Aspectos clínicos e moleculares da criptococose em pacientes não HIV no Estado do Amazonas, Brasil

Abstract

Cryptococcosis is a fungal infection caused by the Cryptococcus neoformans/Cryptococcus gattii species complex. Most cases occur in people with HIV/AIDS, however, this infection also occurs in non-HIV patients with a frequency of 10 to 30% of all cases. However, this is a disease with high morbidity and mortality, being neurocriptococcosis the most severe clinical form. This study investigated clinical and molecular aspects of seven cases of non-HIV patients diagnosed with cryptococcosis at the Tropical Medicine Foundation (FMT-HVD) from July 2016 to June 2019. A questionnaire was used to obtain epidemiological and clinical information from the patients included in the study. Then, to identify the genotypes we used the PCR-RFLP technique of the URA5 gene which compared the genotypes of isolates with previously genotyped standard strains. For the Multilocus Sequence Typing (MLST) molecular technique, sequencing of the seven reference genes recommended by the International Society for Human and Animal Mycology (ISHAM) was performed for the determination of sequence typing (ST). Antifungal susceptibility assays were in accordance with CSLI M27-A3 standard. As clinical and epidemiological results, we observed that most patients were female (57.14%), age range 10-53 years (mean 36.3 years), neurocryptococcosis was the predominant clinical form (100%). The time elapsed from symptoms to diagnosis ranged from 15 to 730 days (average 172.9 days) and the observed mortality was 57.14%. In addition, the comorbidities prior to cryptococcosis observed in the present study were: hypertension, diabetes mellitus and intestinal tuberculosis. Isolates were obtained from cerebrospinal fluid (CSF) samples and the diagnosis was made by direct examination in Nankin ink. Further, initial genotyping by the PCR-RFLP technique of the URA5 gene showed that all clinical isolates were of molecular type VGII. From the MLST assay it was possible to identify the sequence typing ST20 (n = 4), ST5 (n = 3) and ST552 (n = 1). This is the first study to report the existence of ST552. Cultures showed mean MIC for amphotericin B (AMB), fluconazole (FLC) and itraconazole (ITC) antifungals of 0.05, 3.85 and 0.18 µg/mL, respectively. In this study we show the importance of using the MLST molecular tool to identify the genotypes that cause infections in non-HIV patients. We describe the predominance of CNS involvement by C. gattii mainly of molecular type VGIIa, which is widely dispersed in northern Brazil.