Assessment of a Real-Time PCR Assay for the Detection of Dermatophytosis by Sampling With an Adhesive Tape Technique

Abstract

Dermatophytosis is a very common fungal infection [1]. Prompt and accurate diagnosis utilising newer technologies [2, 3] may facilitate early commencement of suitable treatment. We previously reported the development and validation of a real-time polymerase chain reaction (RT-PCR) assay targeting the chitin-synthase 1 gene (CHS-1) specific for common dermatophytes, including species of the Microsporum, Epidermophyton and Trichophyton genera [4]. Since implementation, this assay has been efficacious in screening clinical samples for dermatophytes and for early detection (i.e., < 6 weeks) in filamentous cultures. However, the assay was limited in its ability to detect Microsporum canis due to the inefficient binding of the primers and probe used in the PCR assay [4].

Also developed was an adhesive tape sampling technique to facilitate the collection of genomic material for the RT-PCR assay and circumvent practical issues that can be associated with traditional use of scalpel blades and clippers (e.g., operator injury, patient antipathy to ‘sharps’, suitability of the infected site) [5]. Adhesive tape strips have been used in dermatology research as a minimally invasive technique for epidermal transcriptomics and proteomics, particularly in atopic dermatitis [6-9].

In our initial study of 16 patients, the RT-PCR results for the adhesive tape impressions correlated with those obtained by microscopy and culture [4]. Here, we report an assessment of the utility of the RT-PCR assay for detection of dermatophyte CHS-1 gene on adhesive tape impressions of lesions undertaken from April 2018 to February 2024 (approximately 5.8 years). Impressions were taken from 308 lesions located on a variety of specified anatomical sites (274 lesions) and unspecified sites (34 lesions) in patients attending dermatology clinics at a tertiary teaching hospital and an academic private practice in Australia (Appendix S1). Using fungal culture results as the reference standard, the sensitivity and specificity were calculated for RT-PCR assay alone, microscopic detection of fungal elements alone, and when both methods were used (Table 1).

Fifty-two dermatophyte strains were cultured from 308 samples (16.9%). The most common strain cultured was Trichophyton rubrum (n = 25), followed by Trichophyton tonsurans (n = 11), Trichophyton sp. (n = 8), Trichophyton mentagrophytes (n = 5), Microsporum canis (n = 2) and Epidermophyton floccosum (n = 1). Among the dermatophytes isolated, 46 of 52 (88.5%) were RT-PCR positive (Appendix S1). The remaining six RT-PCR negative isolates included Trichophyton rubrum (n = 2), Trichophyton tonsurans (n = 1), Trichophyton sp. (n = 1) and Microsporum canis (n = 2). As mentioned, failure to detect Microsporum canis by RT-PCR was anticipated. However, failure to detect the other four fungi could be due to inadequate collection of genomic material by the adhesive tape. Excess DNA collected by adhesive tape could also compromise the assay's sensitivity [10], but was excluded as the internal control was co-amplified. It was also noted that 20 of 256 (7.8%) dermatophyte-negative cultures were RT-PCR positive, possibly due to poor sample quality (i.e., scraping/clippings) used to inoculate culture media or previous treatment of the lesion with antifungal agents.

As shown in Table 1, the RT-PCR assay is more sensitive than microscopy for predicting cultural isolation of dermatophyte species. Also evident is the potential compromise to the assay's specificity and positive predictive value, which may be due to sample quality or pre-treatment with antifungals inhibiting viability when cultured. However, of note was marked improvement of the sensitivity, specificity and negative predictive value (NPV) when both microscopy and the RT-PCR assay were used to predict a culture-positive sample, using traditional sampling techniques and adhesive tape impressions. Especially remarkable was the high NPV (with a high probability) which shows the potential benefit of performing both methods on clinical samples as a screen for the likelihood of failure to isolate a dermatophyte species on culture.

We found that the RT-PCR assay is able to detect a wide diversity of dermatophytes. We have also demonstrated the feasibility of molecular testing using adhesive tape impressions of suspect fungal lesions as assessed over 5.8 years. During this period, we have shown this method to allow safe sampling of a wide range of lesion surfaces differing in presentation (i.e., dried or highly suppurative) without the risks and limitations associated with using sharp implements. Furthermore, this adhesive sampling allows the usually limited specimens collected by traditional means to be used exclusively for microscopy and culture. Here, we have shown that, combined with microscopy, the RT-PCR assay using adhesive tape sampling is conducive to reliable, rapid laboratory diagnosis of dermatophytosis if supported by strong clinical evidence, and thus prompt treatment. In this study, we have also demonstrated, with a high level of certainty, that this rapid testing algorithm can be used to exclude the likelihood of cultivating a targeted dermatophyte.

The authors declare no conflicts of interest.