Padronização de método molecular para avaliação da diversidade genética de Histoplasma capsulatum em isolados do Centro-Oeste Brasileiro em PVHIV

Abstract

Histoplasma capsulatum is a dimorphic fungus with global geographic distribution and high genetic diversity. Etiological agent of histoplasmosis, a disease that is of great relevance for the population living with HIV/AIDS, since it is the biggest cause of morbidity and mortality of those patients in the American continent. The present study aims to elucidate, through the standardization of molecular method, the evaluation of genetic diversity and the presence of genetic material belonging to the fungus H. capsulatum through the extraction of DNA from peripheral blood samples. We enrolled 14 patients randomly from a larger project registered at clinicaltrials.gov NCT04059770. The DNA extraction procedure from the samples of this study was performed according to the procedure of Guha et al., 2016. The quantification and purity of the nucleic acids extracted from the blood samples were measured with the aid of a pedestal spectrometer. All steps were confirmed by electrophoresis in 1% agarose. To obtain DNA fragments from the samples of approximately 1000 base pairs by PCR, the oligonucleotide sequences 5'-CTTGGTCATTTAGAGGAAGTAA-3' and 5'-CCGTGTTTCAAGACGGG-3', named OligoITS1F and OligoLR3, described by Raja et al., 2017 . In order to improve the sensitivity of the fungal universal region PCR method, the 1000bp PCR amplicons were resubmitted to PCR. However, to confirm that it is the fungus H. capsulatum, confirmation by DNA sequencing is necessary. The analysis of the sequences by comparison with the sequences deposited in the GeneBank database provides us with the homology for validating the results with the clinical samples. More interesting and opportune, when discussing the diversity of possible manifestations of histoplasmosis, it is not possible to neglect a role that the genetic diversity of the fungus can play in the presented clinic. It is known that in Brazil, nine known genetic groups of Histoplasma capsulatum circulate: LAm A, LAm B, LAm C, LAm D, LAm E, RJ, L, BrHC1 and BrHC3. The genetic diversity presented by the pathogen not only makes possible different clinical manifestations, but also double infection by fungi of different genotypes in the same host. This stage of our work is still in progress.