Genotypic diversity of environmental Cryptococcus neoformans isolates from Northern Portugal

The Cryptococcus neoformans/C. gattii species complex members are the main agents of systemic cryptococcosis. This disease is believed to be acquired from the environment via fungal cell inhalation. Often, isolates recovered from environmental and clinical sources have proven to be genotypically similar. We assessed the occurrence of C. neoformans and C. gattii in environmental substrates collected in a Portuguese region. Twenty‐eight isolates were identified as C. neoformans – five from decaying Eucalyptus leaves and 23 from domestic pigeon droppings. The isolates were genotyped using a URA5‐RFLP approach. The C. neoformans VNIV (53.6%, n = 15) and VNI (32.1%, n = 9) genotypes were abundantly present among environmental isolates. The hybrid VNIII (14.3%, n = 4) genotype was underrepresented and the VNII was not found. Cryptococcus gattii was also not found although some isolates yielded a positive canavanine–glycine–bromothymol blue test.     

Molecular epidemiology and in vitro antifungal susceptibility testing of 108 clinical Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato isolates from Denmark

Cryptococcosis is mainly caused by members of the Cryptococcus gattii/Cryptococcus neoformans species complexes. Here, we report the molecular characterisation and in vitro antifungal susceptibility of Danish clinical cryptococcal isolates. Species, genotype, serotype and mating type were determined by amplified fragment length polymorphism (AFLP) fingerprinting and qPCR. EUCAST E.Def 7.2 MICs were determined for amphotericin B, flucytosine, fluconazole, voriconazole and isavuconazole. Most isolates were C. neoformans (serotype A; n = 66) and belonged to genotype AFLP1/VNI (n = 61) or AFLP1B/VNII (n = 5) followed by Cryptococcus deneoformans (serotype D; genotype AFLP2, n = 20), C. neoformans × C. deneoformans hybrids (serotype AD; genotype AFLP3, n = 13) and Cryptococcus curvatus (n = 2). Six isolates were C. gattii sensu lato, and one isolate was a C. deneoformans × C. gattii hybrid (genotype AFLP8). All isolates were amphotericin B susceptible. Flucytosine susceptibility was uniform MIC₅₀ of 4–8 mg l^?1 except for C. curvatus (MICs >32 mg l^?1). Cryptococcus gattii sensu lato isolates were somewhat less susceptible to the azoles. MICs of fluconazole (>32 mg l^?1), voriconazole (≥0.5 mg l^?1) and isavuconazole (0.06 and 0.25 mg l^?1 respectively) were elevated compared to the wild‐type population for 1/19 C. deneoformans and 1/2 C. curvatus isolates. Flucytosine MIC was elevated for 1/61 C. neoformans (>32 mg l^?1). Antifungal susceptibility revealed species‐specific differential susceptibility, but suggested acquired resistance was an infrequent phenomenon.     

Evaluation of antifungal combination against Cryptococcus spp.

The second cause of death among systemic mycoses, cryptococcosis treatment represents a challenge since that 5‐flucytosine is not currently available in Brazil. Looking for alternatives, this study evaluated antifungal agents, alone and combined, correlating susceptibility to genotypes. Eighty Cryptococcus clinical isolates were genotyped by URA5 gene restriction fragment length polymorphism. Antifungal susceptibility was assessed following CLSI‐M27A3 for amphotericin (AMB), 5‐flucytosine (5FC), fluconazole (FCZ), voriconazole (VRZ), itraconazole (ITZ) and terbinafine (TRB). Drug interaction chequerboard assay evaluated: AMB + 5FC, AMB + FCZ, AMB + TRB and FCZ + TRB. Molecular typing divided isolates into 14 C. deuterogattii (VGII) and C. neoformans isolates were found to belong to genotype VNI (n = 62) and VNII (n = 4). C. neoformans VNII was significantly less susceptible than VNI (P = 0.0407) to AMB; C. deuterogattii was significantly less susceptible than VNI and VNII to VRZ (P < 0.0001). C. deuterogattii was less susceptible than C. neoformans VNI for FCZ (P = 0.0170), ITZ (P < 0.0001) and TRB (P = 0.0090). The combination FCZ + TRB showed 95.16% of synergistic effect against C. neoformans genotype VNI isolates and all combinations showed 100% of synergism against genotype VNII isolates, suggesting the relevance of cryptococcal genotyping as it is widely known that the various genotypes (now species) have significant impact in antifungal susceptibilities and clinical outcome. In difficult‐to‐treat cryptococcosis, terbinafine and different antifungal combinations might be alternatives to 5FC.     

In vitro susceptibility testing of amphotericin B for Cryptococcus neoformans variety grubii AFLP1/VNI and Cryptococcus gattii AFLP6/VGII by CLSI and flow cytometry

Cryptococcus neoformans var. grubii AFLP1/VNI is the main causative agent of cryptococcosis associated with AIDS in the world. Cryptococcus gattii AFLP6/VGII causes mainly endemic primary infection in immunocompetent hosts. To determine the minimum inhibitory concentrations (MICs) of C. neoformans var. grubii AFLP1/VNI and C. gattii AFLP6/VGII against amphotericin B (AMB) in a short period of time, flow cytometry (FCM) with FUN‐1 fluorochrome was used to compare with broth microdilution method (CLSI M27‐A3). The minimum incubation period was evaluated by minimum fungicidal concentration procedure. Seventeen clinical isolates of C. neoformans var. grubii AFLP1/VNI and 18 of C. gattii AFLP6/VGII were analysed. The time for the determination of MICs by FCM was 2 h against 72 h by CLSI M27‐A3 and the comparison of MIC showed a positive significant correlation (P = 0.048). It is important to highlight the role of the FCM as an alternative method to determine the MICs for AMB in within a day, with positive cost‐benefit.     

Molecular types of Cryptococcus gattii/Cryptococcus neoformans species complex from clinical and environmental sources in Nairobi,Kenya

Cryptococcal meningitis infections cause high mortality rates among HIV‐infected patients in Sub‐Saharan Africa. The high incidences of cryptococcal infections may be attributed to common environmental sources which, if identified, could lead to institution of appropriate control strategies. To determine the genotypes of Cryptococcus gattii/C. neoformans‐ species complex from Nairobi, Kenya, 123 clinical and environmental isolates were characterised. Typing was done using orotidine monophosphate pyrophosphorylase (URA5) gene restriction fragment length polymorphism (URA5‐RFLP). The majority of the isolates [105/123; 85.4%] were C. neoformans genotype (AFLPI/VNI) and 1.6% AFLP1A/VNB/VNII, whereas (13%) were C. gattii (AFLP4/VGI). This is the first report on the genotypes of C. gattii/C. neoformans species complex from clinical and environmental sources in Nairobi, Kenya and the isolation of C. gattii genotype AFLP4/VGI from the environment in Kenya.     

Cryptococcal meningitis due to Cryptococcus neoformans genotype AFLP1/VNI in Iran: a review of the literature

Cryptococcal meningitis is the most important opportunistic fungal infection with a high mortality in HIV‐patients in less developed regions. Here, we report a case of cryptococcal meningitis in a 49‐year‐old HIV‐positive female due to Cryptococcus neoformans (serotype A, mating‐type alpha, genotype AFLP1/VNI) in Sari, Iran. In vitro antifungal susceptibility tests showed MICs of isavuconazole (0.016 μg ml^?1), voriconazole (0.031 μg ml^?1), posaconazole (0.031 μg ml^?1), itraconazole (0.063 μg ml^?1), amphotericin B (0.125 μg ml^?1) and fluconazole (8 μg ml^?1). Despite immediate antifungal therapy, the patient died 4 days later due to respiratory failure. Cryptococcal infections have been infrequently reported from Iran and therefore we analysed all published cases of cryptococcosis in Iran since the first reported case from 1969.     

Genotypic diversity and antifungal susceptibility of Cryptococcus neoformans isolates from paediatric patients in China

Cryptococcosis is a life‐threatening mycosis primarily occurring in adult patients particularly those with immunosuppression such as HIV infection/AIDS. The number of reported cases of paediatric cryptococcosis has increased in the last decade around the world, including China. However, current information on the characteristics of cryptococcosis in children, particularly the genotypic diversity and antifungal susceptibility of the isolates, is limited. In the present study, a total of 25 paediatric isolates of Cryptococcus neoformans were genotyped using the ISHAM‐MLST scheme. In vitro susceptibility to antifungal agents of the 22 isolates was tested using the CLSI M27‐A3 method. Our analyses revealed that the genotypic diversity of C. neoformans isolates from Chinese paediatric patients was low, with ST 5 (80%) and ST 31 (12%) being the two major sequence types. Reduced susceptibility to fluconazole (FLU), 5‐flucytosine (5‐FC) and itraconazole (ITR) was observed among C. neoformans isolates from Chinese paediatric patients, particularly among the ST5 isolates, which was similar to observations made on C. neoformans isolates from Chinese adult patients. In addition, the majority of isolates (3/4, 75%) obtained from deceased patients showed decreased antifungal susceptibility, which indicates that further monitoring of antifungal susceptibility of Cryptococcus isolates is warranted in management of paediatric cryptococcosis.     

Molecular characterisation of the causative agents of Cryptococcosis in patients of a tertiary healthcare facility in the state of Amazonas-Brazil

As there are four major molecular types of Cryptococcus neoformans (VNI, VNII, VNIII and VNIV) and four molecular types of Cryptococcus gattii (VGI, VGII, VGIII and VGIV), it is important to identify the specific groups causing cryptococcosis in different geographical regions. Here, we investigated the molecular types of 57 cryptococcal isolates from patients in a tertiary care hospital in the state of Amazonas, Brazil, between 2006 and 2010. The isolates were characterised by PCR fingerprinting using the M13 minisatellite and confirmed by URA5-RFLP analysis, and the presence of specific genes from the mating type locus (MATα and MATa) of these species was analysed by PCR. Most of the patients were male (66.7%), between 16 and 30 years of age (51.7%), and HIV-positive (75.0%). Most isolates were collected from cerebrospinal fluid samples (71.7%). Most of the C. neoformans isolates (n=40) were characterised as members of the VNI molecular group (n=39), a unique isolate was characterised as VNII whereas all isolates of C. gattii (n=17) were members of the VGII molecular group. With regard to mating types, 55 isolates were type 'α', and only two were type 'a'. This study revealed the prevalence of the VNI molecular group and provides the first reported observation of the VNII molecular group in the northern region of Brazil.     

Molecular epidemiology and in vitro antifungal susceptibility of Trichophyton schoenleinii,agent of tinea capitis favosa

Trichophyton schoenleinii is an anthropophilic dermatophyte usually causing tinea favosa. Only few studies have provided data on molecular epidemiology and antifungal profiles of this fungus due to its limited prevalence after 1950s. Forty‐nine strains from Asia (n = 27), Africa (n = 10), Europe (n = 10) and from unknown regions (n = 2) were analysed with amplified fragment length polymorphism fingerprinting (AFLP) to reveal intraspecific genetic diversity in this dataset. Amplified fragment length polymorphism fingerprinting genotyping revealed five clusters which did not correspond to geographic origins or clinical characteristics. Additionally, in vitro antifungal susceptibility to seven antifungals was provided for all strains. Terbinafine, ketoconazole, miconazole and itraconazole proved to be the most effective drugs, followed by griseofulvin. No correlation between genotypes and differences in antifungal susceptibility was observed. It is concluded that the AFLP groups are lineages within a single species.     

Five‐year profile of candidaemia at an Indian trauma centre: High rates of Candida auris blood stream infections

Candidaemia is a potentially fatal infection with varied distribution of Candida species and their antifungal susceptibility profiles. The recent emergence of Candida auris in invasive candidiasis is a cause for concern. This study describes the profile of candidaemia at an Indian tertiary care hospital and reports the emergence of C. auris. All patients diagnosed with candidaemia between 2012 and 2017 were studied. The isolates were identified using conventional methods, VITEK 2 and MALDI‐TOF MS. The isolates not identified by MALDI‐TOF were sequenced. Antifungal susceptibility testing was done by the CLSI broth microdilution method and VITEK 2. A total of 114 isolates of Candida species were analysed. Candida tropicalis (39.4%) was the most common species, followed by C. auris (17.5%), C. albicans (14%) and C. parapsilosis (11.4%). Notably, Diutina mesorugosa isolates (n = 10) were not identified by MALDI‐TOF and were confirmed by sequencing. Furthermore, 45% (n = 9) C. auris strains exhibited low MICs of FLU (0.05‐4 μg/mL) and the remaining 55% (n = 11) isolates had high MICs ≥ 64 μg/mL. Also, D. mesorugosa exhibited high MICs of FLU (32 μg/mL) in 2 isolates. A high rate of errors in antifungal susceptibility was noted with the VITEK 2 as compared to the CLSI method. Candida auris was the second most prevalent species causing candidaemia warranting infection control practices to be strengthened to prevent its spread.     

Mating genotypes and susceptibility profiles of clinical isolates of Candida glabrata from Turkey

The sexual cycle of Candida glabrata is not known; however, genomic evidence is indicative of recombination among subpopulations and the genome harbours genes necessary for undergoing mating and meiosis, which may increase fitness. The relationship between specific mating type‐like (MTL) loci and antifungal susceptibility is not well understood in C. glabrata. We investigated different combinations of clinical C. glabrata isolate mating types and their antifungal susceptibility profiles. Allele profiles of the mating genes of 103 clinical C. glabrata isolates were identified, and their antifungal susceptibility to azoles, echinocandins and amphotericin B were compared. The majority (88.3%) of screened isolates harboured the a allele in the locus. The MTL1, MTL2 and MTL3 loci harboured a (88.3%), a (95.1%), and α (71.8%) alleles, respectively. The C. glabrata isolates were susceptible to echinocandins but displayed high minimal inhibitory concentrations (MICs) for azoles. The MIC ranges and MIC90 values of all isolates were 1.0 to ≥64 and 8.0 μg mL^?1 for fluconazole, 0.06 to ≥16.0 and 0.5 μg mL^?1 for voriconazole, 0.06 to ≥16.0 and 1.0 μg mL^?1 for posaconazole, ≤0.015 to 0.06, and 0.03 μg mL^?1 for caspofungin, ≤0.015 to 0.06 and 0.015 μg mL^?1 for anidulafungin and 0.5‐2 and 2.0 μg mL^?1 for amphotericin B, respectively. The mating gene alleles of the clinical C. glabrata isolates were not associated with differences in the MICs of the tested antifungals, except for the MTL3 α‐allele and echinocandins. The mating genotypes of the clinical C. glabrata isolates had no recognisable common effect on antifungal susceptibility.     

Genotyping of Cryptococcus neoformans isolated from captive birds in Uberaba,Minas Gerais,Brazil

To evaluate Cryptococcus spp. molecular types isolated from captive birds’ droppings, an epidemiological survey was carried out in Uberaba, Minas Gerais, Brazil, from December 2006 to September 2008. A total of 253 samples of bird excreta (120 fresh and 133 dry) were collected from pet shop cages and houses in different neighbourhoods. Cryptococcus neoformans was isolated in 19 (14.28%) dry samples and one fresh sample (0.84%). Cryptococcus laurentii was recovered from seven (5.26%) dry samples, but not in the fresh samples. The canavanine–glycine–bromothymol blue test was positive in all but one of the C. laurentii isolates. Cryptococcus neoformans molecular typing was performed using URA5‐RFLP and the mating type locus using mating type specific PCR. Nineteen (95.0%) presented genotype VNI and one VNII (5.0%). In addition, all isolates presented mating type α. Thus, the genotype of the environmental C. neoformans isolates observed in this study is in accordance with others already reported around the world and adds information about its distribution in Brazil. Cryptococcus laurentii strains were typed using URA5‐RFLP and M13 fingerprinting, which showed similar profiles among them. Thus, despite the low number of C. laurentii isolates analysed, their molecular profile is different from another already reported.     

Molecular characterisation of clinical Cryptococcus neoformans and Cryptococcus gattii isolates from Sichuan province,China

Previous reports on the molecular characteristics of clinical isolates of Cryptococcus species in China have focused on isolates from southeast China. To obtain a more detailed molecular epidemiology, a total of 92 cryptococcal isolates were collected from Sichuan province. A total of 24 isolates from 12 other provinces were collected for comparative study. Genotypes and mating types of 116 Cryptococcus isolates were determined. Among the 116 isolates, 43 isolates (19 isolates from Sichuan and 24 isolates outside of Sichuan) were analysed by multi‐locus sequence typing (MLST). All 116 clinical isolates were mating type α. Most isolates (114/116) were molecular type VNI and the remaining two isolates were VGI and VGII respectively. MLST results revealed five sequence types (STs) of C. neoformans including two novel STs, with most isolates identified as ST5. The two C. gattii isolates identified in our study were ST44 and ST159. Based on our report and previous studies, there are 15 C. neoformans STs in China which can be divided into three subgroups. The C. gattii isolate from Sichuan could be a scattered subtype of VGII (ST44). Our findings demonstrated that C. neoformans isolates in Sichuan are genetically homogeneous, and ST5 is the epidemic clone of C. neoformans in China.     

In vitro antifungal activity of amphotericin B and 11 comparators against Aspergillus terreus species complex

Aspergillus terreus infections are difficult to treat because of the intrinsic resistance to amphotericin B, and higher mortality compared to infections caused by other Aspergillus species. The aim of the present study was to determine the in vitro antifungal activity of amphotericin B and 11 comparators against clinical (n = 36) and environmental (n = 45) A. terreus isolates. In vitro antifungal susceptibility was performed using the CLSI M38‐A2 procedure. Amphotericin B exhibited the highest MICs (MIC range, 0.125‐4 μg/mL; MIC₉₀, 2 μg/mL), followed by terbinafine (MIC range, 0.002‐1 μg/mL; MIC₉₀, 1 μg/mL). Only one isolate (1/81) showed amphotericin B MIC above the epidemiologic cut‐off value (ECV; 4 μg/mL). None of the isolates had a MIC of ≥ ECV for voriconazole, itraconazole and posaconazole. The reasons for the difference in amphotericin B susceptibility patterns between studies remain unknown. The genetic and species diversity, clinical, environmental and ecological factors in Terrei section on various amphotericin B susceptibility profiles in different countries should be considered more as the main reasons associated with these differences.     

Multidrug‐resistant Fusarium in keratitis: a clinico‐mycological study of keratitis infections in Chennai,India

In this study, we aimed to present the first molecular epidemiological data from Chennai, India, analyse keratitis cases that have been monitored in a university hospital during 2 years, identify the responsible Fusarium species and determine antifungal susceptibilities. A total of 10 cases of keratitis were included in the study. Fusarium isolates were identified using the second largest subunit of the RNA polymerase gene (RPB2) and the translation elongation factor 1 alpha (TEF1). Antifungal susceptibility was tested by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) methodology. The aetiological agents belonged to Fusarium solani species complex (FSSC) (n = 9) and Fusarium sambucinum species complex (FSAMSC) (n = 1), and the identified species were Fusarium keratoplasticum (n = 7), Fusarium falciforme (n = 2) and Fusarium sporotrichioides (n = 1). All strains showed multidrug resistance to azoles and caspofungin but exhibited lower minimum inhibitory concentration (MIC) to natamycin and amphotericin B. Fusarium keratoplasticum and Fusarium falciforme belonging to the Fusarium solani species complex were the major aetiological agents of Fusarium keratitis in this study. Early presentation and 5% topical natamycin was associated with better patient outcome. Preventative measures and monitoring of local epidemiological data play an important role in clinical practice.     

Electrophoretic karyotyping of Cryptococcus neoformans AD-hybrid strains

This study investigated the differences in genome structure between haploid serotype A and D isolates and AD-hybrid strains of Cryptococcus neoformans , and the correlation between the karyotype of A and D strains with their mating ability. The electrophoretic karyotyping of 16 AD-hybrid, eight haploid serotype-A MATα, and eight haploid serotype-D MATα C. neoformans isolates was performed. These 32 isolates presented, two by two, the same genotype and flow cytometry profile. Five clusters were identified, each including VNI (serotype A), VNIV (serotype D) haploid strains and VNIII AD hybrids. Similarly, mating types were also randomly distributed in the five clusters. In addition, AD-hybrid isolates, with double content of DNA, showed only a slight increase in both the number of chromosomal bands and the calculated genome size compared with haploid isolates. Data support the hypothesis that hybrid isolates are aneuploids (2n+x) rather than eudiploids (2n). In addition, a set of six mating type a strains were karyotyped and then used for mating experiments carried out crossing the haploid isolates with similar or different karyotype profile strains. Isolates with completely different karyotype were able to mate confirming that meiosis occurred even in the presence of chromosomes of different lengths.     

Susceptibility profile and epidemiological cut‐off values of Cryptococcus neoformans species complex from Argentina

Epidemiological cut‐off values (ECVs) based on minimal inhibitory concentration (MIC) distribution have been recently proposed for some antifungal drug/Cryptococcus neoformans combinations. However, these ECVs vary according to the species studied, being serotypes and the geographical origin of strains, variables to be considered. The aims were to define the wild‐type (WT) population of the C. neoformans species complex (C. neoformans) isolated from patients living in Argentina, and to propose ECVs for six antifungal drugs. A total of 707 unique C. neoformans isolates obtained from HIV patients suffering cryptococcal meningitis were studied. The MIC of amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole and posaconazole was determined according to the EDef 7.2 (EUCAST) reference document. The MIC distribution, MIC₅₀, MIC₉₀ and ECV for each of these drugs were calculated. The highest ECV, which included ≥95% of the WT population modelled, was observed for flucytosine and fluconazole (32 μg ml^?1 each). For amphotericin B, itraconazole, voriconazole and posaconazole, the ECVs were: 0.5, 0.5, 0.5 and 0.06 μg ml^?1 respectively. The ECVs determined in this study may aid in identifying the C. neoformans strains circulating in Argentina with decreased susceptibility to the antifungal drugs tested.     

In vitro postantifungal effect,adhesion traits and haemolysin production of Candida dubliniensis isolates following exposure to 5‐fluorocytosine

The phenomenon of postantifungal effect (PAFE), which is the suppression of candidal growth following brief exposure to antifungal agents, is linked with candidal pathogenicity. Adhesion to buccal epithelial cells (BEC), germ tube (GT) formation and relative cell surface hydrophobicity (CSH) are all adhesion traits of candidal pathogenicity. Ability to produce haemolysin by Candida species is also a determinant of its pathogenicity. There is no information on either the PAFE or its impact on adhesion traits and haemolysin production of oral Candida dubliniensis isolates following exposure to 5‐fluorocytosine (5‐FC). Hence, the focus of this investigation was to research the in vitro PAFE, adhesion to BEC, GT formation, relative CSH and haemolysin production on 20 C. dubliniensis isolates following exposure to 5‐FC. Following obtaining the minimum inhibitory concentration (MIC) of 5‐FC, isolates of C. dubliniensis were exposed to sub‐lethal concentrations (×3 MIC) of 5‐FC for 1 h. After this brief exposure, the antimycotic was removed and PAFE, adhesion to BEC, GT formation, relative CSH and haemolysin production was determined by formerly described in vitro methods. MIC (μg/ml) of C. dubliniensis isolates to 5‐FC ranged from 0.002 to 0.125. The mean PAFE (hours) elicited by 5‐FC on C. dubliniensis isolates was approximately 1 h. Exposure to 5‐FC suppressed the ability of C. dubliniensis isolates to adhere BEC, GT formation, relative CSH and haemolysin activity by a mean percentage reduction in 50.98%, 29.51%, 36.79% and 12.75% (P < 0.001 for all) respectively. Therefore, brief exposure of C. dubliniensis isolates to 5‐FC appears to exert an antifungal effect by subduing its growth, adhesion traits as well as haemolysin production.     

Central nervous system infection due to Cryptococcus gattii sensu lato in India: Analysis of clinical features,molecular profile and antifungal susceptibility

Cryptococcus gattii species complex has evolved as a pathogen in the last two decades causing infection among both immunocompetent and immunocompromised hosts. We aimed to analyse the clinical features of CNS infection caused by C. gattii sensu lato, molecular and antifungal susceptibility profile of this pathogen. Cases diagnosed to have CNS cryptococcosis were included in the study. Cryptococcus recovered from patient's specimen was identified by standard protocol. Species confirmation, mating type and molecular type determination were performed by PCR based methods. Antifungal susceptibility was tested in VITEK2C to amphotericin B, 5‐flucytosine, fluconazole and voriconazole. Among 199 cases, 20 (10%) were due to C. gattii, comprising of 75% cryptococcal meningitis and 25% cryptococcoma cases. Young adult males were commonly affected. Headache and vomiting were prominent symptoms and 50% were immunocompromised. Among the isolates, 75%, 20% and 5% were C. tetragattii, C. gattii sensu stricto and C. bacillisporus respectively and all had mating type α. Four (20%) isolates of C. tetragattii and the only isolate of C. bacillisporus were resistant to fluconazole. The most common species isolated from south India is C. tetragattii. The study contributes to the epidemiology of C. gattii and reiterates the need for genotyping and antifungal susceptibility testing.     

Molecular typing of environmental Cryptococcus neoformans/C. gattii species complex isolates from Manaus,Amazonas, Brazil

Cryptococcus neoformans and Cryptococcus gattii are the main causative agents of cryptococcosis, a systemic fungal disease that affects internal organs and skin, and which is acquired by inhalation of spores or encapsulated yeasts. It is currently known that the C. neoformans/C. gattii species complex has a worldwide distribution, however, some molecular types seem to prevail in certain regions. Few environmental studies of Cryptococcus have been conducted in the Brazilian Amazon. This is the first ecological study of the pathogenic fungi C. neoformans/C. gattii species complex in the urban area of Manaus, Amazonas, Brazil. A total of 506 samples from pigeon droppings (n = 191), captive bird droppings (n = 60) and tree hollows (n = 255) were collected from June 2012 to January 2014 at schools and public buildings, squares, pet shops, households, the zoo and the bus station. Samples were plated on niger seed agar (NSA) medium supplemented with chloramphenicol and incubated at 25°C for 5 days. Dark‐brown colonies were isolated and tested for thermotolerance at 37°C, cycloheximide resistance and growth on canavanine‐glycine‐bromothymol blue agar. Molecular typing was done by PCR‐RFLP. Susceptibility to the antifungal drugs amphotericin B, fluconazole, itraconazole and ketoconazole was tested using Etest^® strips. In total, 13 positive samples were obtained: one tree hollow (C. gattiiVGII), nine pigeon droppings (C. neoformansVNI) and three captive bird droppings (C. neoformansVNI). The environmental cryptococcal isolates found in this study were of the same molecular types as those responsible for infections in Manaus.