ANTIFUNGAL SUSCEPTIBILITY TESTING AND GENOTYPING CHARACTERIZATION OF Cryptococcus neoformans AND gattii ISOLATES FROM HIV-INFECTED PATIENTS OF RIBEIRÃO PRETO, SÃO PAULO, BRAZIL

Abstract

Cryptococcosis is a leading invasive fungal infection in immunocompromised patients. Considering the high prevalence and severity of these infections in immunocompromised patients attended at HC-FMRP-USP, the present research aimed to characterize the clinical isolates of Cryptococcus strains by biochemical and molecular methods and evaluate antifungal susceptibility of clinical isolates. Fifty isolates from 32 HIV-positive patients were obtained at HC-FMRP-USP. Most of the isolates (78.1%) were identified as C. neoformans, and 100% of C. neoformans and C. gattii strains were susceptible to amphotericin B, ketoconazole and fluconazole. All isolates were classified as serotype A (grubbii variety) by PCR and most of them were characterized in mating type MATa. PCR analysis of specific M13 microsatellite sequence revealed that VNI type was predominant among C. neoformans, while VGII was predominant among C. gattii. The strains did not show a significant resistance to the antifungals tested, and Canavanine-Glycine-Bromthymol Blue Agar (CGB) proved to be a reliable test presenting a good correlation with the molecular characterization. C. neoformans isolated from disseminated infections in the same patient showed molecular identity when different anatomical sites were compared; besides, the studied strains did not present a significant increase in resistance to antifungal agents. In addition, the homogeneity of the molecular types and detection of the mating types suggested a low possibility of crossing among the strains.

Fig. 1. Group I Cryptococcus isolates. Agarose gel of PCR reactions used for determination of sexual type and serotype. PCR products were obtained using specific primer pairs (MFa1, MFa2 B/C, SOD1 B/C, Aa, Da, Aa and Da). Molecular weight markers are shown on the left side; the lane on the right side indicates the (+) control of each reaction. The remaining lanes represent the studied isolates, identified at the top of the figure by numbers.

Fig. 2. Agarose gels showing sexual types and serotypes of Cryptococcus isolates. 2A (on the left) corresponds to Group II samples and 2B (on the right) to Group III samples. PCR products were obtained using specific primers (MFa1, MFa2 B/C, SOD1 B/C, Aa, Da, Aa and Da). The first lane on the left side shows the molecular weight markers; the right lane on the right side shows the (+) control of each reaction. The remaining lanes represent the studied isolates, identified at the top of the figure by numbers.