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.

Fig 1

Overview of the PCR-TRFLP assay developed for fungal identification in onychomycosis. First, fungal DNA was extracted from nail samples. Then, 28S rDNA was amplified using a labeled forward primer. A single-step digestion of PCR amplicons with AvaI, AvaII, and StuI was completed. The digested PCR products were purified, and finally, the purified, digested, and labeled PCR products were separated using a DNA analyzer, and output data were analyzed with GeneMapper.

Fig 2

RFLP profiles of 28S ribosomal DNA PCR products used to prepare the TRFLP reference ladder. DNA was loaded onto a 1.5% agarose gel and stained with ethidium bromide. The PCR low ladder set (Sigma) was used as a molecular size marker. The different profiles were obtained from one strain representing each of the common infectious fungi in onychomycosis. Lane 1, T. rubrum; lane 2, F. oxysporum; lane 3, F. solani; lane 4, A. flavus; lane 5, A. versicolor; lane 6, A. strictum; lane 7, A. alternatum; lane 8, P. citrinum; lane 9, S. brevicaulis; lane 10, C. parapsilosis; lane 11, Alternaria spp. The 28S ribosomal DNA sequences and fragment sizes obtained by AvaI, AvaII, and StuI digestion are listed in Table 1. The fragments that are red labeled in the reference ladder are indicated by asterisks.

Fig 3

Identification of nail-infectious fungi in onychomycosis (panels 1 to 10) by TRFLP analysis. Fragments of the reference ladder were labeled with Red-ATTO565 (red). Fragments used to discriminate infectious fungi in onychomycosis by TRFLP analysis were labeled with Yellow-ATTO550 (black). Eleven fungi were discriminated: Trichophyton spp., A. versicolor, Candida spp., F. oxysporum, P. citrinum, A. alternatum, Alternaria spp., S. brevicaulis, A. flavus, A. strictum, and F. solani. Mixed infections are highlighted by multiple peaks. Panels 9 and 10 are examples where the detected peak did not correspond to any of the peaks in the reference ladder. Scytalidium spp. and Microascus spp. were identified by sequencing of the amplicons.