Diagnosis of dermatophytes: from microscopy to direct PCR

Keywords: dermatophyte, Epidermophyton, fungal culture, medical mycology, microscopy, Microsporum, onychomycosis, real-time PCR, tinea pedis, Trichophyton.

Dermatophyte fungi are a common cause of skin, nail and hair infections globally. The most common agents are Trichophyton rubrum and T. interdigitale,^1–3 but other dermatophytes belonging to the genera Arthroderma, Trichophyton, Epidermophyton, Microsporum, Nannizzia, Paraphyton, and Lophophyton (the latter three genera formerly classified within Microsporum) also cause infection. Infections commonly present as tinea pedis and onychomycosis, but can affect any keratinised area of the body, having low clinical acuity. However, some infections may be debilitating or invasive in immunocompromised or elderly patients.^4,5 The prevalence of onychomycosis is approximately 10% in the general population, but may increase to 50% in those aged >70 years.⁶ Infections are usually transmitted by direct or indirect human contact, but may also be acquired from animal sources or soil, depending on the species. The prevalence of these infections and the species that cause them appears to vary significantly by geographic region, and is well reviewed by Nowicka and Nawrot.³

Occasionally, yeasts such as Candida spp. and non-dermatophyte moulds such as Scopulariopsis spp., Aspergillus spp., Fusarium spp., and Acremonium spp. may cause onychomycosis, but the diagnosis is complicated by these fungi also being common environmental contaminants.^7,8 Diagnostic guidelines require at least two subsequent isolations of these fungi in the absence of a dermatophyte and in the setting of direct microscopy exhibiting fungal hyphae not resembling dermatophytes.⁹

Effective antifungal agents are available for treatment of dermatophyte infections in topical or oral formulations, with the latter being necessary to treat refractory nail infections. However, for oral formulations to be prescribed through the Pharmaceutical Benefits Scheme (PBS), patients need to fulfil clinical requirements and laboratory diagnostic criteria including microscopy and dermatophyte culture.¹⁰ Therefore, these infections form a significant laboratory workload and reliable, sensitive, and specific diagnostic methods are needed for effective treatment.

While much of mycology continues to utilise conventional methods such as microscopy and culture, the diagnosis of dermatophyte infections is increasingly becoming modernised through the use of real-time PCR assays. Here we review current best practices and future prospects for laboratory diagnosis of dermatophyte infections, including the role of microscopy, culture and direct PCR, and Table 1 summarises the advantages and disadvantages of each.

Table 1.  Summary of available diagnostic methods for dermatophyte infections.

Direct microscopy of specimens is an essential component of the diagnostic pathway for cutaneous fungal infections. It provides a relatively fast result demonstrating the presence or absence of fungal elements indicative of infection. More importantly, a skilled microscopist can differentiate dermatophyte hyphae from that of non-dermatophyte moulds and yeast pseudohyphae, and the budding yeast cells of Candida spp. from those of Malassezia spp. This alone may be sufficient for a diagnosis of dermatophyte and dermatophyte-like infections. However, observation of fungal elements does not necessarily indicate causation of infection or demonstrate viability of the fungal elements and it is not possible to determine the genus or species. Therefore, microscopy is a low-specificity diagnostic technique.

Traditionally, skin and nail specimens are digested in 10–20% potassium hydroxide (KOH) as a clearing agent for 3–16 h prior to microscopy, having a false-negative rate of 5–15%.¹¹ Staining with chlorazol black E can provide additional contrast for visualising the fungal elements (Fig. 1a). However, optical brightener stains such as Calcofluor White or Blankophor, which bind to cellulose and chitin in the fungal cell wall and fluoresce under UV light (Fig. 1b), significantly improve the sensitivity of microscopy (82–91% vs 74–85% KOH preparation alone)^12,13 and allows more rapid scanning of slides. Optical brightener stains are recommended in laboratory guidelines for dermatophyte studies.¹⁴

Fig. 1.  KOH wet mounts of nail tissue containing dermatophyte hyphae with arthroconidia, stained with (a) chlorazol black, viewed under a light microscope, and (b) calcofluor white, viewed under a UV microscope. Image credit: (a) courtesy of David Ellis, Mycology Online (www.mycology.adelaide.edu.au).

Culture remains the gold standard for diagnosis of dermatophyte and other fungal infections, yielding a specific aetiological agent that can be identified to species level. However, culture has low sensitivity,¹⁵ requires 2–4 weeks for growth, and potentially a further 2 weeks for identification. Species identification needs detailed examination of colony and microscopic morphology, requiring specialised media and significant expertise.

Culture media for dermatophyte studies should include a specialised isolation medium such as Lactritmel or Dermatophyte Test Medium, containing antibiotics and cycloheximide; this minimises growth of fast growing bacterial and fungal contaminants, allowing the slower growing dermatophyte to grow. Sabouraud’s dextrose agar containing antibiotics but not cycloheximide is also recommended for nail specimens to allow growth of potential causes of non-dermatophyte onychomycosis.

The identification of dermatophytes and other fungi requires specialised training and expertise, something of a dying art in the era of laboratory automation. Commercial matrix assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-ToF) databases have the potential to identify common dermatophyte species; however, many species, for example, Trichophyton benhamiae, T. equinum, T. erinacei, T. eriotrephon, T. interdigitale, T. mentagrophytes and T. tonsurans, cannot be reliably differentiated.¹⁶ Optimised protein extraction methodologies combined with supplemented databases may be far more accurate,^17,18 although significant initial setup is required. DNA sequencing for species identification using the Internal Transcribed Spacer (ITS) rDNA region may be useful in some cases, but sequence databases contain numerous entries for dermatophytes with incorrect identifications that complicates making a reliable identification, and particularly between T. interdigitale and T. mentagrophytes.¹⁹

Given the slow and labour-intensive nature of conventional methods, many laboratories are looking towards real-time PCR for detection of dermatophytes in clinical samples.^20–26 The primary advantage of this approach is the decreased turnaround time over culture, with results typically available within 1–3 days of receipt in the laboratory. Additionally, the sensitivity of PCR is around 20–30% higher than culture, in part because dermatophyte DNA can be detected even in the presence of fast-growing fungal contaminants that might overgrow the slower-growing dermatophytes in cultures.^15,20,21,25 PCR-based identification reduces the need for morphologists, who could be utilised more effectively diagnosing fungi from life-threatening infections.

PCR assays may be commercial or in-house designed. In-house assays have the benefit of being customisable to existing laboratory platforms, as well as to include species that best represent the local epidemiology,²³ and fungi involved in non-dermatophyte infections of the skin and nails. Commercial assays typically have the benefit of regulatory approval, lessening the validation requirements on the laboratory. The sensitivity of dermatophyte PCR assays has invariably been shown to exceed that of culture.^20–26

Some commercial assays include pan-dermatophyte detection and/or specific primers for detection of a limited number of species. For example, the Dermatophyte PCR Test (SSI Diagnostica, Denmark), utilises pan-dermatophyte primers targeting the CHS1 gene as well as T. rubrum-specific ITS2 primers, the DermaGenius Nail multiplex assay (PathoNostics, The Netherlands) contains species-specific primers for T. rubrum, T. interdigitale and C. albicans only, and the Dermatophytes Real-Time PCR (EurobioPlex, France) is designed to detect six dermatophyte species. The ‘Dermatophytes and other Fungi’ multiplex tandem PCR (MT-PCR) (AusDiagnostics, Sydney) detects at least 14 dermatophyte species, as well as four Candida species and two non-dermatophyte moulds, with species identification made possible by specific primers and melt curve analysis (Fig. 2). This assay does not currently differentiate all of the species that it detects,^21,22 but differences in melt curve may be suggestive of certain species, requiring further validation. Given the limitations of species differentiation with PCR assays, it may only be possible to report the species complex, genus, or simply ‘dermatophyte detected’. This loss of species resolution limits insight into the potential origins of infection (i.e. anthropophilic vs zoophilic), but for most clinicians the faster turnaround time coupled with increased sensitivity, appears to be an acceptable trade-off.

Fig. 2.  Screenshot of Dermatophytes and other Fungi MT-PCR result display (AusDiagnostics Multiplex-Tandem PCR Results Software ver. 1.7.14) from a nail scraping, demonstrating melt curve detection of Trichophyton species (red), further identified as T. rubrum complex (green), trace quantities of a Candida species (pink) likely representing skin flora, and an internal PCR control (black).

PCR assays may not detect non-dermatophyte fungi that cause onychomycosis, which have an incidence of around 2–20%.^7,8,27,28 The AusDiagnostics panel includes targets for Aspergillus spp., Scopulariopsis spp., and four Candida species but not Fusarium spp., Acremonium spp., and Neoscytalidium spp. Since these may be environmental contaminants or opportunistic skin flora, they may be detected in the absence of infection and should be reported with caution, potentially utilising reflex cultures where non-dermatophyte onychomycosis is suspected.

Real-time PCR detection of squalene epoxidase mutations conferring terbinafine resistance has recently been described.²⁹ However, the significance and clinical utility of such assays are currently limited, as susceptibility breakpoints for dermatophytes remain tentative or unavailable.³⁰

Despite the superior sensitivity of PCR compared to culture, dermatophyte detection in nail specimens requires confirmation by either microscopy or culture in order to satisfy Australian PBS requirements for prescription of oral terbinafine. This represents additional work, and does not always provide the necessary confirmation. Some of these cases may be attributed to the detection of DNA from non-viable dermatophytes (i.e. already inactivated by antifungal treatment).

PCR detection of dermatophyte infection brings many benefits to laboratories that are increasingly stretched for resources. However, currently, such advances come at the cost of accurate speciation, and the associated ecological and epidemiological information that accompanies it. Current PBS requirements and the increasingly apparent role of non-dermatophyte fungi in cutaneous infections means that there remains a role for microscopy and culture in routine diagnostic laboratories.

Data sharing is not applicable as no new data were generated or analysed during this study.

The authors declare no conflicts of interest.

This work did not receive any specific funding.