Histoplasmose disseminada em pacientes com AIDS no estado do Ceará: aspectos epidemiológicos, diagnóstico e análise in vitro da sensibilidade antifúngica e do biofilme de Histoplasma capsulatum.

Abstract

Histoplasma capsulatum is the etiologic agent of histoplasmosis, an endemic mycosis in the Americas, common in individuals with HIV / AIDS. In the past years, in addition to studies aiming at analyzing the clinical and laboratory patterns of histoplasmosis, some researches have sought to characterize this fungus. The objective of this study was to evaluate the epidemiological aspects of disseminated histoplasmosis in HIV/AIDS patients, from the state of Ceará (2011-2015) and to analyze the RPY1 gene, a conserved transcriptional regulator, as a target for the molecular diagnosis of histoplasmosis.In addition, epidemiological cutoff values (ECVs) were established for classic antifungals, based on the antifungal susceptibility of the isolates, and the biofilm-forming ability of H. capsulatum in the mycelial form was analyzed.Socio-demographic and clinical-laboratory data were collected to characterize the population and identify the factors that are mostly related to unfavorable outcomes (relapse or death), by ODDS ratio analysis. The results showed an average of 42.2 cases/year. The population consisted mainly of young men with clinical signs of fever (91.9%), cough (76.3%), weight loss (74.9%), dyspnea (65.2%). The rate of relapse and lethality was 14.7% and 39.3%, respectively. Among the factors that are mostly related to relapse and lethality, the occurrence of histoplasmosis as AIDS-defining disease, the presence of pulmonary comorbidities and elevated levels of urea and creatinine are highlighted. As for RYP1 gene, detection by PCR was observed in 18/18 cultures of H. capsulatum and in 13/13 clinical specimens from HIV-positive patients with disseminated histoplasmosis. Susceptibility to antifungals was evaluated by the broth microdilution method with mycelial (n =70) and yeast (n=07) forms and the minimum inhibitory concentration (MIC) ranges were 0.0078-1 μg/mL for amphotericin B and voriconazole; 0.0005-0.0625 μg/mL for itraconazole; 1-≥256μg /mL for fluconazole and ≤0.0156-16 μg/mL for caspofungin. ECVs were established against the mycelial form for the classification of the isolates as wild type or non-wild type. We obtained ECVs of 0.5; 0.5; 0.0313; 128 and 16 μg/mL for amphotericin B, voriconazole; itraconazole; and caspofungin, respectively, with more than 96% of the isolates classified as wild-type for all drugs. The kinetics of H. capsulatum biofilm formation in the filamentous form in RPMI, YNB and urea broth was evaluated using crystal violet (0.3%) assay, XTT assay and microscopic analysis.RPMI was the best medium, in which all isolates formed biofilms, after 8 days of incubation. Of the 46 evaluated isolates, 43 were strong and 3 were moderate biofilm producers. In summary, the prevalence and lethality of histoplasmosis in Ceará emphasize the need to adopt measures to facilitate early diagnosis, adequate treatment and improve prognosis. In this context, the RYP1 gene is a promising target region for the rapid diagnosis of histoplasmosis. In addition, more than 96% of the isolates are wild-type and these ECV values are a valuable tool for monitoring antifungal susceptibility. Finally, H. capsulatum in filamentous form forms biofilm in vitro, which may be an important way of adaptation to the environment for long periods.