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.

Caracterización de aislamientos clínicos del complejo Cryptococcus neoformans-Cryptococcus gattii, del Estado Amazonas - Brasil / Characterization of clinical isolates of the Cryptococcus neoformans-Cryptococcus gattii species complex from the Amazonas State in Brazil

Antecedentes. La diferenciación y clasificación de las especies patógenas del género Cryptococcus aporta datos importantes para la asistencia clínica y para estudios epidemiológicos. Objetivos. El objetivo de este trabajo fue caracterizar 40 aislamientos clínicos del complejo Cryptococcus neoformans de pacientes que fueron atendidos en la Fundación de Medicina Tropical de Amazonas desde 2006 hasta 2008. Métodos. Se utilizaron métodos fenotípicos (producción de enzimas y pruebas de sensibilidad a los antifúngicos) y moleculares (URA5-RFLP). Resultados. Los pacientes con VIH/sida fueron los más afectados de criptococosis. Se observó que 31 (75,5%) y 9 (22,5%) de los aislamientos fueron Cryptococcus neoformans y Cryptococcus gattii, respectivamente. La producción de proteasa y fosfolipasa fue alta en la mayoría de las cepas. Utilizando la prueba de difusión en disco (CLSI M44-A) se observó que el 81, 35 y 100% de los aislamientos de C. neoformans fueron sensibles al fluconazol, itraconazol y amphotericin B, respectivamente, mientras que 78, 56 y 100% de los aislamientos de C. gattii fueron sensibles a estas sustancias. El valor promedio de la concentración mínima inhibitoria (CMI) para C. neoformans y C. gattii fue de 0,26 y 0,58mg/ml, respectivamente. Todos los aislamientos (9) de C. gattii presentaron un patrón de electroforesis compatible con el genotipo VGII, y todos los aislamientos (31) de C. neoformans presentaron el genotipo VNI. Conclusiones. Este estudio confirma la importancia del HIV/sida para la epidemiología de la criptococosis, la sensibilidad de los aislamientos a la anfotericina B y la alta prevalencia de los genotipos moleculares VNI y VGII en el norte de Brasil(AU)

Background. The differentiation and classification of pathogenic Cryptococcus species provides useful data for epidemiological studies and for the clinical diagnosis and treatment of patients. Aims. The aim of this study was to characterise 40 clinical Cryptococcus isolates obtained from patients at the Tropical Medicine Foundation of Amazonas (FMTAM) from 2006 to 2008. Methods. It was used phenotypic (i.e., enzyme production and antifungal resistance) and molecular biological (URA5-RFLP) experiments. Results. Patients with HIV/AIDS were most affected with cryptococcosis. Thirty-one (75.5%) of the clinical isolates were classified as Cryptococcus neoformans and 9 (22.5%) as Cryptococcus gattii. High amounts of protease and phospholipase enzymes were produced by most of the isolates. Using the disk diffusion test (CLSI M44-A), 81, 35 and 100% of the C. neoformans isolates were characterized as susceptible to fluconazole, itraconazole and amphotericin B, respectively, whereas 78, 56 and 100% of the C. gattii isolates were susceptible to these antimicrobial agents. The average of Minimal Inhibitory Concentration (MIC) for C. neoformans and C. gattii isolates was 0.26 and 0.58µg/mL, respectively. The 9 isolates of C. gattii had a fingerprint pattern comparable with the VGII molecular type, while all 31 isolates of C. neoformans presented with a pattern consistent with the VNI type. Conclusions. This study confirms the importance of HIV/AIDS for the cryptococcosis epidemiology, the susceptibility of the isolates to amphotericin B and the high prevalence of the molecular genotypes VNI and VGII in the north of Brazil(AU)

Characterization of clinical isolates of the Cryptococcus neoformans-Cryptococcus gattii species complex from the Amazonas State in Brazil.

BACKGROUND: The differentiation and classification of pathogenic Cryptococcus species provides useful data for epidemiological studies and for the clinical diagnosis and treatment of patients. AIMS: The aim of this study was to characterise 40 clinical Cryptococcus isolates obtained from patients at the Tropical Medicine Foundation of Amazonas (FMTAM) from 2006 to 2008. METHODS: It was used phenotypic (i.e., enzyme production and antifungal resistance) and molecular biological (URA5-RFLP) experiments. RESULTS: Patients with HIV/AIDS were most affected with cryptococcosis. Thirty-one (75.5%) of the clinical isolates were classified as Cryptococcus neoformans and 9 (22.5%) as Cryptococcus gattii. High amounts of protease and phospholipase enzymes were produced by most of the isolates. Using the disk diffusion test (CLSI M44-A), 81, 35 and 100% of the C. neoformans isolates were characterized as susceptible to fluconazole, itraconazole and amphotericin B, respectively, whereas 78, 56 and 100% of the C. gattii isolates were susceptible to these antimicrobial agents. The average of Minimal Inhibitory Concentration (MIC) for C. neoformans and C. gattii isolates was 0.26 and 0.58 µg/mL, respectively. The 9 isolates of C. gattii had a fingerprint pattern comparable with the VGII molecular type, while all 31 isolates of C. neoformans presented with a pattern consistent with the VNI type. CONCLUSIONS: This study confirms the importance of HIV/AIDS for the cryptococcosis epidemiology, the susceptibility of the isolates to amphotericin B and the high prevalence of the molecular genotypes VNI and VGII in the north of Brazil.