Comparison of Fungal Fluorescent Staining and ITS rDNA PCR-based Sequencing with Conventional Methods for Diagnosis of Onychomycosis

Author:

Makled Amal F.ORCID,Ghonaim Mabrouk M.ORCID,Ali Sahar A.M.,ElHefnawy Sally Mohammed,Sabal Mona Salah,Elbrolosy Asmaa MohammedORCID

Abstract

Accurate identification of fungal causes for onychomycosis is essential for proper treatment. Presently available laboratory methods show unreliable sensitivity; so there is a requirement for innovative detection techniques. The aim for this work was to assess the efficiencies of fluorescent staining and internal transcribed spacer (ITS) ribosomal DNA (rDNA) polymerase chain reaction (PCR)-based sequencing in comparison to conventional techniques for diagnosis of onychomycosis. Nail specimens obtained from 100 patients with clinically- diagnosed onychomycosis were analyzed. Nail scrapings or clippings were subjected to direct microscopic examination by KOH mount, culture by using Sabouraud’s dextrose agar and histopathological examination with periodic-acid Schiff (PAS). Collected specimens were subsequently examined by fluorescent staining and PCR-based sequencing (30 specimens only) to compare the feasibility, sensitivity and diagnostic accuracy for these two methods. The most frequently isolated fungi were yeasts (39/76: 51.3%), dermatophytes (24/76; 31.6%) and non-dermatophyte molds (NDMs) (13/76; 17.1%). Mixed mycotic infections were recovered from 6% of the collected nail specimens. The positive detection rates were significantly different between KOH examinations (52%), nail plate histology (55%), fungal culture (70%) and fluorescent staining (80%). Considering fungal culture as the gold standard, the most sensitive technique was PCR (100%) followed by fluorescent staining (89%), PAS staining (69%) while the least sensitive technique was KOH mount (53%). Fluorescence staining can be used as a rapid and high-yield technique for identification of fungi in the specimens. PCR-based sequencing was highly sensitive and faster compared to culture. Whenever possible, it enables species identification with higher adequacy.

Publisher

Journal of Pure and Applied Microbiology

Subject

Applied Microbiology and Biotechnology,Microbiology,Biotechnology

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