Identification of Infectious Agents in Onychomycoses by PCR-Terminal Restriction Fragment Length Polymorphism

Abstract

A fast and reliable assay for the identification of dermatophyte fungi and nondermatophyte fungi (NDF) in onychomycosis is essential, since NDF are especially difficult to cure using standard treatment. Diagnosis is usually based on both direct microscopic examination of nail scrapings and macroscopic and microscopic identification of the infectious fungus in culture assays. In the last decade, PCR assays have been developed for the direct detection of fungi in nail samples. In this study, we describe a PCR-terminal restriction fragment length polymorphism (TRFLP) assay to directly and routinely identify the infecting fungi in nails. Fungal DNA was easily extracted using a commercial kit after dissolving nail fragments in an Na 2 S solution. Trichophyton spp., as well as 12 NDF, could be unambiguously identified by the specific restriction fragment size of 5=-end-labeled amplified 28S DNA. This assay enables the distinction of different fungal infectious agents and their identification in mixed infections. Infectious agents could be identified in 74% (162/219) of cases in which the culture results were negative. The PCR-TRFLP assay described here is simple and reliable. Furthermore, it has the possibility to be automated and thus routinely applied to the rapid diagnosis of a large number of clinical specimens in dermatology laboratories.

Evaluation of a commercial PCR test for the diagnosis of dermatophyte nail infections

“…In our settings, specific detection of T. rubrum in parallel with pan-dermatophyte primers is sufficient as the vast majority of positive samples were identified as T. rubrum [40 out of 44 (91 %) by culture, and 122 out of 132 (92.4 %) by the combination of culture and PCR]. Similar results were obtained in recent studies (Bergman et al, 2013;Verrier et al, 2012). Nevertheless, dermatophytes belonging to the less terbinafine-susceptible genus Microsporum are unanimously reported to be very rare agents of onychomycosis.…”

“…However, real-time PCR is less laborious, may enable a broader spectrum of simultaneous species detection and as a closed system reduces contamination risk (Alexander et al, 2011;Bergman et al, 2013; Bergmans et al, 2010;Miyajima et al, 2013;Paugam et al, 2013;Wisselink et al, 2011). (Bergman et al, 2013;Verrier et al, 2012). Nevertheless, dermatophytes belonging to the less terbinafine-susceptible genus Microsporum are unanimously reported to be very rare agents of onychomycosis.…”

“…Finally, post-PCR strategies, e.g. nested PCR (Verrier et al, 2013), PCR-ELISA (Beifuss et al, 2011;Pankewitz et al, 2013), PCR-RFLP (Elavarashi et al, 2013;Ghannoum et al, 2013;Verrier et al, 2012) and DNA microarray assays (Sato et al, 2010), may increase the number of species identified; however, they prolong the turnaround time (Jensen & Arendrup, 2012). In our settings, specific detection of T. rubrum in parallel with pan-dermatophyte primers is sufficient as the vast majority of positive samples were identified as T. rubrum [40 out of 44 (91 %) by culture, and 122 out of 132 (92.4 %) by the combination of culture and PCR].…”

Terbinafine Resistance of Trichophyton Clinical Isolates Caused by Specific Point Mutations in the Squalene Epoxidase Gene

“…Trichophyton total DNA was extracted from fresh fungal cultures on SD agar medium and nail samples as previously described using a DNeasy Plant minikit (Qiagen) (31). A diameter of approximately 0.5 cm of growing mycelium was used.…”

Oral Terbinafine and Itraconazole Treatments against Dermatophytes Appear Not to Favor the Establishment of <b><i>Fusarium</i></b> spp. in Nail

“…as well as Acremonium spp. and Scopulariopsis brevicaulis identified in culture were in most cases the infectious agent in onychomycosis [6,7,8]. Fusarium spp.…”