Desenvolvimento de reação de qPCR para detecção de H. capsulatum em amostras de solo de grutas e cavernas do Município de Presidente Figueiredo

Abstract

Histoplasma capsulatum is a fungi found in the environment that causes a deep mycosis, which presents itself in the asymptomatic form until disseminated. In its disseminated form, it mainly affects AIDS patients. In South America, this disease is one of the three main causes of death for AIDS patients. Despite these facts, few studies have evaluated its environmental distribution. This study aimed to develop a qPCR reaction to detect H. capsulatum in soil samples from caves in the county of Presidente Figueiredo. Initially, an in silico study was performed with the Geneious software to develop a primer targeting the ITS region of the DNAr of the H. capsulatum, then the primer and probe set was validated with the execution of the standard curve with target fungal culture DNA, experimental error, tested to check cross reactions with other pathogens and sensitivity and specificity test. Finally, with this primer and probe, the presence of H. capsulatum was verified in 30 soil samples from 3 caves of Presidente Figueiredo that had their DNA extracted by two different methodologies. As a result, a set of primers and probe was developed to detect H. capsulatum for use in real-time PCR (qPCR) targeting the ITS region of DNAr. The design demonstrated in silico to be equal or more appropriate than other proposals that targeted this same region. The set demonstrated satisfactory results in terms of detection limit, 10 fg/μL of sample (20 fg/reaction), adequate standard curve (r2 0.97), low experimental error, absence of cross reaction with other pathogenic organisms, adequate sensitivity and specificity. The experiments applying the new primer and probe set in 30 soil samples from caves in the Presidente Figueiredo demonstrated the presence of DNA from H. capsulatum in one sample. The qPCR proposal is a new detection and diagnosis option that can help in understanding the environmental distribution of the pathogen as well as in the clinical diagnosis in biological samples. The detection of H. capsulatum in a sample of cave soil in the municipality of Presidente Figueiredo reinforces the need for recommended care for tourism in the region.