Molecular identification of Histoplasma capsulatum using rolling circle amplification.

Histoplasmosis is a systemic fungal disease that occurs worldwide, causing symptomatic infection mostly in immunocompromised hosts. Etiological agent is the dimorphic fungus, Histoplasma capsulatum, which occurs in soil contaminated with bird or bat droppings. Major limitation in recognition of H. capsulatum infections is the low awareness, since other diseases may have similar symptomatology. The molecular methods have gained importance because of unambiguous diagnostic ability and efficiency. The aim of this study was to develop and evaluate a padlock probe in view of rolling circle amplification (RCA) detection method which targets ITS (Internal Transcribed Spacer) rDNA of H. capsulatum enabling rapid and specific detection of the fungus in clinical samples. Two padlock probes were designed and one of these (HcPL2) allowed specific amplification of H. capsulatum DNA while no cross-reactivity was observed with fungi used as negative controls. This method proved to be effective for H. capsulatum specific identification and demonstrated to be faster than the traditional method of microbiological identification.

Seroepidemiology of Toxoplasma gondii infection in bats from São Paulo city, Brazil.

Toxoplasmosis is a parasitic zoonosis caused by Toxoplasma gondii protozoan with a worldwide distribution. The aim of this study was to determine the frequency of IgG anti-T. gondii antibodies in bats from São Paulo city, Brazil. A total of 616 serum samples were collected from 22 species of bats. Anti-T. gondii antibodies were searched using the indirect fluorescent antibody test (IFAT ≥ 1:16) and IgG anti-bat antibodies produced in sheep on samples collected during 2006-2011; 32.62% (201/616) of bats had T. gondii antibodies. The modified agglutination test (MAT ≥ 1:25) was performed on samples collected during 2010-2011; 18.61% (35/188) were seropositive. The concordance between IFAT and MAT (serum samples from 2010 to 2011) by Kappa (95% CI) was 0.144, resulting in a low agreement between the techniques. The specificity and sensitivity of MAT and IFAT have not been evaluated for the diagnosis of toxoplasmosis in bats. Thus, it was verified that bats are exposed to T. gondii during their lifetime and they are also part of the toxoplasmosis epidemiology.

Genetic diversity of Histoplasma capsulatum isolated from infected bats randomly captured in Mexico, Brazil, and Argentina, using the polymorphism of (GA)(n) microsatellite and its flanking regions.

The genetic diversity of 47 Histoplasma capsulatum isolates from infected bats captured in Mexico, Brazil, and Argentina was studied, using sequence polymorphism of a 240-nucleotides (nt) fragment, which includes the (GA)(n) length microsatellite and its flanking regions within the HSP60 gene. Three human clinical strains were used as geographic references. Based on phylogenetic analyses of 240-nt fragments achieved, the relationships among H. capsulatum isolates were resolved using neighbour-joining and maximum parsimony methods. The tree topologies obtained by both methods were identical and highlighted two major clusters of isolates. Cluster I had three sub-clusters (Ia, Ib, and Ic), all of which contained Mexican H. capsulatum samples, while cluster II consisted of samples from Brazil and Argentina. Sub-cluster Ia included only fungal isolates from the migratory bat Tadarida brasiliensis. An average DNA mutation rate of 2.39 × 10(-9) substitutions per site per year was estimated for the 240-nt fragment for all H. capsulatum isolates. Nucleotide diversity analysis of the (GA)(n) and flanking regions from fungal isolates of each cluster and sub-cluster underscored the high similarity of cluster II (Brazil and Argentina), sub-clusters Ib, and Ic (Mexico). According to the genetic distances among isolates, a network of the 240-nt fragment was graphically represented by (GA)(n) length haplotype. This network showed an association between genetic variation and both the geographic distribution and the ecotype dispersion of H. capsulatum, which are related to the migratory behaviour of the infected bats studied.