Identification of Clinical Mold Isolates by Sequence Analysis of the Internal Transcribed Spacer Region, Ribosomal Large-Subunit D1/D2, and beta-Tubulin

BACKGROUND: The identification of molds in clinical laboratories is largely on the basis of phenotypic criteria, the classification of which can be subjective. Recently, molecular methods have been introduced for identification of pathogenic molds in clinical settings. Here, we employed comparative sequence analysis to identify molds. METHODS: A total of 47 clinical mold isolates were used in this study, including Aspergillus and Trichophyton. All isolates were identified by phenotypic properties, such as growth rate, colony morphology, and reproductive structures. PCR and direct sequencing, targeting the internal transcribed spacer (ITS) region, the D1/D2 region of the 28S subunit, and the beta-tubulin gene, were performed using primers described previously. Comparative sequence analysis by using the GenBank database was performed with the basic local alignment search tool (BLAST) algorithm. RESULTS: For Aspergillus, 56% and 67% of the isolates were identified to the species level by using ITS and beta-tubulin analysis, respectively. Only D1/D2 analysis was useful for Trichophyton identification, with 100% of isolates being identified to the species level. Performances of ITS and D1/D2 analyses were comparable for species-level identification of molds other than Aspergillus and Trichophyton. In contrast, the efficacy of beta-tubulin analysis was limited to genus identification because of the paucity of database information for this gene. CONCLUSIONS: The molecular methods employed in this study were valuable for mold identification, although the different loci used had variable usefulness, according to mold genus. Thus, a tailored approach is recommended when selecting amplification targets for molecular identification of molds.

[ effect of variable CO[2] concentrations on HSP70 gene in Trichophyton rubrum and Microsporeum canis]

Trichophyton rubrum is considered as the most common causes of dermatophytosis in human skin and nail tissues. Microsporeum canis is a zoophile dermatophyte which can be transmited to human. HSP70 is a 70 KD heat shock protein in fungi. In this study, the effects of variable CO[2] concentrations were examined on HSP70 expression in T. rubrum and M. canis. Strains used in this study were obtained from skin scales and nails of the patients who were suffering from onychomycosis. Samples were cultured on Sabouraud dextrose broth [SDB] and incubated at 25°C for 2, 4 and 7 days under 3%, 5%, and 10% of CO[2] concentrations. Control cultures maintained for 7 days without CO[2] concentrations. Then, RNA was isolated from the harvested mycelia mass, and HSP70 gene expression was studied in T. rubrum and M. canis by RT-PCR. The obtained results were compared to the Beta actin as a house keeping gene. The results of this study revealed the maximum variations under 3%,5%, and 10% of CO[2] concentrations in maximum 7 days incubation period, and the expression of HSP70 gene showed different variations under different CO[2] concentrations. Our results showed a negative effect of CO[2] concentrations in the expression of HSP70 in T. Rubrum and a positive effect in M. canis comparing to the controls

Molecular characterization of GTP binding protein gene in dermatophyte pathogen trichophyton rubrum

Trichophyton rubrum [T. rubrum] is an anthropophilic dermatophyte that is distributed worldwide and causes common cutaneous disease such as mycosis. Although several properties of this fungus have been investigated so far, however a few studies were carried out in the field of molecular biology of this fungus. In the present study we tried to identify its molecular characterization of the goanosin three phosphat [GTP] binding protein gene. Pairs of 21 nt primers were designed from highly conserved regions of the gene in other fungi. The primers were utilized in PCR by using isolated genomic DNA tem-plate as well as cytoplasmic RNA of T. rubrum and the PCR and RT-PCR fragments were then sequenced. About 645 nucleotides have been sequenced which encodes a polypeptide with 214 amino acids. Nucleotide sequence comparison in gene data banks [NCBI, NIH] for both the DNA and its deduced amino acid sequence revealed significant homology with GTP binding protein genes and proteins of other eukaryotic cells. The amino acid sequence of the encoded protein was about 64% identical to the sequence of GTP binding protein from other fungi. In summary, we have cloned the first GTP binding protein of dermatophytes and characterized it as a member of this gene family in other eukaryotic cells