Assessment of a pan-dermatophyte nested-PCR compared with conventional methods for direct detection and identification of dermatophytosis agents in animals

Conventional direct microscopy with potassium hydroxide (KOH) and culture were found to lack the ability to establish a fast and specific diagnosis of dermatophytosis. A pan-dermatophyte nested-PCR assay was developed using a novel primer pair targeting the translation elongation factor 1-α (Tef-1α) sequences for direct detection and identification of most veterinary relevant dermatophytes in animal samples suspected to dermatophytosis. A total of 140 animal skin and hair samples were subjected to direct microscopy, culture, and ITS-RFLP/ ITS-sequencing of culture isolates for the detection and identification of dermatophytosis agents. Nested-PCR sequencing was performed on all the extracted DNAs using a commercial kit after dissolving the specimens by mechanical beating. Nested-PCR was positive in 90% of samples, followed by direct microscopy (85.7%) and culture (75%). The degree of agreement between nested-PCR and direct microscopy (94.4%) was higher than with culture (83.3%). In 105 culture-positive cases, the measures of agreement for the identification of dermatophytosis agents were as follows: 100% between nested-PCR sequencing and ITS-RFLP/ ITS-sequencing and 63.8% between nested-PCR sequencing and culture. The developed nested-PCR was faster as well as more sensitive and specific than conventional methods for detection and identification of dermatophytes in clinical samples, which was particularly suitable for epidemiological studies. m

Analysis of clinical and environmental Candida parapsilosis isolates by microsatellite genotyping – a tool for hospital infections surveillance

Candida parapsilosis emerged as an important opportunistic pathogen, causing candidaemia worldwide. Nosocomial outbreaks triggered by this species have been frequently described, particularly in cancer patients. For a better understanding of its epidemiology, several typing methods are used and microsatellite analysis has been reported as highly discriminant. The main objective of this work was to study C. parapsilosis isolates by application of microsatellite genotyping to distinguish epidemiologically related strains, compare clinical and environmental isolates and determine possible routes of dispersion of the isolates in the hospital setting. A total of 129 C. parapsilosis isolates from different origins, including hospital environment and hands of healthcare workers, were genotyped using four microsatellite markers. The isolates were recovered from different health institutions. Analysis of C. parapsilosis isolates from hospital environment showed great genotypic diversity; however, the same or very similar genotypes were also found. The same multilocus genotype was shared by isolates recovered from the hand of a healthcare worker, from the hospital environment and from patients of the same healthcare institution, suggesting that these could be possible routes of transmission and that infections due to C. parapsilosis may be mainly related with exogenous transmission to the patient. Examination of sequential isolates from the same patients showed that colonizing and bloodstream isolates had the same multilocus genotype in the majority of cases. We demonstrate that this typing method is able to distinguish clonal clusters from genetically unrelated genotypes and can be a valuable tool to support epidemiologic investigations in the hospital setting.This research was supported by FCT/MEC, Portugal through Portuguese funds (PIDDAC) - Pest-OE/BIA/UI4050/2014 (CBMA), University of Minho. Raquel Sabino was financially supported by a fellowship from FCT, Portugal (contract BD/22100/2005).info:eu-repo/semantics/publishedVersio