Genotipagem, sorotipagem e determinação de mating-type de isolados clínicos de Cryptococcus neoformans do Estado de São Paulo, Brasil

The basidiomycetous yeast Cryptococcus neoformans is an important fungal pathogen mainly in immunocompromised patients. In this study, 47 clinical isolates of C. neoformans from regions of São Paulo State were studied serologically by using the Crypto Check Iatron RM 304-K kit, their genetic diversity was estimated by PCR-fingerprinting with a microsatellite-specific sequence (GACA)4, RAPD with primer 6 (Amersham Pharmacia Biotech), PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PLB1) digested with AvaI and mating type analysis by PCR. All 47 strains isolated from HIV positive patients included in this study were serotype A and MATalpha. The majority of the isolates (45/47) were VNI and only two were VNII by PCR-fingerprinting and PCR-RFLP analysis. High degree of homogeneity was observed when (GACA)4 was used, being highly correlated (>; 0.9). In contrast, the RAPD analysis was more heterogeneous with higher number of molecular profiles. By PCR-RFLP, no new molecular type was found, enhancing the suggestion that the differences based on conserved gene as PLB1, can be resultant of ongoing divergent evolution within the C. neoformans complex, into the current eight subtypes. Our results furnish new information on the molecular epidemiology of C. neoformans in the southeast region of Brazil.Cryptococcus neoformans, pertencente à classe dos basidiomicetos, é um importante patógeno, principalmente em pacientes imunocomprometidos. Neste estudo, 47 isolados clínicos de C. neoformans de várias regiões do Estado de São Paulo foram avaliados quanto aos sorotipos e ao mating-type por PCR. A diversidade genética foi analisada por PCR-fingerprinting com a seqüência iniciadora específica para regiões microssatélite (GACA)4, RAPD com o iniciador 6 (Amersham Pharmacia Biotech) e por RFLP do gene PLB1 digerido com AvaI. Todos os isolados foram obtidos de pacientes HIV positivos e identificados como sorotipo A e MATalfa. A maioria dos isolados pertencia ao tipo molecular VNI (45/47) e apenas dois foram VNII quando analisados por PCR-fingerprinting e PCR-RFLP. Homogeneidade alta foi obtida com o iniciador (GACA)4, com a maioria dos isolados apresentando correlação alta (>; 0.9). Os resultados do RAPD, por sua vez, revelaram maior heterogeneidade com número maior de perfis moleculares. Por PCR-RFLP, nenhum tipo molecular novo foi encontrado, realçando a idéia de que em genes conservados como PLB1, as diferenças podem ser resultantes de divergências evolutivas dentro do complexo C. neoformans, separando os isolados nos oito subtipos moleculares já estabelecidos. Nossos resultados fornecem novas informações sobre a epidemiologia molecular de C. neoformans na região sudeste do Brasil

Tipagem molecular, perfis de sensibilidade e caracterização de transcritos diferencialmente expressos durante a infecção de Cryptococcus neoformans

Cryptococcus neoformans é patógeno importante, principalmente em pacientes imunocomprometidos. A principal porta de entrada deste patógeno é pela via respiratória, disseminando-se posteriormente e atingindo principalmente o sistema nervoso central, provocando a meningite criptocóccica. A primeira parte deste estudo teve como objetivos determinar, nos 106 isolados clínicos de C. neoformans obtidos de dois Estados (São Paulo e Rio de Janeiro), (1) as variedades e (2) os mating-types por PCR (Polymerase Chain Reaction), (3) analisar a diversidade genética por PCR-fingerprinting com a seqüência iniciadora específica para regiões microssatélite (GACA)4, (4) por RAPD (Random Amplification of Polymorphic DNA) com o iniciador 6 e (5) por PCR-RFLP (Restriction Fragment Length Polymorphism) do gene da fosfolipase B (PLB1) digerido com a enzima de restrição AvaI e (6) determinar a sensibilidade a quatro antifúngicos (fluconazol, itraconazol, 5-fluorocitosina e anfotericina B) seguindo o método de referência (documento M27-A2) do CLSI (Clinical and Laboratory Standards Institute). A segunda parte teve como objetivo, analisar transcritos diferencialmente expressos durante a infecção pulmonar de C. neoformans, pela técnica de RDA (Representational Difference Analysis). Entre os 106 isolados, 104 foram identificados como C. neoformans e apenas dois foram C. gattii (=C. neoformans var. gattii), todos MATa. O tipo molecular VNI (C. neoformans var. grubii, sorotipo A) foi o mais prevalente entre os isolados (97/106), seguido do tipo VNII (C. neoformans var. grubii, sorotipo A) (7/106) e VGII (C. gattii, sorotipos B ou C) (2/106) quando analisados por PCR-fingerprinting e PCR-RFLP. Homogeneidade alta foi obtida com o iniciador (GACA)4, com a maioria dos isolados apresentando correlação em torno de 0,9. Os resultados do RAPD, por sua vez, revelaram maior heterogeneidade com número maior de perfis moleculares.Cryptococcus neoformans is an important pathogen, mainly in immunocompromised patients. The pathogen penetrates mainly by respiratory way, disseminate afterward and reach specially the central nervous system causing the cryptococcal meningitis. The first part of this study had the objective to determine, in the 106 clinical isolates of C. neoformans obtained from São Paulo and Rio de Janeiro State, (1) the varieties and (2) mating-types by PCR (Polymerase Chain Reaction), (3) analyze the genetic diversity by PCR-fingerprinting with specific primer to microsatellite regions (GACA)4, (4) by RAPD (Random Amplification of Polymorphic DNA) with primer 6 and (5) by PCR-RFLP (Restriction Fragment Length Polymorphism) of the phospholipase B gene (PLB1) digested with restriction enzyme AvaI and (6) to determine the susceptibility to four antifungal (fluconazole, itraconazole, 5-flucytosine and amphotericin B) following the reference method (document M27-A2) from CLSI (Clinical and Laboratory Standard Institute). The second part had the goal to analyze differentially expressed transcription during pulmonary infection of C. neoformans, by RDA (Representational Difference Analysis) technique. Among 106 isolates, 104 were identified as C. neoformans and only two were C. gattii (=C. neoformans var. gattii), all MATa. The molecular type VNI (C. neoformans var. grubii, serotype A) was the most prevalent among the isolates (97/106), followed by VNII (C. neoformans var. grubii, serotype A) (7/106) and VGII (C. gattii, serotypes B ou C) (2/106) when analyzed by PCR-fingerprinting and PCR-RFLP. High homogeneity was obtained with the primer (GACA)4, with most of the isolates showing correlation around 0.9. By contrast, the RAPD analysis revealed more heterogeneous with more numbers of molecular profiles.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

A flow cytometry method for testing the susceptibility of Cryptococcus spp. to amphotericin B

Human fungal infections have increased at an alarming rate in recent years, particularly in immunocompromised individuals. Cryptococcosis is the second most prevalent systemic fungal infection worldwide, and the most prevalent systemic infection in immunocompromised individuals, representing more than 70% of cases. The incidence of cryptococcosis is high in people with HIV/acquired immunodeficiency syndrome (AIDS), with recent estimates indicating that there are one million cases of cryptococcal meningitis globally per year in AIDS patients. The aim of this research was to develop a rapid flow cytometric antifungal susceptibility test and to compare the results with the standard methods. A reference strain and clinical isolates of Cryptococcus neoformans and Cryptococcus gattii were tested for susceptibility to amphotericin B by flow cytometry using propidium iodide as indicator of viability. Flow cytometry (FC) results were compared with the minimum inhibitory concentration (MIC) values determined by microdilution. The antifungal activity of amphotericin B ranged from MICs of 0.06 to 2μg/ml for the 11 isolates studied. The same results were found by FC. The FC method allows same-day results, assisting in the selection of appropriate antifungal therapies. These results demonstrate an excellent correlation between FC and the classic methods of testing for susceptibility to antifungal agents. This rapid diagnosis method makes it possible to quickly administer effective therapeutic interventions, often saving lives.Antecedentes: En los últimos anos, ˜ las infecciones por hongos en el ser humano, particularmente en individuos inmunodeprimidos, han aumentado a un ritmo alarmante. La criptococosis es la segunda infección fúngica sistémica más frecuente en todo el mundo, y también la infección sistémica más frecuente en personas inmunodeprimidas, lo que representa más del 70% de los casos. La incidencia de la criptococosis es alta en las personas con VIH/síndrome de inmunodeficiencia adquirida. Estudios recientes indican que hay un millón de casos de meningitis criptocócica en pacientes con síndrome de inmunodeficiencia adquirida por ano˜ en todo el mundo. Objetivos: El objetivo de esta investigación fue desarrollar una prueba rápida de sensibilidad antifúngica por citometría de flujo (CF) y comparar los resultados con los métodos estándar. Métodos: Se estudió la sensibilidad a la anfotericina B por CF, con yoduro de propidio como indicador de la viabilidad, de una cepa de referencia y varios aislamientos clínicos de Cryptococcus neoformans y Cryptococcus gattii. Resultados: La actividad antifúngica de la anfotericina B varió de una concentración mínima inhibitoria de 0,06 a 2 g/ml para los 11 aislamientos estudiados. Los mismos resultados fueron encontrados por CF. Conclusiones: El método de la CF permite resultados el mismo día, lo que hace posible una rápida selección de los tratamientos antifúngicos adecuados. Estos resultados demuestran una excelente correlación entre el método de la CF y los procedimientos clásicos de pruebas de sensibilidad a los agentes antifúngicos. Este método de rastreo ágil hace que sea posible realizar rápidamente intervenciones terapéuticas eficaces y, a menudo, salvar vida

Genotyping, serotyping and determination of mating-type of Cryptococcus neoformans clinical isolates from São Paulo State, Brazil

Cryptococcus neoformans, pertencente à classe dos basidiomicetos, é um importante patógeno, principalmente em pacientes imunocomprometidos. Neste estudo, 47 isolados clínicos de C. neoformans de várias regiões do Estado de São Paulo foram avaliados quanto aos sorotipos e ao mating-type por PCR. A diversidade genética foi analisada por PCR-fingerprinting com a seqüência iniciadora específica para regiões microssatélite (GACA)4, RAPD com o iniciador 6 (Amersham Pharmacia Biotech) e por RFLP do gene PLB1 digerido com AvaI. Todos os isolados foram obtidos de pacientes HIV positivos e identificados como sorotipo A e MATalfa. A maioria dos isolados pertencia ao tipo molecular VNI (45/47) e apenas dois foram VNII quando analisados por PCR-fingerprinting e PCR-RFLP. Homogeneidade alta foi obtida com o iniciador (GACA)4, com a maioria dos isolados apresentando correlação alta (> 0.9). Os resultados do RAPD, por sua vez, revelaram maior heterogeneidade com número maior de perfis moleculares. Por PCR-RFLP, nenhum tipo molecular novo foi encontrado, realçando a idéia de que em genes conservados como PLB1, as diferenças podem ser resultantes de divergências evolutivas dentro do complexo C. neoformans, separando os isolados nos oito subtipos moleculares já estabelecidos. Nossos resultados fornecem novas informações sobre a epidemiologia molecular de C. neoformans na região sudeste do Brasil.The basidiomycetous yeast Cryptococcus neoformans is an important fungal pathogen mainly in immunocompromised patients. In this study, 47 clinical isolates of C. neoformans from regions of São Paulo State were studied serologically by using the Crypto Check Iatron RM 304-K kit, their genetic diversity was estimated by PCR-fingerprinting with a microsatellite-specific sequence (GACA)4, RAPD with primer 6 (Amersham Pharmacia Biotech), PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PLB1) digested with AvaI and mating type analysis by PCR. All 47 strains isolated from HIV positive patients included in this study were serotype A and MATalpha. The majority of the isolates (45/47) were VNI and only two were VNII by PCR-fingerprinting and PCR-RFLP analysis. High degree of homogeneity was observed when (GACA)4 was used, being highly correlated (> 0.9). In contrast, the RAPD analysis was more heterogeneous with higher number of molecular profiles. By PCR-RFLP, no new molecular type was found, enhancing the suggestion that the differences based on conserved gene as PLB1, can be resultant of ongoing divergent evolution within the C. neoformans complex, into the current eight subtypes. Our results furnish new information on the molecular epidemiology of C. neoformans in the southeast region of Brazil.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Genetic diversity and antifungal susceptibility testing of Trichosporon asahii isolated of Intensive Care Units patients

Trichosporon asahii é um patógeno oportunista que apresenta altos índices de mortalidade em pacientes imunocomprometidos. No presente trabalho, dez cepas foram isoladas da cavidade bucal e urina de pacientes internados na Unidade de Terapia Intensiva (UTI) por seis meses. Todos os isolados foram identificados por métodos clássicos e moleculares, tipados por RAPD e testados in vitro quanto à sensibilidade ao fluconazol, itraconazol, 5-fluorocitosina e anfotericina B. Houve concordância total entre a identificação de Trichosporon sp por PCR baseado na seqüência da região ITS (Internal Transcribed Spacer) e na seqüência da subunidade menor do DNA ribossômico (rDNA). Os perfis genéticos foram determinados por RAPD utilizando dois iniciadores P6 e M13. A análise do dendrograma mostrou que a maioria das amostras apresentou alta similaridade entre elas (>0.9) e todas foram multidrogas resistentes. Este estudo traz novos resultados em relação à identificação e genotipagem de isolados brasileiros de T. asahii em pacientes internados em UTI, bem como dados sobre o perfil de sensibilidade aos antifúngicos.Trichosporon asahii is an opportunistic pathogen, associated with a high mortality rate in immunocompromised patients. In this study, ten isolates, recovered from oral cavity and urine of patients in Intensive Care Units (ICU) over six months, were identified by classical and molecular methods, typed by RAPD and tested in vitro for susceptibility to fluconazole, itraconazole, 5-flucytosine and amphotericin B. A total agreement between the identification of Trichosporon sp by PCR based on sequences of the Internal Transcribed Spacer Regions (ITS) and on the sequences of small-subunit (SSU) ribosomal DNA (rDNA) was found. Randomly amplified of polymorphic DNA (RAPD), with primers P6 and M13, was used to determine the genomic profiles. The dendogram analysis indicated that almost all strains showed similarity >0.9 among them and all strains were multidrug-resistant. This study brings new results on the identification and genotyping of T. asahii isolated from Brazilian ICU patients and information about their antifungal drugs susceptibility.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Genetic relatedness of commensal strains of Candida albicans carried in the oral cavity of patients' dental prosthesis users in Brazil

The aim of this study is to describe the degree of yeast-colonization in diabetic and hemodialysed-users of dental prostheses. Individuals (306) were examined using an oral rinse technique in order to evaluate the incidence of yeast-carriage, and genotype of C. albicans. Yeasts were isolated from 68.4% (91/133) individual's dental prostheses users. Dental prostheses were found to be a significant factor for the yeast colonization (P < 0.05). Overall, the intensity of carriage was higher in diabetic patients as compared with health and hemodialysed individuals (P < 0.05). The isolation rates were: C. albicans (51.7%), C. parapsilosis (20.9%), C. tropicalis (14.3%), C. glabrata (6.6%), C. krusei (3.3%), C. rugosa (1.1%), and Pichia (Pichia ohmeri, 2.2%). Ready-To-Go RAPD Analysis Beads were used and primer OPJ 6 distinguished the C. albicans isolates found in prostheses users. All the isolates were grouped into 11 RAPD profiles in four main clusters and, the average S (AB) for the entire collection of 47 C. albicans isolates were 0.779 +/- 0.178. Over 85% of isolates had a similarity level higher than or equal to 0.8 reinforcing the idea that the use of dental prostheses, independently of the host's clinical condition, probably provides the necessary conditions for these strains to gain a growth-specific advantage over others

Differential gene expression analysis of Paracoccidioides brasiliensis during keratinocyte infection

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, one of the most important systemic fungal diseases in Latin America. This initiates in lung tissue and can subsequently disseminate to other tissues. Clinical manifestations range from localized forms to disseminated disease that can progress to lethality, probably depending on the relationships among the virulence of the fungus, the immune response and the ability to interact with the surface structures and invade epithelial cells and mononuclear cells of the host. It is generally regarded as a multifocal disease, with oral lesions as the prominent feature. The aim of this study was to evaluate P. brasiliensis yeast infection in normal oral keratinocytes (NOKs). The differential expression of mRNAs and proteins was also determined when the fungus was placed in contact with the cell in order to characterize differentially expressed genes and proteins during P. brasiliensis infection. After contact with NOKs, the fungus appeared to induce alterations in the cells, which showed cellular extensions and cavitations, probably resulting from changes in the actin cytoskeleton seen at 5 and 8 h after infection. Levels of protein expression were higher after reisolation of the fungus from infected NOK culture compared with culture of the fungus in medium. The analysis identified transcripts related to 19 proteins involved in different biological processes. Transcripts were found with multiple functions including induction of cytokines, protein metabolism, alternative carbon metabolism, zinc transport and the stress response during contact with NOKs. The proteins found suggested that the yeast was in a stress situation, as indicated by the presence of RDS1. Nevertheless, the yeast seemed to be proliferating and metabolically active, as shown by the presence of a proteasome, short-chain acetylator, glucosamine-6-phosphate isomerase and ADP/ATP carrier transcripts. Additionally, metabolic pathways may have been activated in order to eliminate toxic substances from the cell as a zinc transporter was detected, which is a potential target for the development of future drugs.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Molecular typing and antifungal susceptibility of clinical sequential isolates of Cryptococcus neoformans from São Paulo State, Brazil

The antifungal susceptibility profiles and the genetic variability of 83 sequential clinical isolates of Cryptococcus neoformans, including four Cryptococcus gattii isolates, obtained from 38 São Paulo AIDS patients with cryptococcal meningitis were assessed by electrophoretic karyotyping and random amplified polymorphic DNA (RAPD) analysis. The majority of the Cryptococcus neoformans isolates were highly susceptible to amphotericin B and fluconazole. Twenty percent of the minimum inhibitory concentration values for amphotericin B varied from 0.5 to 1 mu g mL(-1). For fluconazole, 22% occurred in the range 8-16 mu g mL(-1). Sequential isolates from nine patients showed a trend towards lower susceptibility to fluconazole, flucytosine, itraconazole and amphotericin B. The results of molecular typing by electrophoretic karyotyping and RAPD analysis showed the presence of 22 electrophoretic karyotypes (EK) and 15 RAPD profiles that were highly correlated. Our results provided evidence for the occurrence of genetic changes in some strains associated with microevolution during the course of infection. We also observed both microevolution and simultaneous coinfection with two distinct Cryptococcus neoformans strains in one patient. In some patients, we found changed EK- and RAPD patterns in association with increased MIC values