Evaluation of A PCR Based Microarray Assay for the Diagnosis of Superficial Fungal Infections

Dermatomycosis is an infection of the skin, hair and nails, mainly due to dermatophytes, with occasional involvement of yeasts and moulds. Its global prevalence is 20%–25% [1], and 30%–70% [2] of adults are asymptomatic carriers. Onychomycosis accounts for 50% of nail diseases [3]. The positive culture rate in onychomycosis, even in the presence of positive direct microscopy, varies from 30% to 50% [4]. Current diagnostic methods include direct microscopy (sensitivity 70%–80%, turnaround time < 2 h) and fungal culture (sensitivity 50%–60%, turnaround time 2–6 weeks). Both are time-consuming, prone to false negatives, lack species-level identification, and rely on manual, subjective interpretation by experienced laboratory personnel [3]. Polymerase chain reaction (PCR)- based assays have emerged as valuable tool in the diagnosis of onychomycosis. Potential benefits include shorter turn-around-time (TAT) [5], greater positive results than cultures and increased sensitivity of detection of dermatophytes in nail specimens [6]. We evaluated the use of a commercially available PCR-based microarray assay (EUROIMMUN, Lübeck, Germany) in the diagnosis of superficial fungal infections. This was a single-centre prospective study at the National Skin Centre, Singapore, from March 2021 to February 2022, recruiting patients from general clinics with clinical features of superficial fungal infections (tinea capitis, tinea corporis, or onychomycosis) and positive direct microscopy. Patients at extremes of age (< 3 or > 90 years) were excluded. PCR results were compared with conventional fungal culture for concordance. Prior ethical approval was obtained from the National Healthcare Group Domain Specific Review Board. Of the 66 subjects recruited, 5 were excluded (4 due to discarded DNA samples, 1 ineligible), leaving 61 for analysis. The sensitivity of fungal culture was 50.82% (31/61), while PCR demonstrated a higher sensitivity of 93.44% (57/61). There were a total of 50 nail samples, 10 skin samples and 1 hair sample. Among pathogens identified via fungal culture, T. rubrum was the most common, isolated in 48.39% patients (15/31), followed by T. mentagrophytes var. interdigitale in 29.03% patients (9/31). Similarly, PCR detected T. rubrum and T. mentagrophytes var. interdigitale most frequently, in 64.91% (37/47) and 21.05% patients (12/57), respectively (Figure 1). Twenty-nine patients tested positive for both PCR and culture. Concordant pathogens were identified in 82.76% of samples (24/29). Discordant pathogens were identified in five samples, shown in Table 1. Two patients tested positive on the culture but tested negative on the PCR. These pathogens included Fusarium spp on the nail and Candida albicans on the skin, which were both confirmed with microscopy. A total of 28 nail specimens tested positive on the PCR but negative on cultures. Out of these patients, 71.4% (20/28) patients comprised of T. rubrum, 17.9% (5/28) with mixed infections and 10.7% (3/28) patients of T. intergitale (Table 2). There were two patients that tested negative for both PCR and culture, but tested positive on microscopy for Ectothrix spores and Mycelium. PCR demonstrated higher detection rate (93.4%) compared to fungal culture (50.8%) in detecting pathogens, with concordant results in 82.8% of cases. Our findings are consistent with a previous study [7], that showed PCR having a combined diagnostic yield of 100%. Notably, it may also be superior in identifying mixed infections. Culture-negative findings may be attributed to several factors such as prior antifungal use, which can suppress fungal growth [8]; slow-growing (e.g., T. rubrum) or fastidious fungal species that may not thrive in standard culture conditions, or the presence of non-viable hyphae, which are detectable by PCR but not culturable. Contamination from other fungal species can interfere with fungal growth in the culture. In patients with discordant results, mixed infections or overgrowth of commensals (e.g., non-dermatophyte moulds, Candida parapsilosis) may lead to detection of only the predominant organism in culture [9]. The culture yield may also vary with specimen type. Of the 61 samples, 50 (81.9%) were nail clippings, 10 (16.4%) skin and 1 (1.64%) hair. The predominance of nail samples likely contributed to the lower culture positivity observed, given their dense keratinised structure and limited viable fungal content. Study limitations include the small sample size and inability to assess assay specificity. Rare genetic variants may not be detected if their sequences are not represented in the PCR primers and probes used. Hence, fungal culture remains necessary when PCR is negative but clinical suspicion persists. The cost of approximately SGD 180 per sample may limit routine use. However, the 5-h turnaround and commercial availability are likely to reduce practical barriers to uptake. The PCR-based microarray showed higher sensitivity and rapid identification, with improved detection of mixed infections. Larger studies and cost-effectiveness analysis are needed to support wider adoption. This work was supported in part by Euroimmun, who sponsored the PCR assay utilised in this study. We thank Ms. Neo Sin Hui, from the Department of Research, National Skin Centre, Singapore, for her assistance in biostatistical analysis. Ethics approval by NHG DSRB. The authors declare no conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request.