Comparative antifungal susceptibility analyses of Cryptococcus neoformans VNI and Cryptococcus gattii VGII from the Brazilian Amazon Region by the Etest, Vitek 2, and the Clinical and Laboratory Standards Institute broth microdilution methods.

Early diagnosis, efficient clinical support, and proper antifungal therapy are essential to reduce death and sequels caused by cryptococcosis. The emergence of resistance to the antifungal drugs commonly used for cryptococcosis treatment is an important issue of concern. Thus, the in vitro antifungal susceptibility of clinical strains from northern Brazil, including C. neoformans VNI (n = 62) and C. gattii VGII (n = 37), to amphotericin B (AMB), 5-flucytosine, fluconazole, voriconazole, and itraconazole was evaluated using the Etest and Vitek 2 systems and the standardized broth microdilution (CLSI-BMD) methodology. According to the CLSI-BMD, the most active in vitro azole was voriconazole (C. neoformans VNI modal MIC of 0.06 µg/ml and C. gattii VGII modal MIC of 0.25 µg/ml), and fluconazole was the least active (modal MIC of 4 µg/ml for both fungi). Modal MICs for amphotericin B were 1 µg/ml for both fungi. In general, good essential agreement (EA) values were observed between the methods. However, AMB presented the lowest EA between CLSI-BMD and Etest for C. neoformans VNI and C. gattii VGII (1.6% and 2.56%, respectively, P < .05 for both). Considering the proposed Cryptococcus spp. epidemiological cutoff values, more than 97% of the studied isolates were categorized as wild-type for the azoles. However, the high frequency of C. neoformans VNI isolates in the population described here that displayed non-wild-type susceptibility to AMB is noteworthy. Epidemiological surveillance of the antifungal resistance of cryptococcal strains is relevant due to the potential burden and the high lethality of cryptococcal meningitis in the Amazon region.

Determination of the minimum inhibitory concentration of Cryptococcus neoformans and Cryptococcus gattii against fluconazole by flow cytometry.

Recent studies have used flow cytometry (FCM) as an important alternative method to determine the antifungal susceptibility of yeasts compared to the broth microdilution Clinical and Laboratory Standards Institute (CLSI) reference procedure. We present a comparative study of the broth microdilution method and flow cytometry to assess the in vitro antifungal susceptibility of Cryptococcus neoformans (n = 16) and C. gattii (n = 24) to fluconazole. The minimum inhibitory concentration (MIC) assays by flow cytometry were defined as the lowest drug concentration that showed ∼50% of the count of acridine orange negative cells compared to that of the growth control. Categorical classification showed all C. neoformans isolates were susceptible to fluconazole. Three isolates of C. gattii were susceptible dose-dependent and the remaining 21 isolates were classified as susceptible. MICs comparison of both methodologies demonstrated 100% categorical agreement of the results obtained for C. neoformans and C. gattii. The MICs obtained with the CLSI-approved method and flow cytometry were compared by the Spearman correlation test and a significant Pv = 0.001. The flow cytometric method has the advantage of analyzing a large and constant number of cells in less time, i.e., 9 h incubation for fluconazole using acridine orange versus 72 h for broth microdilution method. In conclusion, the two methods were comparable and flow cytometry method can expedite and improve the results of in vitro susceptibility tests of C. neoformans and C. gattii against fluconazole and also allows comparative studies in vitro/in vivo more rapidly, which along with clinical data, could assist in selecting the most appropriate treatment choice.

Identification of a Candida parapsilosis strain producing extracellular serine peptidase with keratinolytic activity.

A yeast strain isolated from feather waste from a chicken processing plant was identified as Candida parapsilosis by biochemical tests and morphological studies. The yeast was able to grow in phosphate-buffered saline supplemented with 1% native feather as the sole carbon and nitrogen source. A keratin substrate was obtained from the feathers by dimethylsulphoxide extraction. A 20-fold concentrated culture supernatant from Candida parapsilosis grown on feathers was analysed by SDS-PAGE electrophoresis containing either 1% gelatin or 1% keratin as copolymerised substrates. The presence of a single band with an approximate molecular mass of 60 kDa with gelatinolytic and keratinolytic activities was observed. This proteolytic activity was fully inhibited by phenylmethylsulphonyl fluoride. These results suggest that the extracellular enzyme belongs to the serine peptidase class. This is the first report of an extracellular serine peptidase produced by C. parapsilosis with keratinolytic activity. The role of this enzyme in yeast-host interactions is discussed.

Indoor Dust as a Source of Virulent Strains of the Agents of Cryptococcosis in the Rio Negro Micro-Region of the Brazilian Amazon.

Cryptococcosis, a potentially fatal mycosis in humans, is acquired via exposure to exogenous environmental sources. This study aimed to investigate the frequency, genetic diversity, and virulence of cryptococcal strains isolated from indoor dust in the Rio Negro micro-region of the Brazilian Amazon. A total of 8.9% of the studied houses were positive, recovering nine Cryptococcus neoformans VNI and 16 C. gattii VGII isolates, revealing an endemic pattern in domestic microenvironments. The International Society for Human and Animal Mycology (ISHAM) consensus multilocus sequence typing (MLST) scheme for the C. neoformans/C. gattii species complexes identified two sequence types (STs), ST93 and ST5, amongst C. neoformans isolates and six STs amongst C. gattii isolates, including the Vancouver Island Outbreak ST7 (VGIIa) and ST20 (VGIIb), the Australian ST5, and ST264, ST268 and ST445, being unique to the studied region. Virulence studies in the Galleria mellonella model showed that five C. gattii strains and one C. neoformans strain showed a similar pathogenic potential to the highly virulent Vancouver Island outbreak strain CDR265 (VGIIa). The findings of this study indicate that humans can be exposed to the agents of cryptococcosis via house dust, forming the basis for future studies to analyze the impact of early and continuous exposure to indoor dust on the development of subclinical or clinical infections.

Morphological and biochemical characterization of the aetiological agents of white piedra.

The Trichosporon genus is constituted by many species, of which Trichosporon ovoides and Trichosporon inkin are the causative agents of white piedra. They can cause nodules in genital hair or on the scalp. At present, Brazilian laboratory routines generally do not include the identification of the species of Trichosporon genus, which, although morphologically and physiologically distinct, present many similarities, making the identification difficult. The aim of this study was to identify the aetiological agents at the species level of white piedra from clinical specimens. Therefore, both the macro and micro morphology were studied, and physiological tests were performed. Trichosporon spp. was isolated from 10 clinical samples; T. ovoides was predominant, as it was found in seven samples, while T. inkin was identified just in two samples. One isolate could not be identified at the species level. T. inkin was identified for the first time as a white piedra agent in the hair shaft on child under the age of 10.

Regional pattern of the molecular types of Cryptococcus neoformans and Cryptococcus gattii in Brazil.

The molecular types of 443 Brazilian isolates of Cryptococcus neoformans and Cryptococcus gattii were analyzed to determine their geographic distribution within Brazil and their underlying host conditions. The following data, imported from previous epidemiological studies as well as two culture collections, were analyzed for: place of isolation, source (clinical or environmental), host risk factors, species, serotype, mating type, and molecular type. Molecular typing by PCR-fingerprinting using primers for the minisatellite-specific core sequence of the wild-type phage M13 or microsatellites [(GACA)4, (GTG)5], restriction fragment length polymorphism of URA5 gene analysis, and/or amplified fragment length polymorphism (AFLP) identified eight major genotypes: VNI/AFLP1, VNII/AFLP1A, VNIII/AFLP2, and VNIV/AFLP3 for C. neoformans, and VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5, and VGIV/AFLP7 for C. gattii. The most common molecular type found in Brazil was VNI (64%), followed by VGII (21%), VNII (5%), VGIII (4%), VGI and VNIV (3% each), and VNIII (< 1%). Primary cryptococcosis caused by the molecular type VGII (serotype B, MAT alpha) prevails in immunocompetent hosts in the North and Northeast regions, disclosing an endemic regional pattern for this specific molecular type in the Northern Brazil.

Primary endemic Cryptococcosis gattii by molecular type VGII in the state of Pará, Brazil.

In order to study the infectious agents causing human disseminated cryptococcosis in the state of Pará, North Brazil, 56 isolates of Cryptococcusspp. (54 isolated from cerebral spinal fluid and two from blood cultures) from 43 cases diagnosed between 2003-2007 were analysed. The species were determined through morphological and physiological tests and genotypes were determined by URA5-RFLP and PCR-fingerprinting (wild-type phage M13). The following species and genotypes were identified: Cryptococcus neoformans VNI (28/56, 50%), Cryptococcus gattii VGII (25/56, 44.64%) and C. gattii VGI (3/56, 5.26%). The genotype VNI occurred in 12 out of 14 HIV-positive adults, whereas the genotype VGII occurred in 11 out of 21 HIV-negative adults (p < 0.02, OR = 6.6 IC95% 0.98-56.0). All patients less than 12 years old were HIV negative and six cases were caused by the VGII genotype, one by the VGI and one by VNI. Therefore, endemic primary mycosis in HIV-negative individuals, including an unexpectedly high number of children, caused by the VGII genotype deserves further study and suggests the need for surveillance on cryptococcal infection in the state of Pará, Eastern Amazon.

Trichosporon isolation from human ungueal infections: is there a pathogenic role?

BACKGROUND: Although dermatophytes are considered the major cause of onychomycosis, many reports have incriminated non-dermatophyte moulds and yeasts in the disease's etiology. Successive Trichosporon isolation from onychomycosis has led to the genus being suspected as a nail primary pathogen. OBJECTIVE: To determine the prevalence of Trichosporon isolation in onychomycosis patients who attended a mycology diagnostic service in Rio de Janeiro, Brazil, between January 2003 and December 2006. The study also includes a worldwide review on Trichosporon isolation prevalence in ungueal disease, emphasizing T. ovoides. METHODS: This retrospective study was conducted with the support of staff from the Mycology Laboratory at the Dermatological Service of Rio de Janeiro's Santa Casa da Misericórdia (MLDS). RESULTS: Mycological analysis provided positive results equaling 47/5036 (0.93%) for Trichosporon spp.; obtained mainly as a single agent (72.35%), and from mixed cultures (27.65%; X2= 6.397; p= 0.018). The great majority belongs to the T. ovoides species (91.5%; n=43), obtained as a single isolate (74.41%; n= 32/43; X2 = 7.023; p= 0.014). CONCLUSIONS: Although T. ovoides is classically associated as an etiologic agent of white piedra, this study highlights its potential as a human nail disease pathogen. Our study opens doors for future epidemiologic and virulence factors aimed at determining whether T. ovoides is an important causative agent of onychomycosis in Brazil.

Environmental isolation of Cryptococcus gattii VGII from indoor dust from typical wooden houses in the deep Amazonas of the Rio Negro basin.

Cryptococcosis is a human fungal infection of significant mortality and morbidity, especially in the meningoencephalitis form. Cryptococcosis is distributed worldwide and its agents, C. neoformans and C. gattii, present eight major molecular types-VNI-VNIV and VGI-VGIV respectively. The primary cryptococcosis caused by molecular type VGII (serotype B, MAT alpha) prevails in immunocompetent patients in the North and Northeast of Brazil, revealing an endemic regional pattern to this molecular type. Since 1999, C. gattii VGII has been involved in an ongoing outbreak in Canada, and is expanding to the Northwest of the United States, two temperate regions. Exposure to propagules dispersed in the environment, related to various organic substrates, mainly decomposing wood in and around dwellings, initiates the infection process. The present study investigated the presence of the agents of cryptococcosis in dust from dwellings in the upper Rio Negro, municipality of Santa Isabel do Rio Negro in Amazonas state. Indoor dust was collected from 51 houses, diluted and plated on bird seed agar. Dark brown colonies were identified phenotypically, and genotypically by URA5 restriction fragment length polymorphism analysis and multilocus sequence typing (MLST). The mating type was identified using pheromone-specific primers. Three of the 51 houses were positive for C. gattii molecular type VGII, MATα and MATa, showing a high prevalence of this agent. MLST studies identified eight subtypes, VGIIb (ST7), VGIIa (ST20), (ST5) and 5 new subtypes unique to the region. For the first time in the state of Amazonas, C. gattii VGII MATα and MATa were isolated from the environment and correlates with endemic cryptococcosis in this state. This is the first description of MLST subtypes on environmental isolates in the Brazilian Amazon, indicating domiciliary dust as a potential source for human infection with different subtypes of C. gattii VGII MATα and MATa.

Trichosporon isolation from human ungueal infections: is there a pathogenic role?

Abstract BACKGROUND: Although dermatophytes are considered the major cause of onychomycosis, many reports have incriminated non-dermatophyte moulds and yeasts in the disease’s etiology. Successive Trichosporon isolation from onychomycosis has led to the genus being suspected as a nail primary pathogen. OBJECTIVE: To determine the prevalence of Trichosporon isolation in onychomycosis patients who attended a mycology diagnostic service in Rio de Janeiro, Brazil, between January 2003 and December 2006. The study also includes a worldwide review on Trichosporon isolation prevalence in ungueal disease, emphasizing T. ovoides. METHODS: This retrospective study was conducted with the support of staff from the Mycology Laboratory at the Dermatological Service of Rio de Janeiro’s Santa Casa da Misericórdia (MLDS). RESULTS: Mycological analysis provided positive results equaling 47/5036 (0.93%) for Trichosporon spp.; obtained mainly as a single agent (72.35%), and from mixed cultures (27.65%; X2= 6.397; p= 0.018). The great majority belongs to the T. ovoides species (91.5%; n=43), obtained as a single isolate (74.41%; n= 32/43; X2 = 7.023; p= 0.014). CONCLUSIONS: Although T. ovoides is classically associated as an etiologic agent of white piedra, this study highlights its potential as a human nail disease pathogen. Our study opens doors for future epidemiologic and virulence factors aimed at determining whether T. ovoides is an important causative agent of onychomycosis in Brazil.

A 25-kDa serine peptidase with keratinolytic activity secreted by Coccidioides immitis.

Coccidioides immitis is the causative agent of coccidioidomycosis, a systemic mycosis that attacks humans and a wide variety of animals. In the present study, we showed that the C. immitis mycelial form is able to release proteolytic enzyme into the extracellular environment. Under chemically defined growth conditions, mycelia secreted seven distinct polypeptides ranging from 15 to 65 kDa and an extracellular peptidase of 25 kDa. This enzyme had its activity fully inhibited by phenylmethylsulphonyl fluoride, a serine peptidase inhibitor. Conversely, metallo, cysteine, and aspartyl peptidase inhibitors did not alter the 25-kDa enzyme behavior. This extracellular serine peptidase was able to degrade keratin, a fibrous protein that composes human epidermis. Additionally, this peptidase cleaved different protein substrates, including gelatin, casein, hemoglobin, and albumin. Curiously, an 18-kDa serine peptidase activity was evidenced solely when casein was used as the co-polymerized protein substrate into the gel. The existence of different secreted peptidases could be advantageous for the adaptation of C. immitis to distinct environments during its complex life cycle.

Avaliação de um Sistema de Banco de Dados para Coleção de Fungos de Referência / Evaluation of a Database System for Reference Fungi Collection

De acordo com o Guia para Operação de Centros de Recursos Biológicos da Organização para Cooperação e Desenvolvimento Econômico (OCDE), os Centros de Recursos Biológicos devem dispor os dados descritivos do e sua origem ao Global Biological Resource Center Network. Essa deliberação ratifica a preocupação da validação de ferramentas utilizadas no tratamento de dados pelas coleções e laboratórios prestadores de serviços no escopo indireto de suas rotinas, visando à qualidade. A avaliação do Sistema de Banco de Dados da Coleção de Culturas de Fungos de Referência (INFOGER_FUNGOS), do Instituto Nacional de Controle de Qualidade em Saúde (INCQS), de acordo com a norma ISO/IEC nº 17.025:2005, estabelece um marco na qualidade e integridade das informações, economizando tempo na elaboração das estruturas do sistema permitindo ao profissional diretamente envolvido com a coleção e suas particularidades seja seu administrador. Este trabalho teve como objetivo demonstrar um método de avaliação de um sistema de gerenciamento de dados no cumprimento de sua finalidade atingindo níveis de qualidade satisfatórios. Ele servirá de modelo para avaliações de sistemas utilizados em coleções de micro-organismos dentro das normas da Qualidade e Acreditação de seus serviços e produtos, e colaborar no estabelecimento de padrão de dados baseado em experiências brasileiras, adaptado e estendido a modelos existentes.

Morphological and biochemical characterization of the aetiological agents of white piedra

The Trichosporon genus is constituted by many species, of which Trichosporon ovoides and Trichosporon inkin are the causative agents of white piedra. They can cause nodules in genital hair or on the scalp. At present, Brazilian laboratory routines generally do not include the identification of the species of Trichosporon genus, which, although morphologically and physiologically distinct, present many similarities, making the identification difficult. The aim of this study was to identify the aetiological agents at the species level of white piedra from clinical specimens. Therefore, both the macro and micro morphology were studied, and physiological tests were performed. Trichosporon spp. was isolated from 10 clinical samples; T. ovoides was predominant, as it was found in seven samples, while T. inkin was identified just in two samples. One isolate could not be identified at the species level. T. inkin was identified for the first time as a white piedra agent in the hair shaft on child under the age of 10.

Regional pattern of the molecular types of Cryptococcus neoformans and Cryptococcus gattii in Brazil

The molecular types of 443 Brazilian isolates of Cryptococcus neoformans and Cryptococcus gattii were analyzed to determine their geographic distribution within Brazil and their underlying host conditions. The following data, imported from previous epidemiological studies as well as two culture collections, were analyzed for: place of isolation, source (clinical or environmental), host risk factors, species, serotype, mating type, and molecular type. Molecular typing by PCR-fingerprinting using primers for the minisatellite-specific core sequence of the wild-type phage M13 or microsatellites [(GACA)4, (GTG)5], restriction fragment length polymorphism of URA5 gene analysis, and/or amplified fragment length polymorphism (AFLP) identified eight major genotypes: VNI/AFLP1, VNII/AFLP1A, VNIII/AFLP2, and VNIV/AFLP3 for C. neoformans, and VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5, and VGIV/AFLP7 for C. gattii. The most common molecular type found in Brazil was VNI (64 percent), followed by VGII (21 percent), VNII (5 percent), VGIII (4 percent), VGI and VNIV (3 percent each), and VNIII (< 1 percent). Primary cryptococcosis caused by the molecular type VGII (serotype B, MAT) prevails in immunocompetent hosts in the North and Northeast regions, disclosing an endemic regional pattern for this specific molecular type in the Northern Brazil.

Primary endemic Cryptococcosis gattii by molecular type VGII in the state of Pará, Brazil

In order to study the infectious agents causing human disseminated cryptococcosis in the state of Pará, North Brazil, 56 isolates of Cryptococcusspp. (54 isolated from cerebral spinal fluid and two from blood cultures) from 43 cases diagnosed between 2003-2007 were analysed. The species were determined through morphological and physiological tests and genotypes were determined by URA5-RFLP and PCR-fingerprinting (wild-type phage M13). The following species and genotypes were identified: Cryptococcus neoformans VNI (28/56, 50 percent), Cryptococcus gattii VGII (25/56, 44.64 percent) and C. gattii VGI (3/56, 5.26 percent). The genotype VNI occurred in 12 out of 14 HIV-positive adults, whereas the genotype VGII occurred in 11 out of 21 HIV-negative adults (p < 0.02, OR = 6.6 IC95 percent 0.98-56.0). All patients less than 12 years old were HIV negative and six cases were caused by the VGII genotype, one by the VGI and one by VNI. Therefore, endemic primary mycosis in HIV-negative individuals, including an unexpectedly high number of children, caused by the VGII genotype deserves further study and suggests the need for surveillance on cryptococcal infection in the state of Pará, Eastern Amazon.