Molecular strategy for the direct detection and identification of the most common fungal community in cerumen specimens by multiplex PCR

Abstract

Introduction. Otomycosis is a superficial fungal infection that is responsible for approximately 9–27 % of otitis externa. However, fungal communities in otomycosis are varied, but Aspergillus spp. and Candida spp. are the most common causes of this infection. Hypothesis Statement. The multiplex PCR assay is postulated to be able to directly detect more than one fungal genus in cerumen specimens. Aim. This study aimed to develop and evaluate the role of the multiplex PCR assay in detecting the most common genus of fungi that cause otomycosis directly from the cerumen specimens. Methodology. To detect Candida and Aspergillus/Penicillium genera, three pairs of primers, including pan-fungal, pan-Candida, and pan-Aspergillus/Penicillium, were used in a multiplex PCR. In order to evaluate the performance and reproducibility of the multiplex PCR. the cerumen of 140 patients suspected of otomycosis were investigated. Results. Pan-Candida and pan-Aspergillus/Penicillium primers were designed to amplify the ITS1–5.8S–ITS2 region and the β-tubulin gene, respectively. In the multiplex PCR assay, 64 (47.40 %) and 118 (87.40 %) specimens were positive with pan-Candida and pan-Aspergillus/Penicillium primers, respectively. Double amplicon bands of Candida and Aspergillus were obtained in 51 (37.77 %) specimens. In the culture method, yeast (n=18, 13.33 %) and mould (n=117, 86.66 %) were isolated from 135 cerumen specimens. The sensitivity, specificity, and positive and negative predictive values of the multiplex PCR assays using culture method results as the gold standard were determined to be 94, 33, 97, and 22 %, respectively. Conclusion. In our study, multiplex PCR assays enabled simultaneous detection of two common genera of the causative agent of otomycosis in a cerumen specimen. Regarding the high sensitivity of the first step of the multiplex PCR assay, this assay may be used for the direct detection of Candida and Aspergillus genera in other clinical specimens.