Development and evaluation of a pan-dermatophyte polymerase chain reaction with species-level identification using sloppy molecular beacon probes.
Journal
The British journal of dermatology
ISSN: 1365-2133
Titre abrégé: Br J Dermatol
Pays: England
ID NLM: 0004041
Informations de publication
Date de publication:
06 2019
06 2019
Historique:
accepted:
03
12
2018
pubmed:
14
12
2018
medline:
22
8
2020
entrez:
8
12
2018
Statut:
ppublish
Résumé
Conventional laboratory diagnosis of dermatophyte infection is cumbersome and time-consuming. We aimed to establish a simple, robust and rapid molecular diagnostic assay for the detection of dermatophytes and optionally nondermatophytes in clinical specimens. We developed a two-tube pan-dermatophyte polymerase chain reaction (PCR) assay using six sloppy molecular beacon (SMB) probes. The first PCR uses dermatophyte-specific primers and enables detection and identification of most dermatophyte species. The second PCR with pan-fungal primers allows further differentiation of Trichophyton interdigitale and T. mentagrophytes/T. quinckeanum, T. violaceum and T. soudanense, and T. tonsurans and T. equinum, and detection of nondermatophytes. The test was evaluated with 306 clinical specimens by comparing it with the results of microscopy and culture. In melting-curve analyses, species-specific melting temperature signatures of the SMBs were defined, and thus, our new PCR enabled detection and species-level identification of at least 19 dermatophyte species. Sensitivity and specificity of PCR for detection of dermatophytes in clinical samples were estimated to be 96·9% and 90·4%, for culture 46·7% and 98·7%, and for microscopy 91·4% and 84·0%, respectively. The detection of nondermatophytes by PCR and culture did not correlate. The new assay showed excellent performance characteristics for the detection of dermatophytes and is significantly faster than culturing techniques, which makes it very promising for routine diagnostics of dermatophytosis. We noted that the detection of nondermatophytes in our assay currently has no benefit.
Sections du résumé
BACKGROUND
Conventional laboratory diagnosis of dermatophyte infection is cumbersome and time-consuming.
OBJECTIVES
We aimed to establish a simple, robust and rapid molecular diagnostic assay for the detection of dermatophytes and optionally nondermatophytes in clinical specimens.
MATERIALS AND METHODS
We developed a two-tube pan-dermatophyte polymerase chain reaction (PCR) assay using six sloppy molecular beacon (SMB) probes. The first PCR uses dermatophyte-specific primers and enables detection and identification of most dermatophyte species. The second PCR with pan-fungal primers allows further differentiation of Trichophyton interdigitale and T. mentagrophytes/T. quinckeanum, T. violaceum and T. soudanense, and T. tonsurans and T. equinum, and detection of nondermatophytes. The test was evaluated with 306 clinical specimens by comparing it with the results of microscopy and culture.
RESULTS
In melting-curve analyses, species-specific melting temperature signatures of the SMBs were defined, and thus, our new PCR enabled detection and species-level identification of at least 19 dermatophyte species. Sensitivity and specificity of PCR for detection of dermatophytes in clinical samples were estimated to be 96·9% and 90·4%, for culture 46·7% and 98·7%, and for microscopy 91·4% and 84·0%, respectively. The detection of nondermatophytes by PCR and culture did not correlate.
CONCLUSIONS
The new assay showed excellent performance characteristics for the detection of dermatophytes and is significantly faster than culturing techniques, which makes it very promising for routine diagnostics of dermatophytosis. We noted that the detection of nondermatophytes in our assay currently has no benefit.
Substances chimiques
DNA, Fungal
0
Molecular Probes
0
Types de publication
Evaluation Study
Journal Article
Validation Study
Langues
eng
Sous-ensembles de citation
IM
Pagination
1489-1497Commentaires et corrections
Type : CommentIn
Informations de copyright
© 2018 British Association of Dermatologists.