MALDI-TOF mass spectrometry - a rapid method for the identification of dermatophyte species.
ABSTRACT Altogether 285 dermatophyte isolates of 21 different species - including both Trichophyton rubrum and T. interdigitale, but also eight additional Trichophyton species, Microsporum canis and seven other Microsporum species, as well as Epidermophyton floccosum and Arthroderma spp. - were analyzed using Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and the AnagnosTec 'SARAMIS' (Spectral Archiving and Microbial Identification System) software. In addition, sequence analysis of the internal transcribed spacer (ITS) of the ribosomal DNA was performed for a high number of the tested strains. Sufficient agreement was found between the results obtained with standard identification methods and those with the MALDI-TOF MS for species identification of dermatophytes. A mass spectra database was constructed which contained the species identifications of all 285 isolates. The results were confirmed for 164 of the isolates by sequence analysis of the internal transcribed spacer (ITS) of the ribosomal DNA. Statistical analysis of all 285 dermatophyte strains showed that conventional identification matched the results of MALDI-TOF MS for 78.2% of the isolates tested. In the case of the 164 isolates for which the identifications were confirmed by PCR, the results of their conventional diagnosis and MALDI-TOF MS were in agreement for only 68.9 % (113 of 164 strains) of the test isolates. In contrast, there was agreement of 99.3 % or 98.8 % in the identifications obtained with PCR and MALDI-TOF MS techniques (283/285 or 162/164). The two exceptions were isolates that proved to be T. violaceum which could not be identified by the MALDI-TOF MS technique. In conclusion, the MALDI-TOF mass spectroscopy represents a fast and very specific method for species differentiation of dermatophytes grown in culture.
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ABSTRACT: A novel strategy for the molecular identification of fungal agents of onychomycosis (including Trichophyton rubrum) has been designed based on the use of species-specific and universal primers in conjunction with a commercial kit that allows the extraction of DNA directly from the nail specimens. The microsatellite marker T1, which is based on a (GT)n repeat, was applied for the species-specific identification of Trichophyton rubrum. To evaluate how often Scopulariopsis spp. are detected in nail specimens, a second primer pair was designed to amplify specifically a 336-bp DNA fragment of the 28S region of the nuclear rRNA gene of S. brevicaulis and closely related species. Other fungal species were identified using amplification of the internal transcribed spacer (ITS) region of the rRNA gene, followed by restriction fragment length polymorphism analysis or sequencing. In addition, polyacrylamide gel separation of the T1-PCR product allowed subtyping of T. rubrum strains. We studied 195 nail specimens (the "nail sample") and 66 previously collected etiologic strains (the "strain sample") from 261 onychomycosis patients from Bulgaria and Greece. Of the etiologic agents obtained from both samples, T. rubrum was the most common organism, confirmed to be present in 76% of all cases and serving as the sole or (rarely) mixed etiologic agent in 199 of 218 cases (91%) where the identity of the causal organism(s) was confirmed. Other agents seen included molds (6% of cases with identified etiologic agents; mainly S. brevicaulis) and other dermatophyte species (4%; most frequently Trichophyton interdigitale). Simultaneous infections with two fungal species were confirmed in a small percentage of cases (below 1%). The proportion of morphologically identified cultures revealed by molecular study to have been misidentified was 6%. Subtyping revealed that all but five T. rubrum isolates were of the common type B that is prevalent in Europe. In comparison to microscopy and culture, the molecular approach was superior. The PCR was more sensitive (84%) than culture (22%) in the nail sample and was more frequently correct in specifically identifying etiologic agents (100%) than microscopy plus routine culture in either the nail or the strain samples (correct culture identifications in 96% and 94% of cases, respectively). Using the molecular approach, the time for diagnosing the identity of fungi causing onychomycosis could be reduced to 48 h, whereas culture techniques generally require 2 to 4 weeks. The early detection and identification of the infecting species in nails will facilitate prompt and appropriate treatment and may be an aid for the development of new antifungal agents.Journal of Clinical Microbiology 05/2006; 44(4):1419-27. · 4.07 Impact Factor
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ABSTRACT: We have focused on the DNA topoisomerase II genes of pathogenic fungi and have previously applied polymerase chain reaction (PCR)-based identification of several species including the some of the major dermatophyte species. To identify the dermatophytes (18 species) to a species level by PCR and PCR-restriction fragment length polymorphism (RFLP) techniques, without determining the nucleotide sequence. The genomic DNAs of the dermatophytes (ten species of Trichophyton, seven species of Microsporum, and Epidermaphyton floccosum) were amplified by PCR using a common primer set (dPsD1) for the dermatophytes, followed by nested PCR using other primer sets (dPsD2, PsT and PsME) that contained primers specific for the DNA topoisomerase II genes of the dermatophytes. PCRs using PsT and PsME were used for the species-identification of Trichophyton, Microsporum and E. floccosum. The PCR products generated by dPsD2 were digested with restriction enzymes (Hinc II, Hinf, Afl II and PflM I), and the restriction profiles were analyzed. Of the eighteen species of dermatophytes, five species (T. rubrum, T. violaceum, M. canis, M. gypseum and E. floccosum) were specifically identified by the PCR using PsT and PsME to the species level, and the remaining species were identified by the unique restriction profiles for each species in the PCR-RFLP analysis, except that the restriction profile of T. mentagrophytes var. interdigitale was identical to that of T. mentagrophytes var. quinckeanum. PCR and PCR-RFLP techniques targeting the DNA topoisomerase II gene are simple and rapid, and quite useful as tools for the identification of dermatophytes to the species level.Journal of Dermatological Science 11/2003; 33(1):41-54. · 3.52 Impact Factor
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ABSTRACT: The dermatophytes are among the most frequently observed organisms in biomedicine, yet there has never been stability in the taxonomy, identification and naming of the approximately 25 pathogenic species involved. Since the identification of these species is often epidemiologically and ethically important, the difficulties in dermatophyte identification are a fruitful topic for modern molecular biological investigation, done in tandem with renewed investigation of phenotypic characters. Molecular phylogenetic analyses such as multilocus sequence typing have had to be tailored to accommodate differing the mechanisms of speciation that have produced the dermatophytes that are commonly seen today. Even so, some biotypes that were unambiguously considered species in the past, based on profound differences in morphology and pattern of infection, appear consistently not to be distinct species in modern molecular analyses. Most notable among these are the cosmopolitan bane of nails and feet, Trichophyton rubrum, and the endemic African agent of childhood tinea capitis, Trichophyton soudanense, which are effectively inseparable in all analyses. The molecular data require some reinterpretation of results seen in conventional phenotypic tests, but in most cases, phylogenetic insight is readily integrated with current laboratory testing procedures.Mycopathologia 06/2008; 166(5-6):239-56. · 1.49 Impact Factor