Since the beginning of the HIV epidemic, there has been a significant increase in the number of histoplasmosis cases in Ceará , a state in north-east Brazil. The lack of epidemiological data on the genotypes circulating in the north-east region shows the importance of more detailed studies on the molecular epidemiology of Histoplasma capsulatum var. capsulatum in this region. Different molecular techniques have been used to better characterize the genetic profile of H. capsulatum var. capsulatum strains. The aim of this study was to analyse the genetic diversity of H. capsulatum var. capsulatum isolates in Fortaleza, the capital of Ceará , through the sequencing of the internal transcribed spacer (ITS)1-5.8S-ITS2 region, and establish the molecular profile of these isolates, along with strains from south-east Brazil, by RAPD analysis, featuring the different clusters in those regions. The isolates were grouped into two clusters. Cluster 1 included strains from the south-east and north-east regions with separation of isolates into three distinct subgroups (subgroups 1a, 1b and 1c). Cluster 2 included only samples from north-east Brazil. Sequencing of the ITS1-5.8S-ITS2 region allowed the detection of two major clades, which showed geographical correlation between them and their subgroups. Therefore, it can be concluded that the H. capsulatum var. capsulatum isolates from Ceará have a high degree of genetic polymorphism. The molecular data also confirm that populations of this fungus are composed of different genotypes in Brazil and worldwide.
INTRODUCTIONHistoplasmosis is an important systemic mycosis, whose aetiological agent is the dimorphic fungus Histoplasma capsulatum. The natural habitat of this fungus is soil, where it grows as a filamentous saprophyte (Kauffman, 2009). This mycosis is widely distributed in the Americas, with a predominance of cases in some areas of the United States, such as along the valleys of the Mississippi and Ohio rivers, as well as in countries of Central and South America (Rossini & Goulart, 2006;Ferreira & Borges, 2009).In Brazil, the occurrence of histoplasmosis is relatively common, as demonstrated by the observation of autochthonous clinical cases, either in isolated cases or in the Abbreviations: ITS, internal transcribed spacer; UPGMA, unweighted pair group analysis with arithmetic mean.The GenBank/EMBL/DDBJ accession numbers for the ITS1-5.8S-ITS2 region sequences of strains used in this study are given in Table 1. Extraction of genomic DNA. Genomic DNA was extracted from yeast-phase cultures of the micro-organism grown for 5 days at 35 uC in Sabouraud agar (Himedia) supplemented with 10 % sheep's blood. DNA extraction was performed as described by Ausubel et al. (2002). Genomic DNA was extracted by mechanical lysis with glass beads and chemistry lysis using a breaking buffer. The nucleic acids were precipitated with ethanol after exposure to a solution of phenol/ chloroform/isoamyl alcohol to separate the cellular debris. The DNA pellet was then resuspended in TE buffer an...