Molecular identification of Cryptococcus species isolated from pigeon dropping in Shiraz, Southern...

Abstract

Poster Abstracts

P001

Cryptococcosis in hematological patients in Saint-Petersburg, RussiaS. 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

1I.I.Metchnikov North-Western State Medical University, Saint-

Petersburg, 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 withcryptococcosis in St. Petersburg, RussiaMethods The prospective study was conducted during the period2010–2013. Diagnosis of cryptococcosis was made according to EO-RTC/MSG criteria (2008).Results We observed 8 hematological patients with cryptococcosis.The mean age of patients was 24 years (range 2.6–60), male/femaleratio 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 neoformanswere isolated from 25% patients. Diagnosis of cryptococcosis wasconfirmed by histology and direct microscopy of biopsy samples in25% of patients. The main sites of infection were CNS – 62.5%, lungs– 37.5%. In 50% patients after cryptococcosis had invasive aspergil-losis. 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 deoxycho-late – 37.5%. Combination therapy was performed for 37.5% patients(amphotericin B + fluconazole). Surgical treatment (resection of theaffected lung) was conducted in 25% patients. Duration of antifungaltherapy was 72–360 days (median – 199). Overall survival at12 weeks was 87.5%, 1 year – 25%.Conclusion The main underlying diseases in hematological patientswith cryptococcosis – acute leukemia (62.5%). Main locations – CNSand lungs. Twelve weeks overall survival was 87,5%.

P002

Cryptococcosis in Nigeria: the neglect of a giantE. Nnadi,1 M. Cogliati2 and I. B. Enweani3

1Plateau State University, Bokkos, Bokkos, Nigeria; 2University of

Milan, Milan, Italy and 3Nnamdi Azikiwe University, Nnewi,

Nigeria

Background Cryptococcosis is one of the most severe opportunisticinfections in patients with HIV/AIDS and other underlying immuno-compromising disease conditions having grave complications andprognosis especially in developing countries. It is estimated to causeabout one million cases of meningitis per year globally, prevalentlyin HIV infected subjects. It has been associated with 17% of alldeaths among HIV-infected patients in sub-Saharan Africa and there-fore dubbed a ‘sleeping disease’ emerging as an ‘awakening giant’.Although cryptococcosis incidence has risen dramatically in the lasttwo decades especially within the HIV positive population, there is a

dearth of published article about the pathogen diversity, epidemiol-ogy and virulence potential in Nigeria.Aim This paper represents a compendium on this disease in theNigerian clinical setting, highlighting areas of need for futureresearch and collaborative structures within the country as well asthe 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 inNigeria’, ‘Cryptocococcus neoformans + Nigeria’, ‘Cryptococcus gattii +Nigeria’, and ‘Cryptococcus + Nigeria’ regardless of date ofpublication.

Less than 20 papers were found reporting few data concerningCryptococcus and cryptococcosis in Nigeria. A study carried out in2010 reported a frequency of 36% of cryptococcal meningitis in acohort of 100 HIV positive patients. In another study a prevalence of18.8% of pulmonary cryptococcosis among HIV patients wasreported. Other papers reported a prevalence of 13% of cryptococco-sis in HIV pregnant and 4% among other HIV patients. Cryptococcusspecies were not identified in the results and none of the papersreported C. gattii isolation. There is also a need for comprehensivereports on the antifungal susceptibility patterns of Nigeria’s clinicalisolates of C. neoformans as there are no information on the suscepti-bility profile. In the environment, some studies have reported isola-tion of C. neoformans serotype A and D from pigeons and otherhealthy birds including bats.Conclusion On the basis of the few results reported in the literatureit is clear that cryptococcosis epidemiology in Nigeria is far to be eluci-dated. Therefore, a great effort of collaboration between hospitals andresearch centers of the country connected with international referencecenters is needed. The main goals in the next future to improve theknowledge of this life-threatening disease are the following:

(1) to establish a network involving clinician, microbiologists andresearchers of the main hospitals and research centers in Nigeriaunder the coordination of a local reference center connected with aninternational Cryptococcus reference laboratory;

(2) to report clinical and microbiological data concerning eachcase of cryptococcosis in a standard form collected by the coordinatorcenter;

(3) to collect Cryptococcus isolates for biochemical and molecularanalysis as well as determination of their antifungal susceptibility;

(4) to analyze clinical and microbiological data to improve crypto-coccosis diagnosis and to determine distribution of Cryptococcusgenotypes in Nigeria;

(5) to survey the environment by sampling trees, soil, and birdexcreta;

(6) to compare clinical and environmental data.

P003

Second prospective survey on cryptococcosis in Italy after10 yearsM. Cogliati,1 A. M. Tortorano1 and FiMUA Cryptococcosis

Network1Universit�a degli Studi di Milano, Milano, Italy

Background The first prospective survey on cryptococcosis in Italycarried in 1997–1999 drew a picture of the epidemiology of this life-threatening disease confirming that it was mainly associated to AIDSpatients. Cryptococcus neoformans var. grubii, C. neoformans var. neo-formans, and AD-hybrids were isolated with a similar frequency butwith a different geographical distribution.Aim In the present study we carried out a new prospective surveyon cryptococcosis in Italy with the aim to compare the clinical andmicrobiological data collected to those obtained ten years ago. Thestudy represents an update of epidemiological changing of cryptococ-cosis in the recent period.Methods An Italian network including 18 hospitals was establishedand cryptococcosis cases were recorded from January 2010 to

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mycosesDiagnosis,Therapy and Prophylaxis of Fungal Diseases

December 2013. Clinical information were collected using the sameform adopted ten years ago to enable data comparison. One or moreisolates from each case were collected for molecular type and matingtype identification.Results During the survey 58 cases of cryptococcosis were recorded.Males represented 74% of the cases. The median age was 47.5 yearsranging from 24 to 81 years. Twenty-four (41%) patients were HIVnegative with different predisposing conditions. Diagnosis of crypto-coccosis was mainly made by positive GXM antigen and cultures. Inthree cases diagnosis relied only on positive antigen in serum and/orcerebrospinal fluid. Sixty-four isolates were available from 58 cases.Molecular typing showed that 22 patients were infected by C. neofor-mans 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 thenumber of recorded cases is significantly reduced compared to theprevious survey (156 cases) although the period and geographicalcoverage were different. The percentage of HIV negative patients ischanged rising from 2.8% to 41%. This result confirms that, afterthe introduction of HAART, HIV infected patients are less susceptibleto Cryptococcus infection. The ratio male/female is changed from5.5 : 1 to 2.9 : 1 as a consequence of the higher percentage of non-HIV infected patients. The molecular analysis of Cryptococcus isolatesshowed that VNI was the molecular type predominantly isolated(50%) followed by VNIII (36%) and VNIV (14%), in contrast withthe percentages observed in the previous survey (35%, 35%, 30%respectively). In conclusion our results highlight the need of a con-stant monitoring of cryptococcosis in Italy since the epidemiology ofthis pathogen is changing.The FIMUA cryptococcosis networkAncona: E. Manso; Bergamo: M. Passera; Genova: M. Mikulska; Mi-lano: A. Grancini, R. Grande, C. Ossi, M. Cainarco; Bolzano: P. Inno-centi; 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.

P004

Epidemiological and clinical features of aids patients withcryptococcosis confirmed or diagnosed at necropsy in ateaching hospital in BrazilM. 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

Triangulo Mineiro Federal University, Uberaba, Brazil

Background Yearly, one million cases of cryptococcosis in AIDSpatients are diagnosed around the world, of which two thirds dieduring the second week on therapy. Commonly, cryptococcosis is thefirst AIDS defining condition in these patients and concerns aboutthe high mortality rates observed are intimately associated to lateAIDS diagnosis and advanced immunodeficiency in most patients atadmission. Consequently, a severe and disseminated fungal infectionis commonly seen, mainly in poor-resource settings where diagnosisand specific therapy are far to be ideal.Aim To evaluate epidemiological, clinical and anatomopathologicalfeatures of AIDS patients with cryptococcosis confirmed or diagnosedat necropsy in a Teaching Hospital in Brazil.Methods Retrospectively, medical and necropsy records of 45 AIDSpatients with cryptococcosis diagnosed from 1993 to 2012 in theTeaching Hospital in Uberaba, Brazil, were reviewed. Demographic,epidemiological, clinical, laboratory and necropsy data of these caseswere obtained and compared to those of 24 AIDS patients with cryp-tococcosis who presented a good outcome over the same period.Results A total of 45 AIDS patients with cryptococcosis who per-formed necropsy were included. Of these, 35 (77.8%) were male withmean age of 31.7 years. Cryptococcosis was the first AIDS definingcondition in 30 (66.7%). The evolution of symptoms in 33 (73.3%)

cases was of 1–3 weeks. At admission altered consciousness andintracranial hypertension were present in 23 (52.3%) cases whereasCSF assessment showed hypocellularity in 17/31 (54.9%). CSF cul-tures yielded Cryptococcus neoformans in 29/29 (100%) cases. TheCD4 cell baseline count ≤100 cells mm�3 was present in 13/15(86.6%) cases. Of 29 (64.4%) patients who started antifungal ther-apy, 23 (79.3%) died over the second week on therapy. Most patientswere antiretroviral naive and in the remaining 16 (35.5%), the cryp-tococcosis diagnostic was performed at necropsy. Disseminated infec-tion involving mainly CNS 38 (84.4%), lungs 31 (68.9%), lymph-nodes 25 (55.6%), kidney 22 (48.9%), liver and spleen 21 (46.7%),adrenal gland 10 (22.2%), pancreas 10 (22.2%), prostate (20%) andbone marrow (20%) was evidenced in 31 (68.9%) cases. Similar pro-portion of disseminated cryptococcosis was found among necropsiedpatients who had received or not antifungal therapy. The remaining15 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 wereconcomitantly observed. Demographic, epidemiologic and most clini-cal features observed in these patients were similar to those noticedin survivors. However, the presence of altered consciousness, intra-cranial hypertension and severe malnutrition at admission were sta-tistically associated with a poor outcome.Discussion/conclusion This cryptococcosis case series in AIDSpatients confirmed or diagnosed at necropsy, evidenced the epidemio-logical and clinical profile already described by others in poor-resource settings. The severity of fungal infection showed by multipleorgan involvement in most cases and the presence of clinical poorprognostic factors are directly associated to late AIDS diagnosistogether with the advanced immunodeficiency observed at admission.Commonly, this infection is the first AIDS defining condition andmost patients are not receiving ART. Nowadays and despite specificdiagnosis and antifungal therapy, cryptococcosis mortality rate of55% is still observed in Brazil and other Latin American countries.The necropsy performance in teaching hospitals is a valuable tool toknow the outcome of AIDS patients and to improve the accuracy ofclinical diagnosis.

P005

Hydrocephalus as the first clinical feature of cryptococcosisin an aids patient on antiretroviral therapyM. 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


Background Clinical signs and symptoms of cryptococcosis are com-monly related to meningeal and or encephalic involvement. Globally,most cases of Cryptococcal meningitis occur in AIDS patients whousually present a subacute clinical picture and are severely immuno-compromised. Hydrocephaly is a common complication of intracra-nial increased pressure, but to our knowledge, it is uncommon as afirst clinical presentation of this infection.Aim To describe an AIDS patient with uncommon clinical picture ofcentral nervous system cryptococcosis.Case report A 30-year-old Brazilian woman was admitted at theteaching hospital in March 2013. She complained of holocranialheadache for several months which became more intense persistentduring the last month. Besides, she referred ataxia and lack of bal-ance. No other general symptoms or neurological complains weredescribed. She was found HIV positive in 2004 during a pregnancyand remained without medical care until 2010 when she was admit-ted with Pneumocystis jirovecii pneumonia, Herpes zoster infection andlymphocytic meningitis which were adequately treated. The skull CTwas normal at the time. Her CD4 T cell baseline count was19 cells mm�3 and the viral load of 356.322 RNA copies/ml Shestarted antiretroviral therapy with AZT + 3TC + efavirenz and was

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Poster Abstracts

discharged. During the follow up, she remained asymptomatic, herCD4 T count increased to 250 cells mm�3 and the viral load waspersistently undetectable. At admission she presented slow thinkingand mild ataxia. The skull CT scan evidenced moderate non commu-nicant hydrocephalus and signs of intracranial hypertension. TheMRI imaging confirmed these features. The CSF assessment: cells:25 mm3, neutrophils 7%, lymphocytes 90%, monocytes 3%, glucose17 mg dl�1 and proteins 90 mg dl�1. CSF smear was negative tobacteria, mycobacteria and fungal structures. India ink and Cryptola-tex tests were negative. CSF culture was negative to bacteria, myco-bacteria and fungus. VDRL and FTA-abs were non-reactive,Toxoplasma gondii IgG antibodies 1 : 16, ELISA test to cysticercusantibodies was non-reactive. Patient performed ventriculostomy andwas discharged. One week later, she was readmitted with worseningof headache, vomits, photo and phonofobia and right side paresthe-sia. A new CT scan showed the same findings and mild hemorrhagicfoci in frontal lobe on the craniotomy site. A external shunt was per-formed and CSF obtained showed: Cells 21 mm3, glucose17.9 mg dl�1 and proteins 102.5 mg dl�1. The Cryptolatex andIndia ink tests were negative. CSF culture yielded Cryptococcus neofor-mans on day five. Amphotericin B + fluconazole were prescribed anda ventriculoperitoneal shunt was performed due to the obstruction ofexternal derivation. Her clinical picture progressively improved andafter 27 days, she was discharged to continue the antifungal therapyat day hospital which was kept for 10 weeks. During the follow up,patient remains asymptomatic and the CT brain scan performed fivemonths later showed a significant reduction of hydrocephalus andventricular asymmetry.Discussion Hydrocephalus is a complication of cryptococcal menin-gitis as a consequence of intracranial increased pressure whichoccurs due to CSF outflow obstruction caused by the sum of severalfactors such as: aggregation of capsular polysaccharide, inflammationimmune-mediated and osmotic effect of mannitol derived of fungus.Most cases of hydrocephalus are associated to other signs and symp-toms of meningitis or meningoencephalitis, different from the presentreport. This patient presented a chronic headache which led to evi-dence moderate ventricular dilatation and CSF findings similar tothose seen in fungal and tuberculosis infection. Probably, she pre-sented a very low fungal burden which explains the insidious clinicalpicture, and diagnosis difficulty and could be the consequence of herpartial immune reconstitution after three years on regular antiretro-viral therapy. To our knowledge it is an uncommon first clinical pre-sentation of cryptococcal meningitis in AIDS patients.

P006

Ocular cryptococcosis in an AIDS patient withdisseminated infection: case reportM. 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


Background As opportunistic infection in immunocompromisedpatients, cryptococcosis can potentially involve any organ or system.Ocular primary cryptococcosis seems to be a rare event, and mostcases already reported occurred associated to disseminated infectionin patients with or not underlying immunodeficiency due to corticos-teroids, hematologic malignancy, vascular collagen disease and AIDS, among others. According to the literature reviewed, most cases pre-sented a presumptive clinical diagnosis of ocular cryptococcosis.Aim To describe an AIDS patient with cryptococcal choroiditis con-firmed at necropsy.Case report A 20-year female Brazilian patient was admitted at theTeaching Hospital with fever, progressive dyspnea, dry cough andweight loss of 10 kg during the last two months. Moreover she com-plained of holocranial headache during the last two weeks. Due to thesymptoms related to severe anaemia, she had been previously

hospitalized in her town, received blood transfusion and then wastransferred because she had worsened and the ELISA anti-HIV testwas positive. At admission, her clinical status was critic, presentingsevere malnutrition, dyspnea and acrocyanosis. Axillary temperaturewas 37.5 °C and the body weight of 30 kg. White lesions in the oralcavity suggestive of thrush were seen. Cardiac and pulmonary auscul-tations were normal, and no evidence of lymph-nodes, hepatomegalyand/or splenomegaly was found. No meningeal signs were present.Ophthalmological exam showed multiple white oval patches aroundoptical nerve and mild retinal hemorrhagic points. The clinical diag-nosis was retinochoroiditis by Pneumocystis jirovecii. Complete bloodcount showed pancytopenia. At Chest X-ray, bilateral interstitial infil-trate and pneumonic consolidation in both lung basal regions wereevidenced, and confirmed at chest CT. Arterial blood gases measure-ment evidenced severe hypoxemia, the CD4 cell count was30 cells mm�3, and no viral load was available at the time. At theemergency room intravenous trimetoprim-sulfamethoxazole plushydrocortisone were prescribed to treat a presumptive P. jiroveciipneumonia. A lumbar puncture was also performed and the CSFanalysis showed: 5 cells mm�3, of which 98% were lymphocytes and2% monocytes, glucose 27.7 mg dl�1 and protein 43.0 mg dl�1;India ink and Crypto-latex tests were positive and CSF culture yieldedCryptococcus neoformans. Amphotericin B deoxycholate plus Fluconaz-ole were then prescribed. During the following days, her clinical pic-ture worsened, presenting unresponsive cardiorespiratory arrest atday 8. Necropsy was performed, and showed severe and disseminatedcryptococcosis involving meninges and brain, eyes, bone marrow,lungs, stomach, colon, liver, spleen, pancreas, hypophysis, adrenalgland and kidney. At histopathology exam of both eyes, a mild inflam-matory infiltrate formed by lymphocytes and macrophages was evi-denced through the choroids which presented multiple yeasts stainedwith Mayer’ Mucicarmin. The retina, optical nerve and sclera werenot involved.Discussion This case illustrates how difficult it is to establish the eti-ology of choroiditis in AIDS patients. Despite adequate diagnosis andantifungal therapy, patient died with disseminated cryptococcosis evi-denced at necropsy. Due to the presumptive clinical diagnosis of ocu-lar involvement by P. jirovecii, it was asked to exam the eyes, whichpresented fungal infection limited to choroids. This finding is in linewith previous reports showing that this structure is commonlyaffected by Cryptococcus neoformans, different from CMV, Herpes sim-plex and Toxoplasma gondii infections which more frequently involveretina and optical nerve. Of ocular cryptococcosis cases alreadydescribed, few, including the present, were confirmed by histopatho-logical exam.

P007

Cryptococcosis in ferrets from Spain and Portugal: theveterinarians’ roleM. F. Colom Valiente,1 C. Juan Sall�es,2 R. Patricio,3 C. Artigas,4

J. I. Serra,4 F. Hagen5 and N. Morera6

1University Miguel Hernandez, Sant Joan d’Alacant, Spain;2Noah’s Path, Elche, Spain; 3Cl�ınica Veterinaria Alcabidechevet,

Alcabideche, Portugal; 4Cl�ınica 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 dif-ferent areas of the world being the Mediterranean basin one of them.Exposure to infection might be more likely in animals than in humanbeings, given their closer relationship with the natural habitat of theyeast. Animals and especially pets, can act as indicators of the pres-ence of this yeast in a determined area. We present a review of cryp-tococcosis in ferrets in Spain and Portugal, and its relationship withthe findings of Cryptococcus in environmental samples in the samelocations. The work emphasizes the importance of suspecting the dis-ease in ferrets, which can act as sentinels, and performing an

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Poster Abstracts

adequate identification and environmental search to improve ourknowledge of the epidemiology of cryptococcosis.Aim Review of the occurrence of cryptococcosis in ferrets in Spainand Portugal and their possible role as sentinels of the presence ofthe yeast in the environment.Methods We describe three cases of cryptococcal infections in ferretsfrom Spain and Portugal. One of them was identified as the first caseof cryptococcosis by C. gattii in Spain. All of them were exhaustivelystudied with a complete clinical evaluation. The three cases weredetected on the basis of the anatomopathologic findings. Needle aspi-rates from different tissues (nasal mass, thoracic mass and lymphnode) were studied at the mycology laboratory. Cultures onto Sabou-raud agar allowed the development of characteristic yeast coloniesfor all three cases. Asymptomatic carriers detection and environmen-tal search of the yeast in relation with these cases was performed.Nasal swabs from the owners and other animals living in the samehousehold of two of the cases were tested for asymptomatic carriageof Cryptococcus spp. Sampling of trees and soils in the vicinity of theanimals 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 wereprocessed for the search of Cryptococcus spp. Yeast colonies from clini-cal and environmental samples were phenotypically typed by carbo-hydrate compounds auxonograme (Auxonograme2-Biorad), capsuleproduction, urea hydrolysis, growth at 37 °C, melanin productionand growth on canavanine-glycine-bromothymol Blue medium(CGB). Isolates identified as C. neoformans/C. gattii species complexwere subsequently molecularly characterized by Amplified FragmentLength 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 varietygrubii AFLP1/VNI from the nasal mass of the ferret in BalearicIslands; C. gattii AFLP6/VGII from the thoracic mass of the Portu-guese ferret and C. gattii AFLP4/VGI from the lymph node of the fer-ret in Barcelona. In environmental samples five isolates of C.neoformans var. grubii genotype AFLP1/VNI were obtained in thevicinity of the ferret from Majorca (Balearic islands) and 11 isolatesof C. gattii AFLP4/VGI from environmental samples related to theanimal in Barcelona. In Portugal all environmental samples and car-riers detection were negative. Nasal swabs from two persons and twohealthy ferrets related with the case from Barcelona, were positiveand the same species and genotype of the infected ferret wereobtained.Conclusions The molecular characterization of the isolates and theirgenetic profiles evidenced a very close relationship between clinicalstrains and the environmental isolates of the same area, suggestingthe same origin and the acquisition of the disease from naturalsources. Adequate investigation of the genotype helps determiningthe virulence of the yeast and the potential danger for the peoplewho live with the infected animal. Veterinarians should be aware ofthis and carry out the proper tests to identify cryptococcal infections.

P008

Cryptococcosis gattii: a poor recognized fungal infection inBrazilF. Queiroz-Telles and V. A. Vicente

Federal University of Parana, Curitiba, Brazil

Background Cryptococcal meningitis is the most common opportu-nistic fungal infection of the central nervous system in AIDS patients.Among HIV-uninfected patients, several predisposing factors for cryp-tococcosis such as corticosteroid medication, solid organ transplanta-tion and malignancy, etc, have been identified. However,cryptococcal infections in apparently healthy individuals are alsoincreasingly being reported, especially from Asian data. This raisesthe question of whether the so-called healthy hosts are, in fact,accompanied with immune genetic predispositions.

Aim Host immune responses are initiated by pattern recognitionreceptors (PRRs), including Toll-like receptors (TLRs), C-type lectinreceptors (CLRs), NOD-like receptors (NLRs), and others. In the lastyears, several studies have identified genetic polymorphisms in thesereceptors to be associated with susceptibility to Cryptococcus neofor-mans (C. neoformans).Methods We review the contribution of genetic polymorphisms ofthe PRRs to susceptibility to C. neoformans infection and some of ourgenetic studies are included. Basically, the case-controlled geneticassociation studies were conducted and the genotypes were comparedbetween patients and control subjects.Results The FCGR 3A 158 F/V polymorphism was firstly found tobe associated with Cryptococcus infections in patients with pulmonarycryptococcosis and cryptococcal meningitis. Our previous case-con-trol genetic association study also found that the FCGR 2B 232 I/Tgenotype was associated with cryptococcal meningitis in HIV-unin-fected Chinese patients, but no other significant difference was foundamong FCGR 2A, 3A, 3B genotype frequencies. In addition, ourinvestigation showed that the proportion of deficient MBL-producinggenotypes in patients with cryptococcal meningitis was significantlyhigher than in the control group, indicating MBL deficiency may beone of the predisposing factors of cryptococcal meningitis. The differ-ence is even more significant in the subgroup of patients without im-munocompromising factors. Recently, we have found some othergenetic variations predisposing to cryptococcal infection in ourcohort study, including TLRs and NLRs which will be discussed indetail.Conclusion These genetic polymorphisms in PRRs that predispose toC. neoformans infection provide better understanding of the molecularimmunity against cryptococcosis. However, more genetic variationsin immune genes predisposing to cryptococcal infection need furtherscreening, and additional validations are also required in larger, bet-ter designed multicenter studies.

P009

Investigation of environmental sources of a cryptococcosisoutbreak in Maraj�o Island, Par�a state, BrazilL. 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. Laz�era1 and B. Wanke1

1Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; 2SESPA,

Bel�em, Brazil and 3Federal University of Par�a, Bel�em, Brazil

Cryptococcosis by Cryptococcus gattii in Brazil is endemicin the North(N) and Northeast (NE) of the country. In these regions, noteworthywas the emergence of cryptococcosis in immunocompetent (HIV-neg-ative) children in about one fifth of the reported cases, suggesting thatnatural infection occurs early in life. This study was motivated by theoccurrence, in a 3 months period (2013) of two laboratory confirmedcases and one not confirmed case of cryptococcal meningitis in S~aoSebasti~ao da Boa Vista, a municipality on the Maraj�o Island at theestuary of the Amazon River in Brazil. This locality at the coordinates01°43004″S 49°32027″W has an estimated population of 22 890 peo-ple (2010). To investigate possible sources of infection that caused theoutbreak, 27 environmental samples from the wooden houses wherethe patients live and surrounding areas where they work or studywere collected. The samples, constituted mostly of indoor dust anddecaying wood from the houses and surrounding hollow trees, werediluted in sterile saline and plated on bird seed agar. Dark brown colo-nies were isolated and identified phenotypically. From six positivesamples, three harbored C. gattii and C. neoformans, one only C. gattiiand one only C. neoformans. High positivity was observed in dust oftwo houses of patients (>10 000 CFU g�1) and the house of the8 years old patient was negative, but a jambo tree in the cemetery, inwhich the patient used to play, was positive. Eighty colonies wereidentified as C. gattii and five as C. neoformans. The molecular analysis(multilocus sequence typing – MLST) for subtyping is still ongoing.

ª 2014 The Authors


Poster Abstracts

The results reinforce evidence of indoor and outdoor exposure risksfor cryptococcal infection, and of the underdiagnosed cryptococcalrespiratory manifestations.

P010

Molecular characterization of Cryptococcus gattii genotypeAFLP6/VGII isolated from woody debris of divi-divi(Caesalpinia coriaria), Bonaire, Dutch CaribbeanF. Hagen,1 A. Chowdhary,2 A. Prakash,2 J. B. Yntema3 and

J. F. Meis1

1Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands;2Vallabhbhai Patel Chest Institute, Delhi, India and 3Radboud

University Medical Centre, Nijmegen, The Netherlands

Background The basidiomycetous yeast Cryptococcus gattii is a pri-mary pathogen that is mainly restricted to subtropical and tropicalclimate zones. It is an important cause of cryptococcosis amongimmunocompetent subjects and it has emerged as a significant path-ogen in Canada and the Pacific Northwest, USA. There is a lack ofinformation about its environmental presence outside MediterraneanEurope, India, North and South America. Environmental samplingfor C. gattii and molecular characterization of the obtained isolateswill provide an insight into the global spread of the various geno-types. Therefore, environmental sampling was carried out on theisland of Bonaire, Dutch Caribbean.Material and methods Woody debris, collected from inside trunkhollows of living divi-divi trees in April 2013, were cultured on sim-plified niger seed agar as described. The sampled divi-divi trees werelocated in Lagun Goto (N12° 140 3.13440, E-68° 220 6.3660), Rinconvillage (N12° 140 24.11160, E-68° 190 32.56620) and neighboringsurroundings of Hato village (N12° 100 11.87340, E-68° 1701.23660). Plates were incubated at 28 °C and periodically observedfor chocolate brown colonies of C. gattii and C. neoformans for up to7 days. Suspected colonies of Cryptococcus spp. were subcultured bydilution plating and identified by their morphologic and biochemicalprofiles, including development of blue color on L-canavanine-glycinebromothymol blue medium and identification by matrix-assisted laserdesorption ionization-time of flight mass spectrometry.Isolates identi-fied as C. gattii were subjected to amplified fragment length polymor-phism genotyping, mating-type analysis and multi-locus sequencetyping.Results Ten colonies of C. gattii were cultured from different trunkhollows of the same divi-divi tree, molecular characterization showedthat all isolates were genotype AFLP6/VGII and mating-type a.Multi-locus sequence typing revealed that all isolates were geneticallyindistinguishable from each other and that these isolates are geneti-cally closely linked to strain CBS1930 (a strain isolated from a goat,after inoculation of clinical material from a fatal pediatric case fromAruba, Dutch Caribbean in the 1950s).Conclusions Cryptococcus gattii is present in the environment of Bo-naire, which suggests that C. gattii will be present in the environmentof other Caribbean islands too. Puerto Rico is the only Caribbean islandwhere C. gattii has been found to date in the environment.

P011

Clinical and epidemiological profile of Cryptococcalmeningoencephalitis cases in a Reference Center Teresina– Piaui, BrazilH. 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 Eul�alio1

1Instituto de Doenc�as Tropicais Natan Portella, Teresina/Piaui,

Brazil and 2Universidade Federal do Piau�ı, Teresina/Piaui, Brazil

Background Cryptococcosis is an important opportunistic fungal infec-tion, with considerable morbidity and mortality in patients with HIV. Itis the second most common cause of opportunistic CNS infections.Aim of the study To establish an epidemiological clinical profile ofcryptococcal meningoencephalitis patients, and to assess a possiblerelationship between CSF cellularity with immunosuppression and tocorrelate the findings with the literature.Methods A descriptive, retrospective cohort, quantitative studybased on a review of the records of patients admitted to a referralhospital over a period of 7 years.Results In the sample analyzed, 67% were men, with a mean age of33.8 years. Most patients came from the states of Piau�ı, Maranh~aoand Par�a, where the disease is considered to be endemic; 28.5 %were HIV negative and 57.1% HIV positive. It was observed that thepatients analysed showed lymphocytic pleocytosis (<500 cells mm�3

, with a normal CSF glucose and high protein concentration. Of the91 patients, 35% died. The correlations performed showed no statisti-cal significance.Conclusion In this paper we conclude that there is a high frequencyof cryptococcosis patienst in adult young men and HIV-infectedpatients, mainly coming from the states of Piau�ı and Maranh~ao.

P012

Lethality of Cryptococcal meningitis in a ReferenceHospital in Teresine, Piau�ı, BrazilH. 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 Eul�alio1

1Instituto de Doenc�as Tropicais Natan Portella, Teresina/Piaui,

Brazil and 2Universidade Federal do Piau�ı, Teresina/Piaui, Brazil

Background Cryptococcosis is a systemic mycosis caused by speciesof the fungus genus Cryptococcus genus. C. gattii occurs mainly inareas of tropical and subtropical climate affecting immunocompetenthosts, and C. neoformans is a cosmopolitan species affecting mainlyimmunocompromised individuals. The infection is often progressingto pulmonary asymptomatic infection that can spread to variousother organs and organ systems. In the systemic form of the diseasethe most commonly affected organ is the central nervous system(CNS) with meningoencephalitis as the most common clinical mani-festation in our environment.Aim of the study Analysis of clinical cryptococcosis patients duringa 7-year period in Piau�ı, Brazil.Methods This study analysed data from 92 patients hospitalized forcryptococcal meningitis in a referral centre for infectious diseases inTeresina, Piau�ı, 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) inpatients with retrovirus and 30% (12/40) in non-retrovirus carriers.Discussion Cryptococcal meningitis remains a disease of high mor-tality despite the new strategies used for early diagnosis and adjuvanttreatment developed in recent years.

ª 2014 The Authors


Poster Abstracts

P013

Genotypes of Cryptococcus neoformans and Cryptococcusgattii as agents of endemic cryptococcosis in Teresina,Piau�ı (Northeastern Brazil)L. Soares Martins,1 B. Wanke,2 M. Santos Laz�era,2 L. Trilles,2

G. Gonc�alves Barbosa,2 R. C. Lima Macedo,2 M. A. Salmito

Cavalcanti,3 K. Dantas Eul�alio,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

1Universidade Federal do Piau�ı, Teresina-Piau�ı/Brasil, Brazil;2Fundac�~ao Oswaldo Cruz, Rio de Janeiro, Brazil; 3Instituto de

Doenc�as Tropicais Natan Portella, Teresina, Brazil and4Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

In Brazil, cryptococcosis caused by C. neoformans occurs in all regionsand it often causes systemic mycosis in AIDS patients and is the thirdleading cause of opportunistic CNS infection in these patients. How-ever, C.gattii acts predominantly as a primary pathogen, attackingimmunocompetent hosts, including children and young adults inNorth and Northeast, and is therefore considered endemic to thestates of Amazonas (AM), Par�a (PA), Roraima (RR), Piau�ı (PI), Per-nambuco (PE) and Bahia (BA). The southern and southeasternregions show sporadic infections by C. gattii. In PI, previous studieshave shown that cryptococcosis caused by C. gattii is strongly associ-ated with human immunodeficiency virus (HIV)-negative patients,afflicting a significant portion of children and young adults. Here, wereport on the identification of the molecular pattern of the Cryptococ-cus species that caused meningitis in patients in Teresina, which isthe capital of the northeastern state of PI, Brazil, providing a quicktool for diagnosis. In this study, the molecular types of 63 cryptococ-cal isolates recovered from the cerebrospinal fluid of meningitispatients diagnosed between 2008 and 2010 in Teresina, Piau�ı, Bra-zil, were analysed. Out of the 63 patients, 37 (58.7%) were humanimmunodeficiency virus (HIV)-positive and 26 (41.3%) were HIV-negative. URA5-restriction fragment length polymorphism analysisidentified 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-negativepatients (21/26, 80.8%). The occurrence of C. gattii VGII in sixapparently healthy children and in seven adolescents/young adultsin this region reaffirms the endemic occurrence of C.gattii VGII-induced primary cryptococcosis and early cryptococcal infection. Inour study, the occurrence of cryptococcal meningitis caused by C.gattii was high among children and Young adults (not older than20 years) (13/63, 19.9%). This result was similar to studies from1995 to 2003 in this same region, as well as in AM and PA. In allof these studies, it is worth noting that none of the children showedsigns of pulmonar compromise at the time of their diagnosis withcryptococcal meningitis. Therefore, with pulmonary infections over-looked by physicians, the diagnosis of the mycosis usually occurswhen the infection has already spread to the CNS, which certainlyaggravates the prognosis. Lethality occurred in 18/37 (48.6%) of theHIV-positive subjects and in 13/26 (50%) of the HIV-negativepatients. The growing number of recent cryptococcosis cases is irre-futable. In this current study, we showed that the C.gattii VGII geno-type is possibly spreading in several cities of PI and MA (Brazilianmiddle-north). More eco-epidemiology studies are necessary for a bet-ter understanding of its risk factors, virulence and geoclimatic distri-bution, as well as its correlation with the outbreak in VancouverIsland and the extent of the expansion of this pathogen in northeastBrazil. It is likely that the geographical distributionis much widerthan currently documented. Our results provide new information onthe molecular epidemiology of C. neoformans and C. gattii in Brazilianendemic areas.

P014

Azole susceptibility of Cryptococcus and other melanizedyeast isolates from environment exposed to fungicidesJ. 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

1Graduate Program of Coordination for Diseases Control,

Secretariat of Health, Sao Paulo State, Brazil; 2Adolfo Lutz

Institute, Public Health Reference Center, Secretariat of Health,

S~ao Paulo State, Brazil; 3Brazilian Collection of Microorganisms

from Environmental and Industry Cpqba/Unicamp, Paulinia, S~ao

Paulo, Brazil and 4Grupo Fleury, S~ao Paulo, Brazil

Background In fungi some mechanisms of resistance to azole drugsare known. It has been observed that these phenotypes develop in yeastpopulations either due to mutations or selection processes. In clinicalsetting the long azole exposition is observed in Cryptococcosis associ-ated to AIDS-cases. Azoles agents extensible employed for agriculturepurpose could result in azole resistance in environmental isolates. Tak-ing account data from 2008, the amounts of agrotoxic compounds soldfor use in agriculture indicated Brazil ranked first world-wide.Aim Evaluate the in vitro azole-susceptibility of Cryptococcus andother melanized yeast isolates recovered from environmental Brazil-ian 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 PauloState, Brazil. The two studied areas differ regarding the usage ofazole-fungicides. We will measure the occurrence of fungicide in col-lected soil and water samples through gas chromatography method-ology. Melanized yeast were recovered and identified according tomacroscopic and biochemical characteristics. MALDI-TOF wasemployed for speciation of unconcluded identification. Additionally,we performed molecular identification by rDNA sequence analysisusing LSU D1/D2 sequences amplified with the NL1 and NL4 primersand to C.neoformans we used a technique the RFLP and mating-type.The obtained sequences were compared with those existing inGenBank and CBS databases. In vitro antifungal susceptibility wasmeasured according to AFST-EUCAST E.Def 7.2 guidelines for vorico-nazole, fluconazole and itraconazole. The activity of pure powder ofazole-fungicides obtained from the manufacturer (Syngenta, USA)were also assayed against all isolates.Results Among 79 yeast colonies we found seven Cryptococcus iso-lates. The sequences results presented 100% similarity to strainsdeposited 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. Wefound minimum inhibition concentration (MIC) values of azoles usedin clinics up to 1 mg/l�1, among Cryptococcus isolates from bothexposed and not exposed to azole-fungicide areas. The MIC rangewere: fluconazole 0,12 to 1 lg/ml�1; itraconazole 0.015 lg/ml�1

and voriconazole >8 to 0.015 lg/ml�1. The difenocolazole-MIC were8 lg/ml�1 for all isolates and for ciproconazole the MIC ranged from0.25 to 8 lg/m l�1.Discussion and Conclusion It’s a hard task the recovering of mel-anized yeasts from water and soil due to fast growing-moulds habitingfrom soil that overgrown on inoculated agar medium. Furthermore,Candida and Rhodotorula colonies develop faster than Cryptococcus iso-lates on primary culture. Even under such contamination we couldrecover 10% of Cryptococcus species isolates. The fluconazole-MICswere the highest. The fungicide-MICs were higher than clinical azole-MICs being. The less susceptible isolates showed high fungicide-MICand clinical azole-MIC values. Of note, the isolates showing the high-est MICs were obtained from fungicide exposed soil and water. Ourfindings suggest that exposition to azole-fungicide compounds couldexplain highest azole-MICs encountered in this study. We emphasizedthe relevance of monitoring the azole-resistance of environmentalCryptococcus isolates for routine in fungicide exposed area, in particu-lar, in agricultural countries with large usage of those compounds.

ª 2014 The Authors


Poster Abstracts

P015

Report of filamentous forms in a mating type VNI clinicalsequential isolates from a single AIDS patientL. Oliveira,1 M. A. Martins,1 M. W. Szeszs,1 J. E. Vidal2 and

M. S. C. Melhem1

1Instituto Adolfo Lutz, S~ao Paulo, Brazil and 2Instituto de

Infectologia Emilio Ribas, S~ao Paulo, Brazil

Background Molecular type and antifungal resistance profiles varydepending on the causative strain and could result in different clini-cal presentation and outcome. Of note, it is recognized that multiplemolecular types can infect a single patient and serial samples yieldaccurate determination of the etiological(s) agent(s).Aim We reported two distinct molecular profiles in the causativeagent of case of meningeal Cryptococcosis that showed filamentouscells in cerebral spinal fluid (CSF) direct examination.Methods and results A male patient 43-old, HIV-positive, native ofLa Paz, Bolivia, living in S~ao Paulo City, Brazil for 15 years, wasattended at a tertiary public hospital. He presented umbilicated pap-ules skin lesions on the trunk and limbs, besides pulmonary andmeningeal symptoms. CD4 counting was 43 cells mm�3 and CSFshowed qualitative positive latex agglutination for Cryptococcus and618 fungal cells per mm3. Treatment was started on day 1 with1 mg kg�1 day�1 of amphotericin B desoxicolate (AmB) associatedwith 400 mg of fluconazole (FCL) twice/day/4 weeks. Relief punc-tures were performed daily in the first seven days of treatment. Theblood culture, CSF and skin tissue cultures yielded the recovery oftypically 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 sam-ple were collected to speciation, sexual mating typing, PCR-RFLPgenotyping and molecular diversity analysis. Morphological and bio-chemical characteristics, CGB test were performed to speciation.URA5gene RFLP was conducted using primers URA5 and SJ01 fol-lowed by digestion with Sau96I and HhaI enzymes. The minisatellite-specific core sequence of the wild-type phage M13 was used in thePCR-fingerprinting of 19 distinct colonies. Molecular type wasassigned, according to the major bands in the patterns. All visiblebands were included for analysis, independent of their intensity,using Bionumerics� software. Antifungal susceptibility test was per-formed by broth micro dilution reference method (E.Def 7.2,AFST-EU-CAST) for FCL and voriconazole (VCL), and Etest� method for AmB.Results The patient presented good clinical course before hospitaldischarge. The initial quantitative CSF culture showed high initialfungal burden (880 000 CFU ml�1) dropping to 270 and180 CFU ml�1 at day 7 and 14, respectively, followed to negativeresults at day 21. The PCR-RFLP results of 18 tested colonies indi-cated C neoformans molecular type VNI and a mating type. Homoge-neous high MIC-values of FCZ (16 mg l�1) and voriconazole(0.25 mg l�1), but low AmB-MIC (0.064 mg l�1) were found.Discussion and conclusion All colonies showed identical band pat-tern by PCR fingerprinting and RFLP analysis, suggesting the occur-rence of just one single molecular pattern of the etiologic agent evenin distinct samples. The antifungal susceptibility to AmB, FCL,vorico-nazole was identical for all colonies in concordance with the homo-geneous molecular type. The combined therapy resulted inimprovement suggesting the in vivo efficacy of AmB, confirmed by invitro results. Otherwise, the primary and sequential isolates showedhigh FCL-MIC values with no correlation with the good outcome.The success of the therapy was demonstrated by serial quantitativeculture, which showed considerable decrease in fungal load duringtreatment. Little is known about the occurrence of filamentation inC.neoformans cells and no relationship to antifungal susceptibility wasdescribed, although low virulence in filamentous forms was priorreported in murine model. One hypothesis is that hyphal productionis an adaptive mechanism to environmental pressures to yeast devel-opment. There is insufficient data to confirm if morphologic altera-tions could result in different molecular pattern as we observed in

this study. Further studies are necessary to explain the clinical rele-vance of multiple molecular profiles of the etiologic cryptococcosisagent and its relationship with morphological changes.

P016

Childhood Cryptococcosis in Colombia, and literaturereviewJ. L. Lizarazo,1 P. Escandon,2 C. A. Agudelo2 and

E. Castaneda2

1Hospital Erasmo Meoz, Cucuta, Colombia and 2Instituto

Nacional de Salud, Bogota, Colombia

Background In the world literature less than 1000 cases of crypto-coccosis in children have been described including those occurring inimmunosuppressed patients. It has not been established the cause ofthe low presentation of cryptococcosis in children and it is thoughtthat it is not because of the lack of exposure.Aim To do an analysis of the epidemiological manifestations of cryp-tococcosis in Colombian children and compare them with data pub-lished in the literature.Methods Epidemiological data (1993–2010) were obtained bymeans of a survey processed by Colombian clinicians and microbiolo-gists. A search was performed in the Medline database from 1996 toSeptember 30, 2013, additionally, we searched for articles in Spanishand Portuguese in the databases Scielo and Lilacs.Results The average age 8.4 years found in this study together witha slight predominance of the male sex (58.5%), is similar to thatfound in other studies: South Africa, Thailand, China, Brazil and theUnited States. Almost half (46.3%) of the studied patients did nothave a known risk factor, 24.4% had HIV infection and the restother conditions. Series in USA, South Africa and Thailand havebeen described where all or almost all the patients had AIDS; like-wise, series exist with patients without AIDS but with a percentageof underlying disease and series of only immunocompetent patients.It is important to highlight the high percentage of immunocompetentpatients 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 thespecies responsible for cryptococcosis. In South Africa, 7% of thepediatric infections are by C. gattii while in Brazil the percentage ismuch higher, 29.6%. The prevalence of 2.6% found in Colombianchildren is low compared with the prevalence reported in the North-ern and Northeastern regions of Brazil, 32% in the State of Bahia,24% in the State of Par�a and 9.5% in Piau�ı. However, Colombianprevalence is similar to those reported in Uruguay (1.3%), Venezuela(0.91%) and USA (0.85–1.4%). In South Africa it has been estimatedthat between 0.9% and 2% of the cryptococcosis cases occur in chil-dren <15 years. In Ghana, cryptococcosis is responsible for 6.9 % ofthe meningitis with positive culture in children <18 years and inBotswana, 2.36% of the meningeal cryptococcosis with positive cul-ture occur in <13 years. The incidence of 0.017 cases 9 100 000children under 16 years reported for Colombia is very low, howeverin one department, the mean annual incidence is 7 times higher(0.122 9 100 000). In South Africa it was calculated, for the year2007, an incidence of 1 case 9 100 000 children in the generalpopulation and 47 cases 9 100 000 children HIV+. In the Gautengprovince, it is estimated an incidence of 38 cases 9 100 000 chil-dren HIV+. In USA an annual incidence of 0.1 % among the pediat-ric population HIV+ was reported. Recently, China reported anincidence of 0.43 cases 9 100 000 children <18 years.Comments In recent years, interest in cryptococcosis in children hasbeen on the rise. However, epidemiological data are still scarce; fac-tors that determine its relative rarity in the child population are stillpoorly understood. Undoubtedly, new studies are needed to improvethe approximation to the handling of children affected bycryptococcosis.

ª 2014 The Authors


Poster Abstracts

P017

Molecular epidemiological and phylodynamic analyses ofCryptococcus neoformans -Cryptococcus gattii speciescomplex in TaiwanH. K. Tseng,1 W. L. Cho,2 C. P. Liu1 and Y. C. Chen3

1Mackay 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 theencapsulated, basidiomycetous yeast-like fungi Cryptococcus neoformans

-Cryptococcus gattii species complex. The climate of Taiwan, located attropical and subtropical regions, is optimal for the growth ofthem.Recently, we published a nationwide survey to explore the molecularepidemiology [1]. However, phylodynamic analyses of clinical Crypto-coccus 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 6VGII isolates) were chosen for multilocus sequence typing (MLST)analysis according to individual M13 fingerprinting pattern. Ourstudy identified seven sequence types (STs) of clinical C. neoformansvar. grubii, which ST5 (86%, 42/49) as the major genotype. More-over, two strains (T107, T169) identical to Vancouver Island minorstrains VGIIb (R272) were identified which was ST7. Two STs(ST328 and ST329) of C. gattii were novel in this study. Contrast tothe C. neoformans var. grubii, the population of clinical C. gattii iso-lates was diverse in Taiwan.Conclusions The population of clinical C. neoformans var. grubii washighly clonal, and the evolutionary change with time was smallbefore 2009 in Taiwan.

P018

Isolation and identification of Cryptococcus Species fromEucalyptus trees in Shiraz, Southern IranK. 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 Crypto-coccus species that has ecologically relationship with eucalyptus treesand could affected lungs and the central nervous system in healthypeoples.Aim of the study To isolate and identify Cryptococcus species fromEucalyptus trees in Shiraz, Southern Iran, by conventional and molec-ular method.Material and methods A total of 406 samples were collected fromtrunks (200), flowers (100) and leaves (106) of eucalyptus treesaround the city of Shiraz. Suspicious colonies were purified and sub-cultured. Initial identification of the isolates was performed using theurease test, the chlamydoconidia test, presence of capsule, colonycolor on Niger seed agar, and ability to growth at 37 °C. For DNAsequence analysis, the ITS regions of DNA were amplified by univer-sal ITS1 and ITS4 primers and the PCR products were sequenced.Results More than 100 yeasts isolated from the samples and a totalof 54 Cryptococcus species were identified as: Cryptococcus albidus 44,Cryptococcus adeliensis 1, Cryptococcus friedmannii 3 and Basidiomycetesp 6. Cryptococcus gattii was not isolated in this study,Conclusion This is the first study about isolation of Cryptococcus spe-cies from Eucalyptus trees and identification by molecular methods inShiraz, Southern Iran. Unfortunately we could not isolate Cryptococ-cus gattii and more study needs to confirm our results.

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

Figure 1. Dendrogram of the neighbor-joining tree based on the con-catenated seven loci of MLST.

ª 2014 The Authors


Poster Abstracts

P019

Molecular identification of Cryptococcus species isolatedfrom pigeon dropping in Shiraz, Southern IranK. 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 birdsdropping and may be acquired by inhalation of the organism fromthe environment.Aim of the study Isolation and identification of Cryptococcus speciesfrom bird’s guano in Shiraz, southern Iran.Material and methods 139 pigeon droppings samples were col-lected from different areas in Shiraz city over a period of 1 year. Thesamples were cultivated on Staib agar and suspicious colonies werepurified and subcutured. Identification of Cryptococcus species wasdone using the urease test, yeast form growth on chlamydosporeagar, presence of capsule in india ink, colony color on Staib agar,ability of growth at 37 °C and internal transcribed spacer sequencinganalysis. Genomic DNA was extracted by the boiling method andsequence analysis of the ITS regions was used for speciesidentification.Results Forty seven (33.8%) out of 139 samples were positive forCryptococcus species. Result of sequence analysis revealed C. neofor-mans 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 placeswere contaminated by different Cryptococcus species and this is a pub-lic health concern. Pigeons play an important role in the spread ofthese organisms.

P020

Effect of adjunctive sertraline on QTc interval in patientswith Cryptococcal meningitisS. Velamakanni,1 R. Kiggundu,2 J. Rhein,1 E. K. Butler,1

D. B. Meya2 and D. R. Boulware1

1University of Minnesota, Minneapolis, MN, USA and 2Infectious

Disease Institute, Makerere University, Kampala, Uganda

Background The first prospective survey on cryptococcosis in Italycarried in 1997-1999 drew a picture of the epidemiology of this life-threatening disease confirming that it was mainly associated to AIDSpatients. Cryptococcus neoformans var. grubii, C. neoformans var. neo-formans, and AD-hybrids were isolated with a similar frequency butwith a different geographical distribution.Aim In the present study we carried out a new prospective surveyon cryptococcosis in Italy with the aim to compare the clinical andmicrobiological data collected to those obtained ten years ago. Thestudy represents an update of epidemiological changing of cryptococ-cosis in the recent period.Methods An Italian network including 18 hospitals was establishedand cryptococcosis cases were recorded from January 2010 toDecember 2013. Clinical information were collected using the sameform adopted 10 years ago to enable data comparison. One or moreisolates from each case were collected for molecular type and matingtype identification.Results During the survey 58 cases of cryptococcosis were recorded.Males represented 74% of the cases. The median age was 47.5 yearsranging from 24 to 81 years. Twenty-four (41%) patients were HIVnegative with different predisposing conditions. Diagnosis of crypto-coccosis was mainly made by positive GXM antigen and cultures. Inthree cases diagnosis relied only on positive antigen in serum and/orcerebrospinal 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 wasisolated.Discussion The results of this cryptococcosis survey show that thenumber of recorded cases is significantly reduced compared to theprevious survey (156 cases) although the period and geographicalcoverage were different. The percentage of HIV negative patients ischanged rising from 2.8% to 41%. This result confirms that, afterthe introduction of HAART, HIV infected patients are less susceptibleto Cryptococcus infection. The ratio male/female is changed from5.5 : 1 to 2.9 : 1 as a consequence of the higher percentage of non-HIV infected patients. The molecular analysis of Cryptococcus isolatesshowed that VNI was the molecular type predominantly isolated(50%) followed by VNIII (36%) and VNIV (14%), in contrast withthe percentages observed in the previous survey (35%, 35%, 30%respectively).

In conclusion our results highlight the need of a constant monitor-ing of cryptococcosis in Italy since the epidemiology of this pathogenis 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. In-nocenti; 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 cryptococcalmeningitis patients treated with amphotericin B andflucytosine plus fluconazole in Western China from 2006to 2013Y. Liu, H. Ye, C. Zhong, K. Liu and X. L€u

West China Hospital, Sichuan University, Chengdu, China

Background Cryptococcal meningitis is a global invasive mycosisassociated with significant morbidity and mortality. Amphotericin B(AmB) combined with flucytosine were recommended as initial treat-ment for cryptococcosis management in 2010 IDSA guideline. SinceCNS remains a pharmacologic barrier to AmB regardless of formula-tion, the concentration of AmB in CNS was only 2–4% vs. plasmaconcentration. Furthermore, AmB need to be gradually increasedfrom lower dosage because of its significant adverse reactions. So it’svery difficult of AmB reaching effective fungicidal concentration in alimited time. While fluconazole has more safety and efficacy withhigh permeability of the blood-cerebrospinal fluid, it is worthy toevaluate fluconazole added to induction therapy of Cryptococcalmeningitis.Aim To investigate the therapeutic effects of non-HIV infectedpatients with Cryptococcal meningitis treated with not only ampho-tericin B and flucytosine but also fluconazole from beginning.Methods A retrospective study was conducted including 45 cases ofnon-HIV-infected Cryptococcal meningitis in West China Hospitalduring 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 fluco-nazole, n = 27). The dosage of AmB was 0.5 mg–1.0 mgkg�1 day�1, that of flucytosine was 100 mg kg�1 day�1 and fluco-nazole was 400 mg kg�1 day�1. Treatment was considered success-ful if the patient had three consecutive negative CSF cultures. Thenthey were continued with fluconazole till recover. The comparisonbetween two groups was done by t test and Chi-squared test.Results Cure rate of triple therapy group and duplex therapy groupwere 81.48% (22/27) and 66.67% (12/18), respectively. But no sig-nificant statistical differences were found between patients in twogroups with successful treatment (P = 0.257). Nonetheless, therewere significant statistical differences of the mean length of time untiltreatment was successful between the triple therapy group andduplex therapy group (71.9 � 37.32 days vs. 116 � 59.08 days;

ª 2014 The Authors


Poster Abstracts

P = 0.021). The total dosage of amphotericin B of the triple therapygroup was less than the duplex one (3090.6 � 1540.21 mg vs.4889.89 � 2787.48 mg, P = 0.035). Furthermore, the adverseeffects 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 plusfluconazole in induction therapy phase could more effective for cryp-tococcal meningitis. Although we could not improve the cure ratewhen we treat cryptococcal meningitis with AmB, flucytosine andfluconazole at the outset, but it maybe cost patients’ shorter lengthof stay and less adverse effects. Further prospective clinical trials withlarge sample size in multi-center will be worthwhile to confirm theconclusions.

P022

Coupling of nucleotide homeostasis and mRNA decay inCryptococcus neoformans virulence and Amphotericin BsusceptibilityD. Banerjee and J. C. Panepinto

State University of New York at Buffalo, Buffalo, NY, USA

Background Nucleotide biosynthesis pathways are common thera-peutic targets for cancer therapy, antiviral therapy, and anti-parasitetherapy. A combination of Amphotericin B (AmB) and a singlenucleoside analogue, 5-fluorocytosine (5-FC) is the gold standard foranti-cryptococcal therapy. Cryptococcus neoformans encodes the nec-essary enzymes for de novo synthesis and salvage of purine andpyrimidine nucleotides. Mutation of de novo uridine and guanosinesynthesis has been demonstrated to reduce virulence in mouse mod-els of cryptococcosis. Given the requirement of nucleotide and nucle-otide sugars for growth and pathogenesis of C. neoformans,disrupting nucleotide metabolic pathways might thus be an effectivemechanism for the development of novel antifungal drugs. When denovo biosynthesis is inhibited, salvage of nucleotides occur by utiliz-ing recycled bases endogenously within the cell or from exogenoussources. One such source of endogenous precursor is nucleosidemonophosphates (NMPs), the end products of mRNA degradation, aprocess initiated by the deadenylase Ccr4p. Our overall hypothesis isthat nucleotide homeostasis modulates virulence and AmB suscepti-bility in C. neoformans and this balance is maintained partially bymRNA turnover.Aim To demonstrate the role of nucleotide metabolism on virulenceand AmB susceptibility of C. neoformans. To investigate the role ofmRNA turnover in the maintenance of nucleotide pools.Methods E-tests, MIC checkerboard and time-kill assays were per-formed with AmB and mycophenolic acid (MPA) together to perturbpurine synthesis and investigate the drug interactions. Similar exper-iments were performed with a ura- FOA mutant and a ura1-delta(dihydroorotate dehydrogenase) mutant and respective comple-mented strains in addition to capsule detection assay by India inkstaining. In vitro virulence assays- to determine growth at 37 °C,capsule production by India ink staining, cell wall integrity by grow-ing in presence of 0.03% SDS and melanin synthesis by growing inAsparagine agar supplemented with L-DOPA were performed. mRNAstability assays were performed with ccr4-delta mutant duringcarbon starvation and levels of ribosomal protein transcripts weremeasured by Northern blot analyses. Time-kill and growth assayswere done with ccr4-delta mutant in presence of MPA and exoge-nous guanine. Levels of de novo and salvage genes were measuredby qRT-PCR.Results C. neoformans ura- and ura1-delta mutants were more sus-ceptible to AmB, and this was reverted by complementation. Additionof uracil/uridine to the medium did not reverse this hypersensitivityto AmB but the supplementation restored capsule formation in theotherwise acapsular strains. ura1-delta mutant also demonstratedgrowth sensitivity at 37 °C and in presence of SDS. Treatment withMPA in wild type also showed increased susceptibility to AmB, dis-playing synergistic interactions in checkerboard MIC and killing

assays. Results with ccr4-delta mutant showed stabilization of ribo-somal protein (RP) transcripts during carbon starvation. Analysis ofgene expression revealed an up-regulation of the nucleotide synthesismachinery. Time-kill assays in the presence of MPA demonstratedenhanced killing of the mutant compared to wild type that was res-cued by exogenous guanine. It also exhibited increased sensitivity toAmB.Discussion Nucleotide depletion, either due to a drug or a mutation,potentiates the antifungal efficacy of AmB. Our data also suggests arole for mRNA turnover in maintaining cellular nucleotide homeo-stasis, as a ccr4-delta mutant is both AmB sensitive and nucleotide-starved. Future studies will aim to quantify and compare the intracel-lular nucleotide pools in wild type and ccr4-delta mutant during car-bon replete and deplete conditions. We will also conduct experimentsto investigate the mechanism of enhanced AmB efficacy duringnucleotide depletion that may aid in identifying novel antifungaltargets.

P023

The novel fungal Cyp51 inhibitor VT-1129 demonstratespotent in vivo activity against Cryptococcal meningitiswith an improved formulationL. 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

1University 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 mor-bidity and mortality in immunocompromised patients. Even withappropriate therapy, morbidity and mortality associated with this dis-ease remains unacceptably high. Thus, there is a need for new anti-fungal therapies against this invasive mycosis. VT-1129 is a novelfungal-specific Cyp51 inhibitor with potent in vitro and in vivo activ-ity 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 newsolid-state formulation of VT-1129 against cryptococcal meningitis.Methods ICR mice were inoculated intracranially with C. neoformansUSC 1597. Treatment with oral VT-1129 (VT-1129 MIC0.12 lg ml�1, 100% inhibition), oral fluconazole (MIC 1 lg ml�1,50% inhibition), or placebo began 1 day later. In the fungal burdenarm (N greater than equal 10 mice/group), treatment consisted ofVT-1129 0.1 to 20 mg kg�1 day�1 or fluconazole 10 and20 mg kg�1 twice daily. Treatment continued for either 7 or14 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 kg�1 oncedaily by oral gavage, fluconazole at 10 and 20 mg kg�1 twice dailyby oral gavage, or placebo. Treatment continued until day 10 afterwhich mice were monitored off-therapy until day 30 to assess sur-vival. Fungal burden was assessed by colony-forming units per gramof brain tissue (CFU per gram). VT-1129 plasma and brain concen-trations were measured by LC/MS-MS. Survival was assessed byKaplan-Meier analysis and differences in brain tissue fungal burdenwere assessed for significance by ANOVA with Tukey’s post-test formultiple comparisons. Non-linear regression analysis was used toassess the relationship between VT-1129 concentrations and tissueburden.Results VT-1129 at doses of greater than equal 0.3 mg kg�1 day�1

and each dose of fluconazole significantly reduced brain tissue fungalburden compared to placebo control after both 7 and 14 days of dos-ing. Reductions in fungal burden in mice dosed with VT-1129

ª 2014 The Authors


Poster Abstracts

ranged between 0.69 to 6.06 log CFU g�1 in mice dosed for 7 days,with a 3.48 log CFU g�1 reduction achieved with a VT-1129 meanplasma trough concentration of 1.4 lg ml�1. Cryptococcal CFUswere not detected in animals treated with VT-1129 20 mg kg�1 witha mean trough concentration of 21 lg ml�1. After 14 days of dos-ing, reductions in fungal burden from 0.39 to 6.67 log CFU g�1 wereobserved, with a 4.44 log CFU g�1 reduction achieved and a VT-1129 mean trough concentration of 0.95 lg ml�1. CryptococcalCFUs were not detected in mice treated with VT-1129 at 10 or20 mg kg�1, with a mean trough concentration of 15 lg ml�1 inthe 10 mg kg�1 group. Non-linear regression analysis demonstratedan inverse relationship between VT-1129 concentrations on days 8and 15 and reductions in fungal burden (R2 values 0.79–0.92).VT-1129 brain tissue concentrations were ~2-fold higher than thoseachieved in the plasma in all experiments. A survival advantage wasalso observed with VT-1129. Median survival and percent survivalwith VT-1129 20 mg kg�1 (>30 days and 100%) were significantlyimproved compared to placebo (10.5 days and 40%; P < 0.01). Inaddition, the percent survival with VT-1129 20 mg kg�1 was alsomarkedly improved compared to either fluconazole dose group (range40–60% survival).Conclusions The new formulation of the fungal Cyp51 inhibitorVT-1129 demonstrated potent efficacy in this murine model of cryp-tococcal meningitis. Both reductions in brain tissue fungal burdenand improvements in survival were observed, with undetectable CFUsat the higher doses. These data demonstrate the potential utility ofVT-1129 to have a marked impact in the treatment of cryptococcalmeningitis.

P024

Susceptibility testing of VT-1129, a novel fungal CYP51inhibitor, against Cryptococcus neoformans andCryptococcus gattiiS. 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

1Centers for Disease Control and Prevention, Atlanta, GA, USA;2National 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 esti-mated 600 000 deaths worldwide annually. Furthermore, fluconaz-ole which is typically used as maintenance therapy followingmeningitis must be taken daily and has limitations such as drug-drug interactions and a Class D pregnancy warning. VT-1129 is apotent and highly selective novel inhibitor of fungal CYP51 (lanos-terol demethylase). As an oral induction treatment, VT-1129 is supe-rior to fluconazole in the murine model of CM caused by C.neoformans (see adjoining abstract), and has a significantly longerhalf-life than fluconazole which may decrease the dosing scheduleand improve patient compliance.Aim To test the in vitro growth inhibition of VT-1129 against a glo-bal collection of C. gattii and C. neoformans isolates.Methods The collection consisted of 100 isolates of Cryptococcus neo-formans from patients in South Africa and 300 isolates of Cryptococ-cus gattii from Africa, Australia and North America. Cryptococcusisolates were identified to the species level and isolates of C. gattiiwere further characterized to molecular type. Isolates of all four C.gattii molecular types were used in this analysis. Testing was per-formed as outlined in CLSI document M27-A3. MIC testing was per-formed using RPMI broth in 96-well microdilution plates with finaldilution concentrations of VT-1129 ranging from 0.015 to8 µg ml�1 (with final in-assay DMSO concentration of 1%). Dilutionplates were stored at �70 °C until used. All MIC values were deter-mined visually as the lowest drug concentration at which there wasa 50% and a 100% decrease in growth after 72 h of incubation at

35 °C. VT-1129 MIC values were compared to fluconazole MIC val-ues for the same set of isolates.Results C. neoformans MIC values against VT-1129 were very lowwith a 50% inhibition range of ≤0.015 to 0.125 µg ml�1 and a100% inhibition range of ≤0.015 to 2 µg ml�1. The VT-1129 MIC50and MIC90 ranges were much lower (seven Log2 dilutions) than thefluconazole MIC50 and MIC90 ranges against the same set of isolatesat 50% inhibition. The MIC values at 100% inhibition with VT-1129were even lower than the MIC values with fluconazole at 50% inhibi-tion. The C. gattii MIC values against VT-1129 were similarly lowwith a 50% inhibition range of ≤0.015 to 1 µg ml�1 and a 100%inhibition range of 0.125 to 8 µg ml�1), but the MIC50 and MIC90values were two and three log2 dilutions higher, respectively, thanthose of C. neoformans. The C. gattii VT-1129 MIC50 and MIC90 val-ues at 50% inhibition were six and five log2 dilutions lower, respec-tively, than those of fluconazole.

Previous studies have shown that C. gattii antifungal susceptibilitypatterns vary according to the molecular type of the isolate, with VGIIisolates generally having the highest MIC values. This was largely truefor VT-1129 as well. At 50% inhibition, the MIC50 and MIC90 valuesfor VGIV isolates were lowest (0.015 and 0.06 µg ml�1) followed byVGI isolates (0.03 and 0.125 µg ml�1). VGII isolates and VGIII isolateshad the highest MIC50 and MIC90 values, but they were only 1–3 dilu-tions 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 Cryptococcusisolates from Africa where the burden of Cryptococcus remains veryhigh, and against isolates of C. gattii with high MIC values to fluco-nazole. Based on these data and the robust in vivo efficacy in a mur-ine model of CM (see adjoining abstract), VT-1129 is currentlyundergoing IND-enabling studies that will support the clinical inves-tigation of its potential to reduce the high mortality rate of this dev-astating disease.

P025

In vitro antifungal susceptibilities of Ugandan clinicalisolatesK. Smith,1 B. Achan,2 T. McDonald,1 A. Akampurira,2

L. Okagaki,3 D. Meya,2 D. R. Boulware1 and K. Nielsen1

1University 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 deathsin sub-Saharan Africa each year. A significant cause of this highdeath rate in resource limited settings is due to inadequate treatment.According to World Health Organization (WHO) guidelines, the opti-mal treatment for cryptococcal meningitis is amphotericin B supple-mented with 5-flucytosine. In sub-Saharan Africa, 5-flucytosine isexpensive and not licensed for use in many countries, thus fluconaz-ole is often used instead. The WHO also provides that fluconazolemay be administered singularly and is common in rural sub-SaharanAfrica due to limited resources and lack of intravenous treatmentcapacity. Due to the widespread usage of fluconazole monotherapy insub-Saharan Africa, we examined the level of fluconazole andamphotericin B antifungal drug resistance in Ugandan clinical iso-lates prior to treatment for cryptococcal meningitis. In addition,while the CLSI photometric assay is considered the gold standard forMIC determination, the CLSI assay requires equipment not commonlyavailable in resource limited settings. Thus, we also developed a mac-rodilution assay that could inexpensively and effectively be adminis-tered in a resource limited setting. Comparison of fluconazoleresistance results from the macrodilution assay versus the CLSI mic-rodilution assay showed no difference between the two assays. Com-pared to previous studies, both the MIC50 and MIC90 appeared toincrease for fluconazole. While the amphotericin B MIC50 and MIC90increased compared to previous studies, all isolates were still within

ª 2014 The Authors


Poster Abstracts

the susceptible concentrations. This re-affirms that amphotericin Bshould be administered as a primary treatment for cryptococcal men-ingitis. The high rate of fluconazole resistance observed in our studysuggests fluconazole monotherapy is unlikely to be effective at con-trolling cryptococcal infections and could be a causative factor in thehigh mortality observed in sub-Saharan Africa. We developed andvalidated a low cost/infrastructure drug resistance screen assessablein resource limited settings to routinely test the level of susceptibilityof C. neoformans isolates to fluconazole. When compared to previousstudies, the MIC50 and MIC90 in current Ugandan clinical isolateswas higher for both fluconazole and amphotericin B. Human-to-human transmission of cryptococcal meningitis is extremely rare,thus primary resistance, as seen in our study, is likely acquired inthe environment. Aspergillus fumigatus has acquired drug resistancedue to tri-azole fungicides used in farming. Widespread tri-azole fun-gicide use in Uganda to control the banana pathogen, Mycosphaerellafijiensis, could be causing the increased resistance to fluconazole in C.neoformans.

P026

Evaluation of in vitro combination of antifungal drugs andgenotyping of clinical isolates of Cryptococcus spp. frompatients assisted at a University hospital in BrazilF. 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

1State University of Campinas, Campinas, Brazil and 2Chiba

University, Chiba, Japan

Background Despite all the advances in medicine, cryptococcosisremains 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 isnot available and the treatment of cryptococcosis has been madewith AMB alone or in association with fluconazole (FCZ).Aim the aim of this study was to evaluate the in vitro activity of thecombinations of antifungal drugs and to identify the genotypes ofCryptococcus isolates from clinical samples in patients assisted at theUniversity Hospital of UNICAMP.Methods 80 clinical isolates of Cryptococcus spp. collected from 57patients with systemic diseases were studied. The antifungal suscepti-bility testing was performed according the CLSI M27-A3 (2008) forAMB, 5-FC, FCZ, voriconazole (VCZ), itraconazole (ITZ) and terbina-fine (TRB). Drug interaction was evaluated using ‘checkerboard’ mic-rodilution test design and four different combinations wereperformed: AMB + 5-FC, AMB + FCZ, AMB + TRB and TRB + FCZ.Cryptococcus species determination was accomplished using canava-nine-glycine-bromothymol blue agar (CGB). Genotyping was accom-plished by restriction fragment length polymorphism of the URA5gene (URA5 – RFLP).Results 66 isolates were C.neoformans and 14 C.gattii. All C.gattiibelonged to VGII genotype and 62 (94%) isolates of C.neoformanswere in VNI genotype. Minimum inhibitory concentration (MIC)ranges for C.neoformans were: AMB: ≤0.125–1 lg ml�1; 5-FC:≤0.125–2 lg ml�1; FCZ: 0.25–8 lg ml�1; TRB: 0.125–2 lg ml�1;ITZ: 0.03–0.25 lg ml�1 and VCZ: ≤0.015–0.125 lg ml�1. MICs forC.gattii ranged from ≤0.25 to 1 lg ml�1 for AMB; 0.5–4 lg ml�1 for5-FC; 2–16 lg ml�1 for FCZ; 0.5–4 lg ml�1 for TRB; 0.06–0.5 lg ml�1 for ITZ and 0.06–0.25 lg ml�1 for VCZ. C.neoformansVNI genotype showed 75.80% synergistic interaction for AMB + 5-FC; 79.03% for AMB + FCZ; 77.41% for AMB + TRB and 95.16%for TRB + FCZ interaction. VNII genotype had 100% synergism effectfor all combinations. C.gattii VGII genotype showed 85.71% synergis-tic interaction for AMB + 5-FC; 85.71% for AMB + FCZ; 100% forAMB + TRB and 85.71% for TRB + FCZ interaction.Conclusion We found a good performance in all combinations, espe-cially those involving TRB for both C.neoformans and C.gattii. C.gattiishowed more than 80% of synergism for all combinations and

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

P027

3-bromopyruvate as a potent anticryptococcal drugM. Dylag,1 K. Niedzwiecka,1 P. Lis,1 Y. H. Ko,2 P.L. Pedersen,3

A. Goffeau4 and S. Ulaszewski1

1Institute 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, Universit�e 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 identifiedin the laboratory of P.L. Pedersen in the year 2000. 3-BP’s killingefficacy toward liver cancer cells has been successfully demonstratedby Y.H. Ko in animals and humans with no apparent side effects.Inour previous studies we have demonstrated that3-BPenters theyeast Saccharomyces cerevisiae cells through the lactate /pyruvate H+

symporter Jen1 membrane protein. We also have demonstrated that3-BP is not submitted to the PDR network of ABC efflux pumps inyeast.Aim The aim of our present study was to determine the spectrumand molecular mechanism of antifungal activity of 3-BP toward dif-ferent yeast-like and filamentous fungi.Methods The killing effect of 3-BP on various strains was examinedby standard spot tests method and also by a microdilution bioassay(CLSI protocol M27-A2). The influence of sub-MIC concentrations of3-BP on the intracellular ATP level in Cryptococcus neoformans wasdetermined using the ATPliteTM system (PerkinElmer). The 3-BPuptake assays were performed using [14C]-labeled 3-bromopyruvatebased on the method previously described for radioactive L-lactate.The intracellular level of glutathione (GSH) was determined spectro-photometrically according to the published procedure.Results Our studies demonstrate different susceptibilities toward 3-BP even between strains from the same Cryptococcus – like genus.Among the tested fungi, all the C. neoformans strains were particu-larly sensitive to 3-BP (MIC 0.12–0.2 mmol l�1). The best killingactivity of 3-BP toward this fungal pathogen correlates with its intra-cellular high accumulation and also with naturally low level of GSH.A rapid and drastic decrease in intracellular ATP after 3-BP treat-ment leads to fungal cell death. The induction of DOPA-melanin syn-thesis in C. neoformans cells may offer some protection against thetoxic effects of 3-BP.Conclusion Summarizing 3-BP could be a novel promising anticryp-tococcal drug.

ª 2014 The Authors


Poster Abstracts

P028

Evaluation of three commercial methods for antifungalsusceptibility testing of ‘non-grubii’ Cryptococcusneoformans strainsM. F. Colom Valiente,1 C. Linares,1 F. Hagen,2 V. Rosa1 and

M. Torreblanca1

1University Miguel Hernandez, Sant Joan d’alacant, Spain and2Canisius-Wilhelmina Ziekenhuis, Nijmegen, The Netherlands

Background Among the non-Candida yeasts, members of the Crypto-coccus neoformans-Cryptococcus gattii complex have been the mostcommon species recovered among clinical isolates, as well as the sec-ond most common cause of severe fungal infection due to pathogenicyeasts. These cryptococcal infections are associated with high mortal-ity rates. Some previous studies on the in vitro antifungal susceptibil-ity of C. neoformans showed that species and genotype influenced thesusceptibility to antifungal drugs used to treat cryptococcosis. In ourgeographical area (Spain and part of Europe) genotypes VNIII andVNIV of C. neoformans are especially prevalent in contrast to theworld wide most prevalent C. neoformans var. grubii.Aim The evaluation of three different commercial methods for theantifungal sensitivity testing of Cryptococcus neoformans VNIII andVNIV (C. n. var neoformans) isolates.Methods Commercial methods tested were Sensititre Yeast One-SYO- (Trek Diagnostic Systems), Etest (AB Biodisk) and SensiQuattroCandida EU (Liofilchem). A total of 31 isolates of C. neoformans var.neoformans serotype D(genotype VNIV) and C. neoformans hybridsserotype AD (genotype VNIII) were tested. The isolates were previ-ously tested by the reference microdilution standardized method foryeasts (CLSI M27-A3). Amphotericin B, Posaconazole, Fluconazoleand Voriconazole were included in all test performed while Itraconaz-ole was only tested by SYO. Flucytosine, Caspofungin, Micafunginand Anidulafungin were only studied in Sensititre YeastOne (SYO)and Sensiquattro panels.Results As it was expected from previous published works, none ofthe echinocandins tested showed antifungal activity against any ofthe C. neoformans studied. All isolates produced clearly visible growthonly after 72 h of incubation in SYO and E-test and even longer peri-ods (5 days) in SensiQuattro panel. We could not read the MIC end-point clearly at 48 h in most of them because the growth in allisolates and systems was insufficient.

The three commercial methods showed a good correlation withCLSI for Amphotericin B and azoles which are the main drugs forthe treatment of cryptococcosis. Nevertheless, for Fluconazole someremarkable differences were detected: Etest showed higher MICs thanCLSI (GM: 33 lg ml�1 for Etest and 8 lg ml�1 for CLSI) and SYOand SensiQuattro lower values than CLSI (4 and 1.4 lg ml�1 respec-tively). Results for Flucytosine are more difficult to analyze becauseSensiQuattro panel can only discriminate from a minimum value of4 lg ml�1. The geometrical mean (GM) for this drug by SensititreYeastOne was 4.4 lg ml�1 and by SensiQuattro panel 79% of strainsshowed values under 4 lg ml�1.Conclusion Results should be read after 72 h incubation for SYOand Etest and more than 96 for SensiQuattro panels. Sensititre Yea-stOne showed the best global correlation with the reference method.For the three commercial systems, results were coincident forAmphotericin B and most azole drugs. Nevertheless, for FluconazoleMICs values obtained with colorimetric microdilution panels (SYOand SensiQuattro) tend to be lower than the ones from the referencemethod and the opposite situation occurs with Etest.

P029

Non-invasive assessment of intracranial pressure inCryptococcal meningitis using Tonometry and OcularSonographyH. 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

1Infectious Diseases Institute, Makerere University, Kampala,

Uganda; 2University of Minnesota, Minneapolis, MN, USA;3Hennepin County Medical Center, Minneapolis, MN, USA and4Center for Infectious Disease & Microbiology Translational

Research, Minneapolis, MN, USA

Background Cryptococcal meningitis (CM) is associated withincreased intracranial pressure (ICP). Daily therapeutic lumbar punc-tures (LP) are recommended when ICP is >250 mmH2O, yet mano-meters are unavailable in Africa and not always used whereavailable.Aim We assessed whether measuring (i) intraocular pressure bytonometry or (ii) optic nerve sheath diameter using ultrasound couldbe used as non-invasive surrogates for predicting increased ICP in CM.Methods Ninety eight HIV-infected Ugandans with suspected menin-gitis (86% CM) had intraocular pressure measured in both eyes usinga handheld tonometer (CareFusion, McGaw Park, IL, USA) prior toLP (n = 77) and/or optic nerve sheath diameter (ONSD) measured byultrasound (Sonosite, Bothell, Washington) before and after LP(n = 89). We determined the diagnostic performance of these non-invasive techniques for predicting increased ICP in comparison withLP opening pressure using manometry.Results The median ICP was 225 (IQR: 135, 405) mmH2O. The med-ian intraocular pressure was 28 (IQR: 22, 37) mmHg. ICP and intra-ocular 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 (oddsratio = 4.6, 95% CI: 1.8–11.9, P = 0.002). As the intraocular pres-sure increased, the proportion with elevated ICP (i.e. positive predic-tive value) increased. The average intraocular pressure from both eyeshad better diagnostic performance than just one eye. Ultrasound cor-related moderately with ICP (rho = 0.36, P = 0.0006). With an aver-age 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 mea-surement before and immediately after lumbar puncture. There wasan observed difference between the measured ONSD in both eyes.Conclusions Non-invasive intraocular pressure measurements byocular tonometry or ultrasound each correlate moderately withintracranial measurement, but both are a suboptimal replacement foractual ICP measurement using a manometer at time of LP.

Table 1 Summary of diagnostic intraocular pressure thresholds topredict elevated intracranial pressure.

Intraocular Pressure from

both eyes

Performance to detect Intracranial Pressure (> 250 mm H2O)

Sensitivity Specificity PPV NPV C-Statistic (95% CI) P value

Average > 20 mm Hg 95% 34% 57% 88% 0.62 (0.52-0.77) 0.029

Average ≥ 28 mm Hg 70% 66% 65% 71% 0.68 (0.56-0.80) 0.006

Average > 35 mm Hg 49% 90% 82% 66% 0.68 (0.56-0.80) 0.006

Minimum ≥35 mm Hg 40% 95% 88% 64% 0.68 (0.56-0.80) 0.007

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 sen-

sitivity and specificity.

Table 2 Summary of diagnostic ONSD thresholds to predict elevatedintracranial pressure.

ONSD from both eyes

Performance to detect Intracranial Pressure (> 250 mm H2O)

Sensitivity Specificity PPV NPV C-Statistic(95% CI) P value

Average ≥ 5 mm 72% 55% 44% 80% 0.08 (0.28-0.57) 0.0006

Average ≥ 6 mm 58% 58% 79% 34% 0.1 (0.103-0.56) 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 sen-

sitivity and specificity.

ª 2014 The Authors


Poster Abstracts

P030

Scorpion-venom derived antimicrobial peptides withantifungal activity against Cryptococcus neoformansF. 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, vertebratesand invertebrate animals and microorganisms produce antimicrobialpeptides (AMPs) as defense tools against infection. In mammalians,AMPs are components of innate immune system that can act both asantimicrobial agents and as important immune modulators enhanc-ing its protective functions. Our group has been working in the iden-tification of AMPs from arthropod venom glands with potentialantimicrobial or immunomodulatory activity, in special against path-ogenic fungi. Considering the relatively low number of antifungaldrugs currently available and their high cost and toxicity, there is astrong need for the development of therapeutic alternatives especiallywith the increasing rise of resistant strains.Aim The present work aimed to evaluate the antifungal activity ofscorpion-venom derived AMPs against the human pathogenic fungusCryptococcus neoformans. C. neoformans is a ubiquitous distributedencapsulated yeast responsible for more than 600 thousand deathsper year worldwide.Methods Chemically synthetized AMPs derived from sequencesobtained from cDNA libraries of the venom gland of Brazilian Cerra-do scorpions. The sequences were chosen based on structural similar-ities with previous isolated antimicrobial peptides from scorpions andamphibians. Cells of different strains of C. neoformans were grown inthe presence or absence those peptides using the microdilution brothsusceptibility assay described in the M27-A2 guidelines with somemodifications and amphotericin B as a positive control. After 48 h ofincubation fungal growth was evaluated by optical density measure-ment at 630 nm. The MIC50 and MIC90 values for each peptidewere calculated using non linear regression analysis.Results and discussion The MIC50 against C. neoformans strainH99 ranged from 4.4 to 75.2 lmol l�1, although none of the

peptides was more potent than amphotericin B. C. neoformans B3501strain showed lower inhibitory values when compared to the H99strain. Capsule induction assays in the presence of AMP6, the mostpotent peptide in our analysis, revealed a significant decrease in cap-sule size in the presence of this molecule. Additionally, scanning elec-tron microscopy of C. neoformans cells incubated with 13 lmol l�1 ofAMP6 for 1 h demonstrated severe deleterious defects in fungalstructure (Fig. 1 right panels) in comparison with untreated cells(Fig. 1 left panels). Preliminary colony forming unit assays suggestedthat AMP6 activity is fungistatic, rather than fungicidal. Peptidesequence modification to enhance alpha-helix structures abolishedAMP6 antifungal activity suggesting that membrane insertion mightnot be the main mechanism of its antimicrobial activity. Presently,we are evaluating the role of melanin and capsule size in C. neofor-mans susceptibility to this peptide and the possible molecular mecha-nisms of how it acts. We also intend to analyze the effects of thispeptide against C. neoformans biofilms and evaluate its effects in com-bination with currently available antifungals.Conclusion Our results demonstrated that AMPs are promising anti-fungal alternatives. Further characterization of the molecular mecha-nisms involved in their antifungal activity as well as in vivo studiesare essential to establish their true potential in our search for betterantifungal therapy options.

P031

Erg6 is a potential drug target in Cryptococcus neoformansF. 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

1Universidade de Bras�ılia, 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 tothe current drugs used against human fungal pathogens are causesof great concern. Treatment failures lead to the urgent need of devel-oping new antifungal drugs, but this requires the identification andcharacterisation of new potential molecular targets. Erg6, a sterol C-24 methyltransferase, acts on the ergosterol biosynthetic pathway inthe conversion of zymosterol to fecosterol and is of interest as apotential antifungal target, as this reaction occurs exclusively infungi, whereas the mammalian hosts have specific enzymes for theconversion of zymosterol to cholesterol.Aim To characterize Erg6 as a potential antifungal target we haveinvestigated its role in Cryptococcus neoformans through deletion ofthe ERG6 gene and phenotypic analyses of the mutant.Methods A deletion cassette was constructed by DJ-PCRand insertedon theH99 (serotype A) strain by biolistic transformation. The erg6Δstrain was confirmed by PCR and Southern blotting. A reconstitutedstrain was generated by transforming the mutant strain with theERG6 gene alongside the pJAF1 plasmid containing the NEOR resis-tance marker. The mutant and reconstituted strain were tested in vi-tro for common phenotypes associated with virulence: capsuleinduction, melanisation, growth at 37 °C, urease and phospholipasesecretion and resistance to several stressor agents such as CongoRed, SDS, caffeine, Calcofluor White (cell wall), NaCl and KCl (osmo-tic) and H2O2 (oxidative). The strains were tested for virulence in thein vitro J774.A16murine macrophage model of phagocytosis followedby CFU counts. The in vivo virulence was assessed by survival curvesin the Galleria mellonella alternative model of infection at 37 °C. Drugsusceptibility tests were performed in solid medium with Fluconazole,Itraconazole, Cetoconazole, Amphotericin B, Nystatin, FK506, Cerule-nin, Brefeldin A and Terbinafin. The Minimal Inhibitory Concentra-tion was determined for each drug according to NCCLS. The sterolcomposition of the cell membranes of the mutant strain was evalu-ated by HPLC.

Figure 1 Morphological changes in C. neoformans cells induced byAMP6.

ª 2014 The Authors


Poster Abstracts

Results The ERG6 deletion causes several alterations in cellularhomeostasis, such as increased sensitivity to osmotic and oxidativestress and to cell wall stressors. The erg6Δ strain is also more suscep-tible to Azoles and more resistant to Polyene compounds. Themutant was unable to grow at 37 °C on solid medium and a growthcurve 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 andto colonise G. mellonella caterpillars. Finally, HPLC analysis showed achange in membrane sterol composition with a dramatic reductionin ergosterol content.Discussion/conclusion The protein transport and anchoringdepends on membrane integrity and can be affected by the loss ofErg6. Our results confirm yet again the importance of membraneergosterol to the viability and adaptability of the fungus to diverseconditions. Therefore, Erg6 being an enzyme found exclusively infungi that plays an important role on ergosterol biosynthesis, it is apotential molecular target for new antifungal drugs that can be usedalone to impair the fungus survival in the host or in combinationwith established drugs to reduce their dosage, and thus the sideeffects, through synergism. With the present work, Erg6 is confirmedon C. neoformans as a very promising molecular target for the devel-opment of new antifungal drugs.

P032

Clinical trial protocol and report on recruitment:comparing flucytosine-fluconazole combination therapy tostandard of care in western KenyaK. 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

1University 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 secondleading cause of HIV-related deaths in resource-limited settings.Screening asymptomatic HIV-infected individuals with advancedimmunosuppression for serum cryptococcal antigen (sCrAg) clearlyidentifies a population at high risk of cryptococcal meningitis (CM)and death in resource-limited settings. Currently there is wide vari-ation in practice and little evidence to guide the treatment of earlycryptococcal infection, also known as asymptomatic cryptococcalantigenemia, in HIV-infected individuals with advanced immuno-suppression. The mainstay of anti-cryptococcal therapy inresource-limited settings is oral fluconazole, though preliminaryevidence suggests this may not be an effective treatment for earlycryptococcal infection. Thus, there is a critical need for potenttherapies that (i) can be safely administered in resource-limited set-tings and (ii) are effective in a heterogeneous population of HIV-infected individuals with advanced immunosuppression and earlycryptococcal infection who are initiating anti-retroviral therapy(ART).Aim To compare the safety and preliminary efficacy of fluconazoleplus flucytosine in combination to the standard of care, fluconazoletreatment alone, in a population of HIV-infected patients with earlycryptococcal infection in Western Kenya.Methods This is a Phase IIB randomized open label clinical trialbased at two sites supported by Family AIDS Care and Education Ser-vices (FACES) in Western Kenya. We will enroll a consecutive sampleof 100 HIV-infected adults with CD4 + T-cel count ≤100 cells ll�1

and sCrAg titer ≥1 : 2 who have no signs or symptoms of severe,systemic cryptococcal infection.

Individuals who meet inclusion and exclusion criteria and consentto participate in the study will be randomized to combination therapywith oral fluconazole (1200 mg day�1) plus flucytosine(100 mg kg�1 day�1) or fluconazole alone for the fourteen days oftherapy. Subsequently both groups will receive anti-retroviral therapy

as well as fluconazole 800 mg day�1 for 8 weeks followed by200 mg day�1. The primary safety endpoint will be the incidence oftreatment-related adverse events and serious adverse events. The pri-mary efficacy endpoint will be survival at 12 weeks. Several secondaryoutcomes including, but not limited to, survival at 24 weeks, numberof individuals who develop cryptococcal meningitis, number of individ-uals who develop immune reconstitution inflammatory syndrome dueto Cryptococcus, and cryptococcal meningitis-free survival at 24 weekswill also be assessed (ClinicalTrials.gov Identifier: NCT01562132).Results and discussion Patient screening and enrollment began inOctober 2013 and at the time of this report, 297 individuals hadCD4+ T-cel count ≤100 cells ll�1. Among these, 21 individuals hada sCrAg titer ≥1 : 2, and 5 met inclusion criteria and consented toparticipate. The median age of participants was 31 years (rangingfrom 26 to 48) and 40% were female. The median CD4+ T-cell countwas 18 (ranging from 3 to 56), and the lowest sCrAg titer was1 : 640 using the lateral flow assay (LFA). All 5 subjects consentedto an optional lumbar puncture, and all had a positive CSF CrAg titerwith the lowest titer being 1 : 64 by latex agglutination (LA). Thusfar, blood or CSF cultures were positive for Cryptococcus spp. in allparticipants. Importantly, one participant was found to have C. albi-dus in the blood, rarely reported as a human pathogen. While thestudy is ongoing, the preliminary culture results may indicate thatdespite their lack of symptoms, individuals with low CD4+ T-cellcounts and cryptococcal antigen in the serum have central nervoussystem infection.

P033

Anticryptococcal activity of secondary metaboliteslaevicarpin and polygodial isolated from Brazilian plantsS. U. Purisco,1 D. S. A. Maciel,2 J. H. G. Lago,2 A. G. Tempone1

and M. S. C. Melhem1

1Adolfo Lutz Institute, S~ao Paulo, Brazil and 2S~ao Paulo

University, S~ao Paulo, Brazil

Background Cryptococcosis is the second leading cause of deathamong systemic mycoses in Brazil, and 23.9 out of every 1000 AIDSrelated deaths are attributed to cryptococcal infection. Among C. gat-tii molecular types VGII is the most frequent type encountered inLatin America. It is recognized that the clinical findings and thera-peutic management of C. gattii cases present some distinct aspectsfrom those caused by C. neoformans. Cryptococcosis has a narrowtherapeutic arsenal and the antifungal agents used in clinical settingsare limited due to drug toxicity, high costs, low availability, and clin-ical resistance. So, there is an urgent need for the discovery of newdrugs to treat Cryptococcosis. Natural products are a rich source ofantimicrobial compounds and Brazil has a rich plant biodiversitywith 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 spe-cies Piper laevicarpu (Piperaceae) and Drimys brasiliensis (Wintera-ceae), against C. gattii strain-type WM 178 designed molecular typeVGII.Methods Hexane extract from steam bark of D. brasiliensis andCH2Cl2 extract from leaves of P. laevicarpu were obtained usingaccelerate solvent extractor ASE300. After solvent elimination, bothextracts were individually subjected to several chromatographic pro-cedures, including column chromatography over SiO2 and/orSephadex LH-20 to afford, respectively, polygodial and laevicarpin.The in vitro activity of polygodial and laevicarpin against VGIIstrain-type was investigated according to the method (M27-A3,2008) with modifications. We employed 96-well flat-botton platesand range concentrations of 200 to 1.5625 lmol l�1 of each com-pound. 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 wereperformed in triplicate. C. krusei (ATCC 6258) and C. parapsilosis

ª 2014 The Authors


Poster Abstracts

(ATCC 22019) were included as quality control in all tests. Theminimal fungicidal concentration (MFC), minimal inhibitory concen-tration (MIC) of 50% and 50% inhibitory concentration (IC50) weredetermined.Results Structures of polygodial and laevicarpin were elucidatedbased in analysis of NMR and MS spectra and compared to the litera-ture. Polygodial showed an IC50 of 0.51 lg ml�1 (95% confidenceinterval 0.30–0.84 lg ml�1), MIC of 1.46 lg ml�1 and MFC of0.73 lg ml�1. Laevicarpin showed an IC50 of 2.35 lg ml�1 (95%confidence interval 1.67–3.31 lg ml�1), MIC of 7.5 lg ml�1 andMFC of 60 lg ml�1.Discussion and conclusion In the course of selection of noveldrugs candidates we verified for the first time in literature, a highantifungal activity of polygodial and laevicarpin against C. gattii. Fur-ther in vivo experiments using a cryptococcal model could confirmthe potential of these candidates. Our results suggested that polygo-dial and laevicarpin could be used as scaffolds for future drug designstudies against Cryptococcosis agents.

P034

Suceptibility profile of a Brazilian collection of clinicalCryptococcus gattii isolatesL. 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

1Instituto Adolfo Lutz, Arac_atuba, Brazil and 2Centers for

Disease Control and Prevention, Atlanta, GA, USA

Cryptococcosis is a life-threatening disease and remains a difficultmanagement issue in developing countries. Although cryptococcosishas generally being associated with Cryptococcus neoformans speciesworldwide, 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 incidenceof lung and brain granulomas and neurological complicationsin com-parison with C.neoformans infections. Importantly, it is recognizedthat C. gattii isolates usually show high minimum inhibitory concen-trations (MIC) to fluconazole and other azole drugs used in the man-agement of cryptococcosis cases. This study aimed to describe thesusceptibility of 54 clinical isolates of C. gattii molecular type VGIIand 4 of molecular type VGI from a Brazilian culture collection.

A total of 58 unique isolates of C. gattii were analyzed at the Cen-ters for Disease Control and Prevention (CDC -Atlanta, GA, USA). Iso-lates were from human cryptococcosis cases collectedbetween 1994and 2012 in hospitals located in the southeast part of Brazil. All iso-lates were previously identified by conventional and molecular meth-ods to be C. gattii and the molecular types were confirmed bymultilocus sequence typing (MLST). The antifungal drugs tested wereamphotericin B (AmB), fluconazole (FZ), itraconazole (IZ), voriconaz-ole (VZ), flucytosine (5-FC), andposaconazole (PZ). For all drugsexcept AmB, MIC determinations were performed as recommendedby Clinical and Laboratory Standards Institute (CLSI) documentsM27-A3 (CLSI, 2008) using RPMI broth and frozen panels custommanufactured by TREK Diagnostics (Cleveland, OH, USA). For AmB,the MIC was determined by the E-Test� (bioM�erieux, Fr) method andthe result was defined as the point of 100% growth inhibition. Dataare reported as MIC ranges and MICs of each antifungal agent neces-sary to inhibit 50% (MIC50) and 90% (MIC90) of the isolates tested.Quality control isolates C. parapsilosis ATCC 22019 and C. kruseiATCC 6058 were included on each day of testing and were alwaysfound to be within the recommended range. Because no breakpointsare available, to interpret the susceptibility data we used the MIC val-ues 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 MICvalues and were outlined previously as the MIC value encompassingat least 95% of the wild-type distribution. The distribution of MICs of54 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/l�1 and VGII, 32 mg/ l�1). None of the isolates showed MICs abovethe ECV for PZ. The rate of itraconazole non-WT MICs were higher(5/58, 8.6%) than those of the other three azoles. Although wetested only four isolates of molecular type VGI,we didn’t observed adifference in antifungal susceptibility for FZ,IZ, PZ, 5-FC orAmB,when compared to VGII strains. However, one strain ofmoleculartypeVGI (1/4, 25%) showed high FZ, IZ and PZ MICs (8, 0.25 and0.25 mg/ l�1). Our results show that, for the most part, C. gattii iso-lates from Brazil have similar antifungal MIC values to C. gattiiiso-lates from other parts of the world, and they confirm the previouslyestablished ECVs.

Table 1 Distribution of MIC values of 54 VGII and fourVGI C. gattii molecular types.

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

ª 2014 The Authors


Poster Abstracts

P035

Heterorresistance to fluconazole in Brazilian clinical strainsof Cryptococcus gattiiL. 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

Background C. gattii strains have high potential to develop in vitroresistance to fluconazole (FLC), which could explain the constanttherapeutic failures and relapses in patients affected by Cryptococco-sis under FLC long-term therapy. Heteroresistance is the phenome-non described as the emergence of a minor subpopulation ofresistant cells within a single colony of the susceptible strain thatcan tolerate FLC concentrations higher than the strain’s MIC levels.Aim We aimed in this study investigate the level of FLC-heteroresis-tance exhibited by 46 C. gattii VGII and 4 C. gattii VGI clinical strains(one per patient) from the culture collection (1994–2004) of InstituteAdolfo Lutz (SP, Br).Methods Firstly, the MIC of FLC was determined by Etest� methodfor each strain, and then its level of heteroresistance to FLC (LHF)was assessed. Cell suspensions of all strains were inoculated ontoYPD plates containing various concentrations of FLC (4–128 mg l�1). The lowest FLC concentration at which minor resistantsubpopulations emerged was identified as each isolate’s LHF. Toobtain the highly resistant subclones, the heteroresistant colonieswere isolated and inoculated on YPD agar containing stepwise (two-fold) increases in the concentrations of FLC (up to 256 mg l�1). Theculture plates of each passage were incubated at 30 °C for 72–96 h.Finally, we have analyzed if the transferring of the highly resistantsubclones on drug-free media caused reversion to the original level ofheteroresistance. The H99 strain, and VGI-VGIV strain-types (kindlysent by Prof. W. Meyer) with designed LHF were used as control test.Results The MICs for these 50 strains ranged between 2 to32 mg l�1. Similarly to the unique published study we have demon-strated that all C. gattii tested strains manifested FLC-heteroresistancewith LHFs that ranged between 32 mg l�1 and 128 mg l�1. Thehighly resistant subclones have been raised in a stepwise manner to256 mg l�1. All strains reverted to the original LHF upon dailytransfers in drug-free medium in distinct time-period.Discussion and conclusion The study on heteroresistance mayreveal a novel adaptive mechanism for survival under the azole stressand can offer helpful insights into the management of long-termtherapy. We observed that in C. gattii the level of FLC-heteroresis-tance was high (LHF ≥32 mg l�1) and varies between strains. More-over, all 50 tested clinical strains (100%) showed highly resistantsubclones (growth at 256 mg l�1). We confirmed it is a reversibleadaptive response to the presence of the drug.

P036

Antifungal susceptibility profiles assessed by a modifiedbroth macrodilution method using patient-specificinoculum method of clinical isolates obtained fromcryptococcal meningitis cases attended at a tertiary publichospital S~ao Paulo, BrazilL. 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

1Instituto Adolfo Lutz, S~ao Paulo, Brazil and 2Instituto de

Infectologia Emilio Ribas, S~ao Paulo, Brazil

Background Antifungal susceptibility tests are largely employed astool for therapy for an individual patient, but lacking of interpretativeclinical breakpoints for Cryptococcus isolates against practice useddrugs cause controversial issue in Cryptococcosis.Up to date, thedrug-MIC values are not likely to predict the clinical outcome inCryptococcosis.In fact, patient’s pretreatment severity of meningitis isconsidered the major risk factor associated with outcome.Due to drugtoxicity, particularly, AmB and/or 5FC, it is warranted to avoidunnecessary drug administration. In previous studies it was proposeda modified macrobroth dilution assay(MMD) for predicting theresponse of individual patient to treatment with AmB, FCL, or thecombination of AmB plus 5FC.Aim We used the MMD test for assessing susceptibility of clinical iso-lates obtained from AIDS-associated cryptococcal meningitis. Addi-tionally we reviewed the medical records to assess the clinicaldecision and outcome for each patient and correlated with antifungalsusceptibility testing results.Material and methods Data and isolates were from five Brazilianpatients infected by C.neoformans (Cn) VNI and one infected by CnV-NII.Severity of meningitis before antifungal-treatment and the quan-titative responses at 0, 7 and 14 days were measured by CFUcounting per milliliter of cerebrospinal fluid. The inoculum employedfor MMD was matched to number of CFU ml�1 present in pretreat-ment CSF for each individual patient. The range of AmB concentra-tions tested was from 0 to 3.5 mg l�1, FCL from 0 to 500 mg l�1,and 5FC from 0 to 1000 mg l�1. The end-point was based on serialdilutions taken directly from each test tube after 48 h-incubation.The drug-concentration that produced the quantitative in vitroresponse that corresponded to patient’s quantitative biologic responsein the CSF at day-14 of treatment was determine as previouslydescribed (relevant in vitro drug concentration = RivDC).Additionally,AmB-MIC, 5FC-MIC and FCL-MIC were determined by E-test�method, and time kill-curves were performed to assess cidalendpopint of 1 mg l�1 of Amb. Checkerboard method was employedfor analysis of drug interactions through FICIvalues.Result All patients (21–45 years-old) showed CD4 < 150 cellsmm�3 and were treated with AmB desoxycolate(1 mg kg�1 day�1)plus FCL(400 mg 12/12 h) for 4 weeks. In one patient the desoxyco-late formulation was changed at Day 5th to liposomal AmB

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

ª 2014 The Authors


Poster Abstracts

(165 mg1x/day for 2 weeks). One patient received also 5FC (200 mgdaily) after the first week. Successful treatment was observed in allpatients 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 ml�1 of CSF.Sterile CSFculture after day-7 of treatment was observed for two patients andday-14 for one patient.Mycological cure was obtained for remainthree patients only after a month of therapy. Using isolates from fourpatients, we found that the estimated RivDC for AmB was0.5 mg l�1, for the remaining 2 the RivDC was 1 mg. In all, but one(CnVNII) case the etiological agent was CnVNI showing AmB-MIC of0.5 mg l�1, FLC-MIC > 8 mg l�1. The AmB cidal-effect was similarfor all isolates (6–12 h).Checkerboard method showed indifferentinteraction (2 > FICI > 1.25) between AmB and 5FC in tests with allisolates.Discussion and conclusions All patients evolved to cure, evenwith causative isolates showing high FCL-MIC values indicating lowsusceptibility. Antifungal susceptibility testing with AmB suggestedgood clinical-laboratory correlation (high cidal-effect by low TKvaluesand high inhibitory action by low MICs-AmB). Drug regimens in50% of our patients did not render CSF sterile after 14 days of treat-ment suggesting therapy should be modified early in the course oftreatment to improve probability of survival to 10th week.Of note, allcases presented significant fungal burden exceeding 2500 CFU ml�1

in pretreatment CSFsample. This value represents the maximuminoculum volume recommended in M27-A-CLSI method. In agree-ment with previous authors we found suitable additional studies toevaluate an alternative parameter incorporating the measure ofseverity of meningitis corresponding to number of organisms foundin the pretreatment CSFsample aiming predict response in individualpatients.

P037

Correlation of CLSI and EUCAST in-vitro antifungalsusceptibility with clinical outcome of patients with AIDSassociated cryptococcosis from IndiaA. Chowdhary,1 S. Kathuria,1 A. Prakash1 and J. F. Meis2

1Vallabhbhai Patel Chest Institute, Delhi, India and 2Canisius-

Wilhelmina Hospital, Nijmegen, The Netherlands

Background Cryptococcus neoformans, is the most common etiologicagent of cryptococcosis in immunosuppressed hosts and associatedwith significant morbidity and mortality. India has world-wide thesecond largest burden of cryptococcosis due to an estimated popula-tion of 3.1 million to 9.4 million persons living with HIV-AIDS. Thedata on antifungal susceptibility profiles along with the outcome oftherapy in patients of cryptococcosis helps in the development ofeffective treatment strategies.Objective To investigate the statistical correlation of MICs obtainedby CLSI and EUCAST antifungal susceptibility testing of C. neoformansvar. grubii from 45 HIV-positive Indian patients with their therapeu-tic outcome.Materials and methods A total of 45 patientswith culture provencryptococcosis were included in a prospective study during 2008–2012. Of 45 patients, 96 C. neoformans isolates comprising 75 fromCSF, 9 blood, 8 sputum, 2 urine and one from a bronchial aspiratewere analysed for AFST by CLSI and EUCAST methods. Twenty ninepatients received combination therapy of amphotericin B(1 mg kg�1) and fluconazole (400 mg day�1) whereas 16 patients inaddition received flucytosine (100 mg kg�1). Genotyping was doneusing microsatellite typing. Susceptibility was determined for ampho-tericin B (AMB), flucytosine (FC), fluconazole (FLU), voriconazole(VRC), posaconazole (POS), isavuconazole (ISA) and itraconazole(ITC) by CLSI and EUCAST methods. Statistical differences betweenmean MICs of CLSI and EUCAST were assessed using Student’s t-test.Discrepancies of more than two dilutions among MIC results wereused to calculate the essential agreement (EA).

Results All 96 isolates were found to be C. neoformans var. grubiiAFLP1/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 knownMC type. All isolates showed good in vitro activities for all antifungalsexcept two which had high MICs of FC. The mean MICs were signifi-cantly higher by CLSI for AMB, (P = 0.0001), ITC (P = 0.035) andPOS (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 treat-ment groups with higher fatalities among patients with disseminatedcryptococcosis involving two or more sites.Conclusions The present study reports on the comparison of in vitroantifungal susceptibility profiles of C. neoformans var grubii by usingCLSI and EUCAST methods revealed good concordance (97% EA)between the two methods. Two microsatellite types dominate in Indiaand exhibit low MICs of FLU and AMB. The low MICs were associ-ated with successful treatment in 67% of cases.

P038

Drug delivery in C. neoformans using nanoparticlesC. B. Nichols, C. Vazquez and J. A. Alspaugh

Duke University Medical Center, Durham, NC, USA

Background Many compounds with potential efficacy againstmicrobial pathogens are ineffective because of poor intrinsic ability toenter the target cell. The cell surface of C. neoformans possesses twostructures that inhibit drug entry: the cell wall and polysaccharidecapsule. In prior studies we demonstrated that the farnesyl transfer-ase inhibitors manumycin and tipifarnib have a higher efficiencyagainst capsule mutants of C. neoformans compared to wild typestrains, suggesting that the capsule offers protection against both ofthese drugs. In recent years, the field of nanomedicine has developedpolysaccharide-based nanoparticles as drug delivery devices. Chito-san, a principal cell wall component of C. neoformans, is also one ofthe polysaccharides capable of forming nanoparticles. Exogenouschitosan also has antifungal properties against C. neoformans.Aim We hypothesize that drugs packaged into chitosan-derivednanoparticles may be able to traverse the C. neoformans capsule/cellwall barrier into the cell resulting in increased activity against C.neoformans.Methods Purified chitosan was mixed with heparin to generatechitosan-based nanoparticles. In control samples, chitosan nanoparti-cles were prepared in the absence of drug. In experimental samples,various concentrations of compounds with varying antifungal activ-ity were incorporated into the nanoparticles. These preparations weretested against a wild type C. neoformans grubii strain (H99).Results We observed minimal but measurable growth inhibition ofC. neoformans in samples containing chitosan/heparin nanoparticles.In addition, there was increased growth inhibition in samples con-taining manumycin- and tipifarnib-containing chitosan/heparinnanoparticles.Discussion/conclusion As we hypothesized, chitosan-derived nano-particles containing various antifungal agents inhibited growth of C.neoformans. This demonstrated that chitosan nanoparticles were ableto incorporate these compounds. In addition, the chitosan/heparinnanoparticles without drug also had a subtle inhibitory effect on C. neo-formans. This demonstrates that the chitosan polysaccharide packagedinto a nanoparticle is able to inhibit growth and also that the combina-tion of chitosan and drugs may have a synergistic effect against C. neo-formans. In conclusion, our results indicate that chitosan-derivednanoparticle drug delivery in C. neoformans is a viable strategy toenhance cell entry for varied compounds, potentially bypassing thebarriers provided by the cell wall and polysaccharide capsule.

ª 2014 The Authors


Poster Abstracts

P039

High-dose fluconazole for the treatment of Cryptococcalmeningitis in HIV-infected individualsR. 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

1USC 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;7University 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 theonly antifungal agents available for the management of AIDS-associ-ated cryptococcal meningitis. Fluconazole treatment at doses>1200 mg day�1 have only been tested in a small cohort of humansubjects which has limited its use at high doses due to potential con-cerns with both safety and efficacy.We report the pre-specified safetyanalysis of stage 1 of the 2 stage ACTG A5225 phase I/II study oforal fluconazole for the initial management of AIDS-associated cryp-tococcal meningitis.Aim To address safetyand tolerability of high dose fluconazole andobtain more robust estimates of 10 week efficacy.Methods Anti-retroviral na€ıve subjects with AIDS-associated crypto-coccal meningitis were sequentially enrolled into three fluconazolecohorts (1200, 1600 and 2000 mg day�1) with a 3 : 1 randomiza-tion of fluconazole vs. AmB based treatment regimen. Quantitativecerebrospinal fluid (CSF) cultures were obtained every 2 weeks.Treatment with the assigned dose of fluconazole (adjusted for base-line weight) was continued for 2 weeks after the first negative quan-titative CSFculture or to a maximum of 10 weeks. Fluconazole wasreduced to 400 mg daily if CSF cultures became negative prior to10 weeks. Antiretroviral therapy was permitted after 4 weeks ofanti-fungal therapy with an efavirenz based anti-retroviral regimen.The subsequent fluconazole cohort opened if 3 or fewer fluconazoledose limiting toxicities (DLTs) were observed in the first 14 days of afluconazole dose cohort.Results 96 participants enrolled -29 in cohort 1 (22fluconazole1200 mg and 7 AmB), 35 in cohort 2 (26 fluconazole 1600 mg and9 AmB) and 32 in cohort 3 (24 fluconazole 2000 mg and 8 AmB).Participants enrolled in the fluconazole 1200 and 2000 mg day�1

cohorts were more ill based upon observed baseline mental statusand quantitative cryptococcal CSF cultures (Table 1). There were nochanges in renal function, hemoglobin, or liver test abnormalitiesattributed to fluconazole. QTc interval changes were identified in 10

participants receiving these doses fluconazole. In four participantsthere was QTc interval prolongation within the normal range whilein six participants (three on 1200 mg and three on 2000 mg day�1)the QTc internal was prolonged beyond the normal range. None witha prolonged QTc had significant arrhythmias and all resolved withdose reduction of fluconazole. Three participants assigned amphoteri-cin B had a QTc interval change >70 ms but none had a prolongedQTc. Concomitant medications (azithromycin, quinolone antibiotics,and the antiemeticsgranisetron and ondansetron) known to prolongthe QTc interval were prescribed in four participants and appeared toexacerbate the QTc prolongation associated with fluconazole in two.Discussion All three dose levels of fluconazole met safety criteria ofthree or fewer DLTs. After review by an ACTG Safety MonitoringCommittee, Stage 2 of the clinical trial will randomize an additional72 participants in a 1 : 1 : 1 treatment ratio among fluconazole at1600 mg day�1, fluconazole 2000 mg day�1 and amphotericin B.

P040

A modified loop-mediated isothermal amplificationmethod for diagnosis of Cryptococcal meningitisM. 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

1Shanghai 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 systemicmycosis with high morbidity and mortality, particularly in immuno-compromised patients in resource-limited countries. The Cryptococcusneoformans/C. gattii species complex is the primary causative agent ofCM. However, the low sensitivity, accuracy, and time involved ofconventional diagnosis approaches hinder further reduction of themortality rate of CM. Loop-mediated isothermal DNA amplification(LAMP) is a potential technology platform that could meet clinicalrequirements in both the developed and developing world.Aim We established a modified LAMP assay for direct identificationof all members of the C. neoformans/C. gattii species complex, evalu-ated its specificity and sensitivity and explored its suitability for clini-cal diagnostics of CM patients in two university hospitals inShanghai, China.

Table 1 Median Baseline Quantitative Values of Study Participants.

Treatment

Assignment

Amphotericin B Fluconazole 1200

Fluconazole

1600 Fluconazole 2000

N=24 N=22 N=26 N=24

Glasgow Coma

Scale†14 (4.2%) 12 (22.7%) 14 (11.5%) 12 (20.8%)

Intracranial Pressure

(mm CSF)

190 (120-300) 200 (105-300) 200 (150-290) 350 (180-550)

CSF Protein (mg/dL) 61 (13,144) 101 (50-180) 68 (30-145) 64 (13-185)

Quant. CSF Culture

(#/mL)

22,900 (2,130-

171,000)

152,000 (16.000-

780,000)

27,500 (96,000-

107,000)

144,000 (18,500-

443,000)

CD4 Cell Count

(#/mm3)

24 (8-53) 20 (9-55) 59 (21-131) 23 (9-64)

HIV Viral Load

(log10/mL)

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. Figure 1

ª 2014 The Authors


Poster Abstracts

Methods The LAMP primers were generated by Primer Explorer V4software (http://primerexplorer.jp/e/) based on the rRNA intergenicspacer 1 (IGS1) sequence (GenBank accession number: EF211264) ofthe dominant C. neoformans type strain H99. Cryptococcus DNA wasextracted from clinical CSF using the ZR Fungal/Bacterial DNA Mini-PrepTM Kit (ZYMO Research Co., Ltd, Los Angeles, USA) according tothe instructions. The LAMP reactions were carried out twice in the spe-cial reaction tube (Eiken Chemical Co., Ltd, Tochigi, Japan) at 63 °Cfor 90 min in Loopamp real-time turbidity meter (LA-320C). TheLAMP 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 63tested isolates of the C. neoformans/C. gattii species complex. Resultswith DNA of related species was negative. Thus, the analytical speci-ficity of our method was 100% in our study. The detection limit ofDNA of all eight genotypes of the C. neoformans/C. gattii species com-plex was approximately 20 fg per PCR. Furthermore, our modifiedLAMP assay detected cryptococcal DNA of 95.7% (90/94) of clinicalCSF in 90 min.Discussion/conclusion Compared to previously published studies ofLAMP assays on the C. neoformans/C. gattii species complex, ourmodified LAMP method can directly identify all members of the C.neoformans/C. gattii species complex with high specificity (100%). Thedetection limit of 20 fg DNA per PCR revealed a sensitivity of approx-imately 30 Cryptococcus genomic DNA copies per PCR. Thus, ourresults are comparable to a real-time PCR assay (5 cells per run), anested-PCR assay (10 cells per run), and the Luminex xMAP assayon Cryptococcus (101–103 cells per run). Furthermore, our modifiedLAMP assay detected cryptococcal DNA from 95.7% (90/94) clinicalCSF. Thus our method meets the ASSURED criteria [Affordable, Sen-sitive, Specific, User-friendly, i.e. simple to perform in a few steps withminimal training, Robust and Rapid, i.e. results available in 30 min,Equipment free, and Deliverable to the end user] for clinical diagnos-tics and surveillance of CM at the point of care in resource limitedcountries.

P041

Chitin-like structures and cryptococcal physiologyF. L. Fonseca,1 L. Kmetzsch,2 G. Ara�ujo,1 S. Frases,1

M. H. Vainstein2 and M. L. Rodrigues3

1Federal University of Rio de Janeiro, Rio de Janeiro, Brazil;2Federal University of Rio Grande do Sul, Porto Alegre, Brazil and3Center for Technological Development in Health, Oswaldo Cruz

Foundation, Rio de Janeiro, Brazil

Chitin and chitooligomers are glycans composed of beta-1,4-linkedN-acetylglucosamine. These glycans interact with glucuroxyloman-nan (GXM), the major capsular polysaccharide of Cryptococcus speciesforming immunologically active hybrids. We have recently demon-strated that chitin-related structures participate in the pathogenesisof C. neoformans, but the general role of these structures in the physi-ology of cryptococci remains obscure. In addition, the functions ofchitin-like structures in C. gattii cells have not been explored. In thiswork, we compared a number of general properties of chitin-likestructures in C. neoformans and C. gattii. ELISA revealed that chitoo-ligomers bind C. neoformans and C. gattii GXM with similar affinity.Supplementation of fungal cultures with soluble chitooligomers fol-lowed by analysis by scanning electron microscopy resulted in pro-found alterations in the capsular architecture of C. neoformans, butnot of C. gattii. These alterations included increased dimensions andformation of intracapsular areas that were apparently unfilled withcapsular components. In C. neoformans, blocking of chito-oligomerswith the wheat germ lectin (WGA) affected capsule formation andGXM release to extracellular space. The lectin had no effect on cap-sule formation or GXM secretion in C. gattii. Lectin treatment down-regulated the expression of eight capsule-related genes in C. neofor-mans, but stimulated expression of PKA1 in C. gattii. This observationwas validated in experimental models using inhibitors of proteinkinase A. These results support the supposition that chitin-relatedstructures have key roles in the physiology of cryptococcal cells. Inaddition, they reveal an important functional divergence in C. neofor-mans and C. gatti that might impact pathogenic mechanisms.

P042

Detection of high CSF levels of (1-3)-Beta-D-glucan incryptococcal meningitisJ. R. Rhein,1 N. C. Bahr,1 Y. Zhang,2 M. Finkelman2 and

D. R. Boulware1

1University 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 diagno-sis of many invasive fungal infections. It is not conventionallythought to be useful in cryptococcal disease, however, and is specifi-cally FDA-labeled as not being approved for detecting Cryptococcus.This assumption is based on limited data from HIV-negative personswith cryptococcal pulmonary disease.Aim We evaluated the utility of BDG levels in CSF as an adjunctdiagnostic strategy for patients with HIV-associated cryptococcalmeningitis (CM).Methods We determined the BDG levels in the CSF of 45 HIV-infectedUgandan subjects with suspected CM using the Fungitell� assay. Wealso assessed whether BDG levels in CSF correlate with quantitativecryptococcal cultures, cryptococcal antigen (CRAG) titers, the levels of18 different cytokines in CSF, and clinical outcomes.Results A cut-off value of ≥80 pg ml�1 provided 97% sensitivity in39 cases of confirmed CM, with one false negative result occurring inan individual with very low CSF CRAG titer (1:2) and sterile CSF cul-ture who later developed culture positive CM. BDG levels were<80 pg ml�1 in all 5 individuals without evidence of cryptococcal

Figure 2. Sensitivity test of the modified LAMP assay using the geno-mic DNA of type strain of C.neoformans var. grubii.

ª 2014 The Authors


Poster Abstracts

disease. One individual that was CRAG+ in serum but without evi-dence of meningitis (CSF culture and CRAG negative) had a CSF BDGlevel of 130 pg ml�1. BDG levels correlated with CSF fungal burdenby quantitative culture (r = 0.824, P < 0.001) as well as CRAG LFAtiters (r = 0.842, P < 0.001). CSF BDG levels also correlated with theCSF 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 deathwithin 1 week of diagnosis (Mann–Whitney U, P = 0.030).Conclusions BDG can be detected in the CSF of HIV-infectedpatients with CM and may provide useful diagnostic and prognosticinformation. Further studies are needed to better define the role ofBDG in the immunology and management of cryptococcal disease.

P043

Cell wall-associated polyphosphates and acidocalcisome-like compartments in Cryptococcus neoformansC. L. Ramos,1 F. M. Gomes,1 S. Frases,1 M. L. Rodrigues2 and

K. Miranda3

1Federal 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 neo-formans is the presence of a polysaccharide capsule, which coats thecell wall and entraps divalent cations, including calcium and magne-sium. These ions are required for capsular enlargement, which isdeterminant for fungal pathogenesis. In this context, mechanismsregulating the concentration of divalent cations at the cell surfaceare likely essential for fungal pathogenesis. Acidocalcisomes are cal-cium storage acidic organelles that contain several polyphosphate-bound cations. These organelles have been described in a variety oforganisms. Acidocalcisomes are acidified through the action of pro-ton pumps such as the vacuolar proton ATPase and the vacuolarproton pyrophosphatase. As described in other microorganisms, po-lyphosphates can participate in ion homeostasis, microbial growthand in cellular responses to environmental stresses. In this study, weanalyzed the presence of cell wall-associated polyphosphates and aci-docalcisome-like organelles in C. neoformans. Intracellular acidicgranules were observed after staining of fungal cells with acridineorange. Cell wall-associated and intracellular polyphosphates wereobserved after staining fungal cells with DAPI followed by analysisby fluorescence microscopy. Transmission electron microscopy com-bined with X-ray microanalysis revealed the presence of phosphorusin both the cell wall and in the intracellular space. Short- and long-chain polyphosphates were extracted from yeast cells and theamount of inorganic phosphate was determined spectrophotometri-cally after treatment of polyphosphate fractions with S. cerevisiae po-lyphosphatase (PPX1). The suggestion of intracellular and cell wallpolyphosphates in C. neoformans may lead to future studies involvingtheir participation in capsular architecture and ion homeostasis. Con-sidering the essential role of the cryptococcal capsule in fungal path-ogenesis, surface-associated polyphosphates might also participate inthe interaction of C. neoformans with host cells.

P044

Cryptococcus neoformans glucuronoxylomannan fractionsof different molecular masses are functionally distinctP. C. Albuquerque,1 F. L. Fonseca,1 F. F. Dutra,1 M. T. Bozza,1

S. Frases,1 A. Casadevall2 and M. L. Rodrigues3

1UFRJ (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 polysaccharidecomponent of Cryptococcus neoformans. We evaluated in this studywhether GXM fractions of different molecular masses were function-ally distinct.Materials and methods GXM samples isolated from C. neoformanscultures were fractionated to generate polysaccharide preparationsdiffering in molecular mass. These fractions were used in experimentsfocused on the association of GXM with cell wall components of C.neoformans, as well as on the interaction of the polysaccharide withhost cells.Results and conclusions GXM fractions of variable molecularmasses bound to a C. neoformans acapsular mutant forming punctatepatterns of surface distribution, in contrast to the usual annular pat-tern of surface coating observed when GXM samples containing thefull molecular mass range were used. The polysaccharide sampleswere also significantly different in their ability to stimulate cytokineproduction by host cells. Our findings indicate that GXM fractionsare functionally distinct depending on their mass.

P045

Association of CD44 polymorphism with Cryptococcalmeningitis in HIV-uninfected Chinese patientsX. Wang, R. Y. Wang and J. Q. Wu

Huashan Hospital, Fudan University, China

Background Hyaluronic acid (HA), one of the capsule componentsof Cryptococcus neoformans, has been validated to play a role as anadhesion molecule during infection by the yeast. CD44 is one of themost common membrane HA receptors. In vitro and animal experi-ments have demonstrated that the interaction between C. neoformansHA and CD44 on human brain endothelial cells plays an importantrole in brain invasion.Aim In this study, we identified the association between CD44 poly-morphism and cryptococcal meningitis in HIV-uninfected Chinesepatients.Methods A case-controlled genetic association study was conducted.We genotyped 42 single nucleotide polymorphisms (SNPs) in theCD44 gene in 150 patients with cryptococcal meningitis and 300control subjects by multiplex SNaPshot technology. The distributionsof allele frequency, genotypes, and haplotypes were comparedbetween patients and control subjects.Results Among the 150 patients with cryptococcal meningitis, 84did 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/Tof rs353626 (OR = 1.81, 95% CI [1.15–2.85]; P = 0.009) wereunder-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 150patients with cryptococcal meningitis. In cryptococcal meningitispatients without predisposing factors, the genotype C/T of rs353626was 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 CD44rs353626 genetic variants have significant effect in susceptibility ofcryptococcal meningitis.

ª 2014 The Authors


Poster Abstracts

P046

Phagocytosis and dissemination of Cryptococcusneoformans sporesN. M. Walsh,1 M. R. Botts2 and C. M. Hull1

1UW Madison, Madison, WI, USA and 2University of California -

San Deigo, San Deigo, CA, USA

Background Aerosolized Cryptococcus is inhaled into the lung andthen disseminates by an unknown mechanism to the brain, causingmeningoencephalitis that is uniformally fatal without treatment.Cryptococcus exists in multiple cell types, including yeast, whichreproduce by budding, and spores, which are the products of sexualdevelopment.Aim In a mouse model of infection, we discovered that spores froman avirulent yeast pair caused uniformly fatal disseminated disease.This study examines the mechanisms by which spores cause crypto-coccosis. We tested the hypothesis that spore-mediated disease isfacilitated by a Trojan Horse mechanism in which spores are phago-cytosed, germinate into yeast, reproduce vegetatively, and escape thelung within host phagocytes to infect the brain.Methods For survival studies, C57Bl/6 mice were infected intrana-sally with a suspension of spores (purified from a cross of B3501 andB3502) or yeast (B3501 + B3502), and survival was tracked. Toexamine dissemination, mice were sacrificed at various time pointsfollowing infection, and organs were homogenized and plated todetermine CFUs. Phagocytosis of spores and yeast was observed byco-incubation with immortalized murine macrophages (RAW 264.7)or dendritic-like cells (JAWS II). Intracellular location of calcofluorwhite labeled spores following phagocytosis was investigated bystaining of a phagolysosomal protein with a FITC-LAMP1 antibody.

Results In a mouse intranasal model of infection, spores isolatedfrom a cross of the type strains B3501 and B3502 caused uniformlyfatal disease by 70 days post-infection, while the yeast parents wereavirulent. Through tracking fungal burdens over time in mouse tis-sues, we discovered that spores (but not yeast) are able to dissemi-nate to the draining lymph node of the lung shortly after infection,and spore-derived cells continue to colonize other organs, eventuallyleading to a fatal CNS infection. Both immortalized and primaryphagocytes robustly phagocytose spores, while their yeast counter-parts are largely resistant to phagocytosis. Fluorescence microscopyrevealed that spores are trafficked to the harsh environment of thephagolysosome following phagocytosis. Surprisingly, the vast major-ity of phagocytosed cells are able to survive in this environment evenwhen the macrophages were activated.Discussion/conclusion Following uptake, spores enter the phago-lysosome and are able to survive, germinate into yeast, and replicate.The ability of spores to germinate and disseminate in the host isessential for disease progression as cryptococcal disease culminates inan overwhelming fungal burden composed of yeast. These data sup-port the hypothesis that phagocytes are providing a protected envi-ronment and vehicle for spores to grow and spread within themammalian host. Future studies will focus on further mapping therelationship between spores and phagocytes in the host; includingidentifying phagocytic receptors and how specific phagocytes mayinfluence dissemination.

P047

Pathogenic profiles in serotype C isolates of Cryptococcusgattii are influenced by chitin-like structuresJ. Rodrigues,1 F. L. Fonseca,1 L. Kmetzsch,2 G. Ara�ujo,1

S. Frases,3 M. H. Vainstein2 and M. L. Rodrigues4

1Universidade 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 and4UFRJ & Fiocruz/CDTS, Rio de Janeiro, Brazil

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

Figure 1. Trojan horse model of C. neoformans spore mediatedinfection.

Figure 2. Early dissemination to the mediastinal lymph node (MLN)in spore mediated infection.

ª 2014 The Authors


Poster Abstracts

surface architecture and pathogenesis in the C. gattii serotype Cmodel, with focus on the participation of chito-oligomers in fungaldissemination. The serotype C cells studied here included strains106.97, ATCC24066, HEC40143 and WM779. Scanning electronmicroscopy (SEM) revealed typical capsular morphologies for allstrains, but immunofluorescence analysis demonstrated that strains106.97 and WM779 had low reactivity with monoclonal antibodiesagainst GXM. Analysis of other virulence factors indicated that ure-ase activity was similar in all strains. Pigmentation tests, however,revealed that strains ATCC24066 and HEC40143 failed to producemelanin in the presence of L-DOPA. In vivo studies in mice models ofinfection confirmed two different pathogenic profiles. Strains 106.97and WM779 efficiently colonized the brain of infected animals. TheATCC24066 and HEC40143 isolates, however, were contained inthe lung and took significantly longer than 106.97 and WM779strains to kill mice. Since chito-oligomers have been recently associ-ated with fungal dissemination in the cryptococcosis model, we askedwhether these structures would influence the different pathogenicprofiles observed in our study. The higher-virulence strains (106.97and WM779) were significantly more efficient than the low pathoge-nicity isolates (ATCC24066 and HEC40143) in inducing chitinaseactivity in the lung of infected animals. This observation was associ-ated to an increased detection of fungal chito-oligomers in lung tis-sues infected with strains 106.97 and WM779. Blocking of chito-oligomers using the wheat germ lectin (WGA) prior to infectiondelayed dissemination of both strains to the brain and resulted inincreased association of these fungal cells with macrophages. WGAtreatment, however, had no effect on phagocytosis profiles or animalpathogenesis of strains ATCC24066 and HEC40143. These resultsdemonstrate different pathogenic profiles in serotype C isolates of C.gattii and suggest a correlation between chitinase-induced chito-oli-gomer production and fungal dissemination. The diversity in thepathogenic potential of serotype C strains of C. gattii reveals the needfor a detailed exploration of the relationship between virulence andsurface architecture in the Cryptococcus genus.

P048

Identification of a role for virulence-related Sec14-1 ofCryptococcus neoformans in export of cell wall enzymesand cell separation using proteomicsJ. 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

1Westmead Millennium Institute, Sydney, NSW, Australia;2University 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 neo-formans (Cn), and are key mediators of host-pathogen interaction.Secretion of the fungal invasin, phospholipase B1 (Plb1)1, is partiallyregulated by the putative phosphatidylinositol transfer protein,Cnsec14-11. However, the composition of the Sec14-1-dependent sec-retome is unknown. In addition to reduced secretion of Plb1, thesec14-1delta mutant has a compromised cell wall and is less virulentin mice, despite increased expression of its closely related homologue,SEC14-21. Although deletion of SEC14-2 has no effect on the viru-lence composite it cannot be disrupted in combination with SEC14-11.The aims of this study were to (i) identify the SEC14-1-dependentand WT secretomes (ii) compare the efflux routes of selected Sec14-dependent proteins and (iii) determine whether SEC14-1 and SEC14-2 are functionally redundant.

To address aim 1, the secretomes of WT (strain H99) and sec14-1delta were analyzed by mass spectrometry. 105 proteins were identi-fied in WT secretions, 27 of which are canonically secreted (containsignal-peptides). The abundance of 25 proteins was reduced in sec14-

1delta secretions. Of these, 7 are cell wall-associated and/or cell wall-modifying enzymes, including canonically-secreted Plb1, laccase(Lac1) and a-1,3-glucan synthase (Ags1), which have proven rolesin pathogenesis, and acid phosphatase (Aph1). Using an APH1 dele-tion mutant, Aph1 was confirmed to be the major phosphate-repress-ible, extracellular acid phosphatase in Cn, and to contribute to thevirulence composite. Comparison of the subcellular localization ofSEC14-1-dependent Plb1 and Aph1, revealed that Plb1 was trans-ported directly to the periphery, while Aph1 accumulated in endo-some-like structures en route to the plasma membrane and vacuoles.Both proteins were enriched in bud necks, implicating a role for themin bud formation and/or septation. Microscopic examination ofsec14-1delta revealed that, in contrast to WT, mother and daughtercells often remained connected via the septum following mitosis.

To address Aim 2, we placed SEC14-2 under the control of a galac-tose-inducible promoter in sec14-1delta. When SEC14-2 gene expres-sion was suppressed by glucose, growth of sec14-1deltaGAL7SEC14-2was inhibited, demonstrating that SEC14-1 and SEC14-2 are func-tionally redundant. We are now in a position to observe the effect offull loss of sec14 function on Aph1 and Plb1 subcellular localization.

Taken together, our findings demonstrate that SEC14-1 regulatesexport of cell wall enzymes via endosome-dependent and -indepen-dent secretion routes to promote cell wall integrity and bud forma-tion, and ensure timely dissolution of the septum, and that loss ofthe 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 deletionmutants related to traverse the blood-brain barrierH. K. Tseng,1 W. L. Cho,2 C. P. Liu,1 J. Y. Jong3 and

J. R. Perfect4

1Mackay 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 nervoussystem (CNS) fungal infection these years. This pathogen has a predi-lection for the brain and causes devastating meningoencephalitis. Inorder to cause the brain invasion, C. neoformans must cross theblood-brain barrier (BBB). However, the brain invasion mechanism islargely unknown. We utilized a mutant library of signature-tagged,targeted gene deletion C. neoformans mutants to decipher how C. neo-formans enter into brain. We identified two genes, FNX1 and PUB1,related to BBB crossing (PLoS ONE 7:e45083) and the strategies toidentify mutants related to transmigration to CNS were described.Briefly, screen strategy to find mutants with significant signature tagloss mutants on mice brain transmigration assay (MBTA) model;competition strategy to show statistically significant differencemutants from parental strain CMO18 on MBTA. The project toscreen the whole mutant library with 1201 strains accordingto above strategy is going and we plan to establish brain cell strategyto confirm BBB transmigration related mutants. Currently twentymutants (Figure) were identified through MBTA in vivo. The in vitrotranscytosis assay by human brain microvascular endothelial cells(HBMECs) for these mutants are in progress. This research helps tounderstand the molecular level of Cryptococcal meningitis patho-physiology and may discover new treatment targets for this medicalimportant fungus.

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Poster Abstracts

P050

Consequences of SIR2 regulation on the pathogenesis ofCryptococcus neoformansT. 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 pathogen-esis of the debilitating fungus, Cryptococcus neoformans. However, theunderlying mechanisms for this unique contribution have not beencompletely elucidated. Previous work has shown that age-relatedresilience is acquired by C. neoformans cells as they undergo asym-metric divisions. The sum of these divisions, which is known as theirreplicative life span (RLS) can impact the outcome of chronic infec-tion when these cells are selected and become the dominant patho-gen population in the host.Aim This work investigated whether C. neoformans strains that dem-onstrate a variable RLS either from gene deletion or drug manipula-tion affect its virulence.Methods A mutant (sir2D) and its complement were generated in aserotype A (H99) and D (RC2) strain, and their phenotypes wereexamined in vitro and in vivo.Results and discussion/conclusion As expected loss of SIR2 atten-uated doubling time and mating ability of C. neoformans. The mutanthad a 33–68% shortened RLS compared to the wildtype (wt). Impor-tantly, it was hypovirulent in Galleria mellonella (waxworm) and inmice. Notably in mice, the mutant was only hypovirulent after intra-tracheal, not intravenous infection, as supported by histopathologyand fungal burden found in the lungs and brain. Since the mutantcrossed the blood-brain barrier equally well compared to the wt, wehypothesized that this was due to the altered nutritional growth con-ditions in the infection sites. Indeed, RLS was dependent on levels ofglucose as calorie restriction (CR) either shortened (RC2) or extended(H99) RLS in different strains; however, the extension was not seenin the absence of SIR2. Also, RT-PCR analysis supported this obser-vation as SIR2 was upregulated in H99 cells and was downregulated

in RC2 cells under CR. RNA sequencing of the mutant showeddownregulation 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, iso-nicotinamide, which has been shown to act on SIR2 in Saccharomy-ces cerevisiae to extend RLS. We found that when it extended RLS inC. neoformans (H99), it rendered it hypovirulent, and when it short-ened RLS (I65), it rendered it hypervirulent. Thus, we provide furthersupport that a shortened RLS may be beneficial to the pathogen dur-ing chronic infection, especially if it helps it attain age-related resil-ience faster. In conclusion, SIR2 could serve as a potential target fortherapeutic inventions against cryptococcosis.

P051

Innate immune cell activation of a copper-dependent anti-cryptococcal agentR. A. Festa,1 M. E. Helsel,2 K. J. Franz2 and D. J. Thiele1

1Duke University Medical Center, Durham, NC, USA and 2Duke

University, Durham, NC, USA

Background The increasing number of cryptococcal infectionsworldwide demands new approaches for treating Cryptococcus neofor-mans. The host immune system uses a variety of weapons in anattempt to eliminate infectious agents, such as production of reactiveoxygen and nitrogen species as well as iron sequestration. In addi-tion, evidence is mounting to support a role for elevated copper (Cu)during infection. In fact, under control of the Cu responsive tran-scription factor, Cuf1, C. neoformans mounts a strong defensiveresponse to elevated Cu in the lung through expression of twoCu-specific metallothioneins. Without these metallothioneins, C. neo-formans is unable to establish a successful infection. This responsehighlights Cu as an important player in the lung, and presents a pos-sible target for anti-Cryptococcal therapies.Aim We seek to perturb Cu homeostasis of C. neoformans both in vi-tro and in vivo to determine the effects on cell viability as well as theoutcome of infection. To do this we use a protected form of the Cu-binding molecule, 8-hydroxyquinoline (8HQ), to selectively harnessits antifungal activity in the context of either activated macrophagesin vitro or in the murine lung infection model. The nontoxic pro-tected form of 8HQ, called QBP, contains a pinanediol boronic esterthat blocks metal coordination and is deprotected via reactive oxygenspecies that are produced by activated immune cells to elicit Cu-dependent killing of the fungal pathogen C. neoformans. This strategyis aimed at minimizing off-target effects, by focusing the antimicro-bial properties of 8HQ to activated macrophages or sites ofinflammation.Methods In this work, we utilize in vitro culture based assays toassess the effects of 8HQ and QBP in the presence or absence of Cuon both C. neoformans and macrophage-like cells. We assess theimpact of these molecules on cell viability, gene expression, andmetal content. LC-MS analysis was utilized to observe the conversionof QBP to 8HQ by activated, but not na€ıve macrophages. Further-more, we used in vitro co-culture experiments to determine theimpact 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 of8HQ bound to copper are fungicidal rather than fungistatic, and themechanism of 8HQ-fungal killing is through its action as a Cu iono-phore, leading to a large increase in cell-associated Cu, highly ele-vated expression of the fungal metallothioneins, and ultimately celldeath. While 8HQ is cytotoxic to mammalian cells, QBP is toleratedat much higher concentrations. However, when activated, macro-phages are able to convert QBP to 8HQ, and this process is able tostimulate killing of C. neoformans when co-cultured in vitro. Further-more, we show that QBP treatment is able to reduce fungal burden

Figure 1. Cryptococcus mutants related to transmigration in MBTAmodel.

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Poster Abstracts

during lung infections, suggesting that QBP is converted to active8HQ in infected lungs where it can exert its antifungal activity.Discussion/conclusion There is a delicate balance of Cu betweenthe host and C. neoformans and we begin to address how this aspectof infection can be targeted and perturbed. As Cu is a critical compo-nent of innate immunity, the activation of Cu ionophores or otheranti-fungal compounds, in concert with immune cell activation, mayprovide first in class antimicrobial therapeutics.

P052

The role of tryptophan biosynthetic genes TRP3 and TRP5on the survival of C. neoformans and their applicability asdrug targetsJ. D. Fernandes,1 V. Tofik,2 J. Machado-Jr,2 M. A. Vallim2 and

R. C. Pascon2

1Universidade de S~ao Paulo, Sao Paulo, Brazil and 2Universidade

Federal de S~ao Paulo, Diadema, Brazil

Defects in amino acid biosynthetic pathways generated by gene dele-tion in C. neoformans lead to auxotroph strains which often have novirulence or attenuated virulence, in other cases the block in thiskind of metabolic route may create lethal phenotypes, impairinggrowth and survival. Therefore, nutrient biosynthesis and/or acquisi-tion pathways offer interesting opportunities as drug targets. Out oftwenty-three amino acids synthesized in plants, microbes and para-sites, nine are essential in animal cells, among these there are thearomatic amino acids, including tryptophan, that differently fromtyrosine and phenylalanine, has only one route of synthesis, increas-ing its interest as target for development of novel antifungal agents.According to bioinformatics analysis, four genes (TRP2 to TRP5) areexpressed and necessary to convert chorismate into tryptophan in C.neoformans. TRP3 encodes a triple function protein that acts on thefirst, third and forth step of this pathway. Whereas, TRP2 proteinacts on the first, TRP4p on the second and TRP5p on the last stepof the pathway. In this work we attempted to delete TRP3 andTRP5 genes to evaluate the impacts of lack of tryptophan biosynthe-sis on virulence factors and survival. An extensive search formutants bearing homologous integration leading to tryptophan aux-otrophy was unsuccessful, therefore, we assumed the hypothesis thatthe tryptophan biosynthetic genes are essential. To address this issuewe used RNAi to modulate TRP3 and TRP5 expression. All strainsgrew well under RNAi repression (Glucose). However, during RNAiinduction (Galactose) Trp3i and Trp5i strains did not grow in richor poor medium with or without tryptophan supplementation. Syn-thetic medium supplemented with proline as the sole nitrogen sourceand tryptophan at 25 °C was the only condition we observedmutant growth, even though growth rate was poor. qPCR was per-formed to confirm TRP3 and TRP5 mRNA suppression under RNAiinduction. This far, tryptophan is the second report of an essentialamino acid biosynthetic pathway in C. neoformans. One possibleexplanation for this fact is that extracellular amino acids are ineffi-ciently transported into the cell, due to, either fewer or lower affinitypermeases or both. To test this hypothesis we searched all aminoacid permeases in C. neoformans genome using 24 query relatedsequences from S. cerevisiae and found only 8 putative permeases(AAP1 to AAP8). By qPCR their gene expression was checked andamong these, only six had expression above the threshold. Compara-tive analysis under various growth conditions applied in these exper-iments showed a distinct expression pattern for AAP2, AAP4 andAAP5 in response to preferred nitrogen source and the presence of apool of amino acids added to the medium. These results suggest thatC. neoformans, in fact, has fewer amino acid permeases, which couldexplain low assimilation. The expression profile suggests permeasesare induced by a pool rather than single amino acids and also, theymay be controlled by global amino acid response, nitrogen catabo-lism repression and a signaling pathway that responds to the pres-ence of the substrate, similar to SPS-sensing in S. cerevisiae. In

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

P053

Characterisation of Cryptococcus strains isolated frommammals living in an environmental site with highcryptococcal presence in South-Western AustraliaP. D. Danesi,1 R. M. Malik,2 M. K. Krockenberger2 and

W. Meyer3

1Istituo 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 systemicfungal infections of mammals in Australia. Investigations of veteri-nary isolates and surveillance studies of captive koalas identified awildlife park in Perth, WA with an exceedingly high environmentaloccurrence of Cryptococcus gattii. Furthermore, there is a high rate ofnasal 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 associatedgenetic variability within the Cryptococcus neoformans/gattii speciescomplex in animals living in this park. Specifically, we were inter-ested in whether colonisation of individual animals or focal environ-mental sites is clonal, or involved more than one cryptococcal strain/molecular type.Methods In October 2012, 58 samples, comprising swabs from cap-tive koalas (n = 27), dingoes (n = 2), a wombat (n = 1) and the envi-ronment (n = 18) were collected at Caversham Wildlife Park. Aftertransport to the laboratory (at 5 °C; for 5 days), swabs were inocu-lated onto birdseed agar plates and incubated at room temperature(approx. 21 °C). The extent of cryptococcal colonisation or environ-mental presence was codified as being of low, intermediate and highgrade according to the number of colonies showing a brown-colour-ing per plate (low < 50; intermediate 50–100; high grade >100 colo-nies). Following DNA extraction, restriction fragment lengthpolymorphism (RFLP) analysis of the URA5 gene and a mating typespecific PCR were performed to determine the molecular type and themating type, respectively, for one colony from each plate, represent-ing isolates from an individual animal or a single environmental site.Genetic variation among Cryptococcus isolates was studied using theISHAM consensus multi-locus sequence type (MLST) scheme for theC. neoformans/C. gattii species complex. Additionally, to determinewhether different Cryptococcus clones were present amongst browncolonies grown from a single plate from an individual animal orenvironmental site, 74 colonies isolated from four plates (n = 18/plate from one koala; n = 19/plate from another koala; n = 20/platefrom an environment sample; n = 17/plate from a different environ-mental 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 extentof colonisation or environmental presence was low (8 koalas and 3environmental samples), intermediate (5 environmental samples) andhigh grade (6 koalas and 2 environmental samples). Among the posi-tive cultures, C. gattii VGII (n = 16; 67%), C gattii VGI (n = 5; 21%)and C. neoformans VNI (n = 3; 12%) were identified. VNI was identi-fied only from koalas. All isolates were of the a-mating type. MLSTanalysis revealed six distinct sequence types (ST) among the 24 iso-lates: ST7 (14), ST23 (2), ST48 (2), ST51 (1), ST58 (1) and ST 154(4). PCR fingerprinting profiles showed identical patterns among colo-nies from the same plate for the four plates so analysed.Discussion/conclusions C. neoformans and C. gattii were both iso-lated from nasal swabs of koalas, whereas only C. gattii was found in

ª 2014 The Authors


Poster Abstracts

the environment. The wide variety of MLST types of C. gattii isolatessupported the possibility of genetic recombination at this location,consistent with earlier work and the previous finding of rare a mat-ing types in this environment. On the other hand, PCR fingerprintingprofiles suggest that within an individual animal or environmentalsite, clonal replication prevails, based on the number of plates stud-ied. The persistent high environmental presence of VGII in Caver-sham Park, with corresponding high prevalence of asymptomaticnasal colonisation, subclinical infection and clinical disease in ani-mals, especially koalas and wombats, emphasizes the need for on-going surveillance at this location. Clonal outbreaks might develop inthis location after genetic recombination, possibly giving rise to aVancouver Island-like outbreak in the vicinity.

P054

Leucine biosynthesis is required for iron homeostasis andvirulence in Cryptococcus neoformansE. S. Do,1 G. Hu,2 L. Oliveira,2 M. Caza,2 W. Kronstad2 and

W. Jung1

1Chung-Ang University, Anseong-Si, South Korea and 2University

of British Columbia, Vancouver, BC, Canada

Amino acid biosynthesis that is absent in mammals is considered anattractive target of antifungal treatment. Leucine biosynthesis is sucha target pathway, consisting of a five-step conversion process startingfrom the valine precursor 2-keto-isovalerate. Isopropylmalate dehy-drogenase (Leu1) is an iron-sulfur cluster protein that is required forleucine biosynthesis in Saccharomyces cerevisiae and is highly homolo-gous to the iron regulatory protein Irp1 in mammalian cells. More-over, our previous transcriptome data showed that the expression ofLEU1 is regulated by iron availability in Cryptococcus neoformans. Inthis study, we aimed to characterize the role of leucine biosynthesisin iron homeostasis and the virulence of the human pathogenic fun-gus C. neoformans. We found that deletion of LEU1 caused the cellsto become leucine auxotroph and that intracellular iron levels weresignificantly distorted in the leu1 mutants. The leu1 mutants also dis-played increased susceptibility to oxidative stress and cell wall/mem-brane disturbing agents. Furthermore, our results suggest that thefunctions of superoxide dismutases, Sod1 and Sod2, are closely asso-ciated with the expression of LEU1 and that LEU1 is required for vir-ulence in a mouse model of cryptococcosis. A mutant lacking thebeta-isopropylmalate dehydrogenase gene (LEU2), which encodes anenzyme catalyzed in the subsequent step of leucine biosynthesis,showed not only similar phenotypes to the leu1 mutant but attenu-ated virulence. Overall, our results suggest that leucine biosynthesisis required for iron homeostasis and virulence in C. neoformans.

P055

Cryptococcus neoformans 14-3-3 is an essential gene forvirulenceY. Jong,1 Y. C. Chang,2 K. J. Kwon-Chung,2 S. Huang,1

S. Shimada1 and X. Fu1

1Children’s Hospital Los Angeles, USC Keck School of Medicine,

Los Angeles, CA, USA and 2NIAID, Bethesda, MD, USA

Background Cryptococcus neoformans invades into brain and causessevere meningoencephalitis. The mechanism of cryptococcal braininvasion is largely unknown, and recent studies suggest that itsextracellular microvesicles (MV) may be involved in the invasion pro-cess. The 14-3-3 protein is abundant in the extracellular MVs of C.neoformans, but the physiological role of 14-3-3 has not beenexplored.

Aim To explore the 14-3-3 gene functions and its potential for viru-lence, we intended to generate a 14-3-3 mutant strain. We investi-gated the mutant characteristics by examining its roles in growth,morphological alterations, MV biogenesis, and its adhesion ability tohuman brain microvascular endothelial cells.Methods C. neoformans 14-3-3 is apparently a single-copy, essentialgene. To explore the functions of 14-3-3, we replaced the promoterregion of the chromosomal 14-3-3 gene with the copper-controllablepromoter CTR4. The growth rate and its morphological changes wereexamined to characterize the alterations of mutant strain. The rolesof 14-3-3 on the biogenesis of MVs were scrutinized by the yield ofisolated MVs, NanoSight display and 3-D plot, and proteomic analy-ses. In vitro BBB adhesion assay was also used to test its roles duringinvasion.Results The knockdown strain C1617 showed a reduction in growthrate, slightly enlarged cell size, drastic change in morphology andthe reduction in the thickness of the capsule under copper repressedconditions. The mutant cells became temperature-sensitive. Further-more, the mutant cells produced lower amount of total proteins in itsextracellular MVs and reduced adhesion to the HBMEC in vitro. Pro-teomic analyses of the protein components under induction and sup-pression conditions reveals that the MV biogenesis may be tightenedwith the 14-3-3 function(s).Discussion/conclusion The 14-3-3 protein is highly conserved pro-tein which plays pleitropic roles among organisms. Our studies of C.neoformans 14-3-3 protein suggest that, as in other organisms, playsseveral important roles relevant to its growth, morphology, cell divi-sion, and other functions. The 14-3-3 protein was first identified inbovine adrenal chromaffin cells as a cytosolic protein Exo1, requiredfor exocytosis. In our 14-3-3 mutant studies, both of total MV pro-teins and activities of two enzymes associated with MVs, laccase andacid phosphatase, were reduced. As secretion of MVs play a role inthe building block transport for its extracellular capsule biosynthesis,it is possible that reduction of 14-3-3 protein level in C1617 resultsin the decline of MV secretion, and subsequently drops the suppliesfor capsule biosynthesis which results in the reduction ofcapsule size.

The temperature-sensitive nature of 14-3-3 knockdown cells pre-vents from doing animal studies. However, the in vitro adhesion stud-ies show that the reduction of 14-3-3 protein levels in the mutantstrain causes a reduction in its ability to bind to the HBMEC. Thisreduction 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 MVsecreted in the mutant since MV can affect the binding of cryptococ-cal cells to HBMEC, or others. Given the fact that the 14-3-3 func-tions are relevant to its growth, morphology, possible cytokinesis,capsule and MV biogenesis, it is perceivable that the C. neoformans14-3-3 functions are closely associated with C. neoformans physiologyand pathogenesis.

P056

Towards understanding cell cycle control and hypoxicadaptation in Cryptococcus neoformansS. Kawamoto,1 E. Virtudazo,1 Z. Moranova,2 M. Ohkusu,1

K. Shimizu,1 A. Suganami,3 Y. Tamura3 and V. Raclavsky2

1Chiba 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

Cryptococcus neoformans is an opportunistic pathogen of worldwidedistribution and responsible for life-threatening infections amongimmunocompromised persons. We have been studying the molecu-lar mechanisms of the cell cycle control in C. neoformans and havereported that the cell cycle behavior of this yeast is different fromthe cell cycle control model exhibited by the common buddingyeast, Saccharomyces cerevisiae. C. neoformans exhibits a delay in

ª 2014 The Authors


Poster Abstracts

budding as the growth phase progresses into late log to early sta-tionary phase, resulting in a tendency to accumulate unbuddedcells with G2 rather than G1 DNA content. This was found to bethe organism’s inherent response to stressful cultural conditionssuch as changes in oxygen availability, pH and temperature andwas implicated as a possible additional virulence mechanism duringsurvival within host tissues.

We have reported the molecular characterization and physiologi-cal roles of the two main eukaryotic cell cycle genes, C. neoformanscyclin dependent kinase 1 (CnCdk1) and cyclin homologues. Only asingle Cdk1-related G1 and G1/S cyclin homologue was found inthe genome sequence of C. neoformans and designated CnCln1. Inthe light of the functional specialization of G1 and G1/S cyclins inS. cerevisiae, it was surprising to find that C. neoformans was foundto have a single G1 and G1/S cyclin in the genome. Thus, it isimportant to understand the mechanisms that govern this yeast’sunique cell cycle behavior during G1-S phase transition and therole of this single cyclin in this unique stress response pathway.We investigated the cell cycle control mechanism of CnCln1 bycomparing its activity with G1 and G1/S cyclins of S. cerevisiaefrom a point of view of their structure-function relationship. CnCln1was not only able to complement the function of the G1 cyclins ofS. cerevisiae, such as ScCln3, but also the G1/S cyclins of S. cerevisi-ae, such as ScCln1 and ScCln2. Our in silico analysis demonstratedthat the CnCln1/ScCdk1 complex was more stable than any of theyeast cyclin and ScCdk1 complexes. These results are consistentwith in vitro analysis that has revealed the flexible functionalcapacity of CnCln1 as a Cdk1-related G1 and G1/S cyclin of S.cerevisiae.

In the obligate aerobic yeast C. neoformans, limited aeration hasbeen demonstrated to cause slowdown in proliferation and delayedbudding, resulting eventually in a unique unbudded G2-arrest. Theability to adapt to decreased oxygen levels during pathogenesis hasbeen identified as a virulence factor in C. neoformans. We tried toidentify and characterize genes that are necessary for the prolifera-tion slowdown and G2-arrest caused by limited aeration. Randommutants were prepared and screened for lack of typical slowdownof proliferation under limited aeration. The CNAG_00156.2 genecoding for a zinc-finger transcription factor was identified inmutants showing most distinctive phenotype. Targeted deletionstrain and reconstituted strain were prepared to characterize andconfirm the gene functions. This gene was also identified in parallelstudies as homologous both to calcineurin responsive (Crz1) andPKC1-dependent (SP1-like) transcription factors. We have confirmedthe role of the cryptococcal homologue of CRZ1/SP1-like transcrip-tion factor in cell integrity, and newly demonstrated its role inslowdown of proliferation and survival under reduced aeration, inbiofilm formation and in susceptibility to fluconazole. Our data dem-onstrate a tight molecular link between slowdown of proliferationduring hypoxic adaptation and maintenance of cell integrity in C.neoformans and present a new role for the CRZ1 family of transcrip-tion factors in fungi.

P057

Genomic approaches to inferring recombination rates andpopulation structure of Cryptococcus neoformans vargrubiiJ. Rhodes,1 M. A. Beale,1 C. Cuomo,2 S. Sakthikumar,2

T. Bicanic,3 T. S. Harrison3 and M. C. Fisher1

1Imperial 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 intothe phylogenetic lineages VNB, VNI and VNII. Population geneticssuggests that the Cng VNI lineage has emerged ‘out of Africa’ tocause the main burden of cryptococcal meningitis worldwide; how-ever, the pathway and timing of global proliferation has yet to be

identified, and this hypothesis needs further testing. We are usingwhole genome sequencing (WGS) of isolates sampled worldwide toinfer the population structure and recombination rates of Cng. Thesegenome sequences will also be used to build a scenario of the dis-persal of Cng across the globe, and assess the likely geographicalorigin.

We are sequencing clinical and environmental isolates have beencollected from sites around the world, with a focus on Africa andAsia. Illumina sequencing has been performed on these isolates togenerate sequences with >1009 coverage across the genome,allowing high confidence variant calling following alignment to theCng H99 reference genome. These genome-wide SNP data wereused by the coalescent-based method LDhat ‘interval’ to estimatepopulation-scaled recombination rates within the VNB, VNI andVNII Cng populations. A 4-gamete test (4GT) was also completed toconfirm the existence of recombination in these populations. Sincelinkage disequilibrium is influenced by a number of factors, such asrecombination rate, the rate of mutation and selection, various sta-tistical analyses were used to measure linkage disequilibrium. Thelevel of population differentiation at individual loci was assessedusing pairwise FST statistics amongst pairs of populations. The stan-dardised index of association, rBarD, was also calculated. Softwaresuch as fineSTRUCTURE and ARGweaver, which were initiallydeveloped for inferring the ancestral recombination graphs ofhumans, have also been used to determine the ancestry of Cngpopulations.

Results from resequencing an initial panel of 39 Cng isolates col-lected worldwide confirm the expansion of the VNI lineage. rBardDanalysis confirms that this population is extremely clonal. SNP den-sity plots of individual chromosomes also show the VNI populationas a clonal population, with low genetic diversity, compared to theVNII and VNB populations.

Recombination analyses using LDhat ‘interval’ show the VNB pop-ulation to be highly recombinogenic; this result is confirmed by the4GT, which shows there is three times more recombination occurringwithin the genomes of the VNB population, compared to the VNIpopulation. The calculation of FST statistics also highlighted thatregions of chromosomes 5 and 6 have a significantly lower FST scorewhen comparing VNB and VNI populations.

A significantly lower FST score for specific regions of chromosomes5 and 6 when comparing VNB and VNI populations suggest thatopen reading frames (ORFs) in these regions may be exhibiting puri-fying or diversifying selection. Since the VNB population displaysextensive recombination, it can be hypothesised that this populationis the ancestor of the low diversity VNI population.

P058

Isolation of Cryptococcus gattii VGII from indoor dust inthe deep Amazon of the Rio Negro basinF. Brito-Santos,1 G. B. Barbosa,2 L. Trilles,2 B. Wanke,2

W. M. Wieland,3 F. A. Carvalho-Costa4 and M. S. Laz�era2

1Fiocruz/IPEC, Rio de Janeiro, Brazil; 2Evandro Chagas Clinical

Research Institute, Fiocruz, Rio de Janeiro, Brazil; 3Centre for

Infectious Diseases and Microbiology, Sydney Medical School-

Westmead, Sydney, NSW, Australia and 4Institute Oswaldo Cruz,

Fiocruz, Rio de Janeiro, Brazil

Cryptococcosis is a human fungal infection of significant mortalityand morbidity, especially in the meningoencephalitis form. Crypto-coccosis is distributed worldwide and their agents, C. neoformans andC. gattii, present eight major molecular types - VNI-VNIV and VGI-VGIV respectively. The primary cryptococcosis caused by moleculartype VGII (serotype B, MAT alpha) prevails in immunocompetentpatients in the North and Northeast of Brazil, revealing an endemicregional pattern to this molecular type. Since 1999, C. gattii VGIIhas been involved in an outbreak in Canada, expanding to the

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Poster Abstracts

Northwest of the United States, two temperate regions. Exposure topropagules dispersed in the environment initiates the infection pro-cess, related to various organic substrates, mainly decomposing woodin and around dwellings. The present study investigated the presenceof the agents of cryptococcosis in dust from dwellings in the upperRio Negro, municipality of Santa Isabel do Rio Negro in Amazonasstate. Indoor dust was collected from 51 houses, diluted and platedon bird seed agar. Dark brow colonies were identified phenotypicallyand genotipycally by restriction fragment length polymorphism ofthe gene URA5 and multilocus sequence typing (MLST). The matingtype was identified using specific primers for pheromone. Threehouses were positive for C. gattii molecular type VGII, MAT alphaand MAT a, showing significant density of this agent. MLST studiesidentified eight subtypes, VGIIb (ST7), VGIIa (ST20), (ST5) and 5new unique subtypes of the region. For the first time in the state ofAmazonas, C. gattii VGII MAT alpha and MATa were isolated fromthe environment and correlates with endemic cryptococcosis in thisstate. This is the first description of MLST subtypes on environmentalisolates in the Brazilian Amazon, indicating domiciliary dust as apotential source for human infection with different subtypes C. gattiiVGII 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-transcriptionalregulation in cryptococcus neoformansJ. Kaur and J. C. Panepinto

State University of New York at Buffalo, Buffalo, NY, USA

Background PUF proteins represent a conserved family of RNA-binding factors that are key regulators of mRNA translation, stabilityand localization across the eukaryotic kingdoms. Investigation of theC. neoformans genome has revealed that it encodes four PUF proteinswhich are typically characterized by the presence of 8 consecutivePuf repeats, however variations do occur. The PUF proteins charac-terized to date have been reported to bind to consensus sequencecharacterized by a UGUR tetra nucleotide in their target RNA. Recentphylogenetic studies have demonstrated that RNA binding domain ofPuf3 is conserved and there is significant enrichment of Puf3 bindingelement in genes that are involved in mitochondrial translationmachinery in Saccharomycotina species, which is lost in higherfungi. Our studies indicate that puf3-delta mutants exhibit no mito-chondrial phenotype. Phenotypic characterization of a C. neoformanspuf3-delta strain revealed a defect in filamentation which led us tohypothesize that Puf3 plays a role in C. neoformans morphogenesis.Aim To characterize the role of PUF3 protein as post transcriptionalregulator of cryptococcal morphogenesis.Methodology We performed bilateral mating assays for wild typeand puf3-delta mutants, in which equal numbers of opposite matingcells were cocultured on V8 agar. To determine the ability of puf3Dmutants to produce pheromone, northern blot for MF alpha wasdone. Puf3 was mCherry tagged using fusion PCR and fluorescencemicroscopy was done to study its localization. Recombinant PUF3protein was made using pLysS expression system and purified usingNi-NTA columns. Protein binding activity of Puf3 to its cognate ele-ment was detected using UV-linked RNA EMSA. Site directed muta-genesis was done to mutate the RNA binding domain (RBD) of Puf3as well as the Puf3 element present in the 50UTRResults Bilateral crosses of puf3-delta mutants are defective in theproduction of dikaryotic hyphae as compared to the wild type, butpheromone sensing and fusion were not affected. Using fluorescencemicroscopy, we have shown that mCherry tagged Puf3 specificallylocalizes to areas of hyphal growth, but is not visible in yeast cells.Mating assay revealed that the RBD mutants are defective for fila-mentation as compared to the wild type. Mating spots of strains com-plemented with Puf3 in which the 50UTR Puf3-binding element wasmutated were more filamentous as compared to the wild type

mating. UV cross-linked RNA EMSA has shown that Puf3 proteinspecifically binds to its consensus TGTACATA cis element present in50UTR.Discussion We have demonstrated a role for Puf3 in the regulationof morphogenesis during C. neoformans sexual development. As thereis no defect in pheromone induction and fusant formation, it suggeststhat bilateral mating of puf3D mutants is defective for post-fusionhyphal extension. The intact RBD is essential for the functional roleof Puf3. Visualization of mCherry tagged PUF3 has depicted thatPUF3 localizes to the areas of hyphal growth or septation, but isabsent in vegetative yeast cells. This morphotype-specific productionof Puf3 may be mediated by auto-regulation through a Puf3-cognatecis element in the PUF3 50 UTR. Future studies will determine themechanism of Puf3 regulation on potential target transcripts whichwould enable us to establish a link between the physiology and thePuf3 regulon of C. neoformans.

P060

Elongation of sexual filaments and spore morphogenesisare coordinately regulated by Cryptococcus neoformansCrk1 and Mub1D. Liu and W. C. Shen

National Taiwan University, Taipei City, Taiwan

Cryptococcus neoformans is a heterothallic basidiomycete that growsvegetatively as yeast and filamentous hyphae are produced in thesexual state. The production of sexual filaments is inhibited byCwc1/Cwc2 complex upon light treatment. A genome wide geneticscreen has identified components such as CRK1 and MUB1 genes inthe pathway. C. neoformans CRK1 gene is a homologue of Ustilagomaydis crk1 and Saccharomyces cerevisiae IME2, and contains theconserved Ser/Thr kinase domain and TXY dual phosphorylationsite. In C. neoformans, Crk1 negatively regulates mating differentia-tion 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 mutantcross. C. neoformans MUB1 gene, a homologue of Saccharomyces cere-visiae MUB1 (multi-budding) gene, is a MYND domain-containingprotein required for ubiquitination. Deletion of C. neoformans MUB1gene caused compromised growth at 37 °C. C. neoformans mub1mutants similarly displayed a multiple-budding phenotype andaltered structures of bud scars were observed. Interestingly, elonga-tion of dikaryotic sexual filaments was abnormal and formation ofbasidiospore chain was defective in the mub1 bilateral cross. To fur-ther examine their epistatic relationship, the MATa crk1mub1 andMATa crk1mub1 mutants were created and mating phenotypes werecharacterized. Similar to the crk1 bilateral mutant cross, basidia andbasidiospores were also seen at 24 h in the crk1mub1 bilateralmutant cross; however, spore morphogenesis was ceased at four-spore stage and production of basidiospore chains was impaired asthe mub1 mutants. These results indicated that both CRK1 andMUB1 genes coordinately regulate dikaryotic filament elongation,but they play different roles in the process of spore chain formationin C. neoformans.

ª 2014 The Authors


Poster Abstracts

P061

Molecular typing of environmental Cryptococcusneoformans isolated from pigeon droppings in Tripoli,LibyaM. S. Ellabib,1 M. A. Aboshkiwa,1 R. D’Amicis2 and M. Cogliati2

1Department of Medical Microbiology and Immunology, Faculty

of Medicine, Tripoli University, Tripoli, Libya and 2Lab. Micologia

Medica, Dipartimento di Scienze Biomediche per la Salute,

Universit�a degli Studi di Milano, Milano, Italy

Background Cryptococcus neoformans is the major cause of fungalmeningitis, a potentially lethal mycosis. Bird excreta can be consid-ered a significant environmental reservoir of this species in urbanareas. 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 ofC. neoformans in pigeon droppings in Tripoli urban area as well as toidentify the molecular type and the mating type of the collectedisolates.Materials and methods One hundred samples from pigeon excretawere collected from three different sites in the city of Tripoli, Libya.Samples were cultured on sunflower seed agar supplemented withantibiotics and biphenyl. Identification of C. neoformans was per-formed on the basis of melanin pigment production on sunflowerseed agar, presence of a capsule observed in India ink preparation,urease production on urea agar medium, and ability to grow at37 °C. Vitek2 compact system was used to confirm C. neoformansidentification. Molecular type and mating type allelic pattern weredetermined performing two specific multiplex PCRs as describedelsewhere.Results Thirty-two out of the 100 samples were positive for C. neo-formans. Multiplex PCR amplifications reveal that all isolates belongedto molecular type VNI (C. neoformans var. grubii) and mating typealphaA.Conclusion This study provides for the first time information con-cerning the ecology and genotypes of C. neoformans in the Tripoliregion of Libya.

P062

Cryptococcus neoformans molecular type VNB is associatedwith mortality in HIV associated cryptococcal meningitis inSouth AfricaM. 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

1Research 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 mor-tality amongst HIV-infected individuals in Subsaharan Africa, fromwhere C.neoformans (Cn) is thought to have evolved. The organism isdivided into two subspecies (var grubii and var neoformans), with Cn

var grubii divided into three molecular groups VNI, VNII and VNB;the latter was originally described in Botswana, and demonstratesgreatest evidence of recombination. Using multi-locus sequence typ-ing (MLST) of Cn isolates from a large clinical trial cohort, weexplored the genotypic diversity of Cn in South Africa, its geographi-cal distribution within Cape Town, and its association with clinicaloutcome.Aims To understand the cryptococcal genetic diversity presentwithin the study population of South African clinical trials patients,and to determine relationships between phylogenetic clade and burstgroups with clinical outcome.Methods DNA extracted fromCn isolated from 222 South AfricanHIV-infected individuals with CM was amplified by PCR andsequenced at seven loci to generate MLST profiles (ISHAM typingscheme). Isolates were classified by phylogenetic relationship intomolecular types and further sub-divided into subclades using maxi-mum likelihood analysis. Clinical outcome was analysed using Coxproportional hazards survival analysis, adjusting for known adverseprognostic indicators as well as CM induction drug treatment. Geo-graphical location data was mapped using patient address to obtainGPS coordinates via Google Maps, followed by plotting onto shapefiles using ArcGIS.Results Of 222 isolates,218 were Cn var grubii, with 4 C.gattii.MLST profiling revealed great genetic diversity within South Africanpatients, with 48 different sequence types (STs), including at least 18novel STs. Molecular types were VNI (n = 166), VNII (n = 42) andVNB (n = 7). Patients infected with VNB molecular type had worsesurvival compared to VNI (HR 2.8, 95% CI 1.2–6.5, P = 0.016),even after adjustment for altered mental status, baseline fungal bur-den (CFU ml�1 CSF) and treatment with amphotericin (HR 2.6, 95%CI 1.1–6.3, P = 0.029). Survival comparisons using specific ST typesand ‘Burst Groups’ were also explored and will be presented. Geo-graphical mapping for 185 cases localised in Cape Town showed noclustering by Molecular Type, Sequence Type, or Burst Group.Discussion/conclusion South African clinical C.neoformans strainsare highly genetically diverse. CM caused by the VNB molecular type,though rare, appears to be associated with poorer outcome. The lackof geographical clustering of isolates argues against a common recentenvironmental exposure, and lends support to the reactivation oflatent infection hypothesis. The underlying mechanisms of this asso-ciation will now be explored using a combined genomic and tran-scriptomic approach, and further in vitro phenotypic analysis.

P063

Two evolutionarily conserved post-transcriptionalregulatory pathways converge to regulate the ER stressresponse of Cryptococcus neoformansV. E. Glazier and J. C. Panepinto

SUNY University at Buffalo, Buffalo, NY, USA

Background Cryptococcus neoformans is one of a small number offungi able to transition from ambient environmental temperatures tohuman core body temperature. We previously reported a role for theER stress response in host temperature adaptation of C. neoformans,with post-transcriptional gene regulation controlling the intensityand duration of this response. Activation of the ER stress responseresults in the unconventional splicing of the pre-spliced HXL1umRNA to the active HXL1s mRNA at the ER surface by Ire1. Oncespliced, the HXL1s transcript is translated into Hxl1, a transcriptionfactor that promotes the expression of ER stress transcripts. Studiesin S. cerevisiae have identified a consensus sequence in HAC1 (anHXL1 homolog) that affects the localization of HAC1 mRNA to theER surface where it is spliced by Ire1. The HAC1 consensus sequenceis conserved in several fungal species however is notably absent inthe C. neoformans counterpart, HXL1, suggesting an alternate methodof regulating HXL1 splicing in C. neoformans.Aim Characterize the role of Puf4 in the post-transcriptional regula-tion of the ER stress response

ª 2014 The Authors


Poster Abstracts

Methods We assessed tunicamycin and temperature sensitivitythrough spot plate assays of H99, puf4-delta and puf4-delta: PUF4�.Northern blot analysis was used to measure steady state levels of ERtranscripts during tunicamycin treatment and growth at 37 °C. Tomeasure mRNA stability 1,10 phenanthroline was added prior toharvesting cells to halt transcription then RNA isolation and north-ern blot analysis performed. To determine the rate of HXL1 splicing,we used semi-quantitative PCR amplification of cDNA utilizing prim-ers that span the HXL1 splice site. The electrophoretic mobility shiftassay contained TYE-705 labeled oligonucleotides and recombinantPuf4 electrophoresed on a DNA retardation gel. Virulence studiesincluded tail vein injection, intranasal inoculation and a competitionassay.Results Spot plate assays reveal an increase in temperature sensitiv-ity of puf4-delta, combined with increased resistance to the ER stressinducing drug tunicamycin, suggesting the ER stress response isaltered in puf4-delta. puf4�delta cells exhibit misregulation of ERtranscripts when compared to wt cells during tunicamycin treatmentand growth at 37 °C. The mRNA encoding the ER stress responsetranscription factor, HXL1, has two potential Puf4 consensussequences. EMSA results show the 50UTR PUF element of HXL1 isable to bind Puf4, suggesting a direct regulation of HXL1 by Puf4.Puf4 appears to regulate both the rate of splicing and mRNA stabilityof HXL1 as the puf4�delta mutant has a defect in the rate of HXL1splicing during ER stress, and an increase in HXL1 transcript stabilitywhen compared to wt. Although Puf4 is involved in the regulationof a pivotal component of the ER stress response, HXL1, deletion ofPuf4 has no effect on virulence in a mouse model, but exhibitsreduced fitness in an in vivo competition assay.Conclusions Our results suggest a novel convergence of the PUFpost-transcriptional regulatory module and the ER stress responsesignaling pathway in Cryptococcus. Puf4 appears to regulate the rateof HXL1 splicing, possibly through localization of HXL1 mRNA toIre1 foci. This delay in HXL1 splicing has a downstream effect on ERtranscripts that rely on HXL1 splicing and production of Hxl1. Inaddition to the effects of Puf4 on HXL1 splicing Puf4 also appears tomodulate HXL1 transcript stability. The interaction between Puf4and HXL1 mRNA occurs through binding of Puf4 to a consensussequence in the 50UTR of HXL1. Future studies will be required toidentify other mRNA targets that make up the Puf4 RNA regulon.

P064

A chemical-genetic portrait of a human fungal pathogenJ. 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

1University of California, San Francisco, San Francisco, CA, USA;2University of Minnesota, Minneapolis, MN, USA; 3University of

Rochester Medical Center, Rochester, NY, USA and 4Hadassah-

Hebrew University Medical Center, Jerusalem, Israel

Background and aim Two major questions in microbial pathogene-sis are how to systematically identify targets of anti-microbial drugsand how annotate genes of unknown function involved in the viru-lence process. We sought to develop and adapt efficient, high-throughput methods to perform these functions and create datasetsof use to the Cryptococcus research community.Methods We used chemical-genomic profiling to address the ques-tions of drug target identification and functional annotation of viru-lence genes. This is the systematic measurement of the impact ofsmall molecules on the growth rate of a large number of deletionmutants. We employed a plate-based colony array method to quan-tify the impact of >200 diverse growth-inhibitory compounds on~1500 C. neoformans deletion strains.Results (1) Identification of novel virulence factors: We performed hier-archical clustering on the resultant dataset, then focused our initialstudies on two clusters that each contained 1–2 genes known to be

required for biosynthesis of polysaccharide capsule, along with a halfdozen genes of unknown function. We assessed all of the mutantswithin each cluster for capsule polysaccharide production and foundthat the majority impact capsule production and/or attachment.Mutants attenuated for capsule production are also attenuated forgrowth in a mouse lung.

(2) Development of drug synergy biomarkers: We hypothesized thatgenes whose knockouts show altered growth to compound pairsknown to be synergistic could be used to predict new drug synergies.We tested this hypothesis experimentally for two drugs and foundthis novel approach to be highly successful at identifying synergisticdrug combinations compared to a randomly generated control groupof drugs.

(3) Identification of the target of a drug-like molecule: We discoveredthat deletion of the gene coding for the Wee1 kinase produced dra-matic resistance to an uncharacterized growth-inhibitory drug-likecompound called TDZ8. Wee1 blocks G2/M transition by phosphory-lating Cdk1; the phosphatase Cdc25 opposes this. Our data suggestedthat TZD8 acts by blocking Cdc25. Indeed, TZD8, but not a highlyrelated compound, produces a G2/M cell cycle arrest. We alsoexpressed and purified C. neoformans Cdc25, measured its phospha-tase activity in vitro, and found that TZD8 directly inhibits Cdc25phosphatase activity.Discussion/conclusions Chemical-genomic profiling is a versatilemethod that can efficiently address several rate-limiting questions inmicrobial pathogenesis research, including identification of drugtargets and functional annotation of virulence genes.

P065

Molecular epidemiology of clinical Cryptococcus gattiiisolates from ColombiaC. Firacative,1 J. Lizarazo,2 P. Escandon,3 C. A. Agudelo,3

W. Meyer1 and E. Casta~neda3

1The University of Sydney, Westmead, Australia; 2Hospital

Universitario Erasmo Meoz, Cucuta, Colombia and 3Instituto

Nacional de Salud, Bogota, Colombia

Background Cryptococcosis caused by Cryptococcusgattii isendemicin several countries and affects mostly immunocompetent patients,both their pulmonary and central nervous systems. Worldwide thenumber of cases of this mycosis is increasing mainly because C. gattiiis expanding its environmental niche, which leads to a wider geo-graphic distribution. In Colombia, a national surveillance on crypto-coccosis, including demographic, clinical and microbiologic data, isbeing conducted since 1997, although molecular characterization ofthe 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 provideinsights into the molecular epidemiology of this important pathogenin the country and to contribute to the general knowledge of Crypto-coccus and cryptococcosis in the world.Methods From 1.207 surveys analyzed, 43 C. gattii cases from 15departments were reported, with the majority of the cases (n = 15)from Norte de Santander. Among all isolates, four were recoveredfrom HIV patients, one from a patient with arthritis and one withdiabetes. The remaining 37 patients did not have or report any riskfactor. Molecular type of the isolates was determined using PCR fin-gerprinting with the primer (GTG)5. Mating type a or alpha wasdetermined using specific primers. Multilocus Sequence Typing(MLST) was carried out using the ISHAM consensus MLST typingscheme for C. neoformans/C. gattii species complex, which includesseven genetic loci, CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and theIGS1 region.Results The molecular type VGII was the most frequent among theisolates (55.8%), followed by VGIII (27.9%), and VGI (16.3%).Among the patients with risk factors associated with the develop-ment of cryptococcosis, the molecular type VGII and VGI were

ª 2014 The Authors


Poster Abstracts

identified in two and one HIV+ patients, respectively. One VGII iso-late was recovered from a patient with arthritis and one VGIII isolatefrom a patient with diabetes. Mating type was determined as alphain 23 (53.5%) isolates and as a in 20 (46.5%). Among 42 isolates,16 MLST sequence types (ST) were identified: two STs amongst VGIisolates, nine amongst VGII isolates, with ST25 being the most com-mon one (68%), and five amongst VGIII isolates. Eight STs of theVGII isolates (ST8, ST12, ST31, ST46, ST321, ST322, ST323 andST324) and three of the VGIII isolates (ST59, ST64 and ST85) wereidentified for the first time in this study. The obtained MLST datawas incorporated into the C. neoformans/C. gattii databases accessibleat http://mlst.mycologylab.org.Discussion/conclusion The current study showed that the clinicalC. gattii isolates from Colombia are genetically diverse. Despite thelow number of isolates studied, several genotypes were identifiedwithin each molecular type, opposite to the less diverse and ratherclonal C. gattii populations reported in other countries such as Can-ada, USA, Australia and Thailand, where few genotypes have beenidentified in a much larger number of isolates. The identification ofthe same ST in different departments, like the prevalent ST25 forVGII isolates found in 7 different departments, the ST51 and ST58 inVGI isolates, and the ST64, ST79 and ST146 in VGIII isolates, sug-gests the circulation of some genotypes in the country. The isolationof C. gattii predominantly from otherwise healthy hosts rather thanfrom patients with impaired immune system supports the idea of C.gattii as primary pathogen and as being clinically more pathogenicthan its sibling species C. neoformans. Our data is giving a moredetailed picture of the molecular epidemiology of cryptococcosis inColombia and includes the country as integral part of the global pop-ulation genetics studies of the C. neoformans/C. gattii species complex.

P066

Purine biosynthetic enzymes as antifungal drug targetsR. B. Blundell, S. D. M. Arras, S. J. Williams and J. A. Fraser

University of Queensland, Brisbane, QLD, Australia

Background With increasingly large immunocompromised popula-tions around the world, opportunistic fungal pathogens such as Cryp-tococcus neoformans are a growing cause of morbidity and mortality;C. neoformans is estimated to be responsible for over 600 000 deathsevery year. Fungal infections are difficult to treat owing to the simi-larities of their eukaryotic physiology to that of humans. The limitedantifungal agents available tend to target the few differences betweenthe two systems. To combat the current scarcity of antifungal thera-peutic agents, research into fungal-specific drug targets is required.Adenylosuccinate synthetase (AdSS) is a crucial enzyme in the ATPbiosynthetic pathway, catalyzing the formation of adenylosuccinatefrom inosine monophosphate and aspartate. We have previouslycharacterised another enzyme in the GTP biosynthetic pathway, ino-sine monophosphate dehydrogenase and have developed a pipelinewith which to test the antifungal potential of these enzymes.Aim To characterise AdSS from Cryptococcus neoformans and investi-gate its potential as an antifungal drug target.Methods The AdSS-encoding gene ADE12 was deleted from type-strain H99, and the effects of this deletion on growth, adeninerequirements, the production of several virulence factors and viru-lence in both C. elegans and mice were investigated. Phenotypicchanges were confirmed using knock-out strains complemented withwild-type ADE12 and the homologuous gene from Escherichia coli.Cryptococcal AdSS was also expressed in E. coli and purified vianickel-affinity and size-exclusion chromatography. Purified proteinwas used to complete enzyme kinetic and multi-angle laser light scat-tering (MALLS) studies. Purified AdSS was crystallised both in its apoform and bound to several substrates and inhibitors, and its structuresolved. This crystal structure was used to examine the structural dif-ferenced between human and AdSS to gain an insight unto potentialroutes of antifungal development.

Results We have characterised the affect of ade12Δ deletion ongrowth and virulence factor production, and documented multiplephenotypic changes as well as the resulting changes in virulence inan animal model. The differences between human and CryptococcalAdSS in both crystal structure and enzyme kinetics suggest that itmay be possible to develop a fungal-specific inhibitor.Discussion/conclusion Overall our results indicate that AdSS is apromising antifungal drug target, and pave the way for further inves-tigation into possible inhibitory compounds and species specificity.

P067

Enhancing the complementation process in CryptococcusneoformansS. D. M. Arras and J. A. Fraser

University of Queensland, Brisbane, QLD, Australia

Introduction Just as Koch’s postulates formed the foundation ofinfectious disease study, so do Stanley Falkow’s adapted molecularpostulates which define the approach to be taken in determiningwhether a gene is involved in pathogenesis. Fundamentally, thesemolecular postulates state that if a gene is involved in virulence, itsremoval will compromise virulence. Likewise, its reintroductionshould restore virulence to the mutant. The deletion of genes inCryptococcus neoformans is a well-established technique, with the bio-listic transformation of a dominant selectable marker and subsequentdeletion 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 theoriginal locus and random integration.

Complementation of a deletion mutation by reinsertion of the geneat the original locus is unlikely to interrupt any other genes and maygive a wild type level of expression, however these transformants canbe indistinguishable from a wild type contaminant if direct selection isused, and if it is not it requires integration of a secondary markerwhich may affect adjacent genes. Secondly and most importantly, onemay not be able tell if the original mutant phenotype was due to thegene deletion or unintended affects from adjacent genes.

In contrast, random integration involves the complementationconstruct being biolistically transformed into a random location inthe genome. While this strategy requires little experimental designand results in an abundant number of transformants, as this specieshas a gene rich genome it is probable that other genes are disturbedduring such events. Short of determining the precise insertion site itis impossible to tell what genes are affected, and in turn what unin-tended phenotypic consequences have arisen. Importantly, these sec-ondary phenotypes may only be obvious under certain conditions,such as in a murine model. To counter the drawbacks of the currentapproaches to complementation we have created a new tool to assistin the to complementation mutant strains.Aim To create a new resource that facilitates the complementationof mutant strains in C. neoformans.Methodology To avoid the disruption of random genes duringintroduction of a complementation construct, it was decided to design aplasmid that targets the transformed DNA to a preselected, silent loca-tion. A pBluescript-SK derivative plasmid was created containinghomology for the silent site, as well as a C. neoformans selectable marker.In order to retain blue/white screening, these features were inserted intothe backbone of pBluescript, keeping the multicloning site intact.Results We determined that the complementation plasmid integratesat high frequency in the correct position, effectively complementing amutant strain without causing secondary mutations that complicateanalyses of our mutant strains. Transformants bearing the comple-mentation plasmid at the correct genomic location were easily identi-fied using a multiplex colony PCR assay. Importantly, once theplasmid successfully integrates, qRT-PCR on the flanking genes oneither side of the silent region revealed no changes in their expres-sion, and no secondary phenotypes were observed.

ª 2014 The Authors


Poster Abstracts

Discussion and conclusions Based on the problems faced duringcomplementation of mutants in C. neoformans, we have successfullycreated a new molecular resource for the Cryptococcus communityensuring that unrelated genes and by extension virulence are notaffected during the complementation process. We believe that thisnew strategy helps overcome problems with the current approaches,and while it will be more difficult to integrate into the genome asopposed to random integration, it eliminates any affects on othergenes, ultimately fulfilling Koch’s postulates.

P068

Deletion of PKC1 by biolistic transformation results inaneuploidy in Cryptococcus neoformansM. J. Donlin,1 R. Upadhya,2 W. Lam2 and J. K. Lodge2

1Saint Louis University School of Medicine, St. Louis, MO, USA and2Washington University School of Medicine, St. Louis, MO, USA

Background Cryptococcus neoformans is a fungal pathogen of immu-nocompromised people that causes fatal meningitis. The fungal cellwall is essential to viability and pathogenesis of C. neoformans, andbiosynthesis and repair of the wall is primarily controlled by the cellwall integrity (CWI) signaling pathway. The protein kinase C,encoded by the PKC1 gene is a DAG-activated kinase and the DAG-responsive C1 domain has been shown to be essential for productionof melanin. In a prior study, a pkc1D strain had severe cell wall phe-notypes, sensitivity to a variety of cell wall stressors and requires anosmotic stabilizer for growth in YPD. The pkc1D was confirmed bymultiple PCR screens and a Southern blot analysis for the correcthomologous recombination and single insertion. Complementation ofthe pkc1D strain within the native locus resulted in wild-type growthand loss of sensitivity to cell wall stressors. We conducted an RNAsequencing experiment to compare pkc1D to wild-type KN99 and dis-covered that ~30% of the genes on chromosome 5 were up-regulatedcompared to ~8–10% of the genes on any other chromosome. Wenoted that passage of this deletion strain on YPD only eventuallyresulted in a strain that grew normally even in the absence osmoticstabilization. Taken together, these results led us to hypothesize thatdeletion of PKC1 by biolistic transformation resulted in an aneuploidchromosome and that this aneuploidy may be lost after passagingwithout osmotic stabilization.Aims Our first aim was to conduct a comparative genome-hybridiza-tion to determine if chromosome 5 in the pkc1D strain was aneu-ploid. Our second aim was to characterize the cell wall phenotypes ofany deletion strains that had lost the aneuploid chromosome. Ourthird aim to repeat these experiments with additional isolates of thePKC1 deletion.Methods The pkc1D strain was generated by biolistic transformationof wild-type KN99. A confirmed pkc1D strain was passaged for sev-eral generations on YPD only; this strain is subsequently referred toas the pkc1D adapted. Genomic DNA was isolated from wild-typeKN99, pkc1D and a pkc1D-adapted strains and hybridized to H99

transcript array. The DNA expression levels were compared betweenwild-type and the two pkc1D strains, relative expression levels wereaveraged across seven genes and plotted by chromosome.Results When the levels of DNA hybridized to the transcripts arecompared between wild-type and the pkc1Dand pkc1D-adapted, chro-mosome 5 in the pkc1D is, on average, almost twice that of the wild-type strain (Fig. 1) whereas none of the other chromosomes in thepkc1Dstrain nor any of the chromosomes in the pkc1D-adapted strainshow any differences from wild-type (Fig. 1). The pkc1D-adaptedstrain remains very sensitive to high temperature, SDS and caffeinebut has lost sensitivity to high salt, calcofluor white and Congo red.We are continuing to analyze this strain as well as additional isolatesand that data will be reported.Conclusions This is the first reported case of chromosomal duplica-tion found in a C. neoformans gene deletion strain that is obtainedafter biolistic transformation. We found that passaging on YPDmedia resulted in loss of the aneuploidy and loss of some, but not allcell wall sensitivity phenotypes. It suggests that aneuploidy may rep-resent one potential source of differential phenotypes observed withmultiple isolates of single gene deletion strain.

P069

Effect of histone deacetylases inhibitors on the virulencephenotypes of Cryptococcus neoformansF. Brandao, L. S. Derengowski, P. Albuquerque and

M. J. Poc�as-FonsecaUniversity of Brasilia, Brasilia, Brazil

Cryptococcus neoformans infection is due to expression of various viru-lence factors among which we highlight the ability of the fungus togrow at 37 °C, presence of a polysaccharide capsule and melanin,urease and phospholipases production. However, the mechanismsthat regulate the expression of these virulence factors are not wellknown. Recent studies indicate that a key regulatory mechanism ingene expression is the modulation of chromatin. Histone deacetylaseinhibitors (iHDAC), such as Sodium butyrate (NaBut) and Trichosta-tin A (TSA), have been employed in studies in different cell typesshowing to be able to affect chromatin structure leading to changesin gene expression.Objective In this context, the aim of this work was to study theeffect of these HDAC inhibitors on expression of the major virulencefactors of C. neoformans, as well its effect onhost-pathogeninteraction.Methods Different concentrations of the drugs were applied to C.neoformans cultures grown on several conditions in order to analyzethe expression of virulence factors. Growth curves were performed bymeasuring the cell density at 630 nm at 30 and 37 °C. For assays ofcapsule and melanin production, cells were incubated in minimalmedium with/without L- DOPA. Cell size was evaluated by flowcytometry. Phospholipase activity was estimated in agar base con-taining egg yolk. Urease secretion was analyzed in Christiansen’surea agar medium. For mating assays, opposite mating types (K99aand K99a) were inoculated on agar Filament at room temperature inthe dark and were observed daily in a microscope for hyphae andother mating structures formation. Survival curve were performedinfecting caterpillars of the alternative host Galleria mellonella with C.neoformans cells pretreated or not with iHDAC.Results and discussion NaBut concentrations starting at1 mmol l�1 were able to interfere significantly in C. neoformansgrowth at both 30 and 37 °C. Flow cytometry revealed a fungal cellsize decrease in the presence of 10 mmol l�1 of the NaBut. Therewas also a significant reduction in capsule size at concentrations aslow as 1 mmol l�1 of this drug. A decrease in the cell size in thepresence of 10 mmol l�1 of NaBut was observed by flow cytometry.Secreted phospholipase activity, melanin synthesis, and matinghyphae formation were all affected at NatBut concentrations as lowas 5 mmol l�1. TSA showed very subtle effects compared to NaButin all analyzes performed. It was able to reduce cell growth only at

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

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Poster Abstracts

37 °C at concentrations above 1.5 lmol l�1. Capsule formation wasaffected at concentrations from 1.5 lmol l�1, being more pro-nounced at higher concentrations. There was a decrease in cell sizein the presence of 10 lmol l�1 of TSA and in mating hyphae forma-tion in concentrations starting at 6 lmol l�1. TSA did not interferevisibly on phospholipase activity. Neither drug affected urease pro-duction in the in any of the tested concentrations. Finally, no differ-ence was observed in survival curves of the invertebrate host G.mellonella infected with pre-treated yeasts with NaBut or TSA com-pared with the untreated control. This fact agrees with the recoveryof virulence phenotypes in vitro since iHDAC are removed. The tran-sient effects observed in cell growth curve of the fungus treated withTSA, as well as the less pronounced effects of drug in longer durationexperiments, such as the mating assay, may be explained by the lackof the stability followed by the short duration of TSA action describedby other studies.Conclusion So far, these results indicate that iHDAC induce changesin the expression of several virulence phenotypes of C. neoformans.Further molecular approaches will be employed to reveal the possiblemechanisms involved in these observations.

P070

The major cytoplasmic exonuclease Xrn1p affects virulenceand mating in Cryptococcus neoformansC. Wollschlaeger,1 N. Trevijano,2 X. Wang,3 M. Legrand,1

O. Zaragoza2 and G. Janbon1

1Institut Pasteur, Paris, France; 2National Center of Microbiology,

ISCIII, Madrid, Spain and 3Duke University, Durham, NC, USA

Background Extracellular vesicles (EVs) are membranous structuresused by a number of fungal species’ cells, including Cryptococcus neo-formans, to deliver virulence factors to the extracellular milieu. C.neoformans EVs are immunologically active and can regulate the anti-fungal activity of phagocytes. The interaction with environmentalpredators is essential for the establishment of virulence mechanismsused by C. neoformans. The ability of EVs to modulate the antifungalactivity of environmental phagocytes and the processes that mediateC. 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 phagocyticevents with this predator.Aim To characterize the effect of EVs produced by C. neoformans onthe antifungal activity of the environmental predator A. castellaniiand identify the association of MBR for amoeba phagocytosis andkilling of this yeast.

A B C

E F 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 thecells 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: TSA3 lm, H: TSA 6 lm and I: TSA 10 lm. Boxes represent 75 percentof the data distribution, the median divides the quadrants. Bars rep-resent the minimum and maximum values. Statistical test appliedwas Kruskal-Wallis and post test Dunn. * indicate P < 0.05,***P < 0.001, **** P < 0.0001. Scale bars represent 10 lm. Threedifferent experiments were performed with similar results.

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

ª 2014 The Authors


Poster Abstracts

Methods Our approach included EV purification, analysis of amoebaviability after exposure to EVs and methyl-alpha-D-mannopyranoside(mannose), stimulation of A. castellanii with EVs, and mannose anddetermination of the phagocytosis index and antifungal activity forH99 strain, an encapsulated serotype A of C. neoformans.Results Our initial results demonstrate that C. neoformans EVs witha diameter range between 50 and 550 nm do not impact the viabil-ity of amoebae, but exposure to EVs increases fungal uptake and sur-vival inside the environmental predator, compared to untreatedamoebae. Mannose treated amoebae also exhibited an increase infungal uptake and survival and were more susceptible to death dur-ing 24 h co-cultures with C. neoformans.Discussion Our results to date demonstrate that fungal EVs impactC. neoformans interactions with A. castellanii, indicating that C. neofor-mans vesicle release to the extracellular space may have developed inresponse to interactions with environmental predators. Additionally,our findings suggest that MBR can mediate fungal phagocytosis byamoebae, opening a new field of investigation about evolutionarysteps that could culminate in intracellular pathogenic strategies forC. neoformans to infect and proliferate inside mammalianmacrophages.

P071

Genotypic variation of Cryptococcus neoformans varietygrubii from Asia and South AfricaK. Khayhan,1 J. Vreulink,2 B. Theelen,3 F. Hagen,4 A. Botha2 and

T. Boekhout3

1Faculty of Medical Sciences, University of Phayao, Phayao,

Thailand; 2University of Stellenbosch, Stellenbosch, South Africa;3Fungal Biodiversity Centre (CBS-KNAW), Utrecht, The

Netherlands and 4Canisius-Wilhelmina Hospital, Nijmegen, The

Netherlands

Background Cryptococcus neoformans variety grubii is an opportunis-tic pathogen that causes diseases, particularly cryptococcal meningi-tis, pulmonary cryptococcosis and cutaneous cryptococcosis, in bothimmunocompetent and immunocompromised individuals. In sub-Saharan Africa Asia, the number of cryptococcosis patients is esti-mated more than 720 000 new cases per year, followed by Southand Southeast Asia accounting for 140 000 new cases per year. Inthis study, we used multilocus sequence typing (MLST) to study thegenetic diversity among cryptococcal isolates from different Asianregions and from South Africa.Objectives We determined genotypic variation of C. neoformans var.grubii isolates collected from various geographical locations in Asiaand from Cape Town, South Africa using MLST. Furthermore, wecompared the genotypes obtained to those of C. neoformans var. grubiiisolates from the other continents.Methods Four hundred and seventy-six C. neoformans var. grubiiwere obtained from various countries in Asia, including China, HongKong, India, Indonesia, Japan, Kuwait, Qatar and Thailand, and 31isolates from Cape Town, South Africa. MLST was performed todetermine sequence types (STs). MLST data of C. neoformans var. gru-bii from previously publications were used for comparison at the glo-bal level.Results MLST showed that 4 predominant STs (i.e. STs 4-6 andST93) occurred among 476 Asian C. neoformans var. grubii isolatesand 3 common STs (i.e. ST23, ST40 and ST69) occurred among 31isolates from South Africa. Among the Asian population the distribu-tion of STs was different in each country. The minimum spanningtree of the global MLST C. neoformans var. grubii isolates showed thatmost Asian isolates clustered together in one group and differed fromisolates from the other continents, whereas the South African isolateshad a scattered distribution.Conclusion Multilocus sequence typing revealed a different distribu-tion of genotypes of C. neformans var. grubii in different geographicallocations at the continental (Asia) and global (Cape Town) scale. The

predominate STs among the Asian isolates differed from those amongSouth African isolates. Asian isolates showed limited genetic varia-tion, whereas isolates from South Africa showed more diversity andoccurred in all clusters present in the minimum spanning tree of theset of global MLST data.

P072

Post-transcriptional regulation contributes to translationalprioritization during host-temperature adaptation inC. neoformansA. L. Bloom and J. C. Panepinto

SUNY University at Buffalo, Buffalo, NY, USA

Background In response to the hostile host environment, pathogensundergo rapid reprogramming of gene expression to adapt to stress.In C. neoformans, exposure to host-temperature results in rapidrepression of ribosomal protein (RP) transcripts and simultaneousinduction of ER stress response transcripts. These responses are tran-sient as levels of both functionally related classes of mRNAs return tobasal-like quantities after 3 h. Ccr4-mediated degradation regulatesthe intensity and duration of each of these responses throughenhanced destabilization of these mRNAs at the time at which eachclass undergoes repression during host-temperature adaptation. Inthe ccr4delta mutant, RP-mRNAs are insufficiently repressed, and theER stress response is constitutively active. The dissociable RNA poly-merase II subunit, Rpb4, also contributes to these transient, fast-relaxing responses by coupling mRNA synthesis and degradation.Following a shift to host-temperature, Rpb4 exits the nucleus, pre-sumably bound to mRNAs, and mediates enhanced decay in thecytoplasm.Aim Our goal is to investigate the prioritization of protein translationin response to host-temperature to understand the stress-induced cel-lular reprogramming required for host-temperature adaptation. Wespecifically want to determine the contribution of post-transcriptionalregulation by Ccr4 and Rpb4 to this reprogramming.Methods Stability of RP-mRNAs and ER stress mRNAs was deter-mined by northern blot hybridization of transcript levels during inhi-bition of transcription. To investigate changes in translating pools ofmRNAs, polysome profiling was performed for whole cell lysates fromwt, ccr4delta, and rpb4delta strains during non-stressed conditionsand 1 and 3 h post-temperature shift from 30 to 37 °C. RNA wasextracted from fractions collected during profiling, and distributionsof RPL2 (RP transcript) and KAR2 (ER stress transcript) were exam-ined by northern blot analyses.Results Polysome profiles do not change appreciably throughouthost-temperature adaptation in the wild type. However, polysome-association of RPL2 is reduced 1 hour after the temperature shift,and increases to non-stressed levels by three hours. KAR2 exhibitsmodestly more polysome-association at 1 h. Profiles are aberrant inthe ccr4delta and rpb4delta null mutants. Overall translation isdecreased following a shift to 37 °C in the absence of Rpb4. Whilefluctuation of RPL2 in and out of polysome fractions is maintained inthe rpb4delta mutant, the changes are not to the magnitude of thewild type. In the ccr4delta mutant, RPL2 and KAR2 remain poly-some-associated throughout the time course. Additionally, in contrastto the wild type, as polysomes become heavier, less RNA is associatedwhen Ccr4 is absent.Discussion/conclusion Our previous work demonstrates thatmRNA synthesis and decay are coupled processes. Our current inves-tigations will address how uncoupling of mRNA synthesis and decayimpacts stress-responsive translational prioritization. Polysome analy-ses demonstrate that the association of RP-mRNAs with the translat-ing pool is reduced 1 hour after a shift to 37 °C. We previouslydemonstrated that RP-mRNAs are maximally repressed by Rpb4- andCcr4-mediated enhanced decay at this time. The combinatorial effectof these processes likely results in more available ribosomes to trans-late stress-specific proteins. Indeed, we observe higher levels of stress-induced ER stress transcripts, and these mRNAs become more

ª 2014 The Authors


Poster Abstracts

polysome-associated in the wild type. In the rpb4delta mutant, weobserve reduced translation following the temperature shift indicatinga role for Rpb4 in maintaining translation during host-temperaturestress. In our ccr4D mutant, many classes of mRNAs are stabilized,likely resulting in greater cellular RNA and a larger RNA::ribosomeratio. This may explain the increase in low molecular weight poly-some peaks and loss of heavy polysome peaks. In the ccr4delta andrpb4delta mutants RP- and ER stress transcripts are aberrantly dis-tributed over the polysome profile, suggesting that mRNA degrada-tion contributes to reprogramming of the actively translating mRNApools required for host-temperature adaptation.

P073

A whole population comparative genomics approach tounderstanding the emergence of Cryptococcus gattii in theAmerican Pacific NorthwestD. 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

1Translational Genomics Research Institute, AZ, USA; 2University

of Birmingham, Birmingham, UK; 3Imperial College, London, UK;4Unversity 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 Cryptococcusgattii (type VGII) in the temperate North American Pacific Northwest(PNW) was unexpected, in that C. gattii was previously known to beendemic only to tropical and semi-tropical regions. Beyond a new hab-itat niche, the dominant emerged populations displayed high virulenceand caused primary pulmonary disease, as opposed to neurologic dis-ease often seen in other C. gattii infections. Since the emergence, therehas been speculation about the origin of the outbreak strains.Aim In this study, whole genome sequence analysis was performedon 118 Cryptococcus isolates, primarily C. gattii VGII, to better ascer-tain the genomic changes behind the PNW emergence.Methods Using Illumina next generation sequencing technology, wesequenced 113 C. gattii VGII isolates and one isolate each of VGI,VGIII, and VGIV genotypes. Our analysis also used genomes frompreviously sequenced isolates of C. neoformans var. neoformans and C.n. var. grubii. We assessed all SNP mutations among isolates, andcompared the gene content of the PNW populations to all otherknown global VGII subtypes and other Cryptococcus genomes.Results While the overall VGII population was highly diverse, dem-onstrating large amounts of both mutation and recombination, thethree dominant subtypes in the PNW had low diversity and are com-pletely clonal. Although, VGII was found on at least five continents,all genetic sub-populations had representation, or closest relatives, inSouth America. Numerous (>50) gene content differences were iden-tifiable, including genes potentially related to habitat adaptation, vir-ulence and clinical differentiation. Evidence was also found of a likelyintrogression event, from C. n. var. grubii, of a gene complementrarely seen in the global C. gattii population but found in all PNWpopulations.Discussion The phylogenetic data highlight multiple dispersal eventsto North America and elsewhere, likely originating from South Amer-ica. The identification of novel gene content among and between thePNW populations, while not causal, provides evidence of genomicevolution that allowed for the recent expansion of habitat and disease.The findings here provide greater understanding of C. gattii adapta-tion in North America and its dispersal from South America.

P074

Multilocus sequence typing of clinical and environmentalisolates of Cryptococcus neoformans in Uberaba, BrazilM. 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


Background Cryptococcus neoformans is widely distributed andaccounts for most cases of cryptococcosis mainly in HIV infectedpatients. According to WHO estimatives, yearly, one million cases ofcryptococcosis occur around the world, of whom, 620 000 die asconsequence of this infection. During the last decades, moleculartools contributed to the development of Cryptococcus complex taxon-omy, identifying several serotypes, genotypes and sub-genotypesbetween the two pathogenic species. More recently, the multi locussequence typing (MLST) was defined a gold standard technique toevaluate seven different loci among clinical and environmental iso-lates of Cryptococcus neoformans and Cryptococcus gattii.Aim To characterize clinical and environmental isolates of Cryptococ-cus neoformans by MLST.Methods A total of 100 C. neoformans isolates were analyzed. Ofthese, 61 were recovered from 55 different AIDS patients with cryp-tococcosis, of which, 40 from the cerebrospinal fluid (CSF), 12 fromblood, five from urine, two from skin lesions and one of bronchoalveolar lavage (BAL). The remaining 39 isolateswere obtained fromenvironment surrounding areas of a teaching hospital. The sequenc-ing experiments were done to the loci CAP59, LAC1, IGS1 andURA5 according ISHAM MLST consensus scheme for Cryptococcusneoformans and Cryptococcus gattii.Results The CAP59 locus presented 99 single nucleotide polymor-phisms (SNPs), and five ATs. The isolates identity compared to thestrain H99 ranged from 92.6 to 100% whereas the SNPs rangedfrom 0-37. The locus IGS1 presented 41 SNPs and eight ATs. Identi-ties and number of SNPs ranged from 85.7 to 100% and from 0 to90, respectively. The locus LAC1 presented 48 SNPs and five ATs.Identities and number of SNPs ranged from 90.1 to 99.8 and from 1to 45, respectively. Finally, the locus URA5 presented 39 SNPs andfive ATs. Identities and number of SNPs ranging from 94.2 to 100%and from 0 to 35, respectively. The phylogenetic analysis of the con-catenated regions showed 13 sequence types (STs) and a total of 300polymorphisms with identities ranging from 90.70 to 99.96%. TheIGS1 locus showed the highest number of polymorphic sites with163 followed by LAC1, URA5 and CAP59 with 54, 42 and 41,respectively. The highest efficiency of typing (0.122) was found inthe LAC1 locus and the greatest discriminatory power in the IGS1ones (0.879). The ST with the largest number of isolates was ST13with 57 isolates (87.7% clinical and 12.3% environmental). BothST1 and ST6 consisted of 100% and 96% of environmental isolates.Of five patients with isolates obtained from different sites, two pre-sented more than one ST. Both clinical and environmental isolateswere included in nine different STs.Discussion/conclusion Through MLST it was possible to identifynine STs among clinical and environmental isolates with a total of13 different STs, which suggests high genetic variability of these iso-lates compared with other reports that evaluated a larger number ofloci. However, a whole genetic variability of the isolates herein evalu-ated, it will be obtained after typing the others loci suggested byISHAM MLST consensus. The finding of more than one ST in isolatesof the same patient seems to be an uncommon feature, since mostreports included only one isolate from each patient. A recently studyin Africa, found more than one ST in 16.7% of CSF samples genotyp-ing more than one colony of the same patient, which can suggestthat mixed infections can be found in one or different samples. Thegenetic characterization of clinical and environmental isolates ofCryptococcus spp. by MLST contribute to understand the genetic vari-ability and dynamic of these complex microorganisms around theworld.

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Poster Abstracts

P075

Molecular epidemiology and ecology of environmentalCryptococcus neoformans populations in ZambiaM. V. Vanhove,1 M. Beale,1,2 J. Rhodes,1 T. Bicanic2 and

M. Fisher2

1Department of Infectious Diseases and Epidemiology, St Mary’s

Campus, Faculty of Medicine, Imperial College London, UK and2Saint 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 spreadingworldwide. However, the population structure of the environmentalpathogen remains largely unknown in Africa. This project aims toexplore the population genetic structure of environmental popula-tions of the basidiomycete yeast in southern Africa.

Multiple ecological niches have been screened to isolate Cng iso-lates from the environment. More than 400 sites have been investi-gated across four ecoregions throughout Zambia. Samples from treebark and soil were cultured on Niger-seed (NS) plates. The Cryptococ-cus neoformans/gattii species complex appears as dark-brown colonieson NS agar. The fungal diversity present in these ecological nicheswas 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 differenti-ation between C. neoformans (n = 35) and C. gattii (n = 40) andneighbour joining tree gave insights on the C. neoformans and C. gat-tii diversity present in Zambia. This preliminary work showed thevalidity of the protocol to isolate Cryptococcus species and paved theway to the sequencing of these Cng isolates using whole genomesequencing (WGS) technologies.

Another question of interest is how this environmental yeastacquired its pathogenicity and became an important human patho-gen. Current knowledge suggests that environmental selection pres-sure from invertebrate hosts led to the pathogenicity of C. neoformansobserved in humans. Cryptococcus neoformans is facultative intracellu-lar pathogen and is able to replicate inside macrophages. Previously,an amoeba model, using Acanthamoeba castellanii was successful indescribing phenotypic variations between different strains. This modelwas used using ImageStream imaging flow cytometry to quantifycryptococcal cells uptake. Using different stains, cryptococcal cellscan be visualised and counted inside and outside amoeba. A cohortof both clinical and environmental isolates has been selected depend-ing of geographic and genetic characteristics. Phenotype differencesin Cryptococcus uptakes could lead to interesting observations regard-ing the development of hypervirulent lineages in humans. The func-tional analysis might reveal that local adaptation leads to variationsin Cryptococcus pathogenicity.

P076

Phylogenetic analysis of cryptococcus laurentii isolatesreveals a high frequency of sibling speciesM. 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

1Triangulo Mineiro Federal University, Uberaba, Brazil; 2Adolfo

Lutz Institute, S~ao Paulo, Brazil and 3Duke University Medical

Center, Durham, NC, USA

Background The Cryptococcus genus is comprised by more than100 species, most of them considered saprophytic. At present, Crypto-coccus neoformans and Cryptococcus gattii are considered pathogenic,but some species such as Cryptococcus laurentii have recently beendescribed infecting immunocompromised hosts.

Aim This study aimed to evaluate biological and molecular aspectsof C. laurentii strains from Brazil, United States, Canada and Africathrough the sequencing of three ribossomal regions.Methods A total of 100 environmental isolates phenotipically identi-fied as C. laurentii were evaluated by sequencing of the 50 end ofsmall subunit of 18S gene, D1/D2 region of the large subunit of 28Sgene and Internal Transcribed Spacer (ITS) region. The applicabilityof these markers as DNA Barcodes was also evaluated.Results BLAST search of the sequenced 550, 650 and 550 bpamplicons obtained from the 18S, D1/D2 region of 28S and ITSregion allowed the identification of 75 C. laurentii strains whichshowed 100% of identity with the C. laurentii NRRL Y-2536 strainfrom Taiwan and C. laurentii RY1 strain from India. Nine strainspresented 99% of identity with the Bullera sp. VY-68 strain fromJapan and C. laurentii RY1 through the 18S sequencing. One ofthem, the isolate P482A from S~ao Paulo, showed 99% of identitywith 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 bythe D1/D2 region of LSU-rDNA 28S and ITS region analysis. Sixteenisolates 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 regionsequencing, while the remaining 10 presented 99% of identity withthe Cryptococcus terrestris (CBS 10810) reference strain recentlydescribed in Brazil and United States. Among the three ribosomalDNA markers evaluated, the ITS region presented the highest vari-ability and the most defined Barcode gap, followed by the 28S and18S. Despite the high variability observed for the ITS region, twostrains of C. flavescens clustered near the C. terrestris group. The con-catenated sequence analyses allowed the identification of sevenSequence Types in C. laurentii, three in C. flavescens, one in Crypto-coccus sp., one in C. rajasthanensis and one in C. terrestris isolatesthrough Neighborhood-joining, goeBURST and Median-joining net-work analyses.Discussion/conclusion Since the first description of C. laurentii byKufferath in 1920, its taxonomy have undergone several changes,especially in the last decade with the introduction of the moleculartechniques. The high intraspecific variability of C. laurentii isolateshave already been described by others, despite few strains have beenincluded. Before, the results herein presented,, the variability of thisspecies was unknown. The intraspecific variability of 2.4% found inthis study represents an original result. In addition, other C. laurentiisibling species including one unknown species were described, andreinforce the relevance of the sequencing technique to species identi-fication. Of the three ribosomal regions herein evaluated, the ITS wasthe most suitable for DNA Barcode, which is in accordance with pre-vious studies.

P078

Cryptococcus gattii VGIII isolates causing infections in HIV/AIDS patients in Southern California: identification of thelocal environmental source as arborealD. 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

1Duke University Medical Center, Durham, NC, USA; 2UCLA, Los

Angeles, CA, USA; 3University of Birmingham, Birmingham, UK;4University of North Carolina, Chapel Hill, NC, USA and5University of Southern California, Los Angeles, CA, USA

Background Ongoing Cryptococcus gattii outbreaks in the WesternUnited States and Canada illustrate the impact of environmental res-ervoirs and both clonal and recombining propagation in drivingemergence and expansion of microbial pathogens. C. gattii comprisesfour distinct molecular types: VGI, VGII, VGIII, and VGIV, with noevidence of nuclear genetic exchange, suggesting these represent

ª 2014 The Authors


Poster Abstracts

distinct species. C. gattii VGII isolates are causing the Pacific North-west outbreak, whereas VGIII isolates frequently infect HIV/AIDSpatients in Southern California and Africa. VGI, VGII, and VGIII havebeen isolated from patients and animals in the Western US, suggest-ing these molecular types occur in the environment. However, onlytwo environmental isolates of C. gattii have ever been reported fromCalifornia: CBS7750 (VGII) and WM161 (VGIII). The incongruenceof frequent clinical presence and uncommon environmental isolationsuggest an unknown C. gattii reservoir in California.Aim We sought to collect C. gattii from environmental samples fromareas that had confirmed reports of clinical and/or veterinary infec-tions to identify the local environmental reservoir of C. gattii inSouthern California.Methods Swab and soil samples were collected during the summersof 2011 and 2012 utilizing BD BBL Single application CultureSwabswith Liquid Amies. In 2011 samples were obtained from 9 locations,64 trees (30 different species), and 25 soil samples in the greater LosAngeles area. In 2012 the two sites that had previously yielded C. gat-tii were resampled and 15 additional sites were sampled. From thesetrees 45 trees were swabbed and 33 soil samples were collected. Cryp-tococcus isolates were selected on Niger seed agar containing chloram-phenicol (0.5 g l�1). Yeast colonies producing brown pigmentationwere selected and colony purified. All clinical and environmental iso-lates 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 treatedat the University of Southern California, Harbor-UCLA Medical Cen-ter, or Kaiser Permanente Downey Hospital between February 2008and January 2013. The clinical isolates were de-identified and linkedto major cross streets near the patients’ homes.

Multilocus sequence typing was performed on 12 loci (SXI1a orSXI2a, IGS, TEF1, GPD1, LAC1, CAP10, PLB1, MPD1, CAP59,TOR1, SOD1, and URA5). Ploidy was determined by fluorescenceactivated cell sorting. Whole Genome Sequencing was done by Illu-mina paired end reads. Fertility was assessed with standard matingcompetent strains. Intracellular proliferation rate and tubular mito-chondrial 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 andMATa isolates and infrequent isolation of VGI MATa from environ-mental sources in Southern California. VGIII isolates were obtainedfrom soil debris associated with tree species not previously reported ashosts from sites near residences of infected patients. These isolates arefertile under laboratory conditions, produce abundant spores, and arepart of both locally and more distantly recombining populations. MLSTand whole genome sequence analysis provide compelling evidencethat these environmental isolates are the source of human infections.Isolates displayed wide-ranging virulence in macrophage and animalmodels. When clinical and environmental isolates with indistinguish-able MLST profiles were compared, environmental isolates were lessvirulent. Taken together, our studies reveal an environmental sourceand risk of C. gattii to HIV/AIDS patients with implications for the>1 000 000 cryptococcal infections occurring annually for which thecausative isolate is rarely assigned species status. Thus, the C. gattiiglobal health burden could be more substantial than currently appre-ciated with diagnostic, prognostic, and treatment implications.

P079

Unisexual reproduction reverses Muller’s RatchetK. C. Roach and J. Heitman

Duke University, Durham, NC, USA

Background Cryptococcus neoformans is a pathogenic basidiomyce-tous fungus that engages in outcrossing, inbreeding, and selfingforms of unisexual reproduction as well as canonical sexual repro-duction between opposite mating-types. Long thought to be clonal,>99% of sampled environmental and clinical isolates of C. neoformansare MATa limiting the frequency of opposite mating-type sexual

reproduction. Sexual reproduction allows eukaryotic organisms toexchange genetic information and shuffle their genomes to avoid theirreversible accumulation of deleterious changes that occur in asex-ual populations, known as Muller’s Ratchet.Aims To characterize whether unisexual reproduction, which dis-penses with the requirement for an opposite mating type partner, isable to purge the genome of deleterious mutations.Methods Spores were dissected from unisexual matings of strainscarrying auxotrophic or temperature sensitive mutations. Parentalstrains and F1 progeny were phenotypically characterized forgrowth, competitive growth, and stress responses. Murine and Galle-ria models were infected by parental strains and F1 progeny.Results The unisexual cycle can restore mutant strains of C. neofor-mans to wild-type genotype, phenotype, and growth rate. Further-more, the unisexual cycle allows attenuated strains to purgedeleterious mutations and produce progeny that are returned towild-type virulence. Our results show that unisexual populations ofC. neoformans are able to avoid Muller’s Ratchet and loss of fitnessthrough a unisexual reproduction cycle involving a-a cell fusion,nuclear fusion, and meiosis. Similar types of unisexual reproductionmay operate in other pathogenic and saprobic eukaryotic taxa.Discussion We show that unisexual reproduction between strainsthat carry deleterious mutations generates progeny that havereturned to the wild-type genotype. Unisexual reproduction allows C.neoformans to escape from Muller’s Ratchet and produce phenotypi-cally and genotypically fit offspring. In addition to restoring growthrate, unisex is also able to produce virulent strains from avirulentparents. There are clear implications of these findings for geneticexchange and transmission of drug resistance and these processesmay occur in both the environment and in the host.

P080

The H99 family tree: variation in the common laboratoryreference strains of Cryptococcus neoformans var. grubiicharacterised through whole-genome sequencingK. L. Ormerod,1 E. J. Byrnes III,2 I. A. Wood,1 J. K. Lodge,3

J. Heitman4 and J. A. Fraser1

1University 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 onchoosing a laboratory reference or wild-type strain; this referencestrain provides the background against which phenotypic changes arecontrasted and consequently a better characterised reference aids bothinterpretation and comparison of results. The most widely used refer-ence for Cryptococcus neoformans var. grubii is H99, isolated in 1978from a 30 year old male with Hodgkin’s lymphoma at Duke UniversityMedical Center. Since then, laboratory passage has led to the forma-tion of two distinct lineages with different phenotypic characteristics:the Stud lineage consisting of the Duke University stock H99F (thegenome sequence subculture), Stud (H99S), and the congenic pairsKN99a/a and YL99a/a, and the Wimp lineage consisting of the DukeUniversity 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 isdecreased in the Wimp lineage. Sequencing of strains within both lin-eages and comparison has already uncovered multiple small mutationsseparating them however the picture currently remains incomplete.Aim To fully characterise the genomic variation between laboratoryreference strains used by the Cryptococcus community.Methods The genomes of each of the strains were sequenced toapproximately 30X coverage. Strains H99ED, H99W, H99S weresequenced using Illumina 72 bp paired-end reads. All other strainswere sequenced at BGI using Illumina 90 bp paired-end reads. Reads

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Poster Abstracts

were mapped against the H99 reference genome using BWA andvariation detected using a combination of the Genome Analysis Tool-kit and BreakDancer. Mappings were visualised using IGV and CLCGenomics Workbench (CLC bio). Further strain genotyping was con-ducted using Sanger sequencing performed at BGI and the AustralianGenome Research Facility.Results Analysis of each of the genomes uncovered a small numberof SNPs and indels separating the key strains enabling us to establisha detailed pedigree describing their history. Further analysis was con-ducted by tracing these mutations through a progeny set originatingfrom CM018 and KN99a. A clear association was identified betweentwo linked indels and decreased mating capacity and melanisation.While one of these mutations was predicted to be silent, the otheroccurred within a glutamine rich protein without functional annota-tion. A deletion mutant created in the Stud background revealed arole in both mating and melanisation, with a slight reduction in mel-anisation and a significant reduction in mating proficiency observed.For this reason we dubbed the gene LMP1 for low mating perfor-mance. The lmp1delta mutant was also avirulent in the mouse modelof infection.Discussion/conclusion The process of characterising genes via thecreation of gene deletion mutants relies on understanding of the back-ground against which the mutation is created. The full characterisa-tion of the strains commonly used as references within theCryptococcus community will ensure accurate interpretation of resultsand facilitate collaboration between laboratories. The small number ofSNPs and indels observed means that mutations behind other keyphenotypic variation between these strains can also now be identified.

P081

Evaluation of modified maldi-TOF-based approach toidentification and typing of Cryptococcus neoformansclinical isolatesN. V. Vasilyeva,1,2 A. A. Atsapkina,1 I. A. Riabinin,1,2

T. S. Bogomolova1,2 and G. A. Chilina2

1Mechnikov North-Western State Medical University, Saint-

Petersburg, Russia and 2Kaschkin Research Institute of Medical

Mycology, Saint-Petersburg, Russia

Background MALDI-TOF-mass-spectrometry is a promising methodin the laboratory diagnosis of cryptococcosis. Recent works in thisarea are related not only with the species identification of Cryptococ-cus spp., but also with the recognition of molecular types establishedby MLST. However, with other methods of typing (RFLP; AFLP, ITS-genotype, PCR-fingerprinting) such works are not carried out yet.

S. L. Cohen and B. T. Chait (Anal. Chem., 1996) indicated the pos-sibility of using different solvent systems for the MALDI-matrix in themass-spectrometry of protein with molecular masses >6 kDa, butsince then there is no data on the use of these solvents for microbialidentification in MALDI Biotyper.Aim The aim of this study is to compare results of Cryptococcus neo-formans species identification, obtained by MALDI-TOF-MS with usingdifferent solvents for MALDI-matrix, and to reveal the compliancebetween molecular types of C. neoformans and mathematical classifi-cations of proteome mass-spectra.Methods Seventeen clinical isolates of C. neoformans from RussianCollection of Pathogenic Rungi (RCPF) were previously analyzedusing M13-PCR-fingerprinting and URA5-restriction fragment lengthpolymorphism (RFLP) with HhaI and Sau96 restrictases in MolecularMycology Laboratory of Centre of Infectious Diseases and Microbiol-ogy, Westmead, Australia (W. Meyer et al., Problems in MedicalMycology, 2003, in Russian). Used strains were attributed to threetypes: VNI (11 strains), VNIII (3 stains) and VNIV (3 stains).

For MALDI-TOF-mass-spectrometrical investigation strains weresubcultivated in wort-agar plates during 24 h at 37 °C. Proteinextraction from cell suspensions was performed according to Brukerstandard protocol. MALDI-TOF-mass spectrometry was made with

using Autoflex speed TOF/TOF (Bruker Daltonics). Mass-spectrometrywas performed twice with two different alpha-cyano-4-hydroxycin-namic acid solutions, one on the basis of a standard solvent (TFA/acetonitrile/water), and another on mixture of formic acid/water/iso-propanol (0.2 : 3 : 2) for two points of the target. Thus each strainwas passed eight times during 2 months. Collected spectra were iden-tified with using Biotyper 3.1 database.

Series of 50 top rates of identification obtained with differentmatrix-solvents were compared by Wilcoxon T-test. Best identifiedspectra were converted into MSP and compared by MSP-dendrogramand cluster of principal component analysis (PCA), which were con-structed by different algorithms implemented in MALDI Biotyper OC.Results Using a standard solvent for the MALDI-matrix is signifi-cantly better to obtain the highest results of species identification (T-criterion = 291, critical range = 397, P = 0.01), however for theone strain, C. neoformans var. grubii RCPF Y-1180 the best rate ofidentification (2,358) was derived using formic acid/isopropanolicmixture. In total 14 strains (82%) were identified with rates above2.018 and 3 strains (18%) with rates in range 1.785–1.990.

None of the methods of constructing MSP-dendrograms and PCA-clusters allowed to group the mass-spectra in accordance with themolecular types of C. neoformans (Fig. 1). In PCA-cluster pointsbelong to single strains, but derived from mass-spectra obtained indifferent 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 identificationthan matrix in modified solvent. Wide variation of proteome profilesdetected on PCA-cluster indicates a high phenotypic variability of C.neoformans, that must be considered when identifying and typing ofCryptococcus isolates.

T. Mihara et al. (Med. Mycol., 2013) using isolates of C. neoformansfrom the environment and non-HIV-infected patients showed thatresults of molecular typing obtained by MLST and RFLP/URA5-algo-rithm were not closely correlated. This fact is one of probable reasonscausing the mismatch of molecular types and distribution of prote-ome mass-spectra on MSP-dendrogram.

Further studies are required to clarify the correspondence betweenthe distribution of the proteome mass-spectra and molecular types ofCryptococcus neoformans obtained by various genetic methods.

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

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Poster Abstracts

P082

When could loss be a gain: the two faces of genomeinstability in RNAi deficient Cryptococcus lineagesE. W. L. Chow, S. Clancey, R. B. Billmyre and J. Heitman

Duke university, Durham, NC, USA

Background The RNAi pathway has been shown to play a role insex-induced silencing in Cryptococcus neoformans, and deletion ofRDP1, a key component in the RNAi pathway results in a significantincrease in transposon movement during meiotic reproduction2. Ofthe pathogenic Cryptococcus species, Cryptococcus gattii VGII is missingkey RNAi pathway components. Because this subspecies is responsiblefor the Vancouver Island and Pacific Northwest outbreaks over thepast decade, it is possible that the loss of the RNAi pathway could bebeneficial, allowing for more rapid genetic evolution. Similarly, loss ofRNAi in C. neoformans mutants might also allow higher rates of geno-mic evolution, such as aneuploidy, chromosomal rearrangements andtransposition, during mitotic or meiotic reproduction.

In fungi, aneuploidy has been shown to be an advantageous adap-tative mechanism, conferring antifungal drug resistance and rapidgenetic evolution in response to hostile environments. Earlier studiesin the lab have shown that meiotic reproduction, both unisexual andbisexual, generates aneuploidy in C. neoformans1. Loss of RNAi mayimpact the rate of aneuploidy development during meiotic reproduc-tion, derepress transposon activity, resulting in increased genomeinstability.Aim Given that meiotic reproduction generates aneuploidy and thatthe RNAi pathway is required for centromere function (in Schizosac-charmoyces pombe) and suppresses transposon activity during Crypto-coccus meiotic reproduction, we are investigating if the deletion ofRDP1 leads to increased rates of aneuploidy generation and chromo-somal rearrangements in C. neoformans serotype D.Methods Wild-types, unilateral wild-type x rdp1, and bilateral rdp1 xrdp1 mating crosses were conducted and random spore dissectionperformed to obtain ~100 progeny from each mating cross. Unisex-ual mating assays were also performed for the wild-type and rdp1mutants. Mating assays were performed on MS medium and dis-sected spores were germinated on YPD medium. Mating progenystrains were spotted in 10-fold serial dilutions and grown under thefollowing conditions for 3 days: (1) YPD at 30 °C, (2) YPD plus8 lg ml�1 fluconazole, (3) YPD plus 100 ng ml�1 rapamycin, (4)YPD plus 1 lg ml�1 FK506, and (4) YPD at 37 °C.

Multiplex PCR, as previously described1, was performed as a diag-nostic screen for aneuploid strains. PCR of serotype D STE20a andSTE20alpha MAT genes were used as markers to determine the mat-ing type of the progeny. Whole chromosomal plugs will be made forstrains that display phenotypic variation in comparison to wild-type,and whole chromosomal PFGE separation will be performed to detectany karyotypic changes.

As described previously2, FPR1, the gene encoding FKBP12, canbe used as a transposon trap. A serotype D JEC43-derived rdp1 dele-tion mutant was used in comparison with a wild-type JEC43 isolateto determine the molecular mechanisms resulting in inactivation ofFPR1 with and without the RNAi pathway. Mutants were selectedon FK506 media and the FPR1 locus of mutants cross-resistant torapamycin was sequenced. Similar studies were conducted with thenaturally RNAi-deficient strain VGII C. gattii strain R265.Results Wild-type RNAi proficient isolates developed spontaneousFK506/rapamycin resistance primarily as a result of mutations inFPR1, while in the rdp1 deficient mutants FK506/rapamycin resis-tance was largely the result of transposon insertions into the FPR1locus. In contrast, with R265 we did not observe transposon inser-tion in the locus, and instead the FPR1 locus was inactivated viamutation, suggesting possible long-term adaptative changes to loss ofRNAi and that the consequences of short-term vs. long-term loss ofRNAi may differ. Phenotypic and karyotypic analyses of the progenyfrom the rdp1 bilateral and unilateral bisexual and unisexual matingcrosses 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.

P083

Structure-function analysis of the centromere-kinetochoreand spindle assembly checkpoint machinery inCryptococcus neoformans var grubiiK. Sanyal,1 S. Sridhar,1 V. Yadav,1 J. Heitman2 and

L. Kozubowski3

1JNCASR, Bangalore, India; 2Duke University Medical Center,

Durham, NC, USA and 3Clemson University, Clemson, SC, USA

Background High fidelity chromosome segregation ensures faithfulinheritance of genetic material. Equal chromosome segregationbetween daughter cells requires the linking of the chromosome tothe dynamic pulling and pushing force of the spindle microtubules.Any error in this process, if not detected/corrected, leads to aneu-ploidy - a hallmark of many cancers. Several proteins assemble toform the kinetochore on centromeric chromatin linking the chromo-some to the dynamic plus ends of spindle microtubules. The spindleassembly checkpoint (SAC) detects unattached kinetochores and haltsthe cell cycle at the metaphase-anaphase transition until the errorsare corrected. Intriguingly, the centromere DNA sequence in variouseukaryotes is highly divergent but the basic architecture and func-tion of the kinetochore remains conserved. However, the process ofkinetochore assembly including localization patterns and localizationdependency varies significantly.Results Centromere location, structure and organization: CENP-C is anevolutionarily conserved kinetochore protein found to be associatedwith all functional centromeres studied to date. We identified centro-meres as genome-wide binding sites of CENP-C in Cryptoccous neofor-mans var grubii (H99) by ChIP-seq analysis. A significantly enrichedbinding region (<5 kb) was found to be present on each of the 14chromosomes. These sites are present in 20–65 kb long ORF-freerepetitive regions that are rich in transposons (Tcn1–Tcn6). Kineto-chore architecture and assembly: Towards understanding the kineto-chore structure and the process of its assembly in various stages ofthe cell cycle, we tagged two proteins each from inner (CENP-A,CENP-C), middle (Mtw1, Nuf2) and outer (Dad1, Dad2) layers of thekinetochore with mCherry or GFP. The localization patterns obtainedby time-lapse confocal microscopy reveal that unlike the pathogenicyeast Candida albicans, the kinetochore assembles in a hierarchicalmanner. In contrast to asomycetous yeasts, centromeres were foundto be unclustered until the onset of mitosis in C. neoformans. In addi-tion, while most yeasts undergo closed mitosis, strikingly, we observea partial opening of the nuclear envelope during mitosis in C. neofor-mans suggesting a semi-open mitosis. Kinetochore localization depen-dency: Utilizing a stringent controllable promoter (the GAL7promoter) to drive expression of fluorescently tagged kinetochore pro-teins, we sought to address the essentiality, localization dependencyand regulation of assembly of various proteins that belong to differ-ent layers of the proposed tri-laminar kinetochore network. Ourresults suggest a putative kinetochore architecture wherein depletingthe protein pools of an outer layer (farther from the centromere DNAbut nearer to the microtubule ends) does not appreciably affect thelocalization of underlying layers, while disrupting the inner layers (clo-ser to the centromere DNA) perturbs the entire multi-subunit assemblyof the kinetochore. Spindle assembly checkpoint response: To our surpriseit 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 aspecific kinetochore protein proceeded through the cell cycle withdefective segregation machinery resulting in cellular destruction due tofaulty chromosome segregation. We speculate that this event is a con-sequence of disrupting the platform required for SAC signalling at thekinetochore. We are currently testing this hypothesis.Conclusions This study provides the first insights of the centromere-kinetochore structure-function in a basidiomycete. Long repetitivetransposon-rich centromere organization, ordered kinetochore assem-bly, hierarchical dependency of kinetochore localization, and partialopening of the nuclear envelope exhibited by this organism suggestan evolutionary transition from ascomycetes to metazoan species.

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Poster Abstracts

More work is needed for a definitive understanding of the interplayof kinetochore architecture and the mitotic checkpoint which willprovide us clues as to the origins of aneuploidy conferring azole drugresistance observed in this organism.

P084

Velvet proteins are mating regulators in CryptococcusneoformansL. 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

1Universidade de Bras�ılia, Bras�ılia, Brazil and 2Duke University,

Durham, NC, USA

Background Velvet proteins comprise four highly conserved mem-bers among ascomycetes and basidiomycetes, all of which share thecommon Velvet domain. They regulate different signaling pathwaysin response to environmental stimuli and also coordinate secondarymetabolism and asexual and sexual differentiation in different fungalspecies. Recently, Velvet proteins have been implicated in the viru-lence of some plant pathogens.Aim To identify and elucidate the role of Velvet proteins in C. neofor-mans, the VEL1, VEL2 and VEL3 genes were deleted and the mutantswere analyzed for phenotype.Methods Identification of Velvet genes: The Velvet homologues werefound by using the amino acid sequences of the Velvet proteins ofAspergillus nidulans (VeA, VelB and VelC) as queries for the BLASTPtool of the Broad Institute H99 C. neoformans genome database. Theretrieved best hits, CNAG_02387, CNAG_00564, CNAG_02697 werenamed VEL1, VEL2 and VEL3, respectively. The fourth member ofthis family, VOSA, was also found and pertaining data are presentedelsewhere (Peconick, et al).

Deletion and reconstitution: The knockout strains were generated byDJ- PCR followed by biolistic transformation of the KN99a andKN99a wild types using the selective markers NATR and HYGR,respectively. The reconstituted strains were generated on the KN99abackground by transforming the mutant strains with the deleted genealongside the pJAF1 plasmid containing NEOR. Single insertion intothe appropriate locus was confirmed by Southern blotting or PCR.

Phenotypical analysis: The mutant and reconstituted strains weretested in vitro for common phenotypes associated with virulence ascapsule, melanin, urease and phospholipase production, growth at37 °C and resistance to several cell wall, osmotic and oxidative stres-sors. Strains were mated on SLAD, Filament or MS agar in the pres-ence and absence of light. The mating filaments were analysedmicroscopically.

Virulence assays: The strains were tested for virulence in the in vitroJ774.A16 murine macrophage model of phagocytosis followed byCFU counts.Results and discussion C. neoformans Velvet homologues have theVelvet domain. All Velvet mutants showed no defects related to viru-lence and stress conditions, but presented altered phenotypes on mat-ing assays.

Deletion of VEL1 causes hypermating: the filaments from crossesbetween vel1D mutants occurred two days earlier relative to the wildtype crosses; it also affects the sensing of the light, as mutants pro-duce filaments even in the presence of white light, which is a knownmating repressor. Therefore, Vel1 is a negative regulator of C. neofor-mans mating. In contrast, deletion of VEL2 blocks the production ofhyphae on mating conditions. Furthermore, a defect in cell fusionwas observed on vel2D. The absence of fusion indicates imbalance inthe pheromone pathway in these mutants, which is essential for theinitial cell recognition stages and fusion. The vel2aD vs. KN99alphacross produced shorter filaments, but normal spore chains. Thus,VEL2 is a positive modulator of mating, and a single gene copy issufficient to trigger the process. VEL3 plays a minor role on C. neofor-mans mating as the vel3D mutant crosses presented a slight

acceleration of filament production. VEL3 is possibly the A. nidulansVelC homologue, the least studied member of this family, and its roleis not yet well understood.Conclusion The Velvet regulators in C. neoformans play no role inpathogenicity and virulence, but are directly involved in the sexualstage of the fungus. Although some of the findings are in agreementwith the literature for Ascomycetes, in C. neoformans Velvets proteinsmay have other functions, given the structural differences in theirsequences. Two-hybrid assays are in progress to clarify how thoseproteins interact.

P085

Functional characterization of bZIP-domain containing AP-1like transcription factor, Bap1 in human fungal pathogenCryptococcus neoformansS. Maeng, H. Kim and Y. S. Bahn

Yonsei University, Seoul, South Korea

Background Cellular response and adaptation to oxidative stress areessential for survival and proliferation of a human fungal pathogenCryptococcus neoformans during host infection. Although previousstudies reported that various enzymes are required for antioxidantdefense system against reactive oxygen species, transcription factorsinvolving in this mechanism still remain elusive in C. neoformans. InSaccharomyces cerevisiae, Yap1 (Yeast AP-1 protein) is critical for theoxidative-stress response and adaptation and induces a variety of oxi-dative stress-responsive genes.Aim In this study, we aimed to identify and characterize the functionof Bap1 (bZIP-domain containing AP-1-like transcription factor 1) inoxidative stress defense system and virulence of C. neoformans.Methods BLAST search and protein domain analysis were performedto identify a Yap1 ortholog in C. neoformans. Expression levels ofBAP1 during oxidative stress responsewere measured by Northernblot analysis. The bap1D mutants were constructed by overlap PCRfollowed by biolistic transformation method. Stress sensitivity wasassessed by treating wild-type and mutant cells with diverse stress-inducing agents and antifungal drugs. Production of virulence fac-tors, including capsule, melanin and urease, were measured andmating ability was also monitored.Results The absence of BAP1 increased cellular sensitivity to oxidiz-ing agents, such as H2O2, tBOOH, diamide, and menadione. Support-ing this, the expression of BAP1 was induced by oxidative stress. Thebap1D mutant also showed increased sensitivity to azole drugs andresistance to a polyene drug, amphotericin B. Moreover, the deletionof BAP1 reduced capsule biosynthesis and mating. In contrast, thebap1D mutant exhibited enhanced urease and melanin production.Conclusion/discussion Our data demonstrated that Bap1 playedimportant roles in environmental stress response and modulating vir-ulence factors, proposing that Bap1 could be a potential target fordevelopment of antifungal therapeutic approaches for the treatmentof cryptococcosis.

P086

Distinct and redundant roles of protein tyrosinephosphatases, Ptp1 and Ptp2, in governing thedifferentiation and pathogenicity of CryptococcusneoformansK. T. Lee, B. Byun, J. Jung and Y. S. Bahn

Yonsei University, Seoul, South Korea

Background Mitogen-activated protein kinases (MAPKs) govern aplethora of cellular processes in eukaryotes, such as proliferation, dif-ferentiation, programmed cell death, and stress responses. In

ª 2014 The Authors


Poster Abstracts

response to a variety of chemical and physical stresses, MAPKs sig-naling is activated to sense environmental cues and eventually acti-vate the expression of MAPK-target genes. During this process,negative feedback regulation for controlling the duration and magni-tude of signaling is as important as positive regulation because allsignaling cascades must be properly tuned to avoid deleterious over-activation or constitutive activation and subsequently desensitized torecurrent external cues in a timely manner. However, the functionsof negative regulators or inactivating signaling components in MAPKpathways are less well characterized than those of positive regula-tors. Protein tyrosine phosphatases (PTPs) serve as key negative feed-back regulators of mitogen-activated protein kinase (MAPK)signaling cascades. However, their roles and regulatory mechanismsin human fungal pathogens remain elusive.Aim In this study, we characterized the functions of two PTPs, Ptp1and Ptp2, in Cryptococcus neoformans, which causes fatalmeningoencephalitis.Methods Genes were elucidated by performing 5 and 3 rapid ampli-fication of cDNA ends (RACE). Genes were deleted in C. neoformansvar. grubii strains H99, KN99a or other mutant backgrounds by bio-listic transformation using overlap PCR or split marker/double joint(DJ)-PCR strategies with a set of primers. Various stress response testwas performed by using solid YPD medium containing the indicatedconcentration of stress-inducing agents and antifungal drugs.Results PTP1 and PTP2 were stress-inducible genes, which werecontrolled by the MAPK Hog1 and the transcription factor Atf1.Ptp2 suppressed the hyperphosphorylation of Hog1 and was involvedin mediating vegetative growth, sexual differentiation, stressresponses, antifungal drug resistance, and virulence factor regulationthrough the negative feedback loop of the HOG pathway. In contrast,Ptp1 was not essential for Hog1 regulation, despite its Hog1-depen-dent induction. However, in the absence of Ptp2, Ptp1 served as acomplementary PTP to control some stress responses. In differentia-tion, Ptp1 acted as a negative regulator, but in a Hog1- and Cpk1-independent manner. Additionally, Ptp1 and Ptp2 localized to thecytosol, 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 antifungaltargets.Discussion Ptp1 and Ptp2 played minor and major roles, respec-tively, in the virulence of C. neoformans. While Hog1 was obviously amajor target of Ptp2, other signaling pathways appeared to also beregulated by Ptp2 in C. neoformans. The PTP2 overexpression(oe)strain often showed more severe or opposite phenotypes compared tothe hog1D mutant in response to stresses (e.g. CdSO4, H2O2, and flu-cytosine); however, these potential additional targets are not yetclear. In our study, Ptp1 appeared to be largely dispensable for Hog1signaling and the growth of C. neoformans, despite its Hog1-depen-dent induction. One notable role of Ptp1 is its involvement in sexualdifferentiation. PTP1oe, but not PTP2oe, reduced normal mating effi-ciency and suppressed enhanced mating in the hog1D mutant. How-ever, PTP1oe failed to suppress enhanced MFa1 expression, stronglysuggesting that Ptp1 could modulate mating without direct involve-ment in Cpk1 and pheromone production. In C. neoformans, Ptp1and Ptp2 localized to both the cytosol and the nucleus, but wereenriched in the nucleus. Furthermore, the finding that deletion ofPTP1 or PTP2 reduced transient nuclear accumulation of Hog1under stressed conditions implied that both Ptp1 and Ptp2 may haveHog1-anchoring functions in the nucleus of C. neoformans. However,it remains to be addressed how Ptp1 and Ptp2 modulate the cellularlocalization of Hog1.

P087

Role of the inositol polyphosphate kinases, Ipk1 and Asp1,in the pathogenesis of Cryptococcus neoformansC. Z. J. Li,1 S. Lev,1 A. Saiardi,2 D. Desmarini,1 T. Sorrell3 and

J. T. Djordjevic1

1Westmead 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 andvirulence of Cryptococcus neoformans (Cn)(1). We recently identified aPlc1 signalling pathway in Cn which involves phosphorylation of thePlc1 hydrolysis product, inositol trisphosphate (IP3), by an inositolpolyphosphate kinase (IPK) called Arg1(2). Arg1 (an IP3 kinase), andKcs1, another IPK predicted to function downstream of Arg1 as anIP6 kinase, convert IP3 to inositol polyphosphates (IP)4-5, and IP6 toinositol pyrophosphates (PP-IP)7-8, respectively. IP3-6 have only a sin-gle phosphate at a given position on the inositol ring while PP-IP7-8have 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 inha-lation model of cryptococcosis (unpublished).Aims Using gene deletion analysis, the aims of this study were todetermine the role of two other IPKs, Ipk1 (putative IP5 kinase) andAsp1 (another putative IP6 kinase) in IP homeostasis and their con-tribution 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 weredetermined using inositol radiolabeling and HPLC as described(4).Mutant phenotypes were assessed by spot dilution tests, enzymaticassays, western blots and antifungal susceptibility testing. Virulencewas assessed using mouse inhalation and Galleria mellonella (30 °C)infection models. Mutant phagocytosis by the THP1 cell-line wasassessed by flow cytometry and monocyte activation using a customPCR cytokine array.Results Similar toDarg1 and Dkcs1, Dipk1 showed a similar attenu-ated virulence composite: reduced growth at 37 °C, increased suscep-tibility to antifungals, a cell wall defect, and reduced urease, laccaseand secreted acid phosphatase activity. Dipk1 was hypovirulent inboth animalmodels, with 20% of mice succumbing to infectionwithin 50 days. In the remaining 80%, Dipk1 lung infection persistedfor the duration of the infection period, even though mice main-tained weight and vigour. Dissemination to the brain was alsoobserved. Dipk1 hypovirulence correlated with reduced Dipk1 uptakeand activation ofmacrophages, compared to WT. In contrast, viru-lence phenotypes were not compromised in CnDasp1.Conclusion Ipk1 is required for the phenotypic expression of a simi-lar set of virulence traits to Plc1, Arg1 and Kcs1. This suggests thatthe PP-IP end products of the IPK pathway, which are commonlyabsent in all the IPK mutants, are essential for production of thesevirulence traits in Cn. Absence of an attenuated virulence phenotypein Δasp1 suggests functional redundancy of Asp1 with Kcs1. Ipk1 istherefore another potential antifungal drug target within the Plc1/IPK pathway.References 1. Chayakulkeeree M. et al. Molecular Microbiology2008; 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 ofCryptococcus neoformansH. 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

1Universidade de Bras�ılia, Bras�ılia, Brazil; 2Universidade Cat�olica

de Bras�ılia, Bras�ılia, Brazil; 3Federal University of S~ao Paulo,

Bras�ılia, Brazil and 4Duke University, Durham, NC, USA

Background Cyclic AMP-independent pathways pathways usingGti1/Pac2 transcription factors have been shown to play a major rolein adaptation of ascomycetes to their environment and hosts. Amongother functions, these factors influence secondary metabolism, conidi-ation, mating, dimorphic transition and pathogenesis in fungi rang-ing from plant to human pathogens. However, nothing is knownabout their function in basidiomycetes, despite the fact that their ho-mologues have been annotated in genome projects.Aim To fill a gap in our knowledge of regulatory processes in C. neo-formans, we have deleted the PAC2 homologue and analysed the phe-notype of the mutant.Methods Identification of the target gene: the PAC2 homologue wasfound by using the amino acid sequence of the Ryp1 protein of Hi-stoplasma capsulatum (the only homologue characterised in a humanpathogen) as query for the BLASTP tool of the Broad Institute H99C. neoformans genome database. The retrieved best hit,CNAG_01983.7, was provisionally named RYP1.

Deletion and reconstitution: the ryp1::HYG strains were generated byoverlap PCR followed by biolistic transformation of the H99, KN99aand KN99a wild types. The reconstituted strain ryp1::RYP1 was gen-erated solely on the H99 background by transforming the mutantstrain with the RYP1 gene alongside the pJAF1 plasmid containingthe NEOr resistance marker. In both cases, transformants wereselected in YPD solid medium containing hygromycin or G418,respectively. Single insertion into the appropriate locus was confirmedby Southern blotting or PCR.

Phenotypical analysis: the mutant and reconstituted strains weretested in vitro for common phenotypes associated with virulence,namely capsule induction, melanisation, growth at 37 °C, ureaseand phospholipase secretion and resistance to several cell wall stres-sors. Strains generated on KN99 backgrounds were mated on SLADagar.

Virulence assays: first,the strains were tested for virulence in the invitro J774.A16 murine macrophage model of phagocytosis followedby CFU counts. The in vivo virulence was assessed by survival curvesin the Galleria mellonella alternative model of infection at 37 °C.Results The ryp1::HYG strain has defects in several virulence fac-tors. It fails to induce capsule growth, be it on DMEM/MOPS or onSabouraud/MOPS. In the latter, it also shows a remarkable floccula-tion phenotype (Fig. 1), not quite similar to the one described byWormley et al. (2005). Among cell wall stressors, only Congo Redinhibited growth of the mutant, which had no difficulty growing at37 °C. Melanisation, as well as urease and phospholipase secretion,were normal in the mutant. Of note, the mutant had a significantgrowth deficit on DMEM/MOPS.

Surprisingly, given how important Pac2 homologues are to matingand conidiation in other fungi, in C. neoformans Ryp1 seems to playno role in mating, as mutants generated sexual filaments at the samerate and frequency (on visual inspection) as their parental wild-typestrains.

The loss of Ryp1 impaired both survival of the fungus within mac-rophages in culture and its ability to colonise the invertebrate hostG. mellonella (Fig. 2), in which the mutant was completely avirulent.Discussion The observed loss of virulence in the ryp1D::HYG strainis not surprising given a similar finding in dimorphic and filamentousfungi. In addition to confirming these observations on a murinemodel of infection, we will follow up with an investigation of the roleof kinase signalling on Ryp1 regulation and with expression studies

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

P089

Clinical parameters of cryptococcosis are associated withCryptococcus strain genotypeT. 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

1University 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 sub-Saharan Africa. Previously, we showed a correlation between patientmortality and strain genotype for 140 strains in a cohort of 111patients with AIDS and cryptococcal meningitis. Here, we haveexpanded these studies to include additional patients from multipleregions in sub-Saharan Africa to determine the generalizability of theassociation between fungal genotype and patient mortality. We ana-lyzed a total of 660 clinically isolated strains, including 562 fromKampala, Uganda, 22 from Mbarara, Uganda and 76 from CapeTown, South Africa. We genotyped the strains using multi-locussequence typing (MLST) at eight standard loci. Genotypes were clus-tered using the BURST algorithm. We found 38 genotypes of Crypto-coccus neoformans var. grubii (serotype A), including both VNI andVNII strains. We found six hybrids between C. neoformans var. grubiiand C. neoformans var. neoformans (A/D hybrids) and one C. gattiistrain. Population structures differ between Uganda and South

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

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

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Poster Abstracts

Africa. The population in Uganda is composed of one major clonalgenotype and several minor genotypes, some of which are singletonsin this analysis and some of which are minor clonal clusters. Thepopulation in South Africa has more genotypes, including bothminor clonal clusters and singleton genotypes in this analysis, but nomajor clonal clusters. In addition, clinical parameters of disease, suchas age/sex of the patient, CD4 counts, HIV viral load, cryptococcalcolony-forming units in the cerebral spinal fluid (CSF), cryptococcalcapsule titers in the CSF, opening pressure (via lumbar puncture, ameasure of fluid in the brain), mortality, time to death, and others,were collected for each patient. Cytokine profiles in the CSF weredetermined for a subset of patients upon entry into treatment. Ofthese clinical parameters of disease, we found an association betweenfungal genotype and mortality. Ultimately the genotype and mortal-ity data can be used to select fungal strains for more in-depthsequencing to identify novel virulence factors that differ betweenhigh-mortality and low-mortality fungal genotypes.

P090

Towards better understanding of the molecularmechanism of fluconazole-induced aneuploidy inC. neoformans var grubiiL. Kozubowski,1 S. Altamirano,1 S. Sridhar2 and K. Sanyal2

1Clemson University, Clemson, SC, USA and 2JNCASR, Bangalore,

India

Background Heteroresistance is the intrinsic feature of Cryptococcusneoformans that is characterized by the infrequent emergence of fluco-nazole (FLC) resistant subpopulations within a single colony of thesusceptible strain. Resistant cells are aneuploids and contain increasedcopy number of genes that contribute to their survival in the presenceof FLC. While significant progress has been made to uncover multi-plied genes that are responsible for the FLC resistance, the effects ofFLC on nuclear division and the exact molecular mechanism responsi-ble for the formation of aneuploidy remain poorly characterized. Spe-cifically, the magnitude of the effect of FLC on cell division andchange in ploidy in the overall population has not been characterized.Moreover, it remains unclear whether the resistant aneuploid cellsare derived from initially formed diploids through subsequent chromo-somal loss or are formed de-novo during FLC treatment.Aim The purpose of this study was to examine the effects of FLC onnuclear division of C. neoformans var. grubiiMethods Cells were grown in YPD liquid cultures supplementedwith FLC at concentrations ranging from 8 to 128 microgram/ml.To estimate the effect of FLC on ploidy, samples were collected at var-ious time-points, fixed, stained with propidium iodide, and analyzedusing fluorescence flow cytometry. To assess the effect of FLC on bud-ding, unbudded cells were treated with FLC and budding index wasscored at various time-points. To assess the effects of FLC on mitosis,localization of fluorescently tagged histone H4 and centromeric his-tone variant Cse4 was analyzed after FLC treatment.Results FLC at 32 lg ml�1 caused a significant inhibition of celldivision. However, FLC had no effect on bud initiation and the initialbud growth. FLC treatment for 9 h at 8 lg ml�1 resulted in a signifi-cant fraction of cells with various ploidy levels above 2n. At64 lg ml�1, the effect of FLC was significant already at 6 h andlonger incubation times increased the percentage of cells with higherfluorescence indicative of further increase in DNA content. After 9 hof treatment with 128 lg ml�1 FLC, more than 20% of cells showedsignals corresponding to ploidy above 2n. Interestingly, no clear shiftto the diploid population was observed. At 6 h many cells with irreg-ular shaped chromatin (GFP-H4) and cells with unequal amounts ofGFP-H4 were present, possible indicators of aneuploidy. At these latertime points mCherry-Cse4 intensity was higher in a fraction of thepopulation. Interestingly at time points 3 h and later, cells with twobuds began to emerge, possibly reflecting a post-anaphase and/orcytokinesis defects. To test possible effects of FLC on the spindleassembly checkpoint (SAC), two SAC mutant strains (mad2 and

bub1) were subject to FLC and ploidy was analyzed by flow cytome-try. Strikingly, no difference was observed when compared to the wt.Discussion/conclusions Our study revealed previously unantici-pated significant dose and time-dependent effects of FLC on ploidy.Our results suggest that aneuploidy is formed de-novo as a result ofaberrant chromosomal segregation rather than through chromo-somal loss within FLC induced diploids. An epistatic-like relationshipbetween FLC treatment and the elimination of SAC (deletion ofBUB1 or MAD2) suggests that FLC inhibits SAC while imposing neg-ative effects on nuclear division. We are presently testing this intrigu-ing possibility.

P091

The Cryptococcus neoformans Velvet gene VOSA is apositive regulator of matingL. D. F. Peconick,1 H. C. Paes,1 C. B. Nichols,2 J. A. Alspaugh,2

M. S. Felipe1 and L. F. Fernandes1

1Universidade de Bras�ılia, Brasilia, Brazil and 2Duke University,

Durham, NC, USA

Background Cryptococcus neoformans is an opportunistic basidiomyc-etethat commonly infects HIV-positive and other immunocompro-mised patients. It is the causative agent of cryptococcosis, apotentially fatal and cosmopolitan disease, the world incidence ofwhich is similar to that of tuberculosis. VosAbelongs to the Velvetfamily, which is exclusive of fungi and was first described in Aspergil-lus, is involved in the regulation of sexual and asexual developmentas well as in spore viability. It is also implicatedintrehalosebiosynthe-sis. Proteins of this family are highly conserved among ascomycetesand basidiomycetesand regulate different processes in response toenvironmental stimuli and in secondary metabolism.Aim To elucidate the role of VosA in virulence and morphogenesis ofC. neoformans by gene deletion and mutant phenotypic analyses.Methods The C. neoformans VOSAgene was found using the aminoacid sequences of the A.nidulansVosA protein as query for theBLASTP tool of the Broad Institute H99 C. neoformans genome data-base. The retrieved best hit was CNAG_06580, re-labelledCNAG_07989 in the latest assembly. A VOSA disruption cassettecontaining the Hygromycin B resistance gene (HPH) was constructedby DJ-PCR. The cassette was transformed by biolistics into KN99a/a(seroA) wild type strains, thusgenerating the vosAaDandvosAaDmutants. Likewise, the fragment containing the VOSAnative locuswas transformed into vosAaDto obtain a MATa reconstituted strain(vosAaD::VOSAa). Disruption and reconstitution were confirmed bySouthern Blotting and PCR, respectively. The vosADmutants weretested for various phenotypes: growth at 37 °C, capsule, melanin,urease and phospholipase production and disturbancesin cell wallintegrity.The strains were tested for virulence in the in vitroJ774.A16murine macrophage model of phagocytosis followed by CFUcounts. Ability-to-mate and fusion assays were also performed onMurashige&Skoog (MS) medium to investigate the role, if any, ofVosA 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-termi-nal portion (1–178), a PEST domain(187–200), a bipartite nuclearlocalization signal (NLS) beginning at position 180 and a nuclearexport signal between positions 132–136. The vosAD mutants didnot show any phenotypic changes relative to the parental strainswith regard to any virulence factortested, including thein vitro mac-rophage assay; they also showed no cell wall defect. However, in themating assays, vosAD mutants showed reduced filamentation in uni-lateral crosses (WT vs. vosAD) and dramatic hypha loss in bilateralcrosses (vosAaD vs. vosAaD). Furthermore, a defect in cell fusion wasobserved in the latter. Among the few filaments observed, the apicalbasidia did not show any Basidiospore chains. The absence of fusionin bilateral crosses indicates an altered pheromone pathway in thesemutants. Pheromone sensoringtriggers the mating process that

ª 2014 The Authors


Poster Abstracts

culminates in cell fusion and sexual spore production. A fluorescencemicroscopy assay was performed to evaluate the defects related tolossof VosA further:the inspection of unilateral crosses revealeddefectiveclamp connections that do not seem to attach to adjacent hyphalcells. Clamp connections are formed along the hyphae to ensure thetransfer of nuclei between cells during septation. Finally, no ramifica-tion was observed along the hyphae.Conclusion This study connectsVosA, a member of Velvet family,with the regulation of sexual reproduction in C. neoformansandcon-tributes to a better understanding of its role in the biology of thispathogen. From the observations it can be concluded that VosA is apositive modulator of mating controlling the formation of sexualstructures and sporulation, and a single gene copy is sufficient totrigger mating, even if it is defective.

P092

Mapping functional transcription factor networks toidentify novel capsule regulatorsR. Gish,1 J. Maier,2 C. Haynes,2 M. Williams,1 Z. Wang,1

M. R. Brent2 and T. L. Doering1

1Washington 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 keyvirulence factors and changes dramatically in response to environ-mental conditions. A complete and integrated model of capsule regu-lation is fundamental for a comprehensive understanding of thegenetic networks involved in capsule synthesis.

We recently reported NetProphet, an algorithm we developed forinferring transcriptional regulatory networks in S. cerevisiae (Hayneset al, Genome Research, 2013). We have now applied NetProphet tothe regulation of capsule size in C. neoformans.Aim Our goals were to develop and apply new computational meth-ods to reconstruct the capsule regulatory network, to use the result-ing network model to identify novel transcription factors involved incapsule regulation, and to determine the role(s) of these novel tran-scription factors in cryptococcal biology and virulence.Methods We used RNA-Seq to profile gene expression in a collectionof transcription factor deletion strains and analyzed the data withNetProphet. We used the resulting network model to identify noveltranscription factors likely to act in capsule regulation, deleted thecorresponding genes, and assayed the resulting mutant strains forcapsule size. Mutants were also examined for additional virulencefactors and behavior in a mouse model of infection.Results We used expression data from 30 cryptococcal mutants (3biological replicates and wild type controls for each) to model thecapsule regulatory network. We identified 12 transcription factors aslikely to act in capsule regulation and successfully deleted 10 of thecorresponding genes; of this group 7 had phenotypes of altered cap-sule thickness and most of those were attenuated in virulence in ashort-term model of fungal growth in mice. One hypercapsular strainwas selected for further study and found to have defects in melaninproduction and other traits relevant to infection; this mutant alsohad severely attenuated virulence in mice. For several transcriptionfactors, direct targets were identified by ChIP-Seq. These studies con-firmed NetProphet’s accuracy in mapping direct, functional interac-tions between transcription factors and their promoters.Discussion/conclusion We have modeled the cryptococcal capsuleregulatory network and identified several novel transcription factorsthat act in regulation of capsule synthesis. These will be the subjectsof further study. More generally, our experimental strategy combinedexpression profiling and computational methods to (i) generate acomprehensive network of upstream and downstream interactions ofgenes involved in regulating a process of interest and (ii) predictmutations that are likely to show a given phenotype of interest. This

method is applicable to other pathways in Cryptococcus and to impor-tant questions in other microbial systems.

P093

DNA repair, ubiquitination and virulence in CryptococcusneoformansS. 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 avoidingthe host defense systems to enable them to cause disease. Protec-tion of its DNA from damage in response to environmental stresses,and especially during the course of infection is one of the importantstrategies adopted. An important DNA repair gene, RAD23 hadbeen implicated in the virulence of opportunistic pathogen Crypto-coccus neoformans and knockout for this gene is shown to be hypo-virulent (Liu et al, 2008). RAD23 has dual function: It is involvedin DNA repair as well as protein sorting ubiquitination pathway(acts as ubiquitin receptor protein). We want to uncouple the func-tion of the two roles of this gene in the virulence, to test whichrole is more important for the virulence one or the other or both.We want to figure out if Rad23 is required for nuclear DNA repairor mitochondrial DNA repair? Another interesting aspect aboutRAD23 is its proposed role in transcriptional regulation in yeast.Apparently RAD23 is involved in regulation of almost two-third ofthe UV regulated genes and almost one third of all the yeast genesare mis-regulated in the RAD23 knockout (Wade et al, 2009 andWade and Auble, 2010). Rad23 has four domains, UBL, UBA1,XPC-B, UBA2. The UBL, UBA1 and UBA2 have major implicationin protein sorting and ubiquitination pathway and XPC-B have rolein DNA repair pathway (Dantuma et al, 2009). We have performeda full-length deletion of RAD23 and are also performing individualdomain knockouts to check their role in different DNA damagestress and in virulence of the fungus. We are also checking thesedomain deletion strains for their function in ubiquitination path-way. Gene knockouts for Rad23 are sensitive to UV stress and haveincreased resistance to endoplasmic reticulum stress inducing antibi-otic Tunicamycin. Rad23 -GFP fusion protein localizes to thenucleus and cytoplasm and not to mitochondria, implicating its rolein nuclear DNA damage repair. We are in process of making differ-ent domain deletion strains of Rad23 and checking their survivalunder different stress conditions and also their localization pattern.Wax moth infection studies indicate RAD23 knockout to be hypo-virulent in consistence with previous studies with mice model andhas to be checked for different strains. Completion of these studieswill shed more light on the virulence mechanisms of C. neoformans.We will be able to uncouple the role of two different key pathwaysin 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 humanfungal pathogen Cryptococcus neoformans. Cell 2008; 135: 174–188.

Dantuma NP, Heinen C, Hoogstraten D. The ubiquitin receptorRad23: at the crossroads of nucleotide excision repair and proteaso-mal degradation. DNA Repair (Amst) 2009; 8: 449–460.

Wade SL, Auble DT. The Rad23 ubiquitin receptor, the proteasomeand functional specificity in transcriptional control. Transcription2010; 1: 22–26.

Wade SL, Poorey K, Bekiranov S, Auble DT. The Snf1 kinase andproteasome-associated Rad23 regulate UV-responsive gene expres-sion. EMBO J 2009; 28: 2919–31.

ª 2014 The Authors


Poster Abstracts

P094

DNA repair and ubiquitination in virulence of CryptococcusneoformansS. Verma, P. S. Shakya and A. Idnurm

School of Biological Sciences, University of Missouri- Kansas City,

Kansas City, MO, USA

Background Pathogenic organisms have evolved a number of waysof avoiding the host defense systems to enable them to cause disease.Protection of pathogen DNA from damage in response to environ-mental stresses, and especially during the course of infection, is oneof the important strategies adopted. The DNA repair gene RAD23 isimplicated in the virulence of Cryptococcus neoformans as knockoutfor this gene is shown to be hypo-virulent (Liu et al, 2008). RAD23has dual functions: it is involved in DNA repair and the protein sort-ing ubiquitination pathway (acting as ubiquitin receptor protein).Another interesting aspect about RAD23 is its proposed role in tran-scriptional regulation in yeast. RAD23 is involved in regulation ofalmost two-third of the UV regulated genes and almost one third ofall the Saccharomyces cerevisiae genes are mis-regulated in the RAD23knockout strain (Wade et al, 2009 and Wade and Auble, 2010).Aim We want to uncouple the function of the two roles of this genein the virulence, to test which role is more important for the viru-lence, one or the other, or both. We want to figure out if Rad23 isrequired for nuclear DNA repair or mitochondrial DNA repair.Methods Rad23 has four domains, UBL, UBA1, XPC-B, UBA2. TheUBL, UBA1 and UBA2 domains are involved in protein sorting andthe ubiquitination pathway, and XPC-B has a role in DNA repairDantuma et al, 2009). We have generated strains with a completedeletion of RAD23, and also complemented this mutANT by express-ing RAD23 under its native promoter. We are performing individualdomain knockouts to test their role in different DNA damage stressand in virulence. We are also checking these domain deletion strainsfor their function in ubiquitination pathway.Results and discussion Gene knockouts for RAD23 are sensitive toUV stress and have increased resistance to endoplasmic reticulumstress inducing antibiotic Tunicamycin. Rad23-GFP fusion proteinlocalizes to the nucleus and cytoplasm and not to mitochondria,implicating its role in nuclear DNA damage repair. We are in processof making different domain deletion strains of Rad23 and checkingtheir survival under different stress conditions and also their localiza-tion pattern. Wax moth infection studies indicate RAD23 knockoutto be hypovirulent, consistent with previous studies in a mice model.Completion of these studies will shed light on the virulence mecha-nisms of C. neoformans. We will be able to uncouple the role of twodifferent 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 humanfungal pathogen Cryptococcus neoformans. Cell 2008; 135: 174–88.

Dantuma NP, Heinen C, Hoogstraten D. The ubiquitin receptorRad23: at the crossroads of nucleotide excision repair and proteaso-mal degradation. DNA Repair 2009; 8: 449–60.

Wade SL, Auble DT. The Rad23 ubiquitin receptor, the proteasomeand functional specificity in transcriptional control. Transcription2010; 1: 22–26.

Wade SL, Poorey K, Bekiranov S, Auble DT. The Snf1 kinase andproteasome-associated Rad23 regulate UV-responsive gene expres-sion. EMBO J 2009; 28: 2919–31.

P095

Mitochondrial morphology and virulence in CryptococcusgattiiV. P. Shakya and A. Idnurm

University of Missouri Kansas City, Kansas City, MO, USA

Background Cryptococcus gattii has the potential to infect healthyindividuals along with the immunocompromised hosts [1]. The Van-couver Island outbreak starting in 1999 was caused by C. gattii, andinfection is prevalent mainly in Washington, Oregon and BritishColumbia. In C. gattii pathobiology, the mitochondria likely play animportant role. Mitochondria are very dynamic organelles of eukary-otic cells that undergo fission and fusion to maintain function duringnormal and stress conditions. Fission is required for the production ofnew mitochondria while fusion of mitochondria helps the cell to copewith stresses. In Saccharomyces cerevisiae the fission and fusion ofmitochondria is mediated through DNM1 and FZO1, respectively,belonging to the GTPase family of proteins and are conserved in fliesand mammals [2]. The virulence of C. gattii is attributed to theinduction of tubular mitochondrial morphology upon engulfment inhost macrophages [3]. The individual punctate mitochondria mayfuse to form tubular mitochondria as a result of stress. The tubularmorphology correlated with higher intracellular proliferation in mac-rophages cell line.Aim Our hypothesis is if we can impede the mitochondrial dynamicsof fission and fusion, it should affect the formation of tubular mito-chondria and in turn virulence of C. gattii.Methods To investigate the role of tubular morphology of mitochon-dria as a virulence trait of C. gattii, we have selected for study twogenes that regulate fission and fusion of mitochondria in eukaryotes- DNM1 and FZO1. To test our hypothesis we are examining thephenotypes of the two gene knockout strains. We are also investigat-ing the role of the two genes in mitochondrial genome recombinationto address the issue of generation of recombinant mitochondria andpossible transmission of virulence traits. The generation of new hy-pervirulent stains through recombination events may also lead toincreased incidences of infection.Results and discussion The knockout of DNM1 in C. gattii hasabnormal fused mitochondrial morphology. Moreover DNM1 knock-out 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 strainbackgrounds was performed, and analysis of progeny is in progressto see its role in recombination if any. The role of FZO1 is yet to beverified and studies are in progress. We plan to perform the prolifera-tion assays within macrophage cell lines and animal studies of theknockout strains to investigate the effect of the two genes on the sur-vival and virulence of C. gattii. Our studies will help to understandthe role of mitochondrial morphology in virulence and genomerecombination of C gattii.References 1. Kronstad JW, Attarian R, Cadieux B, Choi J, D’SouzaCA, et al. (2011) Expanding fungal pathogenesis: Cryptococcus breaksout of the opportunistic box. Nat Rev Microbiol 9: 193–203.

2. Hoppins S, Lackner L, Nunnari J (2007) The machines thatdivide 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 byenhanced intracellular parasitism driven by mitochondrial regulation.Proc Natl Acad Sci USA 106: 12980–85.

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Poster Abstracts

P096

Nucleotide sugar transporters: key players in capsuleglycan biosynthesisL. X. Li, Z. Wang and T. L. Doering

Washington University St. Louis School of Medicine, St Louis,

MO, USA

Background Cryptococcus neoformans, an opportunistic fungal patho-gen, produces a capsule that induces immunological unresponsive-ness, interfering with normal phagocytosis, cytokine release, andleukocyte migration. This definitive virulence factor is mainlycomposed of the polysaccharides glucuronoxylomannan (GXM) andglucuronoxylomannogalactan (GXMGal) with trace amounts ofmannoproteins. By molar ratio, mannose and glucuronic acid consti-tute the major sugar subunits, while xylose and galactose are incor-porated into the structure to a lesser degree. Incorporation of thesemonosaccharides into the capsule requires production of activateddonor molecules (e.g. GDP-mannose) in the cytosol followed by trans-port of these highly charged compounds into the Golgi where mostglycan biosynthesis occurs. Specific enzymes then catalyze the trans-fer of the monosaccharide from the nucleotide sugar donor to thegrowing polysaccharide chain.

Although the biochemical pathways that are required to synthesizethe activated nucleotide sugar precursors have been elucidated, theidentity and regulation of the complete set of nucleotide sugar trans-porters (NSTs) responsible for import of these precursors into thesecretory pathway remain elusive. GDP-mannose transport has beenattributed to Gmt1 and Gmt2 (Cottrell et al, 2007), and UDP-galac-tose appears to be transported by Ugt1p (Moyrand et al, 2007). How-ever, the transporters for the donors of other surface componentssuch as glucuronic acid, xylose, or potentially sialic acid have notbeen identified. One possibility is that known NSTs translocate multi-ple nucleotide sugar substrates. Alternatively, these donors may betransported by additional putative NSTs encoded in the cryptococcalgenome.Aim Our long-term goal is to better understand the glycan syntheticsteps by which C. neoformans forms a polysaccharide capsule. Here,our aim is to determine the biological role(s) of three nucleotidesugar 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, andprotein as well as lipid glycosylation defects. Virulence in mice wasassayed by monitoring mouse survival following intranasal inocula-tion. In vitro transport assays were used to determine the specifictransporter substrate(s).Results Gmt1 and Gmt2 demonstrated similar transport kinetics andsubstrate specificities for GDP-mannose in vitro and showed partialfunctional redundancy in vivo. Single gmt deletion mutants exhibiteddefects in cell growth, capsule phenotype, colony morphology, andprotein glycosylation that were compounded in mutants lacking bothgenes; the double mutant was also completely avirulent in mice.Despite their apparently similar functions, however, Gmt1 and Gmt2had distinct expression and localization patterns. Furthermore, swap-ping the coding sequences of the corresponding genes was insuffi-cient to restore wild-type phenotypes. Mutants lacking NSTxdemonstrated capsule and growth defects that correlated with anattenuation of virulence in mice, and heterologous complementationstudies suggested that the substrate of this transporter is an activatedacidic monosaccharide.Conclusion Gmt1 and Gmt2 are GDP-mannose transporters thatplay distinct roles in cryptococcal biology with only partial overlap infunction; this difference may reflect a non-identical requirement forthese transporters in various stress responses. NSTx appears to trans-port the donor of an acidic monosaccharide although further study isrequired to define the exact substrate(s).

P097

Genotypic diversity within clinical and environmentalCryptococcus neoformans var grubii population in IndiaA. Prakash,1 F. Hagen,2 S. Kathuria,1 J. F. Meis3 and

A. Chowdhary1

1Vallabhbhai Patel Chest Institute, Delhi, India; 2Radboud

University Medical Centre, Nijmegen, The Netherlands and3Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands

Background Cryptococcus neoformans is an encapsulated, ubiquitousenvironmental yeast that causes cryptococcosis, a potentially seriousdisease that affects immunocompromised individuals, especiallypatients with AIDS. The studies determining the genotypic diversityin C. neoformans population is important to gain insightful knowledgeof this important pathogen. Microsatellite typing has become a popu-lar molecular tool to study genotypic diversity in fungal pathogensdue to its high discriminatory power and reproducibility.Aim To investigate genetic relatedness and antifungal susceptibilitiesof a large collection of C. neoformans isolates from clinical and envi-ronmental sources in India collected during 2001–2010.Materials and methods Four hundred and fifty three (224 environ-mental and 229 clinical) Indian Cryptococcus neoformans isolates weresubjected to Amplified Fragment Length Polymorphism (AFLP) andgenotyped by microsatellite typing based on nine markers specific forC. neoformans variety grubii (serotype A) or variety neoformans (sero-type D). In vitro antifungal susceptibility was determined for standardantifungals using CLSI M27-A3 guidelines.Results All 453 isolates were AFLP1/VNI genotype, representing C.neoformans variety grubii serotype A. Microsatellite typing revealedthat 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 oth-ers (8 MCs, n = 20; 4.4%). Forty (9.2%) isolates could not be linkedto 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, and0.06 mg l�1, respectively. Geometric mean MICs revealed that iso-lates 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. neoformansvar. grubii reported so far from a single country. Environmental iso-lates were genetically more diverse than clinical isolates comprising alarger number of MC types than clinical MCs. Two microsatellitetypes of C. neoformans var. grubii dominate in India and were uni-formly distributed over clinical and environmental isolates. Fluconaz-ole and flucytosine had high MICs (>8 mg l�1) in 2% and 7% of theisolates respectively, whereas the new azole isavuconazole exhibitedthe lowest GM MIC of 0.06 mg l�1.

P098

Microsatellite typing show mixed infections with multiplegenotypes of Cryptococcus neoformans var. grubii inIndian HIV positive patients with cryptococcosisA. Chowdhary,1 F. Hagen,2 A. Prakash1 and J. F. Meis2

1Vallabhbhai Patel Chest Institute, Delhi, India and 2Canisius-

Wilhelmina Hospital, Nijmegen, The Netherlands

Background Incidence of cryptococcal infection is high in develop-ing countries such as India. Cryptococcal meningitis is the most com-mon, life-threatening, opportunistic, fungal disease in HIV infectedindividuals. So far, only few reports of cryptococcosis due to mixedspecies or serotypes of Cryptococcus neoformans have been reported.Aim To characterize the genotypes of C. neoformans var. grubii fromrecurrent and non-recurrent cryptococcocal infections in HIV positivepatients.

ª 2014 The Authors


Poster Abstracts

Materials and methods A total of 42 Cryptococcus neoformans iso-lates originating from 25 HIV positive patients and one from HIVnegative were studied from whom primary unpurified original cul-tures were stored. The isolates were recovered from CSF (n = 30,71%), sputum (n = 9, 21%), blood (n = 2, 5%) and one from BAL. Ofthese, 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 AMBor FLU therapy. Five single colonies from primary culture plates werescreened and were subjected to Amplified Fragment Length Polymor-phism (AFLP) and were genotyped using multilocus microsatellitetyping (MLMT) based on nine markers specific for C. neoformans vari-ety grubii (serotype A) or variety neoformans (serotype D). The anti-fungal susceptibility was determined for new azoles along withstandard 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 geno-types of C. neoformans var. grubii in 9 (36%) of the 25 patients. Thisincluded7 (78%)patientswith twoand2 (22%)with threedifferentgeno-types. Different strains were found at different anatomical sites (blood,sputum and BAL) in two patients. Mycological failures after 2 weeks ofantifungal treatment were observed in 4 mixed-infection patients. All ofthe isolateswere susceptible toall the tested antifungal drugs.Conclusions The prevalence of multiple strains of C. neoformans var.grubii in the environment may likely result in acquisition of multiplegenotypes by human hosts leading to infection with multiple geno-types in cryptococcosis patients. Our data showed the presence ofmultiple infecting genotypes of C. neoformans confirming previousobservations in other centers.

P099

Genetic diversity and susceptibility study of singlecryptococcal isolates from HIV-infected patients inIndonesiaR. 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

1University 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 infectionin AIDS. Cryptococcus was divided into two species. i.e. Cryptococcusneoformans and C. gattii. C. neoformans is composed of C. n var. grubii(serotype A), C. n. Var. neoformans (serotype D) and the hybrid sero-type AD. C. gattii consists of serotype B and C. Both species differ intheir virulence, geographical distribution, pathogenicity and clinicalappearance. Serotype A is known as the main cause of cryptococcosisin HIV-infected patients and is distributed world wide. The matingtype is known as a virulence factor and plays a role in the epidemiol-ogy and evolution of the organisms.Aim of the study To identify genetic variation, serotype, matingtype and susceptibility patterns of Cryptococcus isolates from HIV-infected patients in Jakarta.Method We investigated 148spinal fluids derived from 122 HIV-infected patients in Jakarta (144 isolates), Bandung (one isolate), Bali(one isolate), Pontianak (one isolate) and Papua (one isolate). Matingand serotype was determined by a PCR method using four specificprimers. The genotype characterization was conducted using primers

derived from the URA5 gene and restriction enzymes HhA1 andSau961. Reference strains were CBS10512 (serotype A, VNI,AFLP1), CBS8710 (serotype A, VNII, AFLP1A), WM 628 (serotypeAD, VNIII, AFLP3), CBS10084 (serotype D, VNIV, AFLP2), WM 179(serotype B, VGI, AFLP4), WM 178 (serotype B, VGII, AFLP6), WM161 (serotype B, VGIII, AFLP5) and WM 779 (serotype C, VGIV,AFLP7). The susceptibilty study was conducted using the disc diffu-sion method for amphotericine B, fluconazole, voriconazole and ke-tokonazole. 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 of122 HIV-infected patients with cryptococcal meningitis. The geno-type identification shows the following results: sero-mating typingrevealed 21 Aa isolates, 115 Aa isolates, and 12 ADa isolates. Geno-type characterization using URA5 - restriction fragment analysisshowed 117 VNI, 8 VN2 and 12 VNIII isolates. 133 from 147 iso-lates were susceptible to amphotericine B and 15 isolates were SDD,133 were susceptible to fluconazole, six isolates were SDD, and sevenisolates were resistant. For Voriconazole 147 isolates were suscepti-ble, while one strain was SDD. For ketoconazole we studied only 120isolates and all were susceptible.Discussion/conclusion Based on the molecular study the predomi-nant cryptococcal species in HIV-patients in Jakarta is Cryptococcusneoformans with serotype A and mating type a. The susceptibiltystudy showed that most Indonesian Cryptococcus strains are suscep-tible to the antifungals available in Indonesia.

P100

AIM-HII: a new method to rapidly identify agrobacterium-mediated insertion sites in C. neoformansS. K. Esher, J. A. Granek and J. A. Alspaugh

Duke University, Durham, NC, USA

Background Agrobacterium-mediated transformation (AMT) is a com-monly used tool to generate mutations in a variety of plants and fungi,including Cryptococcus neoformans. A. tumefaciens is a soil bacteriumwhich has the ability to deliver a portion of its own DNA to plants andfungi. The transfer or T-DNA is carried on a plasmid and flanked byborder repeats. The border repeats define the insert DNA and the por-tion between them can be manipulated, commonly to include a select-able marker. The T-DNA integrates into the host genome, generallyyielding a single, random insertion. Insertional mutagenesis librariescan therefore be generated and screened for phenotypes of interest, fol-lowed by identification of responsible mutation events. Traditionally,the locations of T-DNA insertion in C. neoformans have been identifiedby 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 sequencingand analysis pipeline to facilitate the identification of insertions gen-erated by AMT. Agrobacterium-mediated Insertional MutagenesisHigh-throughput Insert Identification (AIM-HII) combines batch sam-pling, whole genome sequencing, and bioinformatics analysis tools torapidly identify the locations of insertion. This method allows for theidentification of of 20–30 pooled mutants in the time it would taketo identify 1–2 mutants using traditional methods. In brief, a libraryof C. neoformans AMT mutants was screened for phenotypes of inter-est. The genomic DNA of 28 mutants was harvested, pooled, andsequenced. Whole genome sequencing data was generated, and readsincluding the T-DNA insert sequence were extracted from the rawdata files. Next, the T-DNA sequence was clipped off of these reads,yielding only C. neoformans genomic DNA sequence. Finally, thisresulting genomic sequence was aligned to the C. neoformans gen-ome, and locations of insertion were identified.Results With this approach in mind, we screened a library of1500 ATM mutants for defects in growth on high pH, high salt,and resistance to the detergent SDS. 28 mutants of interest were

ª 2014 The Authors


Poster Abstracts

selected and the AIM-HII pipeline was used to identify sites of inser-tion. Of these, we have confirmed insertions in several previouslyidentified genes including ACA1 and SKN7, as well as a number ofunannotated genes. Through our analysis we have also identifiedpreviously undescribed ATM insertion-induced events. These includepossible chromosomal rearrangements, large deletions, and exten-sions and truncations of the integrated T-DNA. Many of theseevents would have been overlooked using the traditional PCR-basedmethods.Discussion/conclusion Our work introduces and utilizes a new toolthat will greatly facilitate insertional mutagenesis screens in thefuture. Through this work we have identified a number of knownand novel genes involved in C. neoformans virulence attributes. Workto further characterize the function of these genes is ongoing.

P101

D-amino acid oxidase genes in Cryptococcus gattii andCryptococcus neoformansY. C. Chang,1 A. Khanal Lamichhane,1 J. A. Bradley,2

L. H. Rodgers,3 P. Ngamskulrungroj4 and K. J. Kwon-Chung1

1NIH, 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 sisterspecies, Cryptococcus neoformans and C. gattii, which differ consider-ably in their utilization of carbon and nitrogen sources. One ofthe diagnostic characteristics that can distinguish between the twospecies is the ability to utilize D-proline and a few other D-aminoacids by C. gattii but not by C. neoformans. The enzymatic mecha-nism of D-proline metabolism, however, has not been studied inthese species.Aim In order to understand the enzymatic mechanism of D-aminoacid utilization by C. gattii but not by C. neoformans, we characterizeda gene required for D-proline utilization and studied the D-amino oxi-dase gene (DAO) in both species.Methods We created an insertional library using Agrobacterium med-iated transformation and analyze the clones that failed to utilize D-proline. Methods used include gene cloning, sequencing, targetedgene disruptions, gene expression and animal infection model. Utili-zation of different D-amino acids was tested using yeast nitrogen basewithout ammonia supplemented with various D-amino acids as thesole nitrogen source.Results Three homologs of the D-amino oxidase gene (DAO) wereidentified in the genome of both the C. gattii strain R265 and the C.neoformans strain H99. Expression profiles of the DAO gene in eachstrain were examined using different D- amino acids as the sole nitro-gen source. The contribution of each DAO gene in D-amino acid utili-zation was determined by deleting the DAO gene in both species.Substrate specificity of the Dao proteins was determined by express-ing the DAO genes in E. coli. We found the DAO2 gene to be impor-tant for growth of R265 in different D-amino acids as the solenitrogen source. Although deletion of each DAO gene individuallyhad no affect on fungal virulence, triple deletions of all DAO genessignificantly affected virulence in a murine model of R265 but not ofH99. This suggested that D-amino acid utilization is important forthe pathobiology of C. gattii. Interestingly, overexpression of RDAO2,a DAO gene from C. gattii in H99 supported the growth of H99 inD-amino acids. We are investigating further the molecular basis inD-amino acid utilization in C. neoformans and C. gattii.Conclusion Our study shows that D-amino oxidase genes areresponsible for D-amino acids utilization in cryptococci. Interestingly,C. neoformans and C. gattii both possess homologs of three D-aminooxidase genes which affect virulence in C. gattii but not in C. neofor-mans. A detailed investigation into the molecular differences of DAOgene expression in the two species will shed light on their biochemi-cal differences that impact their pathogenicity.

P102

Please see GW2.

P104

Characterization of granulomatous response in pulmonaryand cutaneous cryptococcosis in renal transplantedpatientsM. V. Sold�a, G. Ricci, S. Nishikaku, V. Ponzio, A. L. Colombo and

M. Franco

Federal University of S~ao Paulo, S~ao Paulo, Brazil

Cryptococcosis is the second major systemic mycosis associated tokidney transplants, which is the most likely group to develop crypto-coccosis among solid organ transplant recipients. There are few stud-ies reporting the incidence of cryptococcosis in this risk group andcorrelating the different granuloma patterns during infection andpathogen/host interaction in those patients. Thus, the objective ofthis work is to characterize the granuloma pattern in renal trans-plant recipients with cryptococcosis. The casuistic included a total of14 paraffin blocks from 12 patients from the Hospital do Rim e Hi-pertens~ao of S~ao Paulo, who had cryptococcal infection post renaltransplantation, during the period of 2005–2012. The isolates of C.neoformans/C. gattii complex collected from the lesions of the patientswere identified by biochemical and molecular methods. Paraffin HEstain sections from eight skin biopsies and six of lung of thesepatients were used for the histopathological analysis for characteriza-tion of tissue response, and Mayer’s Mucicarmin for visualization ofthe fungal capsule. From a total of 14 cases analyzed, we observedthe pattern of compact epithelioid granulomas in 4 cases and loosemacrophagical granulomas in 10 cases (see Table 1). As observed inTable 2, all cases showed the presence of inflammatory lympho-mononuclear infiltrate cells rich in xanthomatous histiocytes, and ofmultinucleated giant cells. Multifocal lesions were observed in allpatients accompanied by fibrosis and necrosis. Regarding the fungalmorphology, there was predominance of multiple budding yeast cells.Overall, patients who developed compact granulomas had higher sur-vival than those patients with loose granulomas. In conclusion, themorphological findings of the present study show a diversity of gran-uloma patterns in kidney transplant patients with cryptococcosis. Itmight occur possibly due to factors such as stage of infection, level ofimmunosuppression and pathogen virulence. It is worth to empha-size, this is the first study describing the granulomatous response inrenal 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 skin compact low death*

2 lung loose low death

3 skin loose high death

4** skin 1 loose high discharged

4 skin 2 loose high discharged

4 skin 3 compact low discharged

5 lung loose high death

6 skin loose low discharged

7 lung loose high discharged

8 lung loose high death***

9 skin compact low discharged


11 skin compact low discharged


*Death due to tuberculosis.

**Same patient with biopsies collected in different periods.

***Death due to bacterial sepsis.

ª 2014 The Authors


Poster Abstracts

P105

Growth of Cryptococcus neoformans inside phagosomes: amatter of resistance or a well orchestrated escape?H. F. P. Tavares,1 P. H. M. B€urgel,1 C. C. Coelho,2 A. Casadevall2

and A. L. Bocca1

1UnB, Brasilia, Brazil and 2Albert Einstein College of Medicine,

New York, NY, USA

Phagolysosomes are vesicles located in the intracellular portion ofphagocytes, having the function of uptaking microbes and killingthem. The interior of phagolysosomes presents a harsh microenvi-ronment for the microbes internalized. This is obtained by a num-ber of oxidative, hydrolytic and acidifying enzymes present inthese vesicles. Phagolysosomes plays a vital role as part of thematuration of the phagocytes, which in turn present high impor-tance in suppressing the initial infection and recruiting otherleucocytes.

On the counterpart, microbes developed innumerous strategies forescaping and/or impairing the phagocytes and their defenses, includ-ing the ability of survival inside the phagolysosome. In this matter,Cryptococcus neoformans hasvarious virulence factors that allow itssurvival and its escape from the interior of macrophages. Amongthese factors, there are the enzymes of the phospholipase group(PLB) which destabilize membranes by hydrolyzing ester linkages. Itis well known and described that the omission of the PLB in C. neo-formans promotes a weaker infection with a diminished intracellulargrowth inside macrophages.

Another strategy of the C. neoformans is the nonlytic extrusion,which consists in the exocytosis of the fungal cell from the macro-phage with both cells surviving. This interaction with the host isunique and very peculiar, causing minimal host cell damage andtherefore not triggering a pro-inflammatory response. It is knownthat the extrusion involves the damage of phagosome membrane,therefore it has been shown that the omission of PLB causes less exo-cytosis of the fungal cell.

C. neoformans is described as a facultative intracellular pathogenthat doesn´t interfere with the formation and maturation of phago-lysosomes, being able to survive and multiply inside the acidifiedvesicle. However, reports indicate that the constant damage causedby the pathogen in the vesicle membrane impairs the acidificationof the media. It has also been demonstrated that the experimentalacidification of phagosomes altered the extrusion of the fungal cell.

Therefore, it seems that the C. neoformans is, by some extent,affected by the intracellular acidification, justifying the necessity toanalyze more deeply the connections between the acidification ofthe phagosome, the increased extrusion and possible danger to thefungus viability.

For the analysis, wild-type strain H99, mutant strain Dplb1 andreconstituted strain Dplb1/rec were used in an interaction assaywith murine peritoneal macrophages previously stimulated with S.cerevisiae cell wallb-glucan (Zymosan). The viability of the fungalcells was accessed after 3 and 24 hours by CFU evaluation. Theacidification of the phagosome previously stimulated with Zymosanwas measured using confocal microscopy using the Lysotrackerprobe.

Our preliminary results show that the stimulus of macrophageswith Zymosan was able to promote an acidification of the phagosomeand that this acidification was more constant and prominent inphagosomes containing fungal cells. The interaction assay also dem-onstrated that the wild-type strain of C. neoformans has a diminishedintracellular growth when phagocytosed by an already activatedmacrophage. Ultimately, the interaction assay utilizing the mutantstrain showed that further than inhibiting the growth, the activatedmacrophage was able to kill the internalized fungus.

The capacity of the previously activated macrophage to inhibit theintracellular growth and the acidification of the phagosome by thisactivation lead to a hypothesis that the C. neoformans doesn´t abso-lutely thrive in an acidified vesicle environment. The results obtainedwith the mutant strain corroborate with this hypothesis indicatingthat the fungal cell that remains for a longer period inside the acidi-fied phagosome has trouble in proliferate and survive, leading to hisdeath. Therefore, it may be possible that the decrease in the vesicularphagosome pH is sensed somehow by the C. neoformans, triggeringthe phenomenon of the extrusion.

P106

Differences between acapsular and encapsulated strain ofCryptococcus neoformans in NLRP3 inflammasome-dependent activationP. H. M. B€urgel,1 P. H. Saavedra,1 K. G. Magalh~aes,1

R. J. Cordero,2 D. S. Zamboni,3 A. H. Tavares,1 P. Albuquerque,1

A. Casadevall4 and A. L. Bocca1

1Universidade de Bras�ılia UnB, Bras�ılia, Brazil; 2Universidade

Federal do Rio de Janeiro UFRJ, Rio de Janeiro, Brazil; 3USP

Ribeir~ao Preto, Ribeir~ao Preto, Brazil and 4Albert Einstein College

of Medicine, New York, NY, USA

Cryptococcus neoformans is an encapsulated human pathogenic fungusthat affects primarily immunocompromised individuals. One of themost important C. neoformans virulence factors in study is its capsulewhich is involved in immune response evasion and fungal dissemina-tion. Also C. neoformans capsule polysaccharides have been shown todownregulate production of proinflammatory cytokines such astumor necrosis factor (TNF) and interleukin-1b (IL-1b). Recently ithas reported the existence of quorum sensing in C. neoformans, as vir-ulence factor, regulating the cell growth of planktonic and biofilmcells, glucuronoxylomannan (GXM) release, and melanin synthesis.In this study we investigated whether C. neoformans molecules wasable of triggering inflammasome activation and evaluated the role ofits capsule in this event.

For the analysis, wild-type capsulated strain B3501 and wild-typeacapsulated strain were cultivated in minimum media for 5 days.After that the media was filtrated in 0,22 µm for the obtainment ofthe conditioned medium. This medium was further processed usingultracentrifugation for the obtainment of the conditioned medium<1KDa. These mediums were utilized with murine BALB/C peritonealmacrophages previously stimulated with LPS and later with nigericinfor inhibition assays. The results were obtained by the analysis of Il-

Table 2 Description of the histopathological findings in 12 renaltransplant patients with cryptococcosis.

Patient Tissue

Granuloma Cell population

Other

lesions

Fungal

budding

epithelioid

macro-

phagical MGC= LMN§ fibrosis necrosis single multiple

1 skin +* -** + + + + + -

2 lung - + + + + - + -

3 skin - - - + + + - +4*** skin 1 - + - + + + - +4 skin 2 - + - + + + - +4 skin 3 + - + + + + + -

5 lung - + - + - - - +6 skin - + + + + - + -

7 lung - + - + + + - +8 lung - + + + - - + -

9 skin + - + + + + - +10 lung - + + + + + - +11 skin - - + + - + + -

12 lung - + + + + + - +Total 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.

ª 2014 The Authors


Poster Abstracts

1b secretion and caspase-1 activation, by ELISA assay and cytometryanalysis 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 acti-vated 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 macrophag-es, showing IL-1b secretion to be dependent on NLRP3 inflamma-some. In addition, inflammasome components were dispensable for C.neoformans uptake or killing. The mechanisms underlying IL-1b pro-cessing and release were dependent on reactive oxygen species(ROS), lysosomal destabilization, potassium efflux and Syk tirosinekinase signaling. In this model IL-1b signaling is not required torestrict intracellular yeast, but limited the rate of infection and yeastgrowth. Moreover, the data show that acapsular C. neoformans acti-vates the NLRP3 inflammasome and IL-1b secretion in a caspase-1dependent manner and the mechanisms involved in this event relyon ROS, lysosomal damage, potassium efflux and Syk signaling, lead-ing to restriction of infection rate.

Furthermore, our results show that the addition of conditionedmedium of encapsulated C. neoformans is able to decrease inflamma-some activation and IL-1b secretion through small molecules(<1 kDa) secreted during infection rather than by the major capsulecomponent, GXM. Unfortunately, we were not able to identify thespecific molecule responsible for this process during the present studybut this activity was resistant to acid, alkali and the action of prote-ases and is sustained by the polar fraction.

P107

Searching invertebrate model hosts to study theCryptococosis agentsD. 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

1Graduate Program of Coordination for Diseases Control,

Secretariat of Health, Brazil; 2University Estadual Paulista J�ulio de

Mesquita Neto, Brazil; 3Adolfo Lutz Institute, Public Health

Reference Center, Secretariat of Health, S~ao Paulo, Brazil and4Center of Toxins, Immune-Response and Cell Signaling,

Butantan Institute, Brazil

Background Cryptococcus neoformans is a pathogenic yeast that isthe main causative agent of Cryptococcosis. Murine models for farrepresents usefull strategy to study host- C. neoformans interactions.Evaluation of C. neoformans virulence in a number of non-mamma-lian hosts suggests that this speciesis a non-specific pathogen.Recently, non-vertebrate animals have been described as valuable forstudies on C. neoformans virulence factors and host innate immuneresponses mimicking the natural scenario with environmental preda-tors, as amoebae and nematodes.Aim Identify appropriate invertebrate host for the study of aspects ofcryptococcal pathogenesis.Methods We investigated in two proposed non-vertebrate modelsthe installation and progression of Cryptococcosis infection. The inoc-ulation 1 9 105 cell ml�1 was performed by three route of adminis-tration: topical application, oral delivery and injection in five groupsof 10 animals for Apis mellifera, and via injection in two groups of10 animals for Zophobas morio.A control group inoculated with PBSwas included in all experiments. Additionally, we verify the identitybetween the inoculated strain and the animal recovered strain. Thestrain-type WM 148 C. neoformans molecular type VNI was used toinoculate fourth instar larvae for both species. The follow-up of inoc-ulated larvae was made up to adult phase (insect). A group com-posed by three animals was sacrificed weekly unless dead occurred.The follow-up period depends on the species lifecycle. The dead

larvae was sliced for tissue microscopic examination and tissue cul-ture was also performed in Sabouraud dextrose agar plate.Results Larvae of Apis mellifera died due to injection route of admin-istration. Oral delivery and topical application via cause death duringearly follow-up with 100% mortality at the first week. Conversely,larvae Zophobas morio were alive up to the end of experiments. Ofnote, the injection route in this species stimulated the production ofheavy superficial slime. The multiple colonies obtained from the tis-sue larvae culture were submitted to PCR-fingerprinting using M13single primer. All recovered colonies showed PCR fingerprinting pat-terns identical to those of the original inoculated strain-type.Conclusion The Apis mellifera larvae seems to be a candidate for asusceptible invertebrate model of infection, but both the inoculumsvolume and administration via warranted additional studies toimprove the experiments. Our study suggested Zophobas morio as aresistant non-vertebrate model of infection for Cryptococcosis viainjection. Future investigation are need to assess financial, space andtime commitment required for use the studied species, and impor-tantly to determine virulence trait and host response before select themost appropriated host.

P108

Macrophage autophagy in immunity to Cryptococcusneoformans 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 housekeepingmechanism that allows cells to degrade and recycle bulky intracellu-lar material such as protein aggregates and organelles. In addition tothese classical roles, a decade of research has shown that autophagyplay multiple central roles in immunity to pathogens as varied asviruses, bacteria, protozoa and, more recently, fungi. Several groupshave found that when phagocytes internalize fungi the microorgan-isms end up in vacuoles with autophagosome characteristics. How-ever, using different techniques different authors have founddisparate results as to what function this has on host-pathogenimmunity: some suggest autophagy to be host-protective, some con-cluded it makes no difference and some that it is beneficial to thepathogen. Before autophagy modulation can be employed as immu-notherapy for fungal infections, the precise mechanisms of its role inantifungal immunity must be established.

Here we test the formation of autophagosomes surrounding C. neo-formans or Paracoccidioides spp. cells that were phagocytosed by humanor murine macrophages. C. neoformans causes a life-threateningmeningoencephalitis that kills some 600 000 people each year. Fungifrom the genus Paracoccidioides are thermo-dimorphic and cause para-coccidioidomycosis (PCM), which affects lungs and reticuloendothelialsystem and is the most prevalent systemic mycosis in Latin America.Aim In order to further our knowledge onto the antifungal roles ofmacrophage autophagy, this work focused on two specific aims:

1 – Determine if human macrophages recruit the autophagosomemarker LC3 to vacuoles containing C. neoformans.

2 – Test if internalized Paracoccidioides spp. cells induce LC3 recruit-ment in murine macrophages.Methods We cultivated C. neoformans H99 strain in Sabouraud agarmedia. P. lutzi Pb01 and P. brasiliensis Pb18 and Pb265 were main-tained 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 supple-mented with 10% fetal calf serum and incubated at 37 °C with 5%CO2. THP-1 monocytes were differentiated onto macrophages bytreatment with phorbol 12-myristate 13-acetate (PMA). Macrophageswere plated in coverslips and infected with fungi (opsonized withanti-capsule IgG1 in the case of C. neoformans) for 24 h. The cellswere then fixed, stained for LC3 immunofluorescence and observedon an epifluorescence microscope.

ª 2014 The Authors


Poster Abstracts

Results As shown in the figure, human THP-1 monocyte-derivedmacrophages recruited LC3 to the vacuole containing C. neoformansin a similar way to that previously described for murine macrophag-es. RAW264.7 cells infected with Paracoccidioides spp. yeast cells alsocontained the fungi in autophagosomes regardless of the species (P.lutzi or P. brasiliensis) or virulence in animal models (Pb18 – highvirulence; Pb265 – low virulence). Important controls that are notshown in the figure confirm the results: (i) LC3 was recruited asexpected around C. neoformans in RAW264.7 cells and (ii) no similarstructures were found in experiments in which the primary LC3 anti-body was omitted.Discussion Recent publications have shown that LC3 is present onthe phagosomes containing C. neoformans in Drosophila S2 cells, aswell as in the murine cell lines RAW264.7 and J774.16. Our resultsshow that the same phenomenon occurs in human macrophages,which can now be used to shed light in a more clinically relevantfashion 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 formsaround phagocytosed Paracoccidioides spp. yeast confirms how wide-spread this phenomenon is, as it has already been observed with Sac-charomyces cerevisiae, C. neoformans, Candida albicans and Aspergillusfumigatus. This suggests that autophagy may be a universal responseto fungal infection.

P109

Effects of Cryptococcus neoformans extracellular vesicleson the phagocytosis and antifungal activity of theenvironmental predator Acanthamoeba castellaniiA. Rizzo,1 V. Cabral,2 J.M. Wolf,2 J.D. Nosanchuk2 and

M.L. Rodrigues1

1Federal University of Rio de Janeiro, Rio de Janeiro, Brazil and2Albert Einstein College of Medicine, New York, NY, USA

Background Extracellular vesicles (EVs) are membranous structuresused by a number of fungal species’ cells, including Cryptococcus neo-formans, to deliver virulence factors to the extracellular milieu. C.neoformans EVs are immunologically active and can regulate the anti-fungal activity of phagocytes. The interaction with environmentalpredators is essential for the establishment of virulence mechanismsused by C. neoformans. The ability of EVs to modulate the antifungalactivity of environmental phagocytes and the processes that mediateC. 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 phagocyticevents with this predator.Aim To characterize the effect of EVs produced by C. neoformans onthe antifungal activity of the environmental predator A. castellaniiand identify the association of MBR for amoeba phagocytosis andkilling of this yeast.Methods Our approach included EV purification, analysis of amoebaviability after exposure to EVs and methyl-alpha-D-mannopyranoside(mannose), stimulation of A. castellanii with EVs, and mannose anddetermination of the phagocytosis index and antifungal activity forH99 strain, an encapsulated serotype A of C. neoformans.Results Our initial results demonstrate that C. neoformans EVs witha diameter range between 50 and 550 nm do not impact the viabil-ity of amoebae, but exposure to EVs increases fungal uptake and sur-vival inside the environmental predator, compared to untreatedamoebae. Mannose treated amoebae also exhibited an increase infungal uptake and survival and were more susceptible to death dur-ing 24 h co-cultures with C. neoformans.Discussion Our results to date demonstrate that fungal EVs impactC. neoformans interactions with A. castellanii, indicating that C. neofor-mans vesicle release to the extracellular space may have developed inresponse to interactions with environmental predators. Additionally,our findings suggest that MBR can mediate fungal phagocytosis byamoebae, opening a new field of investigation about evolutionarysteps that could culminate in intracellular pathogenic strategies forC. neoformans to infect and proliferate inside mammalianmacrophages.

P110

CaMK4: a host kinase required for cryptococcalengulfment and normal virulenceD. L. Srikanta, M. Williams and T. L. Doering

Washington University School of Medicine, Saint Louis, MO, USA

Background Cryptococcus neoformans, the causative agent of crypto-coccosis,is an opportunistic fungal pathogen which kills over600 000 individuals annually. Despite extensive research on crypto-coccal pathogenesis, host gene products involved in the initial phago-cytosis of C. neoformans and subsequent stages of infection needfurther study. We have identified CaMK4 as playing an importantrole in these events.

CaMK4 is a calcium/calmodulin-dependent protein kinase. It occu-pies a key position in a host signal transduction pathway thatresponds to receptor signals and activates multiple processes, includ-ing cell differentiation, survival and cytokine release. This kinase

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

ª 2014 The Authors


Poster Abstracts

regulates transcription factors with key roles in immune responseand inflammation, and is predominantly found in cells of the nervousand immune systems.Aim Our overall goal is to identify host genes that influence engulf-ment of C. neoformans by mammalian cells and thereby alter thecourse of infection. Our specific focus for this project is one of thesegenes, which encodes CamK4. We aim to determine the specificmechanism by which CaMK4 influences cryptococcal pathogenesis.Methods To identify host genes involved in phagocytosis of C. neo-formans, we used a previously established high-throughput image-based assay (Srikanta et al., 2011) to screen a human macrophage-like cell line (THP-1) subjected to RNAi. We used the same assay toperform secondary screens for specificity, and also performed follow-up studies using primary phagocytes, enzyme inhibitors, and animalinfections.Results In preliminary studies we treated THP-1 cells with siRNAtargeting genes whose products are known to act in phagocytosis. Asexpected, this resulted in decreased uptake of Cryptococcus. Based onthese results, we performed a primary RNAi screen targeting 901human kinase and phosphatase genes in THP-1 cells; this identified77 host factors that influenced fungal adherence and internalization.To rule out non-specific alteration of host processes, we re-screenedthese genes in parallel with both inert particles (latex beads) and C.neoformans. For 25 of them, silencing consistently and significantlyaltered uptake and/or adherence of cryptococcal cells while causingminimal to no perturbation of inert particle (latex bead) phagocyto-sis. For a further subset of 8 genes, silencing specifically affectedcryptococcal uptake, but not uptake of the model yeast S. cerevisiae.

We focused on one gene, encoding CamK4, whose silencing signifi-cantly and specifically altered cryptococcal uptake. We found thatthis result held in primary human and mouse phagocytes as well asTHP-1 cells. Excitingly, we also found that mice lacking this gene aresignificantly less susceptible to C. neoformans in an inhalationalmodel of infection.Discussion/conclusion CaMK4 plays an important role in crypto-coccal pathogenesis and its absence significantly influences the out-come of infection. We are now investigating the precise mechanismby which CaMK4 influences fungal:host interactions and therebycryptococcal disease. Our RNAi screen also yielded multiple otherhost gene products in cryptococcal uptake by phagocytes, several ofwhich have never been implicated in fungal pathogenesis; these willbe the focus of future studies. Our exciting findings will shed light onhost aspects of cryptococcal pathogenesis and potentially other host:microbe interactions as well.

P111

A role for protein palmitoylation in the interactions ofC. neoformans with host cellsF. 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 byphagocytes are key events that are central in cryptococcal pathogen-esis. Fungal engulfment by host cells and their subsequent intracellu-lar proliferation have been implicated in latency, dissemination, andvirulence, but the full complement of C. neoformans gene productsthat participate in these processes has not been defined. To addressthis question, we used an automated high content method (Srikantaet al., 2011) to assay the interactions between a human macro-phage-like cell line and mutant fungi from a partial deletion collec-tion (Liu et al., 2008).

We identified multiple genes whose deletion led to lower or higheradherence and/or phagocytosis compared to wild-type. Several ofthese have been previously reported to have perturbations of cell sur-face structures, validating our screening approach. The gene deletedin one of these mutants encodes a probable protein fatty acyltransfer-ase (Pfa), one of a family of DHHC domain-containing proteins thatcatalyzes lipid modification of proteins.

Aim The goals of this project are to determine the defects associatedwith specific loss of this putative Pfa and to uncover the mechanismby which it alters the fungal-phagocyte interactions.Methods To accomplish our goal, we generated mutant, comple-mented, and epitope-tagged strains; assayed them for phenotypesincluding growth under stress conditions, phagocytosis by macro-phages, intracellular survival, and virulence in mice; and examinedthem by light microscopy. We also characterized proteins by gel elec-trophoresis, immunoprecipitation, and mass spectrometry.Results We confirmed that deletion of the gene of interest, termedPFA4 for its homology to that gene in S. cerevisiae, results inenhanced adherence to and phagocytosis by human macrophagescompared to wild type. Mutant cells lacking the gene exhibit mor-phological defects that are exacerbated under host conditions. Themutant is sensitive to a variety of cell wall stress conditions in vitroand has a profound defect in intracellular growth; it is also avirulentin a mouse model of cryptococcal infection. Interestingly, this mutanthas no obvious defect in capsule synthesis or induction. All of themutant phenotypes are reversed by genomic complementation andare due to the lack of fatty acyltransferase enzymatic activity, aspoint mutants in the enzyme active site phenocopy the gene deletion.Proteomics studies and investigation of putative Pfa4 substrates arecurrently under way to determine the mechanism of the defectsexhibited by the pfa4 mutant.Discussion/conclusion Defects in lipid modification are known tocause mislocalization or degradation of the substrate proteins, leadingto their dysfunction. Our results are consistent with aberrant palmi-toylation of some cyptococcal protein(s) leading to alteration of thecell surface; this could then cause increased recognition, phagocyto-sis, and killing by host macrophages and loss of virulence in mice.Pfa4 is one of a family of proteins with the same biochemical activ-ity. Our current efforts are directed at identifying the specific proteinsubstrate(s) responsible for the changes we observe in the pfa4mutant, so that we can obtain a detailed picture of the molecularmechanisms behind them.

P113

Automated imaging and analysis of C. neoformans forhigh-throughput assaysA.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 haveexpanded to the genomic scale, enabled by the genome sequence anddeletion collections. Initial forays have also been made into large-scale analysis of host factors that influence fungal:host interactions(Qin et al., 2011).

To capitalize on genome-wide experimental approaches, assays pre-viously performed on a limited scale must be scaled up. One powerfultool for this is high content screening (HCS), where high content refersto ‘processes defined spatially and temporally in the context of eachcell within an array of cells’ (Abraham et al., 2004). HCS refers to theautomation of imaging and analysis for screening purposes. It is apowerful tool that can assist in analysis of libraries that range fromcollections of deletion mutants to banks of chemical compounds. Aparticularly exciting option is the application of HCS to probe the hostaspect of fungal:host interactions by using RNA interference (RNAi) totarget individual host genes on a large scale (Prudencio et al., 2009).

Host phagocyte engulfment of cryptococcal cells is central to cryp-tococcal pathogenesis and affects fungal survival, latency, growth,and dissemination. However, details of their interactions remainundefined, suggesting direct and unbiased assessment of key hostand pathogen features by screening as an appealing strategy to probethese events. The current standard for assessing cryptococcal interac-tions with host cells is microscopic examination in multiple focal

ª 2014 The Authors


Poster Abstracts

planes, but this is time-intensive and laborious. Several groups havedeveloped flow cytometry-based analysis methods (Voelz et al., 2010;Alanio et al.; Nicola et al., 2011), but these are difficult to scale up tothe level required for screening. We have applied high content analy-sis to several aspects of cryptococcal biology relevant to pathogenesis,including adherence to and engulfment by host phagocytes and cap-sule thickness.Aim Our broad goal is to facilitate the investigation of cryptococcalbiology and pathogenesis by developing high-throughput imagingand analysis protocols. The specific focus of this project was to opti-mize HCS assays for cryptococcal adherence, engulfment, and capsulegrowth.Methods We compared GE IN Cell, ImageXpress, and BioTek Cyta-tion3 automated imagers with manual light microscopy, followed byanalysis through open-source, collaborator-developed, and proprie-tary image processing software. Cryptococcal phagocytosis, adher-ence, and capsule growth were assessed.Results We examined multiple imaging and analysis tools, assessingimage quality, analysis quality, cost, and speed, and have developedoptimized HCS assays for cryptococcal adherence, uptake, and cap-sule growth. We identified several combinations that are particularlypowerful for high-throughput cell interaction assays. The IN Cell andCytation3 are excellent imaging options, and the GE Developer soft-ware is most powerful for complex analysis of captured images.Discussion/conclusion Image and analysis quality must be bal-anced with efficiency and cost, and the best HCS option depends onthe requirements of the investigator and the assay. However,through this examination of high-throughput screening methods forC. neoformans, we have developed criteria that may be used to assistthe decision-making process. Host genome screens with RNAi haveproven informative, and the availability of cost-efficient, rapid, andaccurate systems will accelerate discovery of host and cryptococcalfactors important in pathogenesis.

P114

Viability of Cryptococcus neoformans/Cryptococcus gattiiin long term stored environmental samples from ColombiaP. Escandon and E. Castaneda

Instituto Nacional de Salud, Bogota, Colombia

Background Several studies carried out in Colombia have providedvaluable data about the ecology of C. neoformans and C. gattii. Exten-sive environmental samplings in different areas of the country haveprovided us with both C. neoformans and C. gattii isolates recoveredfrom Eucalyptus spp, almond trees, Ficus spp, bird droppings, Eucalyp-tus ficifolia (Corymbia ficifolia) detritus, and some other species oftrees. Attempts have also been made in standardizing a moleculardetection technique in environmental samples of this fungus, beingable to extract and amplify Cryptococcus spp DNA from environmentalsamples with adequate PCR specificity; therefore, the present avail-ability of naturally colonized samples will be very useful in the stan-dardization of a new specific C. neoformans/C. gattii detectiontechnique in environmental samples.Aim To determine the viability of members from the C. neoformans/C.gattii species complex in naturally colonized samples associated totrees decaying wood and bird droppings stored since 2003.Methods A total of 964 samples collected between 2003 and 2008were processed. Of them, 274 were samples of filtrates from treesdecaying wood, 653 were samples from trees decaying wood, and37 were samples of bird droppings. The molecular type of isolateswas established with PCR fingerprinting using the single primer(GTG)5.Results An overall positivity of 3.9% was obtained from the 964samples processed; positive samples had been stored as filtrates fromtrees decaying wood (36.9%), as decaying wood in bags at roomtemperature (52.6%) or as bird droppings (10.5%); from these, 161isolates were recovered and identified as members of the C. neofor-mans/C. gattii species complex. Eighty one isolates (50.3%) were

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 sppdecaying wood. Sixteen (19.7%) isolates, recovered mostly from Ficusspp., were typed as VGI; 43 isolates (53.1%) recovered from Eucalyp-tus trees, as VGII, and 22 (27.2%) recovered from C. ficifolia, almondtrees and Ficus spp., as VGIII isolates. C. neoformans isolates wererecovered from Ficus spp., Eucalyptus spp. and bird droppings. Molecu-lar type VNI was observed in 78 (97.5%) isolates recovered from allthe above-mentioned sources; the remaining two isolates recoveredfrom Ficus spp. were VNII. In two samples recovered from Ficus spp.both C. neoformans and C. gattii were found. CFU were estimatedbetween 0.5 9 102 and 11.4v104 CFU g�1 of sample.Discussion/conclusions In this brief observation, it is clear that C.neoformans/C. gattii are able to survive in environmental samplesstored at room temperature either as soil/plant material or as filtratesfrom the samples themselves. When re-processing the samples previ-ously collected during our study, we established that both speciescan be isolated from the same sample and tree species; this showsthe importance of isolating and characterizing as many colonies aspossible from those suspected to be C. neoformans/C. gattii in a sam-ple. Specific environmental conditions are necessary for species toexist in a particular environment, obtain nutrients, elude predatorsand reproduce; therefore, their survival and abundance in a nichedepend on a series of factors that are essential for their viability. Inthis study we found that agents of the C. neoformans/C. gattii speciescomplex can survive in stored samples for as long as nine years. Fur-ther studies are needed to determine how virulence factors express inthese isolates and compare their expression levels with those of thestrains initially recovered.

P115

Anatomy of an outbreak: recombining clonal clusterscomprise the VGII C. gattii populationR. B. Billmyre,1 D. Croll,2 W. Li,1 P. A. Mieczkowski,3

J. Kronstad2 and J. Heitman1

1Duke 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 typicallytropical and subtropical pathogen Cryptococcus gattii has developed inthe temperate Pacific Northwest of the United States and neighboringBritish Columbia in Canada. This outbreak consists of three clonalpopulations, designated VGIIa, VGIIb, and VGIIc. Robust populationsof C. gattii with high similarity to the VGIIb clonal cluster exist inAustralia and related isolates have been reported from South Amer-ica, while VGIIa and VGIIc appear to be more unique to the PacificNorthwest. Recent studies implicate South America as the ancestralorigin of C. gattii, however it is unclear where the more proximal ori-gin of the PNW outbreak is located.Aim We aimed to utilize whole genome sequencing in place of mul-tilocus sequence typing to gain increased resolution to both (i) distin-guish members of clonal clusters of the C. gattii population from eachother and (ii) determine the relationships and origins of these isolatedclonal populations.Methods We utilized whole genome sequencing with the Illuminaplatform, in conjunction with both reference-based assembly to theR265 VGII reference genome and de novo assembly using velvet. Todistinguish the origin of the outbreak, we have sequenced VGIIb andrelated isolates previously identified from both Australia and theCaribbean. In addition, we have sequenced a number of VGII isolatesof all three clonal lineages from the PNW and incorporated previ-ously published genomes from the CDC.Results Whole genome sequencing has allowed dramaticallyincreased resolution of the VGIIa, b, and c groups from the PacificNorthwest. Interestingly, these isolates are primarily differentiated

ª 2014 The Authors


Poster Abstracts

by private polymorphisms and show evidence of a strong foundereffect, followed by radiation. Additionally, the VGIIb group sharessubstantially more similarity with VGIIb isolates from Australia thanwith a Caribbean Islands isolate, and the Australian isolates clusterwithin the same group as those from the PNW. Furthermore, bysequencing the entire genome, we have discovered that the majorityof VGII isolates share various identity islands throughout their ge-nomes. These shared genomic islands are indicative of a robustlyrecombining population, with common ancestry. Whole genomeallele compatibility tests provide robust evidence for recombinationin the population.Discussion/conclusion The population structure of C. gattiirevealed by whole genome analysis is consistent with intermittentmating, followed by periods of clonal expansion. This is most obviousin the Pacific Northwest where three clonal groups have undergonerapid expansion and are causing an outbreak. However, these threeisolates share regions of identity with each other, suggesting thatsexual exchange and recombination were important factors in theorigins of each outbreak lineage. Each of these isolates is MATalpha,and MATa isolates are absent in the outbreak populations andunderrepresented globally. This suggests that unisexual reproductionmay be contributing to the recombination observed in the popula-tion, or isolates are undergoing sexual reproduction with MATa iso-lates in an under-sampled environmental niche and disseminatinginto sampled locations. These are not mutually exclusive processes,and both a-alpha bisexual and alpha-alpha unisexual reproductionmay contribute to both diversify and amplify genotype within thepopulation.

P116

Frequency of morphologic altered strains amongCryptococcus neoformans - C. gattii complex isolates in aBrazilian reference culture collection and antifungalsusceptibility patternL. Oliveira, D. C. S. Santos, D. M. Castro e Silva, M. W. Szeszs

and M. S. C. Melhem

Instituto Adolfo Lutz, S~ao Paulo, Brazil

Background Typically the cryptococci budding cells are round toelongate with 5–9 µm diameter, but the occurrence of titan cells(>10 lm), microforms (<5 lm) and hyphal forms are known. Envi-ronmental and host selection pressure could promote emerging ofmorphological variants, and could accumulate in vivo promoting per-sistence. The extension and impact on the strain virulence and anti-fungal resistance or relevance to clinical outcome of atypical forms isan enigmatic issue.Aim We analyzed the frequency of atypical forms in 177 clinical iso-lates (112 Cryptococcus neoformans and 65 C. gattii) from a ReferenceCulture Collection and correlated with culture melanization. All butone (VN IV) isolates of C. neoformans were VNI and C. gattii isolateswere typed as VGII (61) and VGI (4).Methods Cerebral spinal fluid and blood Cryptococcus cultures werescreening for melanine production onto Guizotia abyssynica agar andfor atypical forms. Atypical forms (Okagaki et al.,2010) was assuredat the medical Mycology Reference Laboratory of Spain, NationalCentre for Microbiology of Instituto de Salud Carlos III. The atypicalisolates were submitted to PCR-RFLP method and antifungal suscepti-bility tests. Etest� method was employed for amphotericin B (AmB)and fluconazole (FCL). AmB fungicidal activity was determined bytime-kill curves method after 6, 12, 24, 48 and 72 h of exposition to1 mg l�1 of drug.Results Among 177 clinical isolates tested 7 (7/177; 3.95%) isolatesshowed titan cells including 3 (3/7; 41.9%) presenting hyphal forms.No isolates containing micro cells were encountered among the stud-ied isolates. All atypical forms were exclusively observed in C. neofor-mans molecular type VN I. The MIC range for the 7 isolates showingtitan cells were as follow: FCL-MIC 0.25 to 14 mg l�1 and AmB-MIC

0.02–0.94 mg l�1. The MIC range for the 170 remained isolateswas: FCL-MIC 0.12 to 16 mg l�1 and for AmB-MIC 0.12–1 mg l�1.High FCL- MIC (>8 mg l�1) were observed in 2 (2/7; 28.6%) atypi-cal-form isolates and in 5.3% of normal-cell isolates. Overall, datafrom the time-kill curves showed fungicidal effect in 56% of culturesin the initial 6 h-exposition to AmB. We observed designed re-growthphenomenon in 2 (2/7; 28.6%) titan-cell isolates.Discussion and conclusions Our findings suggested that morpho-logic changes are more frequent in C. neoformans isolates than C. gat-tii isolates, although we have studied larger number of C. neoformansisolates since it is the main causative species. Previous work indicatesthat being an accidental but successful pathogen with a broad hostrange, C. neoformans has adapted to survive in multiple hosts thatcould explain the morphologic changes.Furthermore, it was enter-tained that strains demonstrated a gradual increase in cell body sizewith generational aging, and there is some evidence on in vivo selec-tion of cells of advanced age in human cryptococcal meningoenceph-alitis. Moreover, older cells are likely accumulated during chronicinfection, and such cells appear to be resistant to macrophage,H2O2, and AmB killing. We confirmed that almost all C. neoformansstrains that are recovered from patients are susceptible to FCL afterin vitro replication including those showing titan cells. The MIC hadnot evaluated properly the cidal effect of AmB, according to low MICvalues obtained in this study. However, time-kill experiments sug-gested that titan cells seem to be more resistant to antifungal-medi-ated killing in vitro by AmB in comparison to our previousexperience (data not showed) in which 5.4% of 56 normal-cell C.neoformans isolates re-growth in similar conditions. We hypothesizedthat AMB would be less effective on atypical cells, conversely to FCLthat showed strong inhibitory activity against C. neoformans typicaland atypical isolates.

P117

First report on the environmental isolation of Cryptococcusneoformans molecular type VNI in Popay�an, ColombiaP. C. Castillo,1 C. A. Anacona,2 F. G. Gonzalez2 and

P. Escandon1

1Instituto Nacional de Salud, Bogota, Colombia and 2Universidad

del Cauca, Popayan, Colombia

Background The importance of cryptococcosis has led to study theecology and the possible ecological niches of Cryptococcus neofor-mans/Cryptococcus gattii species complex. Reports on the isolation ofserotype A from bird droppings in Colombia started around 1968 inMedellin, with a positivity of 18.8%; in the following years (1994),Casta~neda et al. reported a positivity of 53.8% for C. neoformans indifferent cities and later a positivity of 49.6% was reported also fromavian droppings. Additionally, studies on the isolation of the complexfrom Eucalyptus sp have been reported. These environmental studies,together with a sampling that is being carried out in five regions inColombia, as well as the clinical data, are intended to be used todelineate areas where the C. neoformans/C. gattii species complex isestablished, and to predict the regions where the fungus may dis-perse in the future, thus generating an Ecological Niche Modeling(ENM) for our country.

In Colombia the annual incidence rate for cryptococcosis is3.3 9 103 for AIDS patients and in the general population2.4 9 106. The annual incidence rate in the department of Cauca(capital city Popay�an) was 1.3 9 106; no environmental reportshave been known on the isolation of members of the complex in thiscity, being this is the first report on the recovery of C. neoformansfrom bird dropping and trees in the rural and urban areas ofPopayan.Aim To carry out an extensive sampling in the city of Popay�an andcharacterize phenotypically and genotipically environmental isolatesof the C. neoformans/C. gattii species complex recovered, as a toolheading for the creation of an ENM in Colombia.

ª 2014 The Authors


Poster Abstracts

Methods Samples were collected between September 2012 andAugust 2013 from trees (leaf, bark, flowers, soil and fruits) andbird droppings in areas with a high density of trees and birds. Fivehundred 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, dovecotesand parks of the historical area in Popay�an and its rural area, and454 samples of trees. Phenotypic identification of the C. neofor-mans/C. gattii species complex was based on growth of brown colo-nies on Niger Seed Agar (NSA) and biochemical characteristics;genotypic characterization was done using PCR Fingerprinting(GTG)5 and Restriction Fragment Length Polymorphism (RFLP) ofthe gen URA5.Results Out of 1,031 samples of bird droppings and trees, 180 werepositive, 162/577 (28%) of pigeon droppings and 18/454 (4%) fromtrees samples, recovering 515 isolates, 441 (85.6%) from pigeondroppings and 74 (14.4%) from tree samples. C. neoformans var. gru-bii molecular type VNI was found in 99.3% and VNII in 0.7%. C. gat-tii was not recovered. Most of the isolates recovered from pigeondropping were sampled in churches, roofs and dovecotes.Discussion C. neoformans was recovered, confirming the sameresults obtained in others studies in Colombia, and reinforcing thepredominance of this species in environmental samples. Popay�an isconsidered a region with a lower incidence of cryptococcosis thanother cities in Colombia, although a high environmental prevalenceof the fungus was found in droppings from places with high den-sity of pigeons, being this a risk for immunosuppressed and immu-nocompetent patients, reinforcing that pigeon excreta is a favorableenvironment for C. neoformans. Despite these significant findings,there is still a lack of registration for cryptococcosis, not only inPopay�an but in the whole country, where the disease is not ofcompulsory notification, therefore, more strategies have to beenundertaken to improve the passive surveillance of cryptococcosis inColombia.

(Colciencias Project code: 2011-3600115683).

P118

Isolation of the Cryptococcus neoformans/Cryptococcusgattii species complex in five cities of Colombia, andassociation with ecological conditionsN. V. Velez,1 P. C. Castillo,1 M. A. Alvarez,2 B. C.De Bedout,3

F. G. Gonzalez4 and P. Escandon1

1Instituto 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 beendone describing the occurrence of the complex in diverse habitats;isolates of serotypes A, B and C have been recovered from birddroppings, Eucalyptus, Ficus sp and Terminalia catappa among others.The characterization of the ecological conditions possibly related tothe habitat of the complex will allow us to get a closer knowledgeof the yeast.Aim To carry out an extensive environmental sampling in five citiesin Colombia to recover isolates of the complex, characterize themphenotypically and genotypically, and determine some ecologicalconditions that may be related to its distribution in specific habitats.Methods A total of 4501 samples, (avian droppings n = 609; treesamples n = 3892) were collected in five cities: Bogot�a (n = 446),located in the center of the country, the annual rainfall is 952mm and average temperature of 14 °C. C�ucuta (n = 495), locatedNorthwest, the annual rainfall is 806 mm and its average tempera-ture is 30 °C. Cali (n = 447), is located Southwest, the annualrainfall is 900 mm and its average temperature is 23 °C. Medell�ın(n = 1515), located Northwest, the annual rainfall is 1656 mmwith an average temperature of 23 °C. Popay�an (n = 1598),

located Southwest, the annual rainfall is 1991 mm with an aver-age temperature of 18 °C.

The samples were collected between September 2012 and 2013and processed with conventional techniques, fungus identificationand the variety were determined using biochemical testing, molecu-lar type was determined by RFLP-URA5 and PCR fingerprint.

Climatic data (relative humidity, sun light, temperature and rain-fall) was supplied by the National Institute of Hydrology, Meteorologyand Environmental Studies IDEAM.Results From a total of 4501 samples processed, 238 (5.2%) werepositive, 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. neofor-mans 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 Medell�ınand 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 gat-tii). C. neoformans was recovered more frequently in dry bird droppings,while C. gattii was found mainly in samples of bark, soil and leaves oftrees 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 andC�ucuta, while 8.4% (n = 20) were recovered in the rainy season inBogot�a and Medellin.

Further analyses are in progress to determine the association onthe 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 opportunis-tic pathogen; at the same time, VNI is identified as the most frequentmolecular type in C. neoformans.

Our data show that the highest positivity is obtained in the dryseason, and that the fungus was obtained more frequently from birddroppings than from trees.

Ecological studies are relevant in the epidemiology study of thispathogen; therefore the continuity of the research is important forecological studies which may allow us to determinate areas wherethe fungus can find favorable conditions to develop in Colombia.

(Colciencias Project code: 2011-3600115683).

P119

A new taxonomy for the Cryptococcus neoformans andCryptococcus gattii species complexF. 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

1Canisius-Wilhelmina Hospital, Nijmegen, the Netherlands;2CBS-KNAW Fungal Biodiversity Centre, Utrecht, the

Netherlands; 3Hadassah-Hebrew University, Jerusalem, Israel and4The Field Museum, Chicago, IL, USA

Background During the past two decades, considerable genetic het-erogeneity has been demonstrated to occur in the C. neoformans/C.gattii species complex by a plethora of molecular methods, such asamplified fragment length polymorphism (AFLP) PCR-fingerprintingusing M13, (GACA)4 and (GTG)5 primers, random amplification ofpolymorphic DNA, restriction fragment length polymorphism finger-printing based on the genes CAP10, CAP59, GEF1, PLB1 and URA5,Fourier transform infrared-spectroscopy-based phenotyping, multi-locus microsatellite typing and sequence analysis of a large numberof genes as well as whole genomes. The results of these studiesstrongly question the currently used two species classification of thecomplex.Aim To settle the taxonomic status of the genotypic groups whichare recognized in the C. neoformans/C. gattii species complex.Methods Phylogenetic analysis of 114 globally collected isolates ofthe species complex was done using 11 nuclear loci CAP59, GPD1,

ª 2014 The Authors


Poster Abstracts

IGS1, ITS, LAC1, PLB1, RPB1, RPB2, SOD1, TEF1 and URA5. Thesemolecular data were used to perform gene tree analyses in a maxi-mum likelihood (ML) and Bayesian (B/MCMC) framework and coales-cent-based species trees. We employed a combination of methods toaddress the species delimitation, using gene tree estimation from sin-gle-locus and concatenated data sets, and species tree estimations,including a genealogical species recognition method in which pres-ence of clades in the majority of single-locus genealogies is taken asevidence that these represent distinct lineages. We also used the coa-lescent-based general mixed Yule coalescent (GMYC) method, whichaims at locating the nodes that define the transitions between intra-specific (tokogenetic) and interspecific relationships using branchlengths. Identification of the new species using a test set of 425 iso-lates was tested by MALDI-TOF MS.Results Phylogenetic analysis of 11 loci and various genotypingstudies revealed significant genetic diversity with the pathogenicCryptococcus neoformans - C. gattii basidiomycetous yeast species com-plex. Genealogical concordance, cohesion-based, and species treeapproaches all supported the presence of distinct lineages within thecomplex. Consequently, we propose to recognize the current C. neo-formans var. grubii and C. neoformans var. neoformans as separate spe-cies, and within C. gattii five lineages occur. The type strain of C.neoformans CBS 132 represents a serotype AD hybrid and needs to bereplaced. The ‘newly recognized’ species differ in pathogenicity, prev-alence for patient groups, as well as biochemical and physiologicalaspects, such as susceptibility to flucytosine and fluconazole antifun-gals. MALDI-TOF MS proved to be a reliable and easy-to-use identifi-cation tool as no major errors were observed.Discussion/conclusion Based on an extensive phylogenetic analysisthe currently known main genotypes in the C. neoformans/C. gattiispecies complex are best interpreted as species. The type strain of C.neoformans (and the genus Cryptococcus) CBS 132 has to be replacedbecause of its hybrid nature. All species within the complex, and to alarge extent the hybrids as well, can be identified in the routine clini-cal laboratory by MALDI-TOF MS.

P120

Infective capacity of Cryptococcus neoformans andC. gattii in a human astrocytoma cell lineM. C. Olave, J. C. Vargas-Zambrano, A. Celis, E. Casta~neda and

J. M. Gonzalez

Universidad de los Andes, Bogot�a, Colombia

Background Cryptococcal meningo-encephalitis is caused by themembers of the Cryptococcus neoformans/C. gattii species complex. C.neoformans var. grubii is the most common etiological agent of crypto-coccosis in immunocompromised individuals, and notably C. gattiihas emerged as the primary pathogen in immunocompetent popula-tions. This fungal infection is the most important cause of death inpatients with acquired immunodeficiency syndrome (AIDS). Thepathogenesis of the disease in the central nervous system (CNS) is anongoing research topic; however the biology and mechanisms bywhich C. neoformans and C. gattii invade and infect brain cells remainunknown. Astrocytes, the main CNS cells population, play a funda-mental role in the local immune responses. These cells may activelyparticipate during cryptococcosis either by direct infection or byresponding towards fungal antigens.Aim This study evaluated the infection with C. neoformans and C.gattii in a human astrocytoma cell line to determine their infectivityand the induction of major histocompatibility complex (MHC) mole-cule expression.Methods A glioblastoma cell line (ATTC: CRL-1718) with humanastrocytes characteristics was infected with C. neoformans and C. gat-tii yeasts labeled with FUN-1 fluorescent stain. The percentage ofinfection and expression of HLA class I (HLA-ABC) and class II (HLA-DR) were determined by flow cytometry at day three post-infection.Fluorescence microscopy was carried out in order to observe interac-tions between FUN-1-stained yeasts and astrocytes whose nuclei

were stained with DAPI. To explore the viability of both Cryptococciafter infection, astrocytes were lysed and intracellular yeasts wererecovered and cultured in Sabouraud dextrose agar. The yeastgrowth was assessed by colony forming units (CFU) after 48 hours ofculture. Statistical analysis was performed using non-parametrictests.Results The percentage of astrocyte infection with C. gattii was35.3% (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 ofHLA-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. neofor-mans 39.4% (SD � 17.7%) than C. gattii 20.1% (SD � 9.3%). Boththese populations had different expression than control cells 0.44%(SD � 0.1%) (P = 0.004, Kruskal–Wallis). Fluorescence microscopyanalysis demonstrated more live yeasts in C. neoformans-infected as-trocytes than cells infected with C. gattii. This trend was confirmedwith the count of intracellular yeasts recovered after 48 h of culture,C. neoformans 1943 CFU ml�1 (SD � 133.2) and C. gattii 1340 CFUml�1 (SD � 175.2).Discussion/conclusion There was no difference in the percentageof astrocyte infection by both species, but C. neoformans induced ahigher expression of HLA-DR than C. gattii. Since astrocytes can beinvolved in antigen presentation to T-cells, these results suggestthat infection with C. neoformans could activate T-cells locally. Asmore live yeasts in C. neoformans-infected astrocytes were recoveredthan cells infected with C. gattii, C. neoformans could have differentvirulence mechanisms that allow its survival in human glia-derivedcells.

P121

STAT1-induced classical macrophage activation is essentialfor protection against Cryptococcus neoformans in miceC. M. Leopold Wager,1 C. R. Hole,1 K. L. Wozniak,1

M. A. Olszewski2 and F. L. Wormley Jr1

1University of Texas at San Antonio, San Antonio, TX, USA and2University of Michigan Health System, Ann Arbor, MI, USA

Background Protection against pulmonary inoculation with aninterferon-gamma producing strain of C. neoformans (H99 gamma) isassociated with classical activation of macrophages (M1) andenhanced phosphorylation of the transcription factor STAT1 in thesecells. Studies utilizing general STAT1 KO mice inoculated with H99gamma have revealed that STAT1 is essential for polarization ofmacrophages toward an M1 activation phenotype and, coinciden-tally, for the induction of a protective immune response. However,the necessity for M1 macrophage activation for combating cryptococ-cosis is unknown.Aim The present studies were designed to determine the requirementfor STAT1-induced M1 macrophage activation in protection againstC. neoformans H99 gamma.Methods Mice with macrophage-restricted STAT1 ablation (LysMC-reSTAT1flfl mice) and control mice (STAT1flfl) were given an intra-nasal inoculation with H99 gamma and evaluated for survival andpulmonary fungal burden. Pulmonary macrophages from the macro-phage-restricted STAT1 KO mice and control mice were examined forpolarization phenotype by measuring gene expression of macrophageactivation markers. The anti-cryptococcal activity of macrophageswas determined by enumerating the intracellular cryptococci and,following 24 h of culture, measuring nitrite levels in culture super-natants. We also determined the necessity of nitric oxide (NO) pro-duction by macrophages from control mice towards inhibiting theintracellular growth of Cryptococcus.Results Our data reveal that mice with macrophage-restrictedSTAT1 ablation have a significant increase in pulmonary fungal bur-den compared to control mice. This increase correlates with a 10%

ª 2014 The Authors


Poster Abstracts

survival rate in the macrophage-restricted STAT1 KO mice comparedto a 100% survival rate in the control mice, indicating that STAT1signaling in macrophages is required for protection. Macrophagesfrom macrophage-restricted STAT1 KO mice have an increase ingene expression for alternative macrophage activation (M2) markerArg1, but no change in gene expression for M1 macrophage markeriNOS compared to macrophages from control mice. This indicates aniNOS/Arg1 ratio in STAT1 deficient macrophages that favors an M2phenotype which is associated with a non-protective immuneresponse. Furthermore, compared to STAT1 sufficient macrophages,STAT1 deficient macrophages have an increase in intracellular cryp-tococci. This coincided with a decrease in production of NO, abyproduct of M1 macrophages that has been shown to have anti-microbial activity. The inverse relationship of intracellular yeasts toNO production suggests that the macrophages are phagocytizing butnot killing the yeast. Additionally, inhibition of NO production in pul-monary macrophages cultured from inoculated control mice isaccompanied by a diminished capacity for control of cryptococcalgrowth, indicating that NO is required for anti-cryptococcal activityof macrophages.Conclusions Overall, data indicate that STAT1-mediated M1 macro-phage activation is essential for the induction of protective immuneresponses to C. neoformans. Importantly, production of NO by M1macrophages is critical for fungal growth suppression. These studiesprovide the first evidence of a specific effector cell population andmechanism for protection against pulmonary cryptococcosis.

P122

Dectin-2 polymorphism associated with Cryptococcosis inHIV-uninfected Chinese patientsX. 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

1Zhejiang Provincial People’s Hospital, Hang Zhou, China and2Huashan Hospital, Fudan University, Shanghai, China

Background and aim Dectin-2 is a C-type lectin receptor whichcan recognize critical structures of fungi, and previous studies haveindicated an important role of this receptor in antifungal immunity.We did this research to analysis the distribution of Dectin-2 geneticpolymorphisms among Chinese Han patients with cryptococcosis andhealthy controls to investigate the association between Dectin-2 andcryptococcosis.Patients and methodology In this case control study, we geno-typed 2 unknown functional change SNPs of Dectin-2 (rs11045418,rs118059158). Only rs11045418 have polymorphisms in our popu-lation. A total of 252 patients with cryptococcosis and 464 healthycontrols were included in this study. The overall patients weredivided into three subgroups according to the different types of infec-tion, including patients had cryptococcal meningitis and without pul-monary cryptococcosis (CM group), patients had pulmonarycryptococcosis and had no central nervous system infection (PCgroup), and patients had both these two types of infection (PC + CMgroup).Results Compared to the control group, there was a trend ofincreasing 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). Andthe heterozygote was significantly increased in PC patients who hadno predisposed condition (36/53 vs. 231/464, P = 0.016,OR = 2.08, 95% CI: 1.13–3.81). But no such difference was foundbetween controls and overall patients as well as patients with othertwo types of infection. We also found that the heterozygote ofrs11045418 decreased in the 171 CM&PC + CM patients (patientshad cryptococcal meningitis), when compared with 78 PC patients(who had no CNS involved), although without no significant differ-ence (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, anotable decrease of this heterozygote was found in CM&PC+CM

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 forrs11045418 is associated with the susceptibility and disease proce-dure of cryptococcosis, which suggests that Dectin-2 might play animportant role in this infection.

P123

The role of macrophages in interleukin-4 receptor (IL-4R)-dependent pathology in pulmonary cryptococcosisD. Piehler,1 U. M€uller,1 W. Stenzel,2 A. Grahnert,1

M. Protschka,1 G. K€ohler,3 O. Frey,4 J. Held,2 T. Richter,1

M. Eschke,1 T. Kamradt,5 F. Brombacher6 and G. Alber1

1Insitute 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 cryp-tococcosis, susceptibility to infection is associated with alternativelyactivated macrophages (aaMph). IL-4R plays a key role in the induc-tion of aaMph as in IL-4R-deficient mice aaMph are almost absent inseveral models studied. In murine pulmonary cryptococcosis alterna-tive activation of macrophages is dependent on T helper (Th)2 cells,especially polyfunctional Th2 cells, that produce more than one Th2cytokine (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-defi-cient macrophages that are insensitive to IL4 and IL-13 (while othercells are unaffected) in pulmonary cryptococcosis.Methods To study the function of aaMph, mice with geneticallyablated IL-4Ralpha expression on macrophages (Mph IL-4R�/�) wereinfected with C. neoformans intranasally and compared to heterozy-gous littermates with heterozygous IL-4Ralpha expression (IL-4R+/�).The survival rate, lung burden, pulmonary histopathology, activationstate of macrophages, immunoglobulin levels, and cytokine produc-tion (measured by ELISA and intracellular flow cytometry) wasanalyzed.Results Mph IL-4R-/- mice are more resistant to pulmonary crypto-coccosis, resulting in increased survival rates and lower lung burdenthan non-deficient littermates. Interestingly, in Mph IL-4R�/� micethe number of alternatively macrophages is reduced but still detect-able in comparison to IL-4R+/- mice. Moreover, macrophages fromMph IL-4R�/� and IL-4R+/� mice both show the potential to producethe protective effector molecule NO; however, the capacity is higherin macrophages from Mph IL-4R�/� mice. Although Mph IL-4R�/�

mice are more resistant, the pulmonary Th2 response is comparablein both groups. Pulmonary recruitment of eosinophils and mucusproduction are not diminished in the more resistant Mph IL-4R�/�

mice.Discussion/conclusion In a chronic Th 2-dependent inflamma-tory response mice can control pulmonary cryptococcosis by abro-gation of IL-4Ralpha-dependent induction of aaMph. These findingshighlight the importance of IL4Ractivated macrophages in patho-genesis in pulmonary cryptococcosis (Int Immunol 2013; 25(8):459–70).

Funding: German Research Foundation grant DFG AL 371/5-4.

ª 2014 The Authors


Poster Abstracts

P124

The role of interleukin-4 receptor (IL-4R)-dependentpolyfunctional T helper (Th) 2 cells in pulmonarycryptococcosisD. Piehler,1 U. M€uller,1 W. Stenzel,2 A. Grahnert,1 G. K€ohler,3

O. Frey,4 J. Held,2 T. Kamradt,5 F. Brombacher,6 G. Alber,1

M. Eschke1 and T. Richter1

1Insitute 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 path-ogenesis depending on their polarization. A cellular immuneresponse, also called Th1 response, is protective, with classically acti-vated macrophages and IFN-c-producing Th1 cells. On the otherhand, a Th2 response is detrimental in pulmonary cryptococcosisand is associated with alternative activation of macrophages and dif-ferentiation of IL-4-producing Th2 cells. Susceptibility to pulmonarycryptococcosis is associated with loss of fungal growth control andsubsequent 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 pulmonarycryptococcosis model the role of IL4Rdeficient Th cells that are insen-sitive to IL-4 (and IL-13) while other cells are unaffected.Methods Mice with genetically ablated IL-4Ralpha expression on Thcells were infected intranasally and compared to heterozygous litter-mates with heterozygous IL-4Ralpha expression (IL-4R+/-). The sur-vival rate, lung burden, pulmonary histopathology, activation state ofmacrophages, and Th cell cytokine production (measured by ELISAand multiparameter intracellular flow cytometry) was analyzed.Results In this study we show that IL-4R-dependent polyfunctionalTh2 cells simultaneously producing IL-4 and IL-5 or IL-13, areessential in the pathogenesis of pulmonary cryptococcosis. A poly-functional Th2 response enhances pulmonary eosinophil recruitmentand mucus production as well alternative activation of macrophages,resulting in inhibition of fungal growth control. Th cell-specific IL-4Ralpha ablation is able to confer resistance in pulmonarycryptococcosis.Discussion/conclusion Polyfunctional Th2 cells are the main pro-ducers of the pathology-associated Th2 cytokines leading to fatalalternative 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 Cryptococcusneoformans strains in combination with voriconazoleL.V. Filippova,1 N. V. Vasilyeva,1 E. V. Frolova,1

A. E. Uchevatkina1 and E. P. Kiseleva2

1North-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 yeaststhat are not susceptible to earlier azole-type drugs. Clearance offungi depends on the ability of macrophages to destroy the yeastcells, thereby preventing them from spreading. Phagocytosis offungi activates macrophages to production of various microbicidalfactors and cytokines, the balance of which determines the courseof infection.

The aim of this study was to investigate interactions between mur-ine phagocytes and different virulence Cryptococcus neoformans strainsin combination with voriconazole.

Twelve clinical isolates of C. neoformans, received from the RussianCollection of Pathogenic Fungi (RCPF), were investigated. All strainswere isolated from patients with AIDS or after renal transplantation.Virulence of C. neoformans strains was study according to survival timeof Balb/c male mice 8–12 weeks old after intravenous inoculationwith 0.5 9 106 cells per mice. Highly virulent strains have caused50% mortality of mice on 14th day after inoculation. Weakly virulentstrains have caused 50% mortality on 50th day and there was no100% mortality after 65 days of observation. Phagocytosis of C. neo-formans, production of nitric oxide and cytokines were studied in thepresence or absence of voriconazole in a concentration of 2 lg ml�1.

Intact macrophages poorly uptake all C. neoformans strains.Weakly virulent strains were better phagocytosed by macrophages incomparison with results for highly virulent strains. Was establishedthat voriconazole increased the uptake by macrophages highly viru-lent strains of C. neoformans and did not affect their phagocytic activ-ity against weakly virulent strains fungi. These data were obtainedconfirmed the findings of other researchers about the absence of theinhibitory effect of voriconazole on C. neoformans phagocytosis. In thestudy of the macrophages ability to produce nitric oxide after uptakeC. neoformans, established that all strains of fungi in the interactionwith intact macrophages did not induce nitric oxide production, theaddition of voriconazole also did not affect its production. All investi-gated strains activated the synthesis of IL-10, IL-13, and ability offungi to cytokines induction was directly correlated with their viru-lence degree. Thus, we can suppose that the interaction with allstrains of Cryptococcus macrophages acquires alternatively activatedmacrophages signs. Voriconazole did not affect the production ofIL-10 and IL-13 intact peritoneal macrophages, but significantlyinhibited the ability of highly virulent C. neoformans strains inducemacrophages to release IL-13. Perhaps, this is due to the modifyingeffect of on chitin, a part of the fungi cell walls, which could interactwith dectin-1 macrophages. Thus, besides an antifungal action, vo-riconazole may influence on the production of cytokines responsiblefor the antifungal activity of the macrophages. Furthermore, theresults suggest mechanisms by which voriconazole can cooperatewith immune defense systems in controlling of C. neoformansinfections.

P126

Role of an aspartyl protease (PEP1) and anti-PEP1antibodies on the course of experimental cryptococcosisF. Vernel-Pauillac and F. Dromer

Institut Pasteur, Paris, France

Background Cryptococcosis is a severe opportunistic infection stillassociated with a 20% mortality rate despite adequate antifungaltherapy leaving room for improvement. Clinical and experimentaldata suggest that both the host and the pathogen determine the vari-able outcome of infection. In a relevant murine model of dissemi-nated cryptococcosis, we previously found that mice that survive theinoculation of a usually lethal challenge with Cryptococcus neoformans(Cn) were more likely to develop a delayed and monospecific anti-body response against a 40 kDa protein that those which died fromcryptococcosis. The protein was identified as an aspartyl proteasePEP1.Aim To assess whether vaccination with rPep1 and/or serotheraywith anti-PEP1 antibodies could alter the course of the infection.Methods A recombinant protein (rPep1) and several monoclonalantibodies specific for PEP1 (Mab) were produced. The effect of rPep1or Mabs was tested prior to or after inoculation with Cn on survivaland fungal burden in target organs of BALB/c male mice.Results Compared to 100% mortality rate in control mice, activeimmunization with rPep1 prior to Cn inoculation was associatedwith prolonged survival and decreased fungal burden both in H99

ª 2014 The Authors


Poster Abstracts

and NIH52D-infected mice, with 25% and 60% survival rate at day100 post inoculation, respectively and no residual infection in micesurviving NIH52D inoculation. Therapeutic vaccine based on a singleinjection of rPep1 in mice previously infected with Cn provided pro-longed survival with partial control of the infection.

Passive serotherapy with 1 injection of anti-PEP1 Mabs the daybefore, or 1 or 7 days after Cn inoculation led to a prolonged sur-vival (dependent on the Cn strain, the Mab tested, the timing andthe Mab dose) compared to mice treated with an irrelevant Mab oranti-capsular polysaccharide Mab E1. None of the protocol was asso-ciated with sterilisation of the target organs, but reduced fungal bur-den was obtained.Discussion/conclusion These results suggest that immunomodula-tion with PEP1 or anti-PEP1 antibodies may be of benefit during dis-seminated cryptococcosis. Other experiments are ongoing to decipherthe role of the enzyme in the pathogenesis of the infection.

P127

Plasmacytoid dendritic cells and their role during theprotective immune response to experimental pulmonaryCryptococcus neoformans infectionC. 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 basidiomyce-tous fungus that engages in outcrossing, inbreeding, and selfingforms of unisexual reproduction as well as canonical sexual repro-duction between opposite mating-types. Long thought to be clonal,>99% of sampled environmental and clinical isolates of C. neoformansare MATa limiting the frequency of opposite mating-type sexualreproduction. Sexual reproduction allows eukaryotic organisms toexchange genetic information and shuffle their genomes to avoid theirreversible accumulation of deleterious changes that occur in asex-ual populations, known as Muller’s Ratchet.Aims To characterize whether unisexual reproduction, which dis-penses with the requirement for an opposite mating type partner, isable to purge the genome of deleterious mutations.Methods Spores were dissected from unisexual matings of strainscarrying auxotrophic or temperature sensitive mutations. Parentalstrains and F1 progeny were phenotypically characterized forgrowth, competitive growth, and stress responses. Murine and Galle-ria models were infected by parental strains and F1 progeny.Results The unisexual cycle can restore mutant strains of C. neofor-mans to wild-type genotype, phenotype, and growth rate. Further-more, the unisexual cycle allows attenuated strains to purgedeleterious mutations and produce progeny that are returned towild-type virulence. Our results show that unisexual populations ofC. neoformans are able to avoid Muller’s Ratchet and loss of fitnessthrough a unisexual reproduction cycle involving a-a cell fusion,nuclear fusion, and meiosis. Similar types of unisexual reproductionmay operate in other pathogenic and saprobic eukaryotic taxa.Discussion We show that unisexual reproduction between strainsthat carry deleterious mutations generates progeny that havereturned to the wild-type genotype. Unisexual reproduction allows C.neoformans to escape from Muller’s Ratchet and produce phenotypi-cally and genotypically fit offspring. In addition to restoring growthrate, unisex is also able to produce virulent strains from avirulentparents. There are clear implications of these findings for geneticexchange and transmission of drug resistance and these processesmay occur in both the environment and in the host.

P128

Cytokine profile in human blood mononuclear cellsstimulated by GXM from AIDS patients with cryptococcalmeningitisM. 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


Background Globally, Cryptococcus neoformans is the main etiologi-cal agent of cryptococcal meningitis (CM) in AIDS patients. Yearly, itaccounts for one million cases of which 620,000 die particularly insettings where access to anti-retroviral therapy is less available. Cryp-tococcus spp. are yeasts surrounded by a capsule primarily consistedof glucuronoxylomannan (GXM). This capsular polysaccharide is animportant virulence factor able to inhibit opsonophagocytosis, T-cellproliferation and downregulate proinflammatory cytokines produc-tion 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 peripheralblood 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, 32HIV-positive individuals without CM (CM- HIV+) matched by CD4+ T-cells count and age and 47 non-HIV healthy donors (CM� HIV�)paired by gender and age were recruited. Blood was anti-coagulatedwith 20 U ml�1 pyrogen-free heparin and diluted with equal vol-umes of RPMI 1640 medium. PBMC (1 9 106 cells ml�1) were iso-lated by Ficoll-Hypaque density gradient centrifugation. One milliliterwas dispensed in each one of the 24-well plates and incubated at37 °C for 48 h in 5% CO2 with GXM (10 lg ml�1). GXM wasobtained from culture supernatants of serotype A (ATCC 90112) byCTAB-GXM precipitation and identified by Cryptolatex test and GXM-specific monoclonal antibody 18b7. Lipopolysaccharide (LPS)(100 ng ml�1) from Escherichia coli 026:B6 was used as a positivecontrol. Cultures were centrifuged (600 g for 15 min), and the su-pernatants stored at �70 °C until be tested for cytokine concentra-tion by ELISA.Results Of 24 patients, 19 (79.1%) were male, median age of34.2 years. Cryptococcal meningitis was the first AIDS-defining dis-ease in 14 (58.3%) cases, while in 8 (57.1%) both diseases weresimultaneously diagnosed at admission. The CD4+ baseline valueswere <100 cells mm�3 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 proinflam-matory 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 cyto-kines among CM+ HIV+ patients on week 16 when compared tobaseline value (P < 0.05). PBMC stimulated with GXM producedbaseline high levels of IL-4 and IL-10 in CM+ HIV+ patients whichprogressively decreased during treatment (P < 0.05).Discussion These features indicate that soluble GXM is able toinduce cytokine secretion by PBMC in AIDS patients with CM. Puri-fied GXM suppressed the induction of proinflammatory cytokinesbefore and during antifungal therapy. In contrast, GXM stimulatedthe induction of both IL-4 and IL-10 which are detrimental to thecell-mediated immunity and consequent fungal clearance. This maybe clinically relevant, since high concentrations of this capsular anti-gen are frequently found in the body fluids of AIDS patients with CMand are one of the most important negative prognostic factors associ-ated to outcome.

Financial support: FAPEMIG grant BPD0050713.

ª 2014 The Authors


Poster Abstracts

P129

Immunoproteomics and immunoinformatics analysis ofCryptococcus gattii: novel candidate antigens for diagnosisL. 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

1Universidade Federal do Piau�ı, Teresina-Piau�ı/Brasil, Brazil;2Universidade Federal de Minas Gerais, Belo Horizonte, Brazil;3Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil;4Fundac�~ao Oswaldo Cruz, Rio de Janeiro, Brazil and 5Instituto de

Doenc�as Tropicais Natan Portella, Teresina, Brazil

The potential for C. gattii to cause illness in immunocompetentpatients and its rapid spread worldwide justify the implementation ofa public health effort to increase the awareness of both the publicand healthcare professional. Information from the recently publishedC. gattii genome, combined with data obtained via proteomicapproaches, has created opportunities for the development of diag-nostic tools and therapeutic targets in the context of cryptococcosiscaused by C. gattii. The development of such tools has been improvedby the production of antigens, which increases the range of alterna-tive tests for immunoassay-based pathogen detection. In this study,we identify both immunoreactive proteins of C. gattii and predictedB-cell epitopes for their potential use as antigens in new serologictests. We combined 2D gel electrophoresis, immunoblotting and massspectrometry to identify immunoreactive proteins from four strains ofC. gattii genotype VGII (CG01, CG02, CG03 and R265). Next, wescreened the identified proteins to map B-cell epitopes. We identified68 immunoreactive proteins and highlight that only six of themwere reac�tive in three isolates simultaneously without a reactionfor negative sera. Due to their antigenicity in different strains, webelieve that these proteins would be most promising for testing asantigens. Taken together, assessing the specificity of these antigenswill be the next step to validate them for the diagnosis of cryptococ-cosis. The major finding of this work was the identification of C. gattiiproteins recognized as molecular targets by anti�bodies produced bypatients with cryptococcal meningitis. In addition, we mapped 374peptides potentially targeted by B cells. Considering the evolutionaryrelevance of the identified proteins, we may speculate that they couldbe used as the initial targets for recombinant protein and peptidesynthesis aimed at the development of immunodiagnostic tools forcryptococcosis. In conclusion, the applied combination of immuno-proteomics and immunoinformat�ics methods was demonstrated tobe a specific and powerful tool for identifying novel antigens frommycological pathogens. In addition, we identified potential antigensthat may be used for the development of more accurate serologicaldiagnoses in human cryptococcal meningitis. The immunogenic pro-teins and Bcell epitopes identified herein need to be investigated asantigens in serological tests to diagnose cryptococcal infection. Theseproteins could present cross-reaction with cryptococcal infectioncaused by other species. Although these proteins can react with mul-tiple fungal species, some of these fungi may not be present in thepatient. However, this is the first important step to selecting new tar-get antigens to facilitate the immunodiagnosis of cryptococcosis.

P130

Impaired monocyte production of TNF-alpha and reducedHLADR expression on CD14+ CD16+ monocytes areassociated with mortality in cryptococcal meningitisJ. 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

1Liverpool 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

Introduction Cryptococcal meningitis (CM) remains a significantcause of death amongst individuals with HIV-1 infection. Immunecorrelates of protection are lacking. Animal and in vitro studies sug-gest macrophage and monocyte activation plays a key role in deter-mining outcome from infection.Aims To determine whether abnormal monocyte activation is associ-ated with poor outcome in individuals with HIV-associated crypto-coccal meningitis.Methods Patients with HIV-1-associated CM were recruited fromhospitals in Cape Town. Initial anti-fungal therapy was IV amphoter-icin 1 mg kg�1 and oral fluconazole 800 mg daily for 14 days.Monocyte sub-populations and their activation phenotype were char-acterized in whole blood at presentation using flow cytometry. Intra-cellular cytokine responses were assessed following 6-hourstimulation with lipopolysaccharide (TLR4 agonist) and R848(TLR7/8 agonist). Comparisons in baseline monocyte activation weremade between subjects who survived to 14 days and those who died.Student’s T-test was used for normally distributed variables; non-nor-mally distributed variables were either log transformed prior to para-metric testing or a Mann–Whitney U test was used.Results (see table) Fifty nine subjects were enrolled; median CD4count was 34 9 106/l and 14-day mortality was 24%. At presenta-tion, non-survivors had a significantly lower proportion of circulatingmonocytes, significantly reduced expression of HLA-DR on inflamma-tory monocytes (CD14+CD16+), and a non-significant trend towardsincreased surface expression of CD163 on inflammatory monocytes(CD14+CD16+). Non-survivors were also found to have significantlyhigher baseline serum concentrations of soluble CD163 and C-reac-tive protein (see table). In addition, monocytes from non-survivorshad impaired responses to lipopolysaccharide, with significantly fewercells producing TNFa. No differences in monocyte responses wereseen following TLR7/8 stimulation (see Table).Conclusions This study suggests impaired monocyte function maybe an important factor determining poor outcome in cryptococcaldisease. Despite serum markers suggesting significant baselineimmune activation, circulating monocytes from patients who diedfrom cryptococcal meningitis had a less pro-inflammatory phenotypeand appeared to be functionally unreactive. Reversal of this defectmay be a potential immune-based therapeutic strategy in HIV-associ-ated cryptococcosis.

Table 1 Differences in immune parameters at presentation betweenpatients with CM who died before day 14 and those who survived.

Survivor Non-survivor P value

CD4 count (x106/L) (median, IQR) 29 [11-85] 33 [13-55] 0.72

Monocytes, (%WBC) (mean, 95%CI) 8.0 [6.9-9.0] 5.6 [3.5-7.7] 0.04

HLADR MFI (CD14+CD16+ Mo)

(mean, 95%CI)

7272 [6154-8390] 4197 [2340-5653] 0.007

Log10 CD163 MFI (CD14+CD16+ Mo)

(mean, 95%CI)

3.91 [3.82-4.00] 4.10 [3.86-4.32] 0.08

%TNFa +ve Mo (after LPS stimulation)

(mean, 95%CI)

44% [39-50] 26% [15-37] 0.002

%TNFa +ve Mo (after R848 stimulation)

(mean, 95%CI)

68.1 [60.0-76.3] 64.7 [53.1-76.4] 0.66

sCD163 (ng/ml) (median, IQR) 1033.5 (765.5-1480.5) 1410 [1196-1856] 0.02

CRP (mg/L) (median, IQR) 36.4 (13-68.3) 84.9 (46.5-115) 0.01

ª 2014 The Authors


Poster Abstracts

P131

Various expression patterns of cytokines and chemokinescan be observed after interaction with clinical isolates ofCryptococcus neoformansA. Sturny-Leclere, A. Alanio, F. Vernel-Pauillac, M. Gougeon and

F. Dromer

Institut Pasteur, France

Background Macrophages play a central role in cryptococcocispathogenesis, representing the first line of defense, a potential site foryeast replication and dormancy, and also probably a vehicle for yeastdissemination. We previously demonstrated that clinical isolates ofCryptococcus neoformans showed variations in their interaction withmacrophages (phagocytosis and intracellular proliferation index ofthe yeasts) that were associated with differences in clinical outcomeof the patients (1).Objective and methods First, we investigated the diversity of mac-rophage cellular response following their interaction with 9 clinicalisolates previously selected for their phenotype, in comparison withthe reference strain H99 (1). The in vitro response of J774 macro-phage cell line was assessed by quantifying the production of solublemediators (23-plex Luminex�O) including pro- and anti-inflammatorycytokines (TNF-a, IL-1b IL-6, IL-10) and chemokines (MCP-1, MIP-1a, MIP-1b, RANTES) in kinetics experiments. Second, we performedin vivo experiments in OF1 outbred mice with 2 clinical isolates har-boring low and high virulence compared to H99. We evaluated fun-gal load, tissue lesions and mediators production in individual sera,brains and lungs of 7 mice/group.Results and discussion Among the clinical isolates studied, isolatesSC5 and SC6 that differed in terms of virulence (median survival of29 and 9 days post inoculation, respectively with a median CFU/g oftissue in mice sacrificed on dpi 7 differing by more than 1 log respec-tively), and phagocytic index ratio compared to H99 (PI = 0.5 andPI = 1.21, respectively) exhibited drastically different patterns ofcytokine/chemokine production in vitro and in vivo. In addition, in vi-tro analysis of the nine clinical isolates identified two major cytokine/chemokine profiles that were associated with phagocytic index andvirulence. These data provide additional evidence on the influence offungal diversity on the host immune response.Reference 1. Alanio A et al. mBio. 2011; 2(4): e00158–11.

P132

Transient blockade of IL-10 signaling enhances fungalclearance and Th1/Th17 effector responses in mice withearly and established Cryptococcal lung infectionM. A. Olszewski,1 B. J. Murdock,1 G. H. Chen,1 G. B. Huffnagle2

and J. Osterholzer1

1VA Ann Arbor/University of Michigan, Ann Arbor, MI, USA and2University of Michigan, Ann Arbor, MI, USA

Background Pulmonary infection with Cryptococcus neoformans(Cneo), a fungal pathogen acquired by the inhalational route, canresult in progressive infection and death or persistent infection asso-ciated with chronic lung inflammation and features of allergic airwayimmunopathology. Infection of C57BL/6 mice with Cneo recapitulatesthis persistently-infected phenotype. Studies performed in IL-10 defi-cient mice show that IL-10 is a critical determinant of this phenotypeby skewing the adaptive immune response away from the develop-ment of protective Th1/Th17 responses and towards ineffective Th2/Treg responses.Aim To determine whether transient antibody-mediated blockade ofIL-10 signaling during either the: (i) developing, or (ii) establishedphase of infection enhances effector immune responses against Cneoin C57BL/6 mice.

Methods C57BL/6 mice were infected by the intratracheal routewith Cneo (strain 52D). Two cohorts of mice received either: (i) anti-IL-10 receptor antibody or (ii) an isotype control (0.5 mg per 200 mlPBS by the intraperitoneal route) at 3, 7, and 10 days post-infection(dpi) to assess the effect of IL-10 blockade during the developingphase of infection. Two separate cohorts of mice received the sameantibodies at 22, 25, and 28 dpi to evaluate the effect of IL-10 block-ade during the established phase of infection. At 35 dpi, all fourcohorts of mice were euthanized and evaluated for the followingimmune parameters: (i) lung and brain fungal burden (by CFUassay), (ii) the percentage and total numbers of numerous lung leu-kocyte subsets (by specific antibody staining and 8–10 parameterflow cytometric analysis), and (iii) CD4+ T cell subsets (by intracellu-lar cytokine staining and flow cytometric analysis).Results Blockade of IL-10 signaling during either the developingphase or during the established phase of persistent cryptococcal lunginfection significantly improved fungal clearance at 35 dpi. Improvedclearance 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 notalveolar) macrophages in the lungs.Conclusion Blockade of IL-10 signaling promotes the clearance ofcryptococcal lung infection, likely through mechanisms involving theDC-mediated enhancement of Th1 and Th17 immune responses andthe subsequent accumulation of fungicidal exudate macrophages.Thus, IL-10 signaling blockade represents an attractive therapeutictarget in patients with newly-acquired or established fungal lunginfection.

Funding: Biomedical Research & Development Service, Dept. ofVeterans Affairs, Career Development Award (JJO) and Merit ReviewAward (MAO). NIH T32 Training Grant (trainee BJM).

P133

TNF-alpha-induced stability of DC1 programming isrequired for maintenance of protective Th1/Th17 immuneresponse against Cryptococcus neoformansA. 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

1University of Michigan, Ann Arbor, MI, USA and 2University of

Michigan/Ann Arbor VA Hospital, Ann Arbor, MI, USA

Background The cytokine tumor necrosis factor alpha (TNF-a) isrecognized as being critical to mount an effective immune responseto C. neoformans (C. neo): C. neo strains that induce less host-derivedTNF-a are more pathogenic, AIDS patients that do not produce sub-stantial TNF-a have poor prognosis against C. neo infection, andpatients undergoing TNF-a monoclonal antibody therapy are moresusceptible to C. neo infections. Th1/Th17-polarized T cell responsessupport clearance/containment of C. neo, while Th2 polarization isnon-protective. Dendritic cells (DC) that prime a Th1/Th17 responsedisplay DC1/classical activation phenotype characterized by produc-tion of Th1/Th17-driving cytokines and high levels of iNOS expres-sion. DC2 that prime a Th2 response are reported to display featuresof an alternative activation phenotype including increased expressionof Arginase1, Fizz, and YM-2 and increased production of Th2/regu-latory cytokines. While TNF-a has been demonstrated to promote DCmaturation, it is unknown how TNF-a signaling affects the balancebetween these DC1/DC2-activation phenotypes and how TNF-aaffects DC-phenotype stability during C. neo infection.Aim To determine the mechanisms by which early TNF-a signalingpromotes generation and stability of protective Th1/Th17 response toC. neo, we tested whether TNF-a signaling is necessary to generatestable DC1, thereby supporting the development and long-term stabil-ity of a protective Th1/Th17 response.Methods CBA/J mice (resistant) received TNF-a antibody or isotypecontrol antibody injected into the peritoneum at the time of

ª 2014 The Authors


Poster Abstracts

intratracheal infection with C. neo 24067. This treatment neutralizesTNF-a for the initial 7 days of infection with TNF-a levels recoveringby d14. However, the immune response remains non-protective pastd28. We harvested mice at d7, d14 and d28 post-infection and char-acterized DC and T cell activation profiles by qPCR and flow cytome-try and assessed the fungal lung burden and brain and spleendissemination. We then investigated how TNF-a signaling affects DCactivation and stability of DC1 or DC2 phenotypes. We generatedbone marrow-derived DC (BMDC) and subjected them to 24 hours ofinterferon-gamma (IFN-g) stimulation with or without TNF-a, thenswitched the cytokine stimulation to interleukin-4 (IL4) for 24 h andassessed DC activation and phenotype markers by qPCR and flowcytometry. Lastly, we investigated the link between DC epigenetic sig-natures and phenotype stability by measuring expression of histonemodification enzymes and immunoassays for activating and repress-ing histone methylations.Results In vivo we found that the robust Th1/Th17 response in con-trol mice was replaced by a non-protective response with many Th2features and fluctuating cytokine levels in the TNF-a antibody-treatedinfected group. The DC1 signature in CD11c+ cells was suppressedthroughout infection (d28), and similar suppression could be detectedin myeloid DC precursors (CD11b+) early in the TNF-a antibody-trea-ted mice (d7). Furthermore, the activating histone modificationH3K4 trimethylation was diminished in myeloid precursor cells (d7)and lung DC (d7 and 28) of TNF-a antibody-treated mice, suggestingdecreased activation of certain genes at the epigenetic level. Levels ofthe H3K27 methyltransferase EZH2 were also decreased in the DCof TNF-a antibody-treated mice, suggesting less epigenetic silencingof certain genes. In vitro, the combination of TNF-a and IFN-g reducethe induction of the DC2 marker Fizz and DC2 cytokine Interleukin-10 and increase DC1 cytokines Interleukin-12a and 12b when theDC are switched to IL4.Discussion We conclude that TNF-a contributes to programing DCto become stable DC1, which promote and sustain protective Th1/Th17 responses to C. neoformans. Our results suggest that TNF-a-induced chromatin modification in DC precursors and lung DC sup-port DC1 stability. Studies on the effect of TNF-a on the epigeneticsignatures profiles, the specific genes epigenetically silenced or acti-vated, and how this relates to pulmonary DC1 stability are currentlyunderway.

P134

Differences in host response toward C. neoformans andC. gattiiE. 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-asso-ciated opportunistic infections with an estimated global burden ofone million cases with 600 000 deaths annually. Although bothCryptococcus neoformans and C. gattii are the etiologic agents of cryp-tococcosis, the AIDS associated infection is mostly caused by C. neo-formans and only occasionally by C. gattii even in the C. gattiiendemic regions. C. gattii apparently affects immunocompetent hostsmore often than immunocompromised hosts, hence it is consideredas a primary pathogen. To understand the differences in the host-pathogen interactions of the two species, we compared the pathoge-nicity of the two species in mice with defects in the immune systemcritical for defense against infectious agents. During acute infectionwith HIV, several antiviral and immunoregulatory cytokines, particu-larly type I interferon (IFN), are known to be up-regulated and type IIFN has been used in therapy for several viral infections and malig-nancies. We investigated the effect of exogenous induction of type IIFN during infection by the two cryptococcosis agents.

Aim To address this question, we compared the effect of polyinosinic-polycytidylic acid condensed with poly-l-lysine and carboxymethylcel-lulose (Poly-ICLC), a stabilized version of Poly-IC, in murine crypto-coccosis caused by the two species.Methods Experimental models of pulmonary and systemic cryptococ-cosis were established using wild type (C57BL/6) and knock-out (If-nabr�/�, TLR3�/�, MDA5�/�) mice to evaluate survival, tissue fungalload and histopathology with and without Poly-ICLC treatment.Quantitative RT-PCR, enzyme-linked immunosorbent assays wereused for measurements of type I IFN mRNA and cytokine detection,respectively.Results Poly-ICLC is a synthetic analogue of viral double-strandedRNA (dsRNA) which is designed to stimulate prolonged, high-levelproduction of type I IFN. We observed a protective effect of Poly-ICLCin both C. neoformans and C. gattii infection.As expected, the micelacking type I IFN-receptor 1 (Ifnabr KO mice) were significantlymore susceptible to both species than the wild type mice. Interest-ingly, however, while Poly-ICLC was not protective for Ifnabr-defi-cient mice from C. neoformans infection, it protected the mice from C.gattii infection. Our results suggest that Poly-ICLC induced protectionagainst C. neoformans is type I interferon dependent but not againstC. gattii.Conclusion Our study indicates the existence of major differences inhost defense against the two species. In addition, the study suggeststhat Poly-ICLC can be an alternative anticryptococcal agent that canbe used for cryptococcosis maintenance therapy.

P135

Capsule growth in Cryptococcus neoformans iscoordinated with cell cycle progressionR. García-Rodas,1 R. J. Cordero,2 G. Janbon,3 F. Moyrand,3

A. Casadevall4 and O. Zaragoza1

1National 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

Background The most important virulence factor of the fungalpathogen Cryptococcus neoformans is a polysaccharide capsule thatsurrounds the cell. The size of the capsule is variable and it increasesduring infection. However, the pathways and genes involved in thisprocess are unknown.Aim In this work, we investigated the relationship between capsuleenlargement and cell cycle progression in C. neoformans. We hypothe-sized that capsule enlargement was regulated by the cell cycle for thefollowing reasons: (i) During capsule growth, there is a correlationbetween the size of the capsule and the size of the cell body, so webelieve that the factors that regulate the cell body growth (whichoccurs in G1) also regulate the capsule size; and (ii) We argue thatcapsule enlargement stops during G2-M because in these phases, theaccumulation of new polysaccharide at the capsule might interferewith the emergence and separation of the bud from the mother cells.For these reasons, we hypothesized that capsule growth occursmainly in the G1 phase of the cell cycle.Methods We used flow cytometry, time-lapse microscopy and cellcycle inhibitors to study if cell cycle changed during capsule enlarge-ment. Furthermore, we characterized a mutant strain lacking a genethat encodes a G1-S cyclin (cln1). We studied capsular phenotypes ofthis mutant and its reconstituted strain both in vitro and in vivousing the invertebrate model Galleria mellonella.Results Capsule growth occurred primarily during the G1 phase.Real-time visualization of capsule growth demonstrated that this pro-cess occurred before the appearance of the bud and that capsulegrowth arrested during budding. Besides, benomyl, a cell cycle inhibi-tor that arrests the cells in G2/M, impaired capsule growth. However,

ª 2014 The Authors


Poster Abstracts

rapamycin, which causes G1 arrest, increased capsule size. The cln1mutant showed a delay in S entry, and also an increased capacity toenlarge the capsule, both in vivo (using Galleria mellonella as hostmodel) and in vitro. The cln1 mutant displayed morphological altera-tions, such as a different WGA binding pattern and aberrant formsduring budding. Besides, it was avirulent at 37 °C, which correlatedwith growth defects at this temperature.Discusion/conclusion Our results support a model in which cap-sule enlargement is a differentiation process that causes G1 arrest.Our model also suggests that cell cycle progression in these condi-tions does not occur until the capsule has reached a certain size. Inaddition, the reduced virulence of cln1 mutant opens the possibilityto design antifungal strategies using cell cycle regulatory elements astargets.

P136

The role of aspartyl aminopeptidase (APE4) inCryptococcus neoformans virulence and authophagyM. A. Vallim,1 F. A. Gontijo,1 R. C. Pascon,1 J. Machado-Jr1 and

L. F. Fernandes2

1Universidade Federal de S~ao Paulo, Diadema, Brazil and2Universidade de Bras�ılia, Brasilia, Brazil

In Saccharomyce cerevisiae the M18 metaloprotease Ape4 is activatedby nitrogen deprivation, its protease activity is against the aspartateand glutamate aminoacids located at the protein amino-terminus.This protein is engaged in autophagy and also is part of the cyto-plasm to vacuole targeting pathway (Cvt), which is composed by theproteins Ams1, Ape1, Ape4 e Atg19. The Ape4 protein under nitro-gen starving growth condition is located in the autophagosome. Lackof the Ape4 protein in S. cerevisae does not lead to high temperaturesensitivity whereas the ape4 Cryptococcus neoformans mutant is inca-pable to grow at 37 °C. This information was gathered for this yeastwhen a collection of mutants generated by insertional mutagenesiswas subject to a high temperature screening. To confirm that indeedApe4 was the responsible for this phenotype an independent geneinterruption was carried out generating a new mutant which wasunable to grow at 37 °C. We demonstrated that the C. neoformansape4 mutant has defects for important virulence factors as melaninand phospholipase production, capsule formation, and was unable tosurvive inside the macrophage (linage J774A.1) compared to thewild type. These date set suggested that the ape4 mutant have atten-uated virulence. This hypothesis was confirmed by the animal experi-ment where the mutant was unable to kill the infected animals,whereas the wild type and the reconstituted (ape4D+APE4) strainswhere effective in causing the mice death within about 22 days post-infection. In S. cerevisae under nitrogen deprivation the Ape4 proteinmigrates to the vacuole where it is active. We have fused C. neofor-mans Ape4 protein to GFP and we intend to demonstrate the cellularlocalization for this protein. Also, we have cloned the APE4 gene ina protein expression vector and we are going to isolate and purifythis protein to characterize its amino acid target in vitro employing apolypeptide library.

Fapep: 2007/50536-3.

P137

Golgi reassembly and stacking protein is involved in thevesicular traffic of polysaccharides in CryptococcusneoformansL. 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

1Universidade Federal do Rio de Janeiro/Instituto de

Microbiologia Paulo de G�oes, Rio de Janeiro, Brazil; 2Fundac�~aoOswaldo Cruz - Fiocruz, Rio de Janeiro, Brazil; 3Universidade

Federal do Rio de Janeiro - UFRJ, Rio de Janeiro, Brazil;4Universidade Federal do Rio Grande do Sul - UFRGS, Porto

Alegre, Brazil and 5UFRJ & Fiocruz, Rio de Janeiro, Brazil

We have recently described that Golgi reassembly and stacking pro-tein (GRASP) is required for polysaccharide secretion and virulencein Cryptococcus neoformans. We have also observed in previous studiesthat secretion of capsular polysaccharides in C. neoformans involvesextracellular vesicle (EV) formation. Since GRASP is potentiallyinvolved in vesicular secretion in other eukaryotes, we hypothesizedthat EV-mediated polysaccharide export and GRASP are functionallyconnected in C. neoformans. In silico analysis of protein structurerevealed that C. neoformans GRASP contains unique regions, in com-parison to its human homologues. Predictive studies suggested thatthe fungal-specific GRASP regions have low hydrophobicity and highantigenic potential. Wild type C. neoformans cells and a mutant lack-ing GRASP expression (Dgrasp) had comparable levels of EV forma-tion. However, polysaccharide concentration in EVs produced by theDgrasp mutant was decreased, in comparison with WT cells. Vesicu-lar polysaccharide fibers produced by the Dgrasp mutant also showedreduced dimensions, as determined by dynamic light scattering. EVsobtained from cultures of the Dgrasp mutant also differed from thoseproduced by WT cells in diameter distribution. Analysis of the C. neo-formans surface architecture by scanning electron microscopyrevealed that, in comparison to WT cells, the mutant had capsularpolysaccharide fibers with reduced dimensions. These results weresuggestive of defects in polysaccharide traffic. We also evaluatedwhether lack of GRASP would interfere with polysaccharide synthesisin C. neoformans. Analysis of crude cellular extracts revealed that theDgrasp mutant was in fact less efficient than WT cells in synthesizingcapsular polysaccharides. These results demonstrated that GRASP isrequired for both polysaccharide synthesis and EV-mediated traffic inC. neoformans. Since polysaccharides are determinant for virulence inthis fungus, we conclude that EV formation, GRASP and pathogene-sis are directly connected in the C. neoformans model.

P138

Galleria mellonella model identifies highly virulent strainsamong all major Cryptococcus gattii molecular typesC. Firacative and W. Meyer

The University of Sydney, Westmead, NSW, Australia

Background Worldwide cryptococcosis is mainly caused by Crypto-coccus neoformans. However, the number of cases due to C. gattii isincreasing, affecting mainly immunocompetent hosts. By differentmolecular methods C. gattii has been divided into four major molecu-lar types VGI to VGIV, which differ in their host range, epidemiology,antifungal susceptibility and geographic distribution. Besides studieson the Vancouver Island outbreak strains, which showed that thesub-genotype VGIIa is highly virulent compared to the sub-genotypeVGIIb, little is known about the virulence of the other major molecu-lar types.

ª 2014 The Authors


Poster Abstracts

Aim To investigate the virulence potential of C. gattii strains of allmajor molecular types and different MLST genotypes, by comparingtheir pathogenesis and assessing the cryptococcal virulence factors inan invertebrate model of infection.Methods Galleria mellonella larvae were inoculated with ten globallyselected strains per molecular type, which were previously studied byMLST. Larvae were checked daily, deaths were recorded and survivalplots were constructed. Cells of all C. gattii strains were isolated fromthe larvae after inoculation and cell and capsule sizes were mea-sured. Cell and capsule sizes of the strains before inoculation werealso measured. Melanin production and growth at 37 °C was deter-mined in all strains.Results Among the studied strains, one VGII, one VGIII and oneVGIV strains were more virulent (P < 0.05) than the highly virulentVancouver 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 eightVGIV) were less virulent (P < 0.05). Overall, strains of the moleculartype VGIV showed to be the leats virulent. Cell and capsule size of allstrains increased drastically during larvae infection (P < 0.001). Mel-anin production was directly associated with the level of virulence ofthe strains: highly virulent strains produced more melanin that lowvirulent strains (P < 0.05). All strains had the ability to grow at37 °C and there was no difference among the strains regarding theirgrowth rate (P > 0.05).Discussion/conclusion The results indicate that all major C. gattiimolecular types exhibit a range of virulence, with the potential ofsome strains amongst them being highly virulent. As reported in C.neoformans, the present study supports the fact that fungal virulencefactors like capsule, melanin and growth at physiological tempera-ture, which are involved in mammalian pathogenesis, are alsoneeded for G. mellonella infection and killing. This study highlightsthe importance to determine the major molecular type/genotype ofthe isolates as part of the epidemiology of Cryptococcus and crypto-coccosis and warrants further studies to identify specific genotypictraits of highly virulent C. gattii strains that are associated with theirvirulence and which are relevant for the development and progressof cryptococcosis.

P139

Capsule synthesis occurs in a non-polarized manner and itsgrowth depends on mitochondrial activityN. Trevijano-Contador, S. Landin, R. Garc�ıa-Rodas and

O. Zaragoza

National Center of Microbiology, ISCIII, Madrid, Spain

Background The dynamics of the capsule of Cryptococcus neoformansis an important feature to understand the biology of the main viru-lence factor of this pathogen. The capsule is synthesized by additionof new polysaccharide, but it is not known if the synthesis follows apolarized or dispersed pattern. In addition, the size of the capsule isnot constant, and during infection it increases significantly. It isbelieved that capsule growth is an energy-cost process, but thisaspect has never been addressed.Aim We have investigated the dynamics of capsule formation andthe metabolic dependence of capsule growth, the dependence of cap-sule enlargement on mitochondrial activity. In particular, we wantedto determine if capsule synthesis is polarized from a specific site ofthe capsule. Concerning capsule growth, we have investigated theimportance of mitochondrial activity on this process.Methods We used an acapsular strain (cap59) described by Kwon-chung (1) in which the CAP59 gene has been reintroduced underthe control of the GAL7 promoter, so this strain is acapsular in glu-cose and encapsulated when transferred to galactose. In addition, wehave evaluated the role of mitochondrial activity on capsule growth.For this purpose, we have studied the effect of specific inhibitors ofthe electron respiratory chain on capsule enlargement. Furthermore,we have estimated if during this process there are any changes in

mitochondrial activity by measuring cytochrome c oxidase and mito-chondrial 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 togalactose-containing media. When we examined the dynamics ofcapsule appearance, we have observed that the capsule first appearsas spots randomly distributed throughout the cell, suggesting thatthis process occurs in a non-polarized manner. We have also investi-gated the role of mitochondrial activity on capsule enlargement. Forthis purpose, we used different metabolic inhibitors: rotenone, whichinhibits complex I, Antimycin A which inhibits complex III and Sali-cylhidroxamic acid that blocks the alternative cytochrome oxidasepathway. In all the cases, we used concentrations that inhibited celldivision, but that did not affect the viability. We observed that addi-tion of these inhibitors impairs capsule growth, confirming that thisprocess requires full mitochondrial activity. Current experiments areongoing to determine if during capsule growth there are changes inmitochondrial activity.Discusion/conclusion Our results highlight new aspects and ele-ments involved in capsule dynamics in C. neoformans. We have dem-onstrated that capsule synthesis occurs randomly throughout thecells, which is in agreement that capsule synthesis is linked with ves-icle secretion. We have also confirmed that capsule enlargement isan energy cost process that requires mitochondrial activity. This isan important aspect, because capsule growth normally occurs inconditions of nutrient limitation. So our results indicate that in theseconditions, an important amount of the limited energy supply isinvested in the growth of the capsule, which reflects the importanceof this process to survive in stress conditions and in the hostenvironment.Reference 1. Chang and Kwon-Chung. 1994. MCB, 14: 4912–4919.

P140

Role of GTI1 homologue on Cryptococcus neoformansvirulenceL. S. Derengowski, H. C. Paes, L. F. Fernandes, M. S. Felipe and

I. Silva-Pereira

Universidade de Bras�ılia, Brazil

Background Cyclic AMP independent pathways using Gti1/Pac2transcription factors have been shown to play major role in adapta-tion of ascomycetes to the environment and hosts. Among other

Figure 1. Morphology of gti1D::HYG colony grown on Sabouraudmedium at 30 °C for 48 h. The mutant strain grows as donut-shaped colony.

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Poster Abstracts

functions, these factors influence secondary metabolism, conidiation,matting, dimorphic transition and pathogenesis in fungi rangingfrom plants to humans pathogens. However, nothing is known abouttheir function in basidiomycetes, despite the fact that their homo-logues have been annotated in genome projects.Aim In order to fill a gap in our knowledge of regulatory processesin C. neoformans, we have deleted the GTI1 homologue and analyzedthe phenotype of the mutant.Methods Identification of the target gene: The Gti1 homologue wasfound by using the amino acid sequence of Gti1 from Schizosacchar-omyces pombe as query for the BLASTP2 of the Broad Institute H99C. neoformans genome database. The retrieved best hit,CNAG_05835.7, was provisionally named Gti1. Deletion and recon-stitution: The gti1::HYG strain was generated by double-joint PCRfollowed by biolistic transformation of the H99 wild type as describedby Kim et al., 2009. The reconstituted strain was generated by trans-forming the gti1::HYG strain with the Gti1 gene a long side pJAF1plasmid containing the Neor resistance mark. In both cases transfor-mants were selected in YPD solid medium containing hygromicin orG418, respectively. Single insertion into to appropriated locus wasconfirmed by Southern Blot or PCR. Phenotypic analysis: The mutantand reconstituted strains were tested in vitro for common phenotypesassociated with virulence, namely capsule induction, melanization,growth at 37 °C, urease and phospholipase secretion and resistanceto several cell wall stressors. Virulence assays: First, the strains weretested for virulence in the in vitro J774 murine macrophage model ofphagocytosis followed by CFU counts. The in vivo virulence of thestrains was assessed by survival curves in the Galleria mellonella alter-native model of infection at 37 °C.Results The gti1::HYG strain is able to grow at 37 °C similarly tothe wild type strain. In addition, the mutant has no defect on cap-sule induction, melanin production and urease and phospholipasesecretion. Curiously, the gti1::HYG strain grows as donut-shapedcolony on Sabouraud (Fig. 1) and YPD agar (not shown). Interest-ingly, it is not capable of growing on DMEM/MOPS medium.Finally, the knockout of GTI1 gene impaired survival of the funguswithin macrophages in vitro and the mutant also seems to be hypo-virulent in the invertebrate host G. mellonella comparing to thewild type.Discussion The present results suggest a partial loss of virulence ofthe mutant strain in G. mollonella model which we will be follow upby assessment of virulence in the murine model. The donut-shapedcolony is a striking observation, giving that single cells showed no dif-ference in morphology relative to wild type cells. Whether this is dueto different growth patterns of cells in different areas of the colony, orto an altered secretion pattern by the mutant, remains to be identi-fied. Work with the Pac2 homologue is also in progress in our group.

P141

The vacuolar protein Snf7 regulates export of virulencedeterminants in Cryptococcus neoformansR. M. C. Godinho,1 J. Crestani,2 L. Kmetzsch,2 C. C. Staats,2

A. Schrank,2 M. H. Vainstein2 and M. L. Rodrigues3

1Universidade 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 & Fundac�~aoOswaldo Cruz/CDTS, Rio de Janeiro, Brazil

Cryptococcus neoformans (CN) uses a wide range of pathogenic mecha-nisms to cause damage to host cells, including formation of a polysac-charide capsule, secretion of lytic enzymes and ability to melanize.Several of the virulence determinants in CN are exported throughunconventional secretion mechanisms that require exosome-like,extracellular vesicles. Despite their potential importance for pathoge-nicity, the origin of CN extracellular vesicles remains obscure. Exo-some formation requires a number of functional proteins, includingthose operating in the Endosomal Sorting Complex Required for

Transport (ESCRT)-III. In this study, the role of the ESCRT-III vacuolarprotein Snf7 in the export of CN virulence factors was evaluatedthrough the phenotypic characterization of snf7D and snf7D::SNF7(reconstituted) strains generated on the H99 background. The snf7Dmutant showed increased sensitivity to alkaline pH and high LiCl2concentrations, in comparison to wild type (WT) and snf7D::SNF7strains. The snf7D mutant also displayed reduced efficacy to producemelanin when cultivated in both Niger seed agar and L-DOPA at37 °C. In comparison to WT and reconstituted cells, the snf7D strainmanifested severely reduced glucuronoxylomannan (GXM) secretion.This phenotype was linked to a drastic reduction in capsule size, asconcluded from counterstaining of fungal cells with India ink, immu-nofluorescence using an anti-GXM monoclonal antibody (18B7) andscanning electron microscopy. Lack of Snf7p was also associated witha significantly reduced ability of CN to kill mice in an intranasal infec-tion model of cryptococcosis. These results demonstrated that Snf7pregulates unconventional secretion mechanisms involved in therelease of several CN virulence factors, suggesting that the cryptococ-cal ESCRT machinery plays a central role during infection by C.neoformans.

P142

Cryptococcus neoformans clinical isolates from Serbia -genotypes, antifungal susceptibility and virulenceM. G. Pekmezovic,1 A. Barac,2 V. S. Arsic Arsenijevic,2 F. Hagen3

and J. F. Meis3

1Faculty of Medicine, University of Belgrade, Belgrade, Serbia;2Institute of Microbiology and Immunology, Belgrade, Serbia and3Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands

Background Cryptococcus neoformans causes systemic fungal infec-tion mainly in individuals with a suppressed immune system. It isestimated that approximately 1 million cases of cryptococcosis occurthroughout the world that result in 650 000 associated deathsannually. This species consists of varieties: C. neoformans var. grubii(serotype A) and C. neoformans var. neoformans (serotype D), as wellas hybrids between both cryptococcal varieties (serotype AD). Ampli-fied fragment length polymorphism (AFLP) fingerprinting has identi-fied 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 orMAT-alpha cell. MAT-alpha strains have been shown to be muchmore prevalent and more virulent. Besides the presence of MAT-alpha locus, ability to grow at 37 °C, capsule formation and the pro-duction of melanin, urease and phospholipase have been identified asvirulence factors of C. neoformans strains. Studies of cryptococcosis inanimal models infected with different strains of C. neoformans showedthat individual isolates differ in virulence, susceptibility and ecologi-cal characteristics.Aim The purposes of this study were: (i) to identify the genotypes,serotypes and mating types; (ii) to determine antifungal susceptibilityprofile; (iii) to analyze virulence factors of C. neoformans clinical iso-lates from Serbia.Methods This study investigated 34 clinical Serbian C. neoformansisolates from cerebrospinal fluid and blood of 25 immunocompromisedpatients in the 20-year period. AFLP fingerprinting was used for geno-typing, while real-time PCR was used to determine the mating- andserotype. The MICRONAUT plates were used to determine susceptibil-ity to amphotericin B, 5-fluorocytosine, fluconazole, itraconazole,posaconazole and voriconazole, according to the CLSI standardizedprotocol. Each strain was examined for production of: (i) capsule byIndia ink staining; (ii) melanin by culturing of minimal L-DOPA med-ium; (iii) urease by culturing on Christensen’s agar and (iv) extracellu-lar phospholipase by culturing on Sabouraud-egg yolk agar.Results Men predominated among 25 patients (male 92% vs. female8%). HIV/AIDS (84%) was major predisposing factors for cryptococ-cosis, followed by non-Hodgkin lymphoma (12%) and kidney

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Poster Abstracts

transplantation (4%). Strains were isolated from cerebrospinal fluid(82%) and blood (18%). AFLP analysis revealed that the majority ofisolates 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 foundto be susceptible to amphotericin B, posaconazole and voriconazole,while low resistance level was observed for fluconazole (2.9%) and 5-fluorocytosine (5.9%). A high percentage of isolates was found to besusceptible dose-dependent to itraconazole (47.1%). All clinical iso-lates produced phospholipase, capsule and melanin, but these activi-ties varied with individual isolates. Statistical analysis showed thatisolates obtained from HIV-negative individuals are significantly lesssusceptible to 5-fluorocytosine (t test, P < 0.01). In relation to pro-duction of virulence factors, our results showed that AFLP2 isolatesproduced more melanin, comparing to AFLP1 and AFLP3 (Chi-squared test, P < 0.05). No correlation between expression of viru-lence factors and susceptibility profile was observed.Discussion This genotype and mating type distributions are consis-tent with previous European studies. All isolates were found to besusceptible to amphotericin B, posaconazole and voriconazole, as pre-viously shown. Comparing to similar studies, the percentage of iso-lates resistant to fluconazole (2.9%) was lower, while the percentageof 5-fluorocytosine resistant isolates in this study (5.9%) was higher.Statistical analysis showed no correlation between expression of viru-lence factors, as well as between virulence and susceptibility profile.Further studies are needed to investigate host factor contribution inpathogenesis of C. neoformans.

P143

Possibility of infection by Cryptococcus species for workersin organic farmingM. A. Khiyami,1 A. H. Bahklia2 and H. EL-Zefzaf3

1King 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 inorganic farming. Many factories in Saudi Arabia produce composthowever they do not follow the right procedure of preparation. Apilot study illustrated that most of the composts contain pathogens.The workers in the organic farms dealing with organic compost usu-ally have limited education. Therefore, they might unknowinglybecome infected with these different pathogens.Aim of the study Assess health risks of microbial contamination ofindustrial compost production.Methods In this study a hundred samples of compost were collectedfrom five different factories. The compost samples were analysed foringredients and pathogens, including bacteria, fungi, yeast, and para-sites. The microbial isolates were identified by microbiological andmolecular methods. Twenty five workers were selected for septicscreening. Sputum, blood and stool samples were collected to screenfor any pathogen.Results Thirty-four out of one hundred samples of compost containedseveral microbes includes fungi, yeast, bacteria and parasites. Thedominant bacteria in samples were E. coli and Enterobacter sp. Asper-gillus sp, Penicillium sp, Fusarium sp, Mucor sp, and Cladosporium spwere dominant fungi in all 30 samples. One strain of Cryptococcus wasfound in two compost samples. Giardiasis, Isosporiasis and Entamoebahistolytica were also isolated from all compost samples. The analysis ofstool samples collected from nineteen workers contained E. histolytica.Work is still in progress and the rest of the analysis will elucidatewhether 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 pub-lic health risks of compost production.

P144

Cryptococcus neoformans/Cryptococcus gattii speciescomplex in Cuban patientsM. T. Illnait-Zaragozı,1 Y. Rivera Gallego,1 E.X. Monroy Vaca,1

F. Hagen,2 C.M. Fern�andez Andreu,1 G.F. Mart�ınez Mach�ın,1

M.R. Perurena Lancha1 and J.F. Meis2

1Tropical Medicine Institute “Pedro Kourı”, Havana, Cuba and2Canisius-Wihelmina Hospital, Nijmegen, The Netherlands

Background The incidence of cryptococcosis increased with thebeginning of the aids epidemic. Life expectancy and quality of lifeamong HIV-infected Cuban patients have increased due to the wideHAART application during the past decades.Aims To study clinical and microbiological aspects of patients sus-pected of having cryptococcosis who attended the Tropical MedicineInstitute ‘Pedro Kour�ı’ during 2011–2012.Methods Twenty-five yeasts recovered form cerebro-spinal fluid andblood were identified by conventional methods. Identification of Cryp-tococcus isolates were confirmed by URA5 amplification and ITS1/5.8S/ITS2 sequencing. For every Cryptococcus sp. isolate the sero-and mating-type was determined by PCR and the in vitro susceptibil-ity was performed by using the ATB Fungus system. Clinical and epi-demiological data of patients with confirmed cryptococcosis wereretrieved from their clinical records.Results Conventional identification yielded 13 C. neoformans andone C. gattii isolates. Amplification and sequencing confirmed theidentification. All the C. neoformans isolates were alphaA and noresistance to the studied antifungal drugs were found. Clinicalrecords of patients revealed an average age of 38.5 years, the infec-tion was twice more observed among male patients, nine patientswere HIV-infected, steroid treatment and renal transplant were foundin two patients, and main clinical findings were headache, fever andneurological complications. Examination of cerebro-spinal fluidshowed at least one parameter altered in every patient and a positiveIndian ink in 90% of HIV-infected patients and 67% of non-HIVpatients; hematological studied indicated CD4+ less than200 cells ml�1 in 86% of patients; most patients received amphoteri-cin B plus fluconazole during induction therapy followed by fluconaz-ole for at least eight weeks; all isolates were susceptible to the useddrugs, 21.4% suffered from relapsed infections and 28.6% died.Conclusion Cryptococcal infection is still a problem among HIV-infected Cuban patients. C. neoformans alphaA is the main causativeagent. More effective treatment for cryptococcal infections is requiredsince relapses and fatal outcome are frequent despite absence of in vi-tro resistance to recommended antifungal drugs.

P145

Pseudomonas aeruginosa as a potential source of agentsinterfering with capsule formation in CryptococcusneoformansV. Raclavsky,1 R. Novotny1 and V. Kolek2

1Palacky University, Faculty of Medicine and Dentistry, Olomouc,

Czech Republic and 2University Hospital, Olomouc, Czech

Republic

Background Polysaccharide capsule represents a well-establishedvirulence factor in the pathogenic yeast Cryptococcus neoformans.Although it is dispensable for its survival, it plays very importantrole in its ability to evade the immune defense of host by interfer-ing with its several aspects. Because C. neoformans is an opportunistpathogen, possible imbalance between its virulence and theimmune status of host most probably decides whether a colonisa-tion of airways will progress into tissue invasion and possibly life-threatening systemic infection or not. Consistently, cases of

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Poster Abstracts

reactivation of infection being dormant for several years have beendescribed following alteration of the immune status. In a study atMayo Clinic, C. neoformans was isolated from lungs of 43 patientswithout signs of systemic cryptococcal disease; 26 of the 43patients suffered various bronchopulmonary disorders with chronicobstructive pulmonary diseases (COPD) and asthma being the mostcommon [1]. Colonisation of airways by diverse microbes that canworsen the disease during exacerbations is typical in COPD, as wellas in cystic fibrosis (CF), another progressive chronic pulmonarydisease. Chronic colonisation by Pseudomonas aeruginosa strains thatevolve toward an increased mutation rate and increased antibioticresistance plays important role in progression of disease. Concur-rently, the diversity of the microbial community is graduallyreduced, with mucoid P. aeruginosa strains prevailing at the endstage of disease. This course of disease may also be atributed tointerspecies competition where hypermutational P. aeruginosa is ableto fight down its competitors through production of antimicrobialagents. In a broader search for pseudomonadal toxic action wehave also explored its effect on C. neoformans.Aim Check for influence of P. aeruginosa isolates from sputum ofpatients suffering chronic pulmoary disease (CF and COPD) on C.neoformans.Methods Strains were inoculated on plates and checked for visibleinhibition of growth at crossing of inoculation lanes. Micromorphol-ogy was also observed on nutrient-poor media cultured under CO2atmosphere for induction of capsule formation.Results No apparent growth interference was observed, however,some strains of P. aeruginosa were able to reduce the formation ofcapsule measured by capsule thickness under inducing conditions.Discussion So far, antimicrobial peptides [2,3], anti-beta-glucanantibody [4] and viral protease inhibitors [5] have been described tointerfere with capsule formation in C. neoformans. Our observationsuggest that P. aeruginosa may also be a source of agents potentiallyuseful for blocking the production of this important virulence factor.Such agents may possibly shift the balance towards favorable side inthe host-pathogen interaction.Acknowledgements The Ministry of Health (NT/13560-4) and theInternal Grant Agency of Palacky University Olomouc(LF_2013_012) supported this work. The infrastructural part of thisproject (Institute of Molecular and Translational Medicine) was sup-ported by the Operational Programme Research and Development forInnovations (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 Micro-biol Lett 274: 279–86.

3. Silva FD, Rossi DC, Martinez LR et al. (2011) FEMS MicrobiolLett 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 JMicrobiol 58: 932–6.

P146

Cryptococcus neoformans and Cryptococcus gattii isolatedfrom woody debris samples collected from trees within apublic garden in Cape Town, South AfricaJ. Vreulink,1 K. Khayhan,2 F. Hagen,3 A. Botes,1 L. Moller,1

T. Boekhout,4 H. Vismer5 and A. Botha1

1University of Stellenbosch, Stellenbosch, South Africa;2University 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 deathamong HIV/AIDS patients, especially in sub-Saharan Africa. Infection

is acquired through the inhalation of basidiospores produced by Cryp-tococcus neoformans and Cryptococcus gattii in their natural habitat,which seems to be woody debris. Globally, there have been numer-ous studies about the presence of these yeasts in the environment.However, very little is known about the prevalence of these patho-gens in the South African environment.Aim The first aim was to determine if C. neoformans and C. gattii arepresent in trees located in a public garden in Cape Town, SouthAfrica. If these yeasts were present, the second aim was to determinethe genotypes, mating and sequence types (STs) of the isolates.Lastly, in order to determine if infection was acquired from the treeslocated in the public garden, we compared the genotypes, matingtypes and STs of these isolates to that of clinical isolates obtainedfrom the Medical Research Council (MRC).Methods Woody debris samples were collected from trees located ina public park in Cape Town, South Africa. Serial dilutions of thewoody debris samples were plated onto Niger seed agar plates. Repre-sentatives of C. neoformans and C. gattii were randomly isolated andtheir genotypes were determined by restriction fragment length poly-morphism (RFLP) analysis of the phospholipase B gene (PBL1) andamplified fragment length polymorphism (AFLP) analysis. The mat-ing types of the isolates were determined by PCR amplification of theMATalpha/MATa pheromone genes or amplification of the STE12locus. In addition, the sequence types (STs) of the isolates were deter-mined using the ISHAM multi-locus sequence typing (MLST) scheme.The genotypes, mating types and STs of clinical isolates obtainedfrom the former PROMEC Unit of the MRC were also determined andcompared to the environmental isolates.Results During this study 65 woody debris samples were collectedfrom 17 trees located within a public garden in Cape Town, SouthAfrica. Thirteen (20%) of these samples produced pigmented colo-nies on Niger seed agar plates. A total of 40 pigmented colonieswere randomly selected and further characterized. The majority ofthese isolates were C. gattii VGI/AFLP4 (80%), followed by C. neo-formans 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 fiveclinical isolates were VNII/AFLP1B (26%) and one isolate wasVGIV/AFLP7 (5%). Interestingly, three clinical VNI isolates (16%)were AFLP1A and are therefore VNB. The majority of the environ-mental isolates (85%) and all of the clinical isolates were MATal-pha, while six environmental VGI/AFLP4 isolates (15%) were therare MATa mating type. The majority of the environmental isolateswere ST230 (56%), followed by ST23 (17%), while the rest of theisolates were ST249 (7%), ST270 (5%), ST261 (2%), ST271 (2%),ST330 (2%) and ST331 (2%). In comparison, the clinical isolateswere 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 apublic garden in Cape Town, South Africa. These isolates belonged to8 different STs. However, we only obtained one of these STs, i.e.ST23, from a patient. Therefore, more environmental sampling needsto be done in South Africa, in order to determine the location of theother STs that were obtained from the other patients. Yet, our resultsmake a significant contribution to the knowledge about pathogeniccryptococci present in the South African environment.

P147

Prevalence of CNS Cryptococcosis in a tertiary careteaching hospital of Northern IndiaD. 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 majorcause of fungal meningitis and meningoencephalitis especially in

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Poster Abstracts

immunocompromised patients. Its incidence has increased in recentyears. Worldwide, approximately 1 million new cases of cryptococcalmeningitis 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 Daya-nand Medical College and Hospital, Ludhiana, Punjab (India). Allpatients with the clinical diagnosis of chronic meningitis from Janu-ary 2013 to December 2013 were included in the study.CSF sampleobtained from the patients was processed according to standard labo-ratory procedures.Cryptococcal meningitis was confirmedon the basisof India ink examination, culture and cryptococcal antigen latexagglutination test (CALAS by Meridian Biosciences).Results A total of 747 samples were received during the study per-iod 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 was1.9%. A high index of suspicion and routine mycological surveil-lance is required to help in an early diagnosis and appropriate ther-apy as clinical picture may be confusing with viral or tubercularmeningitis.

P148

Synthetic peptides as promising antigens in theimmunodiagnosis of cryptococcosisR. 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

1Federal University of Piau�ı, 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 affectshumans and animals all over the world. Globally, almost 1 millioncases of cryptococcal meningitis in HIV-infected subjects are esti-mated to occur every year and over 600 000 deaths. The diagnosisof cryptococcosis is made through microscopy, culture followed bybiochemical tests, and detection of cryptococcal capsular antigen(CrAg) by enzyme immunoassay or latex agglutination or lateral flowassay. The application of synthetic peptides aiming to improve thediagnosis for infectious diseases has been reported, but little has beendone as regards systemic mycoses. In leishmaniasis, Chagas disease,paracoccidioidomycosis and tuberculosis have demonstrated that thismethodology has a potential for discovering antigenic targets.Aim Test synthetic peptides as new antigenic targets for the develop-ment of a diagnostic test for cryptococcus.Methods Sixty-three B cell epitopes from C. gattii immunoreactiveproteins previously identified were synthesized and evaluated as anti-gen in enzyme-linked immunosorbent assay (ELISA). Peptides werefirst evaluated for their ability to react against sera from immuno-competent subjects carrying meningococcal meningitis. Peptideswhose first test yielded higher sensitivity and specificity were thenretested with sera from individuals with other fungal pathologies, forcross reactivity determination.Results Six synthetic peptides were recognized by antibodies in im-munoassays, getting: (i) specificity of 100%, (ii) sensibility of 78%,and (iii) minor cross-reactions. The recognition by antibodies of seracoming from other pathologies was shown heterogeneous, and withoptical density (OD) values much lower than the ones observed forcryptococcosis (P value ranged from P < 0.0001 to P = 0.0007),except H18 (Fig. 1).Discussion/conclusion From sixty-three synthetic peptides, six pep-tides were identified as good antigenic candidates for developing newdiagnostic tests for cryptococcosis. Out of these, three come from

Hsp70 (H18, H21, and H26). This chaperone shows both regionsconserved along the evolution and varied regions that allow a dis-tinction between species, and it may present immunogenic epitopes.In fact, recent works have shown that Hsps have been characterizedas key antigens inducing humoral responses to C. neoformans and C.gattii and that Hsp70 is involved in the adhesion and phagocytosisprocesses of C. neoformans. The remainder peptides derive from twoproteins whose information in the literature is still scarce: peptidesHy49 and Hy50 (hypothetical protein CGNB 1302), and peptide S4(protein Sks2). The peptides studied showed a high specificity, whichis essential in the clinical practice, as it excludes other morbiditieswith similar clinical manifestations. In addition, the search for analternative to diagnose cryptococcosis in advance, together with thelack of studies using synthetic peptides in developing serological testsin mycoses, makes the validation of synthetic peptides for developinga diagnostic test of cryptococcosis of utmost importance. In conclu-sion, by using immunoproteomics and bioinformatics techniques wewere able to identify new antigenic targets for developing diagnostictests for cryptococcosis and, in this study, six synthetic peptidesshowed that they are promising antigens in the immunodiagnosis ofcryptococcosis.

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

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Poster Abstracts

P149

Role of fungal burden in cerebrospinal fluid in hivmanagement of cryptococcal meningitisF. Concha Velasco and A. Bustamante Rufino

IMTAvH, Lima, Peru

Background Cryptococcal meningitis (CM) in HIV-infected patientshas high mortality despite the use of highly active antiretroviral ther-apy. Previous studies have shown that high fungal burden is animportant factor in mortality. Relation of fungal burden, assessed asnumber of colony forming units (CFU), with colonies growing timeand cerebrospinal fluid (CSF) opening pressure (OP) could help in themanagement of CM.Aims 1. Quantify CFU in CSF culture and evaluate its associationwith OP at start of treatment and after 2 weeks.

2. Determine the time to fungal clearance in CSF and evaluate itsassociation with OP.

3. Compare time-to-growth and number of CFU at baseline andafter 2 weeks of treatmentMethods We performed a retrospective study in HIV patients withCM, hospitalized from January 2000 to September 2013, at HospitalNacional Cayetano Heredia in Lima, Peru. Patients with a positiveCSF culture for Cryptococcus at baseline and alumbar puncture at2 weeks of treatment were included.Results We included 113 patients, 62 (55%) of them had a positiveculture at week 2. We did not find a positive correlation betweenbaseline number of CFU (n = 51) and high OP (≥ 20 cmH2O)(P = 0.06), but at week 2 of treatment there was a significant corre-lation (P = 0.0001). The median CFU was 4.23 � 0.93 log10 per mlat 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 OPand P = 0.04 for severe OP Fig. 1).

Quantitative culture results were available for 51 patients at base-line and 85 at 2 weeks. There was a significant negative correlation(P = 0.0154 and 0.000) between the number of CFU and time topositivity of culture (Fig. 2). The median time for growth was4 � 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 oftreatment, 62.71% grew in less than 7 days, 13.56% between 7 and14 days and 6.78% in over 14 days.

Discussion and conclusions These results support the value ofcryptococcal fungal burden as a tool in the management and evalua-tion of CM disease. They also suggest that the baseline fungal burdenprobably is not the only contributor to high OP, but seems to be oneof the most important factors at week 2. In addition, they highlightthe importance of incubation of fungal CSF cultures for over twoweeks before being considered negative at baseline and week 2 oftreatment.

P150

Prevalence of cryptococcosis in a tertiary care centre innorth India over 15 yearsJ. Chander,1 N. Singla,1 R. Singh,1 M. Kaur,1 U. Singhal1 and

F. Hagen2

1Government Medical College Hospital, Chandigarh, India and2Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands

Background There is an increased prevalence of cryptococcosisworldwide due to ongoing pandemic of AIDS and other circumstan-tial immunocompromised situations. Cryptococcus neoformans, theclassical etiologic agent of cryptococcosis, is currently recognized as aspecies complex, comprising C. neoformans var. grubii, serotype A, C.neoformans var. neoformans, serotype D, and C. gattii, serotypes B andC. The vast majority of cryptococcal infections, particularly in immu-nocompromised patients, are caused by C. neoformans var. grubiiwhereas C. gattii accounts for a smaller proportion of cases, whichfrequently infects immunocompetent patients. Recent outbreak of C.gattii in Canada and northwestern USA has posed a challenging taskbefore the medical community. In cryptococcal disease typing meth-ods are useful in confirming phenotypic identification, resolving tax-onomy, studying molecular epidemiology, improving the diagnosis ofcryptococcosis, identifying the genotype phenotype association andinvestigating the evolution of pathogenic species of cryptococcus.Aim The present study was undertaken to study profile of cryptococ-cosis and antifungal susceptibility testing pattern of isolates fromcases observed during 15 years i.e. 1999–2013 in the GovernmentMedical College Hospital, Chandigarh, India.Methods The yeast isolates were identified based on cultural charac-teristics, biochemical reactions and serotyping. AFLP typing wasdone at Canisius–Wilhelmina Hospital, Nijmegen, The Netherlands.Results Out of a total of 23 cases, twenty were diagnosed as crypto-coccosis on culture basis. The CSF was clear with no turbidity

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

Figure 1. Fungal clearance vs opening pressure.

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Poster Abstracts

however Crypto-LA was positive. Microscopy of CSF revealed lympho-cytic 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) wereCryptococcus neoformans var. neoformans followed by Cryptococcus neo-formans var. grubii (5) and two of the isolates were Cryptococcus gattii.In three cases we could not isolate Cryptococcus species as the cultureturned out to be sterile and diagnosis was made on histopathology.Most of the isolates (14) belonged to AFLP type 1 followed by oneisolates each of AFLP 2 and AFLP 8. As far as serotypes are con-cerned, maximum isolates (15) were serotype A followed by one iso-late of serotype D and one of serotype BD. All the twenty isolates ofCryptococcus species were found to be sensitive to amphotericin B andfluconazole. Eighteen patients were adults and 2 were children (one5 mol l�1 and other 14 F). Among 18 patients, 12 were male and 6were females. Maximum isolation has been from CSF samples 16, fol-lowed by skin 2, 1 bronchoalveolar lavage and one from gelatinouscyst in abdomen after a blocked VP shunt.Discussion Although the genus Cryptococcus contains more than fiftyspecies, only C neoformans and Cryptococcus gattii are considered prin-cipal pathogens in humans. Previously, C neoformans was defined as

having two varieties i.e. var. neoformans and var. gattii. However,based on the elucidation of the genomic sequences, C neoformans andC. gattii are now considered two distinct species. These two specieshave 5 serotypes based on antigenic specificity of the capsular polysac-charide; these include serotypes A, D and AD (C. neoformans) and sero-types B and C (C. gattii). Worldwide, C. neoformans serotype A causesmost of the cryptococcal infections in immunocompromised patients,including patients infected with HIV. Cryptococcus gattii rarely infectspersons with HIV infection and other immunosuppressed patients andusually involves immunocompetent individuals, respond slowly totreatment and are at risk for developing intracerebral mass lesions likecryptococcoma. C. neoformans is distributed worldwide.Conclusion Identification of species complex can help not only inrecognizing the epidemiology of the strains prevalent in an area butalso in being vigilant about the changing antifungal patterns of thoseisolates.

P151

Cryptococcosis and HTLV 1: underdiagnosed casesF. Concha Velasco and A. Bustamante Rufino

IMTAvH, Lima, Peru

Background Cryptococcosis is an opportunistic fungal infectionoften described in HIV patients, who has a higher mortality rate thanin non-HIV patients. HTLV is a retrovirus, which in South Americaaffects mainly population from Brazil, Colombia and Peru. HTLVinfection 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 Cen-tral America, while there is none report from Peru.Aim Report clinical and epidemiological characteristics of cryptococ-cosis in peruvian patients co-infected with HTLV-1.Methods Clinical records of patients with a definite diagnosis ofcryptococcosis (positive culture for Cryptococcus neoformans) andHTLV-1 infection were reviewed.Results All co-infected patients have lived in the highlands or in theAmazonian regions. The clinical and epidemiological characteristicsof four patients are described in Table 1. HTLV-1 diagnosis was per-formed during hospitalization in two patients and in 15–21 daysbefore in other two. All received amphotericin B deoxycholate

Table 1 Characteristics of cases.

Case Gender/Age Co-morbidities Localization

Clinical characteristics and

outcome

1 M/67 Diabetes, high blood

pressure,

corticosteroid

therapy

Meningitis 3 months: fever, headache,

nauseas, vomiting,

neck stiffness. Glasgow: 11,

CrAg* titer: 1/256.

Death due to Acinetobacter sp.

Bacteremia.

2 F/43 Fungoid mycoses T-

cell lymphoma

Scabies

Meningitis 2 weeks: headache, nauseas,

vomiting, neck

stiffness and weight loss of 5

Kg. CrAg titer: 1/8.

Cure.

3 M/54 Disseminated

Strongyloides

stercoralis

Ancylostoma sp.

Disseminated

(lung,

meningeal,

gastrointestinal)

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.

4 F/58 Diabetes, high blood

pressure

Meningitis 7 months: headache, tremor,

weigh loss of 10 Kg,

bladder

incontinency, hyperreflexia and

meningeal signs.

CrAg: 1/8.

Cure

CrAg* Cryptococcal latex agglutination test.

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

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

ª 2014 The Authors


Poster Abstracts

(1 mg kg�1 day�1) plus fluconazole 800 mg day�1 during the induc-tion phase until negativization of cerebrospinal fluid (CSF).Discussion and conclusion The 4 patients come from epidemicareas in HTLV I, also they had present other additional diseases. Bothdiagnoses, crytococcosis and HTLV 1 infection, were lately per-formed. Cause of death in two patients was not associated with thefungal disease.

Search for HTLV infection should be conducted among HIVpatients diagnosed with cryptococcosis and resident in endemic areasof this retrovirus. Participation of this entity as a risk factor for cryp-tococcosis should also be evaluated.

P152

Cryptococcus gattii infections in the Netherlands – aretrospective molecular diagnostic study using formalinfixed paraffin embedded pulmonary coin lesionsF. 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 yeastgenus Cryptococcus are in majority caused by C. neoformans and C.gattii, while the sibling species C. adeliensis, C. albidus and C. laurentiihave rarely been reported as the culprit of disease. Cryptococcus gattiihas a limited geographic distribution pattern and was until recentlyregarded as a pathogen related to tropical and subtropical climatezones. However, the distribution pattern has largely been changeddue the occurrence of C. gattii outbreaks in temperate climate zonesin Europe, North America and Western Australia. Cryptococcus gattiiinfections are exceptionally rare in the Netherlands as observed dur-ing a 30-year retrospective nationwide epidemiological survey. Sur-prisingly, this ‘tropical’ pathogen was recently isolated from theenvironment in the Netherlands, hence suggesting that C. gattii canbe autochthonous acquired.Aim Retrospective investigation of the presence of C. gattii in lungsamples 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 forthe presence of cryptococcal cells. Archived FFPE-tissue samples wereobtained from pathology departments in the Netherlands. GenomicDNA was extracted from two 10 lm FFPE-tissue slices, showing mor-phological presence of yeasts, using the blackPREP FFPE DNA extrac-tion kit (Analytic Jena, Germany). To detect DNA from Cryptococcusspecies, a published real-time PCR assay was applied, while a novelreal-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 pulmo-nary cryptococcoma and that 97 samples from these patients wereavailable. Finally, 18 pathology units participated in the study by pro-viding 62 FFPE-tissue samples from 33 patients. From the 62 FFPE-samples, 34 were found to be positive for Cryptococcus species. DNAcorresponding to 22 patients (66%). From these positive samples, three(9.1%) were positive for C. gattii by real-time PCR, corresponding tothree patients. Two patient were asymptomatic after removal of thecoin lesion, from one patient no follow up was available.Conclusion Although molecular diagnostic investigation of forma-lin-fixed paraffin embedded samples remains a challenge when itcomes to the detection of fungal pathogens we were able confirm acryptococcal infection in 66% (22 out of 33) of the patients with acryptococcoma 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 wasrecently observed during a retrospective study that made use cul-tured cryptococcal isolates from the same period. Unfortunately, itremains a question where these patients acquired the infection.

P153

First isolation of Cryptococcus neoformans genotype VNIalpha mating-type from hollow trunk of HymenaeacourbarilM. 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 theinhalation of contaminated air. Cryptococcus members can be dis-persed in several sources, including fruits, vegetables, soil, insects,honey bees, and decaying wood. Information on Cryptococcus speciesinhabiting plants may be clinically relevant due to the epidemiologi-cal role of these habitats as possible sources of human infection withC. neoformans and C. gattii.Recent data have shown the presence ofC. 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.neoformansgenotype VNI alpha mating-type.Methods During a 12-month period we collected samples from ninetrees in one public park in S~ao Paulo city. All trees were located inthe biggest city Sao Paulo of Brazil. The park offers visitors directcontact with nature, fauna and flora. Hollow specimens of the follow-ing species were sampled quarterly: Casuarina equisetifolia, Alchorneasidifolia, Tibouchina granulosa, Eriobotryajaponica (yellow plum), Rapa-neaumbellata, Machoeniumnictitans, Ocoteapuberula (gooey cinnamon),Ficusmicrocarpa, andHymenaeacourbaril. The inner part of the hollowtrunk was scraped (~15 g) with a sterilized spatula and transportedin sterile plastic bags to the Reference Mycology Center, Adolfo LutzInstitute, to be processed. Approximately 10 g of each sample wassuspended in 40 ml of sterile saline. After vortexing (2 min), the sus-pension sat for 10 min. The supernatant was aspirated (8 ml) andmixed with 2 ml of penicillin solution containing 1.2 million IU(4.5 mg ml�1) streptomycin (10 mg ml�1) solution. After 15 min,10 ll of the resultant mixture was transferred to 10 Petri dishes con-taining bird seed agar (BSA). The plates were incubated at 30 °Cand observed daily for up to 7 days. All brown, creamy and mucoidcolonies were subcultured on BSA and stored for future phenotypicidentification. The presence of capsule, urease production, positivenitrate reduction test, absent sugar fermentation and typical profileassimilation tests confirmed Cryptococcus neoformans identification.Species and molecular typing was determined using URA5-RFLPanalysis after amplification of the URA5 gene from high-molecular-weight DNA. The obtained URA5-RFLP patterns were visuallyassigned by comparison with the patterns of the following referencestrains: 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 yieldedcryptococcal colonies during summer, autumn and winter seasonswith a high (102 CFU g�1) fungal burden. Using PCR fingerprintinganalysis with the minisatellite-specific core sequence of the wild-typephage M13 we could observed 14 distinct molecular patternsamong 60 colonies obtained during sampling. All colonies havebeen identified as alpha mating-type. Additionally, we observedthrough the European reference antifungal susceptibility testingAFST-EUCAST, different minimum inhibition concentration valuesof fluconazole (MIC range 4 to >64 mg l�1) along with differentmolecular profiles.Discussion and conclusions These observations extend the geo-graphic distribution and substantiated new urban environmentalniche of C. neoformans, the principal Cryptococcosis agent. Further-more, our study reinforces the natural genetic complexity of the C.neoformans VNI molecular type, the most implicated in human infec-tions in our region. To our knowledge, this is the first study describ-ing the occurrence of C. neoformans var. grubii VNI in the wood ofhollow trunks of Hymenaea courbaril. The importance of studiesreveng an increase in isolation from native trees around the worldcould contribute to a better knowledge of the lifecycle of C. neofor-mans var. grubii in association with plant material. Further

ª 2014 The Authors


Poster Abstracts

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

P154

Role of cerebrospinal fluid interleukin-6 as a prognosticbiomarker of Cryptococcal meningoencephalitisM. Chayakulkeeree, J. Kobkijcharoen, D. Waywa and

R. Sirijatuphat

Faculty of Medicine Siriraj Hospital, Thailand

Background Interleukin 6 (IL-6) is a pro-inflammatory and anti-inflammatory cytokine secreted by T cells and macrophages to stimu-late the immune response. IL-6 assay has been commercially avail-able and used as a biomarker in several inflammatory diseases.Previous studies showed that IL-6 secretion from peripheral bloodmononuclear cells was stimulated by cryptococcal components and itmay play roles in cryptococcal pathogenesis. However, the usefulnessand clinical relevance of IL-6 in patients with cryptococcal meningitiswere limited.Aim To investigate the role of interleukin-6 in predicting outcomesof HIV-infected patients with cryptococcal maningoencephalitis.Method This is a sub-group analysis of another study. We analyze11 HIV-infected patients with cryptococcal meningitis who had beentested for cerebrospinal fluid (CSF) IL-6. Clinical response, mycologi-cal response and mortality were analyzed in association with CSF IL-6 level.Result Of 11 patients, 8 (73%) were male with mean age of 37 (range23–59) years. Mean CD4 was 44 (range 3–160) cells mm�3. Fungemiawas found in 9 patients (82%). Clinical response was 64% and microbi-ological response was 73%. Overall mortality was 18%. All 5 patients(100%) with CSF IL-6 less than 100 pg ml�1 had a favorable clinicaland mycological response, whereas those with CSF IL-6 more than100 pg ml�1 exhibited a clinical response of 33% (2/6) and a myco-logical response of 50% (3/6). Early mycological response (negativeculture within 14 days) was demonstrated in 60% and 17% of patientswho had CSF IL-6 less than 100 pg ml�1 and more than 100 pg ml�1,respectively. All patients with CSF IL-6 less than 100 pg ml�1 weresurvived more than 6 months but those with CSF IL-6 more than100 pg ml�1 had a 6-month mortality of 2/6 (33%). In fact, 2 patientswho did not survive had CSF IL-6 higher than 500 pg ml�1 (7661 and736 pg ml�1). According to the low number of patients, the differenceswere not statistically significant.Conclusion High level of CSF IL-6 (more than 100 pg ml�1) showeda trend toward a poor clinical response, poor mycological responseand mortality in HIV-infected patients with cryptococcal meningo-encephalitis. CSF IL-6 may potentially be used as a prognostic bio-marker to predict outcomes in patients with cryptococcal meningoen-cephalitis. Further study with a larger number of patients iswarranted.

P155

Study of multiple single-colony isolates of theCryptococcus neoformans/Cryptococcus gattii speciescomplex in ColombiaP. Escandon and E. Castaneda

Instituto Nacional de Salud, Bogota, Colombia

Background The assumption that a determined infection in a hostis caused by a single strain is considered to be valid for some dis-eases, however, some of them have been possibly caused by mixedinfections with multiple strains of a pathogen. For Cryptococcosis,some reports have been made on experimental infection of mice withmultiple strains of the fungus resulting in mixed infections in the

brain or the lungs. This issue has been demonstrated by Dromer etal., who reported that mixed infections in a single patient are com-posed of different mating types, serotypes and/or genotypes. InColombia, the passive surveillance on Cryptococcosis has provided uswith an important collection of strains in which this type of analysiscould be done.Aim To analyze clinical isolates of the C. neoformans/C. gattii speciescomplex to determine if mixed infections are present in our patients.Methods Clinical cultures were collected during the passive surveil-lance on Cryptococcosis that is being held in Colombia since 1997.Original strains correspond to cultures obtained from the biologicalspecimen on agar. The selection was made randomly, from isolatesreceived between 2006–2013, from different regions in Colombia,and not based on the patient’s characteristics. Sixteen C. neoformansisolates and 7 C. gattii isolates were included. A suspension ofapproximately 9x102 CFU ml�1 of the original culture was platedonto Sabouraud agar and Niger seed agar plates. Five colonies perplate were selected based on colony morphology differences (if any),otherwise randomly when the phenotypes were similar. For each iso-lated colony, conventional biochemical test were done, and molecu-lar 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. Moleculartyping has been done in all of the (16 9 5 = 80) C. neoformans iso-lates 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 samemolecular type has been seen in each of the colonies studied, forsome isolates, slight differences observed until now in 3 C. neoformansstrains have been observed in the electrophoretic profiles, thus sug-gesting the need to perform a more precise technique as MLST.Discussion/conclusions This is the first study carried out in Colom-bia on the determination of possible mixed infections in patients diag-nosed with Cryptococcosis. It is widely considered that a singleisolate of the fungus is the responsible for the disease; however, arecent study reported a high frequency of mixed infections (20%) inEuropean patients suggesting that multiple strains could be exoge-nously acquired from the environment, either simultaneously orsequentially. Our previous experience has shown that we can finddifferent serotypes, mating types and molecular patterns in a samegeographical region, making it possible that a patient may beinfected with more than one strain, thus making a mixed infectionby multiple strains a likely scenario. Further studies may be helpfulfor a complete characterization of the strains, as well as studying iso-lates recovered from serial samples of patients with recurrent infec-tions of Cryptococcosis.

P156

Comparison between latex agglutination test and lateralflow assay for the detection of Cryptococcus antigen inserumR. Wahyuningsih,1 H. Herqutanto,1 D. Imran,2 R. Estiasari,2

T. Natriana,3 N. Komari,2 S. Sucipto2 and E. Yunihastuti2

1University of Indonesia, Jakarta, Indonesia; 2University of

Indonesia/Ciptomangunkusumo Hospital, Jakarta, Indonesia and3Drug Dependence Hospital, Jakarta, Indonesia

Background The classic method for the diagnosis of cryptococcalmeningitis the india ink test and culturing from spinal fluid. But,lumbar puncture, to provide spinal fluid is an invasive procedure thatoften in convenient for the patient. Furthermore, the sensitivity andspecificity of both methods are not as high as we expected. Antigendetection can be conducted for spinal fluid and serum. By doing anti-gen detection in serum we have a presumptive diagnosis. Two meth-ods for antigen detection are recommended by WHO i.e. latexagglutination test (LA) and lateral flow assay (LFA). The first wasused for >10 years, while the second was just launched in Indonesia

ª 2014 The Authors


Poster Abstracts

last year. Are both methods equally suitable for the detection of cryp-tococcal antigen in serum?Aim of the study Comparing lateral flow assay (LFA) and latexagglutination test (LAT) for the detection of Cryptococcus antigen inserum.Method Serum tested originated of HIV infected and non-HIVpatients. Clinical samples were sent to our laboratory for the diagno-sis of cryptococcosis using LFA test (IMMY, Lab USA) and LA test(Pastorex - Crypto plus BioRad France) to detect Cryptococcus anti-gen. Both methods were done according to procedures described bythe manufacturer. Fisher exact test was done to test any associationbetween the two methods, with confidence interval (CI) <5%. Inaddition, the clinical condition was also noted.Result We investigated 80 sera, of which 79 were from HIV-infectedand one from non-HIV infected patients for Cryptococcus antigen.Using both methods (LFA and LA methods), 20 out of 80 sera testedwere positive for Cryptococcus antigen. Two samples were positive ofCryptococcus antigen by LFA test but negative by LA test, but twosamples were negative by LFA test but positive by LA test. The resultof Fisher exact test, revealed that both methods have a very strongassociation (P < 0.0001). Thus, there was a an equal capabilityLFAand LA methods to detect cryptococcus antigen in sera. Patients withpositive results were also diagnosed for other opportunistic infectionsuch as toxoplasma encephalitis, tuberculous meningitis and otherbrain infection. Most of the patients were not diagnosed as cryptococ-cal meningitis, only three patients who’s the sample tested were sus-pected as cryptococcal meningitis.Conclusion/discussion Both methods are equally well in detectingCryptococcus antigen in serum. A positive result in serum is a pre-sumptive diagnosis for cryptococcosis which guide us to search forcryptococcal infection in other organs particularly the central ner-vous system.

P157

Synchronous fungal infections in an immunocompromisedhost – sampling of multiple suspect sites is a requisite fordiscrimination and managementY. A. Chai, K. Lim and J. E. Seet

National University Health System, Singapore

Background In immunocompromised patients at risk of opportunis-tic infections, obtaining clinical specimens for microbiology and his-topathology can be challenging for several reasons. The general illstatus of many such patients, the presence of coagulopathies or thepoor accessibility of the site may deem an invasive sampling proce-dure risky. Weighing these considerations, the clinician is oftena-times faced with the difficult decision on choosing the best accessibleand representative lesion to biopsy, particularly in the setting of sus-pected disseminated fungal disease.Aim In most cases, sampling a representative lesion serves well inclinching the diagnosis. However there are circumstances wherebysuch a pragmatic approach may fail.Methods We describe here, a case of patient status-post liver trans-plant who presented with synchronous fungal infections.Results This is a 62 years old diabetic male with Child’s B liver cir-rhosis from Hepatitis C and newly diagnosed 1.3 cm hepatocellularcarcinoma. He underwent a living donor orthotopic liver transplant inApril 2013. In October, he presented with a 2.5 cm nodular and fle-shy lesion over the right forearm after sustaining an abrasion againsta wooden door frame. He also complained of weight loss, lethargy andlow grade fever. Routine chest X-ray had demonstrated a prominentleft hilum. A CT scan that followed revealed mediastinal and left hilarlymphadenopathy as well as left pulmonary nodule. There were nochest symptoms of note. His immunosuppressive regimen then con-sisted of tacrolimus 1 mg daily and myfortic 360 mg twice daily

A skin biopsy of the right forearm nodule was performed. Histopa-thology showed scattered broad filamentous fungi. Requesting earlydischarge from hospital, the patient was started on empiric

posaconazole 200 mg four times daily for disseminated invasive fun-gal infection. The fungal culture subsequently returned as Lichtheimiacorymbifera. The patient was re-admitted and underwent a wide mar-gin excision of the forearm fungal lesion which confirmed the earlierfinding. The excised margins were clear of fungi. Decision was madeto also perform a transbronchial unltrasound-guided lung biopsy dur-ing this hospitalization. Interestingly cytology of the biopsy revealedintracellular yeasts and the culture grew Cryptococcus neoformans.The serum Cryptococcus antigen test was >1 : 1024. There was nogrowth of moulds.

The patient was started on an initial therapy of conventionalamphotericin B and flucytosine targeted for at least 8 weeks. How-ever, this treatment regimen had to be switched after 6 weeks due toprogressive renal impairment. Theorizing that the forearm Lichtheimialesion had been adequately dealt with the above treatment modali-ties, the patient was switched to oral fluconazole. Four months after,he remains to be on fluconazole maintenance therapy and is doingwell. His latest serum Cryptococcus antigen level was 1:256 andthere was no recurrence of the Lichtheimia infection.Discussions/conclusions Sampling an accessible, representativelesion remains a practical approach for the diagnosis of IFI. However,clinicians need to be mindful that immunocompromised patients canpresent with multiple co-infections in the same setting and shouldweigh the benefits versus risks of adopting an aggressive sampling ofmultiple suspect sites for discrimination and effective management ofsuch synchronous fungal infections.

P158

Cryptococcal antigen in the serum of anti-retro viral na€ıveHIV-infected patientsR. Wahyuningsih,1 D. Imran,2 H. Herqutanto1 and C. K. Muda2

1University of Indonesia, Jakarta, Indonesia and 2University of

Indonesia/Ciptomangunkusumo Hospital, Jakarta, Indonesia

Background Host immunity determines the faith of Cryptococcus inhuman body. It can be dormant in immune-competent host, but ablecause’s infection in immune-compromised host. The most prevalentclinical manifestation is meningitis, but the existence of Cryptococcusin the human body can cause an inconspicuous clinical signs, and atthe same time releases antigen into body fluid that can be detected.Aim of the study To detect Cryptococcus antigen in serum of ARV-na€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 wereexamined. Cryptococcus antigen was found in five out of 78 seratested. They consisted of three male and two female and their agerange from 18, 39 years, and only one patient is 43 years. Four sub-jects have CD4 count less than 50 cells ll�1 and they belong to AIDS

Figure 1. Histology sections of skin nodule and pulmonary lymphnode.

ª 2014 The Authors


Poster Abstracts

group, and one subject has CD4 > 200 cells ll�1 who belong to noneAIDS group. All positive patients were suggested underwent lumbarpuncture (LP), but two patients refused and they died within a monthafter antigen was detected. Another three patients were underwentLP, with india ink, culture and antigen detection were negative andfluconazole was given, and they are survived. Two patients, whorefused doing LP, have mental alteration i.e. cognitive disturbances.Conclusion/discussion Cryptococcemia isassociated with mortality,which normally detected by culture. Culture even though definitediagnosis is time consuming, while antigen detection is easy to beconducted and fast and cheap. We suggest to do antigen detectionbefore ARV treatment, since it can prevent mortality which causesby the disease itself or IRISs associated mortality.

P159

Phenotypic switching of Cryptococcus neoformans var.grubii VNI in a case of retroperitoneal diffuse large B-celllymphoma following modified Rituximab-CHOPchemotherapyH. K. Tseng,1 C. P. Liu,1 Y. C. Chen2 and M. W. Cheng1

1Mackay Memorial Hospital, Taipei, Taiwan and 2National

Taiwan University Hospital, Taipei, Taiwan

An 88 year-old woman with past history of breast cancer post mas-tectomy had retroperitoneal diffuse large B-cell lymphoma, germinalcenter type treated by modified rituximab-CHOP chemotherapy (rit-uximab, cyclophosphamide, vincristine, and prednisolone). She hadneutropenic fever and final blood culture and urine culture yield twomorphological phenotypes (smooth and mucoid) of Cryptococcus neo-formans which VNI was confirmed. Central nervous system infectionwas favored but her family member declined lumbar puncture forCSF study due to personal issue. After completion of induction ther-apy with amphotericin-B and flucytosine two weeks, we switched tomaintenance oral fluconazole 400 mg daily. Due to the controlled ofher infection and improvement of her clinical condition, she wasdischarged.

P160

Relationship of susceptibility testing of Cryptococcusneoformans to survival and mycological clearance in HIVassociated cryptococcal meningitisJ. 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

Background Antifungal susceptibility testing of Cryptococcus neofor-mans is cumbersome and its value is uncertain in patients present-ing with cryptococcal meningitis. Susceptibility measured at thepoint of diagnosis has not reliably been linked to clinical outcome.Most studies have been small and have not accounted for differ-ences in disease severity at abseline. The Sensititre Yeastone is arelatively simple and easy to interpret broth microdilution suscepti-bility assay.Aims To determine the effect of antifungal susceptibility as measuredby 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 cryptococ-cal meningitis were enrolled into a 3 arm trial of combination anti-fungal therapy in Vietnam. Patients received induction therapy witheither amphotericin 1 mg kg�1 day�1 monotherapy for 4 weeks,amphotericin combined with flucytosine 100 mg kg�1 day�1 forFigure 1. Cells of smooth and mucoid capsule.

Figure 2. Colonies of smooth and mucoid capsule.

ª 2014 The Authors


Poster Abstracts

2 weeks, or amphotericin combined with fluconazole 800 mg day�1

for 2 weeks. Following induction therapy all patients received fluco-nazole 400 mg day�1 until 10 weeks after randomization. Previousanalyses had identified fungal burden, treatment allocation and Glas-gow coma score less than 15 as independent predictors of mortality.The susceptibility of revived baseline isolates to amphotericin B, flu-cytosine, and fluconazole was determined following both 48 and72 h incubation. Range, MIC50, MIC90 and geometric mean MICswere determined for all drugs. Cox regression was used to determinethe effect of susceptibility on the primary endpoint of 10 weeks postrandomization, and on 2 weeks and 6 months survival. The jointeffect of randomized drugs was determined according to the treat-ment randomly allocated for the combination therapies and foramphotericin alone for patients on monotherapy, and the joint effectof the MICs of amphotericin, fluconazole, and flucytosine for allpatients. Analyses were performed with and without adjustment forbaseline fungal count and Glasgow Coma score. Mixed models andCox regression were used to model CSF fungal decline over the first14 days and time to CSF clearance.Results 276 isolates from 276 patients were included in the primarypopulation analysis. No effect of susceptibility was found on survivalto 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. Simi-larly no effect of susceptibility was detected on the rate of fungalclearance for each of the three drugs tested. Adjustment for previ-ously defined independent baseline predictors of mortality (GCS, fun-gal burden, treatment allocation)did not significantly affect theseresults.Conclusion There is no role for susceptibility testing of isolatesfrom patients with first episodes of HIV associated cryptococcalmeningitis.

P161

Understanding synergy between iron chelators andantifungals using gene co-expression networksA. Carter,1 Y. W. Lai,1 C. N. I. Pang,2 L. T. Campbell,1

M. Truong,3 S. Chen4 and M. Wilkins2

1University 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 infectionto resolve. Current recommendations for induction therapy areamphotericin B (AMB) combined with 5-flucytosine (5FC), but AMBis highly toxic, requiring vigilant monitoring, and 5FC is expensive,putting it out of the reach of the low income countries that need itmost. Finding and developing new antifungals is very difficult, there-fore a promising avenue of antifungal research is to enhance existingtherapies with synergistic agents. Iron chelators administered withcertain antifungals have been found to improve the clearance ofsome fungal infections. However, mechanistic data on exactly howthese work, and why they sometimes do not, are lacking. In addition,iron depletion can be damaging to the host. In this study, we explorevarious chelator-drug combinations and use gene co-expression net-works to identify pathways and processes that are differentially regu-lated during a synergistic response. The goal is to identify targets fornew synergistic therapies without needing to administer chelators.Aims 1. To find antifungals + iron chelator combinations that resultin synergy when used to treat Cryptococcus;

2. To use RNA-Seq and co-expression networks to analyse the syn-ergistic response at the level of transcription and identify importantmediators of synergy.Methods Checkerboard assays were used to determine synergy forcombinations of the following antifungals and iron chelators: Antifun-gals: AMB, fluconazole (FLC), itraconazole (ITZ), voriconazole (VRZ),

caspofungin (CAS). Iron chelators: lactoferrin, deferasirox, deferiprone,deferoxamine, cyclopirox olamine, EDTA. Fungal species/strains: Cryp-tococcus neoformans H99 (genome strain), C. gattii R265 (genomestrain), C. gattii 97/170 (intrinsically resistant to FLC), Saccharomycescereviseae S288C (genome strain; used for interactome construction).

Synergy data were analysed using MacSynergy and the FractionalInhibitory Concentration Index (FICI). High quality RNA was pre-pared from the most synergistic combinations. RNA-Seq data weregenerated using the HiSeq 2000 Illumina platform with biologicaltriplicates multiplexed and randomized across two sequencing lanes.Differential expression analyses were performed using EdgeR. Cyto-scape was used to visualize potential interaction networks, and self-organising maps (SOMs) were used to group transcripts into co-expressed cohorts.Results Significant synergy was seen when AMB was combined withlactoferrin, a milk protein with iron chelating properties. Synergywas not seen with the FDA-approved chelators deferosirox, deferi-prone, and deferoxamine; nor was it seen with EDTA and cyclopiroxolamine. Antagonism occurred when ITZ and VRZ and were com-bined with deferasirox, deferiprone and EDTA to treat C. neoformansH99, but this was not seen with C. gattii or S. cerevisease; in factVOR + EDTA was synergistic for C. gattii.

Transcriptional analysis by RNA-Seq was performed on S. cerevisiaetreated with (i) AMB only; (ii) AMB + lactoferrin, (iii) and (iv) corre-sponding controls matched for growth but without antifungals orchelators.

Cytoscape and SOM analysis suggested the transcription factorsAFT1 and YAP5 were important for AMB + lactoferrin synergy.AMB treatment alone resulted in the down-regulation of nine genesinvolved in ergosterol biosynthesis and the up-regulation of AFT1, atranscription factor involved in iron transport. AMB + lactoferrinhalted the up-regulation of AFT1 and down-regulated genes involvedin iron transport. The latter were co-expressed with YAP5, a secondtranscription factor that co-ordinates the expression of genes control-ling the nuclear localization of AFT1.Discussion/conclusion Synergy between antifungals and iron chela-tors was uncommon and differed among species. The antagonism seenwith C. neoformans highlights significant species-specific differencesand indicates a need for caution when using iron chelators. AFT1 andYAP5 were affected by the addition of lactoferrin to AMB; their influ-ence on drug synergy will be further analysed using SOMs, Q-PCR andgene deletion/complementation. We will explore the role of homolo-gous factors in Cryptococcus using additional RNA-Seq assays.

P162

Clinical features of pulmonary cryptococcosis in non-HIVpatients in JapanS. 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

1Nagasaki 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;4Saitama 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 cryptococco-sis in Japanese non-HIV population.Methods Retrospective investigation of 151 pulmonary cryptococco-sis cases between 1977 and 2012 was executed. The underlying dis-ease (UDs), aggravating factors, radiological characteristics, andtreatment were examined.

ª 2014 The Authors


Poster Abstracts

Results Sixty-seven patients (44.4%) had no UDs. The common UDswere diabetes (32.1%) followed by hematologic disease (22.6%), andcollagen disease (22.6%). Peripherally distributed pulmonary nod-ules/masses were most commonly seen. Lesions in the right middlelobe (P = 0.01) and air bronchogram (P = 0.05) were significantlymore frequent, respectively, in patients with UDs than patients with-out them. Azoles were mainly selected for the patients withoutmeningoencephalitis. Mean treatment duration for patients with andwithout UDs was 2.87 and 6.64 months, respectively. Patientswhose pulmonary nodules improved after treatment continued toexperience gradual reduction of cryptococcosis antigen titers, even ifantigen titers were positive at the time of treatment cessation. Theaverage time for antigen titers to become negative after treatmentcessation was 10.7 and 13.1 months for patients with and withoutUDs, respectively. When groups were compared according to thepresence of meningoencephalitis complications, deaths, and survivals,factors contributing to cryptococcosis prognosis included higher age,hypoproteinemia, hypoalbuminemia, steroid use, high C-reactive pro-tein levels, and meningoencephalitis complications.Conclusions It is crucial to consider the presence of UDs and menin-goencephalitis for the choice of antifungals and treatment durationfor cryptococcosis in non-HIV patients. Three- and six months-administration of azoles for pulmonary cryptococcosis with or with-out UDs, respectively is reasonable.

P163

Antifungal Activities of T-2307 an arylamidine compoundagainst Cryptococcus gattii: an emerging fungal pathogenH. 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

1Toyama Chemical Co., Ltd, Toyoma, Japan and 2Nagasaki Univ.

Graduate Sch. of Biomedical Sci., Nagasaki, Japan

Background Cryptococcus gattii causes life-threatening disease inotherwise healthy hosts and to a lesser extent in immunocompro-mised hosts. T-2307, an arylamidine compound, exhibits broad-spec-trum and potent antifungal activity and is in clinical trials. In thisstudy, we evaluated in vitro and in vivo antifungal activities ofT-2307 against C. gattii in comparison with other antifungal agents.

Methods MICs against 16 isolates were determined according to theCLSI guideline (M27-A2) for the broth microdilution method. Time-kill studies were performed using C. gattii YF2784 (VGII). In murineintranasal pulmonary infection model caused by C. gattii YF2784,the test compounds were administered once daily for 21 days begin-ning at 2 hours postinfection. In addition, to evaluate therapeuticeffect in advanced infection, the test compounds were administeredfrom 14 days postinfection once daily for 7 days in the murinemodel. The viable counts in lung and brain of compound-treatedgroup and control group at 21 days postinfection were comparedusing parametric Dunnett’s multiple comparison test. Statistical sig-nificance was set to P < 0.05.

The MIC range of T-2307 against 16 isolates was lower than thatof any other antifungal agents tested. T-2307 and AMB showed con-centration-dependent fungicidal activity with a reduction of >3Log10 CFU/ml at 4 MIC or higher within 72 hours though FLCshowed no fungicidal activity even at 64 MIC. When the administra-tion was performed once daily for 21 days from 2 hours postinfectionin the murine model, T-2307 at 0.5 mg/kg/day significantly reducedthe viable counts in both lung and brain, which was comparable toFLC at 40 mg/kg/day and AMB at 4 mg/kg/day. In an advancedinfection model in which the administration was performed from14 days postinfection once daily for 7 days, T-2307 significantlyreduced 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 vivoantifungal activity against C. gattii in comparison with other antifun-gal agents. It is a promising candidate for the treatment of cryptococ-cosis caused by C. gattii.

Results

Compound MIC50 (lg/ml) MIC90 (lg/ml) Range (lg/ml)

T-2307 0.0313 0.0625 0.0078 – 0.0625

5FC 8 32 0.5 – >64FLC 4 16 1 – 16

ITC 0.5 0.5 0.125 – 1

VRC 0.125 0.25 0.0625 – 0.25

MFG >128 >128 >128AMB 1 1 1

ª 2014 The Authors


Poster Abstracts

Molecular identification of Cryptococcus species isolated from pigeon dropping in Shiraz, Southern...

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