Histoplasma capsulatum var. capsulatum : taxa de conversão in vitro, detecção do gene ryp1 e estudo da diversidade genética de cepas brasileiras

Abstract

Histoplasmosis, caused by the dimorphic fungus Histoplasma capsulatum, is the most prevalent systemic mycosis in the Americas and it is frequently observed in AIDS patients. Since the beginning of the HIV epidemic, there has been a significant increase in the number of histoplasmosis cases in the Ceará state, Northeast Brazil. The lack of epidemiological data of the genotypes circulating in the Northeast region shows the importance of more detailed studies on the molecular epidemiology of H. capsulatum in this region. Different molecular techniques have been used to better characterize the genetic profile of H. capsulatum strains. It is noteworthy that, most studies of H. capsulatum are performed in the yeast phase due to its parasitic characteristics. Thus, the use of laboratory techniques for in vitro conversion and maintenance is extremely important. Given the above, this study aimed to determine the in vitro conversion rate of H. capsulatum isolates in six different culture media, as well as to detect by PCR the presence of ryp1 gene, an important transcriptional regulator of the conversion from filamentous to yeast phase. In addition, it aimed to establish the molecular profile of H. capsulatum strains, from human and veterinary source, from Ceará and Southeast region of Brazil through RAPD-PCR assay and to assess the genetic diversity of the ITS1-5.8S-ITS2 region of these isolates compared with isolates from other countries, through the sequencing of nuclear ribosomal DNA. In the study of in vitro conversion, all tested media allowed the conversion, however, the Sabouraud agar media supplemented with 10% sheep blood showed the highest conversion capacity in relation to the other tested media. The ryp1 gene was detected in 18 H. capsulatum strains (from human and animal source) and in three tested clinical specimens (whole blood), not being detected, however, in isolates of Candida albicans, Paracoccidioides brasiliensis, Sporothrix schenckii and Coccidioides posadasii. The analysis of the genetic variability of the H. capsulatum isolates, by RAPD-PCR assay, allowed the detection of two clusters circulating in the state of Ceará. The first cluster included strains from Southeast and Northeast regions of Brazil, being observed, within this cluster, the separation of isolates into three distinct subgroups (subgroups 1a, 1b and 1c). The second cluster included only strains from Northeast region of Brazil. There were no differences in clinical and epidemiological characteristics of individuals whose isolates belonged to different groups, obtained by RAPD-PCR. The sequencing of the ITS1-5, 8S-ITS2 region allowed the detection of two major clades. The clade 1 comprised the majority of the isolates tested, including strains from the Northeast, and included isolates from different geographical locations. The clade 2 was composed exclusively of isolates from the state of Rio de Janeiro. Therefore, it can be concluded that the Sabouraud agar supplemented with 10% sheep blood showed higher conversion capacity of H. capsulatum strains compared to the other tested media, and may be considered the medium of choice for converting of H. capsulatum strains. Furthermore, the ryp1 gene can be used to identify and detect H. capsulatum from clinical specimens, may be used for diagnosis of histoplasmosis. Finally, the H. capsulatum isolates from the state of Ceará can be grouped into two main clusters, detected by RAPD-PCR. In addition, phylogenetic analysis of the ITS1-5.8S-ITS2 region revealed that strains from the Northeast presented genetic differences compared with strains from other Brazilian regions, being grouped in a different cluster of them.