P391 Research on molecular diagnostic methods and clinical application of common pathogenic dermatophytes in tinea capitis

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectiveThe main objective of this study was to design and develop a detection system based on qRT-PCR that can quickly and accurately identify the pathogenic fungi of tinea capitis, in order to improve the diagnostic ability of tinea capitis.MethodsA total of 8 isolates of Microsporum spp., 29 isolates of Trichophyton spp., 3 isolates of Nannizzia gypsea, 6 isolates of non-dermatophytic filamentous fungi, Malassezia spp., and Candida spp. were included in this study.Primer Express Software (V3.0) was used to design specific primers and TaqMan probe for qRT-PCR assay. The specificity of each system was validated using the above fungal isolates. The standard curve of each system was constructed by using the DNA of the standard substance of fungal isolates to find the minimum detection.The clinical specimens from confirmed and suspected patients with tinea capitis were collected. Fungal DNA was extracted from clinical samples and detected by a two-step qRT-PCR system. The results of qRT-PCR were analyzed comprehensively and compared with a fungal microscope and fungal culture.As a supplementary interpretation, the next generation sequencing targeted amplicon was conducted in 14 clinical samples that generated objectional results between fungal culture and qRT-PCR.ResultsThe molecular diagnostic system for tinea capitis herein consists of seven single-tube qRT-PCR assays designed on the complex and species level, which include the group of M. canis complex, T. rubrum complex, T. mentagrophytes complex, T. tonsurans, T. schoenleinii, T. verrucosum, and of N. gypsea. The analytical specificity of each group meets the design expectation.The minimum detection limit of the M. canis complex group, T. rubrum complex group, T. schoenleinii group, and N. gypsea group was 100 Colony Forming Units (CFUs), the counterpart of which for T. mentagrophytes complex group, T. tonsurans and T. verrucosum were 10 CFUs.A total of 351 clinical specimens were collected, including 231 cases confirmed of tinea capitis, 100 suspected of tinea capitis, and 20 with non-tinea capitis. Positive fungal microscopy and/or fungal culture were the gold criteria for diagnosis. Compared with the diagnostic gold standard, the sensitivity and efficacy of qRT-PCR, the combination of qRT-PCR and fungal microscopy, the combination of qRT-PCR and fungal culture were 93.1% and 93.6%, 100% and 100%, and 96.1% and 96.4%, respectively. The diagnostic specificity for cases of non-tinea capitis was 100%. The coincidence rate between qRT-PCR and fungal culture was 95.16%. The positive rate of qRT-PCR in suspected cases was 48%.Amplification sequencing results confirmed that dermatophytes existed in 13 of 14 samples. Consistent with qRT-PCR, there were two species of dermatophytes mixed infection in 4 samples.ConclusionsThe seven single-tube qRT-PCR assays validated in this study can rapidly detect a variety of pathogenic fungi causing tinea capitis, with a high level of sensitivity and specificity. The combination of qRT-PCR and traditional mycological identification methods can further improve the diagnostic efficacy of tinea capitis.