5 = 80) C. neoformans isolates which have been typed as VNI; C. gattii typing is underway
(2 9 5 = 10), having found the same molecular type (VGII or VGIII)
in each of the colonies studied for each strain. Although the same
molecular type has been seen in each of the colonies studied, for
some isolates, slight differences observed until now in 3 C. neoformans
strains have been observed in the electrophoretic profiles, thus suggesting the need to perform a more precise technique as MLST.
Discussion/conclusions This is the first study carried out in Colombia on the determination of possible mixed infections in patients diagnosed with Cryptococcosis. It is widely considered that a single
isolate of the fungus is the responsible for the disease; however, a
recent study reported a high frequency of mixed infections (20%) in
European patients suggesting that multiple strains could be exogenously acquired from the environment, either simultaneously or
sequentially. Our previous experience has shown that we can find
different serotypes, mating types and molecular patterns in a same
geographical region, making it possible that a patient may be
infected with more than one strain, thus making a mixed infection
by multiple strains a likely scenario. Further studies may be helpful
for a complete characterization of the strains, as well as studying isolates recovered from serial samples of patients with recurrent infections of Cryptococcosis.

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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

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 who’s 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 Child’s 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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Figure 1. Histology sections of skin nodule and pulmonary lymph
node.

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 na€ıve
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
cause’s 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 ARVna€ıve HIV infected patient and their clinical profile and outcome.
Method Serum tested was from HIV infected who anti retro viral
(ARV) na€ıve. The method used was lateral flow assay (LFA) method.
The clinical profile was also noted.
Result A total of 78sera from ARV na€ıve 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

Figure 1. Cells of smooth and mucoid capsule.

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

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

Poster Abstracts

2 weeks, or amphotericin combined with fluconazole 800 mg day1
for 2 weeks. Following induction therapy all patients received fluconazole 400 mg day1 until 10 weeks after randomization. Previous
analyses had identified fungal burden, treatment allocation and Glasgow coma score less than 15 as independent predictors of mortality.
The susceptibility of revived baseline isolates to amphotericin B, flucytosine, and fluconazole was determined following both 48 and
72 h incubation. Range, MIC50, MIC90 and geometric mean MICs
were determined for all drugs. Cox regression was used to determine
the effect of susceptibility on the primary endpoint of 10 weeks post
randomization, and on 2 weeks and 6 months survival. The joint
effect of randomized drugs was determined according to the treatment randomly allocated for the combination therapies and for
amphotericin alone for patients on monotherapy, and the joint effect
of the MICs of amphotericin, fluconazole, and flucytosine for all
patients. Analyses were performed with and without adjustment for
baseline fungal count and Glasgow Coma score. Mixed models and
Cox regression were used to model CSF fungal decline over the first
14 days and time to CSF clearance.
Results 276 isolates from 276 patients were included in the primary
population analysis. No effect of susceptibility was found on survival
to 14 or 70 days for any of the 3 drugs tested (70 day survival:
AmB HR 0.78 (0.53, 1.13); P = 0.19, Flucytosine HR 0.97 (0.76,
1.25); P = 0.84, Fluconazole HR 0.96 (0.77, 1.19); P = 0.69. Similarly no effect of susceptibility was detected on the rate of fungal
clearance for each of the three drugs tested. Adjustment for previously defined independent baseline predictors of mortality (GCS, fungal burden, treatment allocation)did not significantly affect these
results.
Conclusion There is no role for susceptibility testing of isolates
from patients with first episodes of HIV associated cryptococcal
meningitis.

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),

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108

caspofungin (CAS). Iron chelators: lactoferrin, deferasirox, deferiprone,
deferoxamine, cyclopirox olamine, EDTA. Fungal species/strains: Cryptococcus neoformans H99 (genome strain), C. gattii R265 (genome
strain), C. gattii 97/170 (intrinsically resistant to FLC), Saccharomyces
cereviseae S288C (genome strain; used for interactome construction).
Synergy data were analysed using MacSynergy and the Fractional
Inhibitory Concentration Index (FICI). High quality RNA was prepared from the most synergistic combinations. RNA-Seq data were
generated using the HiSeq 2000 Illumina platform with biological
triplicates multiplexed and randomized across two sequencing lanes.
Differential expression analyses were performed using EdgeR. Cytoscape was used to visualize potential interaction networks, and selforganising maps (SOMs) were used to group transcripts into coexpressed cohorts.
Results Significant synergy was seen when AMB was combined with
lactoferrin, a milk protein with iron chelating properties. Synergy
was not seen with the FDA-approved chelators deferosirox, deferiprone, and deferoxamine; nor was it seen with EDTA and cyclopirox
olamine. Antagonism occurred when ITZ and VRZ and were combined with deferasirox, deferiprone and EDTA to treat C. neoformans
H99, but this was not seen with C. gattii or S. cerevisease; in fact
VOR + EDTA was synergistic for C. gattii.
Transcriptional analysis by RNA-Seq was performed on S. cerevisiae
treated with (i) AMB only; (ii) AMB + lactoferrin, (iii) and (iv) corresponding controls matched for growth but without antifungals or
chelators.
Cytoscape and SOM analysis suggested the transcription factors
AFT1 and YAP5 were important for AMB + lactoferrin synergy.
AMB treatment alone resulted in the down-regulation of nine genes
involved in ergosterol biosynthesis and the up-regulation of AFT1, a
transcription factor involved in iron transport. AMB + lactoferrin
halted the up-regulation of AFT1 and down-regulated genes involved
in iron transport. The latter were co-expressed with YAP5, a second
transcription factor that co-ordinates the expression of genes controlling the nuclear localization of AFT1.
Discussion/conclusion Synergy between antifungals and iron chelators was uncommon and differed among species. The antagonism seen
with C. neoformans highlights significant species-specific differences
and indicates a need for caution when using iron chelators. AFT1 and
YAP5 were affected by the addition of lactoferrin to AMB; their influence on drug synergy will be further analysed using SOMs, Q-PCR and
gene deletion/complementation. We will explore the role of homologous factors in Cryptococcus using additional RNA-Seq assays.

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

Results Sixty-seven patients (44.4%) had no UDs. The common UDs
were diabetes (32.1%) followed by hematologic disease (22.6%), and
collagen disease (22.6%). Peripherally distributed pulmonary nodules/masses were most commonly seen. Lesions in the right middle
lobe (P = 0.01) and air bronchogram (P = 0.05) were significantly
more frequent, respectively, in patients with UDs than patients without them. Azoles were mainly selected for the patients without
meningoencephalitis. Mean treatment duration for patients with and
without UDs was 2.87 and 6.64 months, respectively. Patients
whose pulmonary nodules improved after treatment continued to
experience gradual reduction of cryptococcosis antigen titers, even if
antigen titers were positive at the time of treatment cessation. The
average time for antigen titers to become negative after treatment
cessation was 10.7 and 13.1 months for patients with and without
UDs, respectively. When groups were compared according to the
presence of meningoencephalitis complications, deaths, and survivals,
factors contributing to cryptococcosis prognosis included higher age,
hypoproteinemia, hypoalbuminemia, steroid use, high C-reactive protein levels, and meningoencephalitis complications.
Conclusions It is crucial to consider the presence of UDs and meningoencephalitis for the choice of antifungals and treatment duration
for cryptococcosis in non-HIV patients. Three- and six monthsadministration of azoles for pulmonary cryptococcosis with or without UDs, respectively is reasonable.

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.

108

Methods MICs against 16 isolates were determined according to the
CLSI guideline (M27-A2) for the broth microdilution method. Timekill studies were performed using C. gattii YF2784 (VGII). In murine
intranasal pulmonary infection model caused by C. gattii YF2784,
the test compounds were administered once daily for 21 days beginning at 2 hours postinfection. In addition, to evaluate therapeutic
effect in advanced infection, the test compounds were administered
from 14 days postinfection once daily for 7 days in the murine
model. The viable counts in lung and brain of compound-treated
group and control group at 21 days postinfection were compared
using parametric Dunnett’s multiple comparison test. Statistical significance was set to P < 0.05.
Results
Compound

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.

ª 2014 The Authors
Mycoses ª 2014 Blackwell Verlag GmbH, 57 (Suppl. 1), 33–108