Molecular characterization and antifungal susceptibility testing of Cryptococcus neoformans sensu stricto from southern Brazil.

PURPOSE: Cryptococcosis is acquired from the environment by the inhalation of Cryptococcus cells and may establish from an asymptomatic latent infection into pneumonia or meningoencephalitis. The genetic diversity of a Cryptococcus neoformans species complex has been investigated by several molecular tools, such as multi-locus sequence typing, amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism and microsatellite analysis. This study aimed to investigate the genotype distributions and antifungal susceptibility profiles of C. neoformans sensu lato isolates from southern Brazil. METHODOLOGY: We studied 219 C. neoformans sensu lato isolates with mating- and serotyping, AFLP fingerprinting, microsatellite typing and antifungal susceptibility testing.Results/Key findings. Among the isolates, 136 (69 %) were from HIV-positive patients. Only C. neoformans mating-type α and serotype A were observed. AFLP fingerprinting analysis divided the isolates into AFLP1/VNI (n=172; 78.5 %), AFLP1A/VNII (n=19; 8.7 %), AFLP1B/VNII (n=4; 1.8 %) and a new AFLP pattern AFLP1C (n=23; 10.5 %). All isolates were susceptible to tested antifungals and no correlation between antifungal susceptibility and genotypes was observed. Through microsatellite analysis, most isolates clustered in a major microsatellite complex and Simpson's diversity index of this population was D=0.9856. CONCLUSION: The majority of C. neoformans sensu stricto infections occurred in HIV-positive patients. C. neoformans AFLP1/VNI was the most frequent genotype and all antifungal drugs had high in vitro activity against this species. Microsatellite analyses showed a high genetic diversity within the regional C. neoformans sensu stricto population, and correlation between environmental and clinical isolates, as well as a temporal and geographic relationship.

In vitro susceptibility and molecular characterization of Candida spp. from candidemic patients / Sensibilidad in vitro y caracterización molecular de aislamientos de Candida procedentes de pacientes con candidemia

Background. Candida species are the main cause of hospital acquired fungal bloodstream infections. The main risk factors for candidemia include parenteral nutrition, long-term intensive care, neutropenia, diabetes, abdominal surgery and the use of central venous catheters. The antifungal drugs used to treat candidemia are mainly the echinocandins, however some isolates may be resistant to these drugs. Aims. This work aims to evaluate the in vitro susceptibility patterns of various Candida species isolated from blood samples and provide their identification by molecular characterization. Methods. Antifungal susceptibility testing was performed using the broth microdilution method. The sequencing of the ITS and D1/D2 regions of rDNA was used for molecular characterization. Results. Seventy-four of the 80 isolates were susceptible to anidulafungin, 5 were intermediate, and 1 was resistant. For micafungin 67 were susceptible, 8 were intermediate and 5 were resistant. All isolates were susceptible to amphotericin B. Lastly, 65 isolates were susceptible to fluconazole, 8 were dose-dependent and 4 were resistant. The molecular identification corroborated the phenotypic data in 91.3% of the isolates. Conclusions. Antifungal susceptibility data has an important role in the treatment of candidemia episodes. It was also concluded that the molecular analysis of isolates provides an accurate identification and identifies genetic variability within Candida species isolated from patients with candidemia (AU)

Antecedentes. Los hongos del género Candida son la causa principal de infección micótica del torrente sanguíneo adquirida en el hospital. Entre los factores de riesgo asociados a la candidemia destacan la nutrición parenteral, la estancia prolongada en una unidad de cuidados intensivos, la neutropenia, la diabetes, la cirugía abdominal y la utilización de catéter venoso central. Los agentes antifúngicos más utilizados para tratarla son las equinocandinas, pero determinados aislamientos son resistentes a dichos componentes, por lo que algunos pacientes no responden al tratamiento. Objetivos. Este trabajo tiene como objetivo evaluar la sensibilidad in vitro de varios aislamientos de Candida procedentes de muestras de sangre y realizar su caracterización molecular. Métodos. Se hicieron pruebas de sensibilidad a los antifúngicos mediante el método de microdilución en caldo. Para la caracterización molecular se utilizó la secuenciación de las regiones ITS y D1/D2 del DNAr. Resultados. De los 80 aislamientos evaluados, 74 fueron sensibles a la anidulafungina, 5 mostraron sensibilidad intermedia y solo uno era resistente. Cuando se utilizó la micafungina, 67 aislamientos resultaron sensibles, 8 presentaron sensibilidad intermedia y 5 fueron resistentes. Los 80 aislamientos fueron sensibles a la anfotericina B. Al menos 65 aislamientos eran sensibles al fluconazol, 8 presentaron sensibilidad dependiente de la dosis y 4 se mostraron resistentes. La identificación molecular confirmó la identificación fenotípica en un 91,3% de los aislamientos. Conclusiones. Teniendo en cuenta los resultados obtenidos con las pruebas de sensibilidad a los antifúngicos, estas resultan indispensables para el tratamiento adecuado de la candidemia. Se concluye además que la identificación molecular proporciona una identificación precisa y consigue identificar la variabilidad genética de las especies del género Candida aisladas en pacientes con candidemia (AU)