Contribution of the internal transcribed spacer regions to the detection and identification of human fungal pathogens.

Fungi are morphologically and phylogenetically diverse. There identification is largely based on phenotypic methods. Thus, related species, phenotypic variants and rare species may be unidentified. So, molecular methods have been introduced for identification of pathogenic molds to overcome these problems. In this study, we report the contribution of molecular tools (PCR sequencing) to identify fungal pathogens in both clinical and environmental samples. A total of 82 mold isolates were used (50 clinical samples and 32 environmental samples). PCR and direct sequencing, targeting the internal transcribed spacer (ITS) regions, were performed. We employed comparative sequence analysis to identify molds by using the GenBank database. 89% of isolates were identified by phenotypic methods. PCR- sequencing allowed the fungal identification in all cases. The concordance between molecular and morphological identification was obtained for 33 cases (40.2%). In 36 cases (43.9%), the molecular study gave the exact species identification. PCR sequencing allowed as revising mycological identification for 13 fungi strains (15.9%). The concordance of identification at species level by phenotypic method and by sequence analysis was obtained for 28% of clinical samples and for 59% of environmental samples. The phylogenetic tree for the ITS sequences showed six different clusters that are composed of isolates belonging to the same genus or species. PCR sequencing has been shown to be useful for the detection of the presence of fungal DNA in both environmental and clinical samples. It is rapid and more sensitive for the identification of medically important fungi.

Analysis of ITS1 and ITS2 sequences in Ensis razor shells: suitability as molecular markers at the population and species levels, and evolution of these ribosomal DNA spacers.

Internal transcribed spacer 1 and 2 (ITS1 and ITS2) sequences were analysed in Ensis razor shells (Mollusca: Bivalvia: Pharidae). We aimed to (1) test ITS1 and ITS2 as molecular markers at the population level in the successful alien E. directus (Conrad, 1843); (2) test these spacers at the species level in E. directus and three other Ensis species, E. siliqua (L., 1758), E. macha (Molina, 1782), and E. magnus (Schumacher, 1817); and (3) analyse the evolutionary processes that may be shaping Ensis ITS1 and ITS2 extant variation. In E. directus, despite the intragenomic divergence detected, ITS1 and ITS2 were informative in differentiating the geographic areas considered (Denmark and Canada) by means of both the insertion-deletion polymorphism and the nucleotide polymorphism. In this species, the 5.8S ribosomal gene (5.8S) showed scarce polymorphism. At the species level, maximum parsimony and maximum likelihood analyses revealed that ITS1 and ITS2 may be suitable to reconstruct Ensis phylogenetic relationships. Finally, the evolutionary models that best fit the long-term evolution of Ensis ITS1-5.8S-ITS2 are discussed. A mixed process of concerted evolution, birth-and-death evolution, and selection is chosen as an option that may reconcile the long-term evolution of Ensis ITS1-5.8S-ITS2 and 5S ribosomal DNA.