Validation of PCR–reverse line blot, a method for rapid detection and identification of nine dermatophyte species in nail, skin and hair samples

Abstract

A dermatophyte-specific PCR-reverse line blot (PCR-RLB) assay based on internal transcribed sequences was developed. This assay allows the rapid detection and identification of nine clinically relevant species within the three dermatophyte genera Trichophyton, Microsporum and Epidermophyton in nail, skin and hair samples within 1 day. Analysis of 819 clinical samples (596 nail, 203 skin and 20 hair) revealed a positive PCR-RLB result in 93.6% of 172 culture-positive and microscopy-positive samples. PCR-RLB was superior to culture and direct microscopy, in both detection and species identification. Comparison of identification results of 208 PCR-positive and culture-positive clinical samples showed five discrepancies (2.4%) between PCR-RLB identification and classical microscopic/biochemical identification of isolates. Comparison of PCR-RLB identification and classical identification of 98 other isolates (dermatophytes and non-dermatophytes) revealed 13 discrepancies (13.3%) and five incomplete identifications of Trichophyton spp. Sequence analysis of ITS1 regions of 23 samples with discrepant or incomplete identification results (four Centraalbureau voor Schimmelcultures dermatophyte strains, four clinical samples and 15 clinical isolates) confirmed identification results of PCR-RLB in 21 of the 23 analyzed samples. PCR-RLB proved to be extremely suitable for routine detection and identification of dermatophytes directly in nail, skin and hair samples because it is rapid, sensitive, specific and accurate.

Dermatophytosis, Trends in Epidemiology and Diagnostic Approach

“…After hybridization and several washes, detection is performed using streptavidin peroxidase and chemiluminescence. This method showed good sensitivity and specificity [101]. However, the method is labour-intensive and difficult to standardize in a diagnostic setting, with a high risk of amplicon contamination and false-positive results [98,99].…”

Successful Identification of Clinical Dermatophyte and Neoscytalidium Species by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

“…Phenotypic identification can also be difficult because of intraspecies morphological polymorphism and phenotypic pleomorphism (6). In the Netherlands, between 1991 and 2002, only 56% of 1,171 dermatophyte isolates were successfully identified in a quality control exercise involving 50 laboratories (5). Alternative molecular methods have been developed to provide rapid and accurate dermatophyte identification.…”

“…They include gene-specific PCR, restriction fragment length polymorphism analysis, sequencing of the large-subunit rRNA gene or of the chitin synthase-encoding gene, PCR fingerprinting, DNA hybridization, and sequencing of the internal transcribed spacer regions (ITS1 and ITS2). However, these molecular methods are time-consuming and expensive for the identification of all isolates usually recovered from clinical samples, stressing the need for new strategies for identification of these common fungal pathogens (5,21,22,25,31).Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) represents a new approach to microbial identification and is based on characteristic fingerprints of intact cells (16,30). This technique has already been validated for rapid and accurate identification of bacterial, yeast, and mold species (1, 3, 4, 9-11, 15, 23, 24, 29, 33).…”

“…They include gene-specific PCR, restriction fragment length polymorphism analysis, sequencing of the large-subunit rRNA gene or of the chitin synthase-encoding gene, PCR fingerprinting, DNA hybridization, and sequencing of the internal transcribed spacer regions (ITS1 and ITS2). However, these molecular methods are time-consuming and expensive for the identification of all isolates usually recovered from clinical samples, stressing the need for new strategies for identification of these common fungal pathogens (5,21,22,25,31).…”

Evaluation of Chitine synthase ( CHS1 ) polymerase chain reaction assay in diagnosis of dermatophyte onychomycosis

“…Genotypic approaches such as PCR [9,13,20,22,32,33], PCR fingertyping [13], restriction fragment length polymorphism [10,11,17,25,28,30], arbitrarily primed PCR [23], sequencing [27,28], PCR-Elisa [3], PCR-reverse line blot [4], Nested PCR [12,14], multiplex PCR [8,21,31], oligonucleotide array [9] and real-time PCR [1,5,6], are promising techniques for developing rapid and accurate diagnosis of dermatophytes by allowing to identify species and to discriminate between strains within a comparatively short period of time.…”