Dermatophytosis laboratory diagnosis

Overview and update on the laboratory diagnosis of dermatophytosis

Shivaprakash M Rudramurthy, Dipika Shaw
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Correspondence Address:
Shivaprakash M Rudramurthy
Department of Medical Microbiology, Division of Mycology, Postgraduate Institute of Medical Education and Research, Chandigarh
India

Source of Support: None, Conflict of Interest: None

DOI:10.4103/CDR.CDR_35_17

Dermatophytosis, caused by dermatophytes is becoming difficult to treat due to various reasons. Accurate diagnosis is essential for the accurate management of this infection and prevention of relapse or recurrence. Although this condition is easy to diagnose clinically, due to overlapping signs and symptoms of few dermatological conditions it may be misdiagnosed necessitating laboratory confirmation. Isolation, identification of the dermatophytes and the antifungal susceptible profile may further help to initiate appropriate antifungal agent. The classical conventional techniques such as direct microscopic examination and isolation of fungi from the clinical specimens are still considered as an important modality of diagnosis. With the rise of the molecular era, molecular techniques are increasingly being applied to diagnose dermatophytosis and identify the dermatophytes. The present review provides an overview and update on the laboratory diagnosis of dermatophytosis.

Keywords:Conventional diagnosis, dermatophytes, dermatophytosis, laboratory diagnosis, molecular diagnosis

Superficial fungal infections affect millions of people worldwide with an estimated lifetime risk of 20%25%.[1] One of the major causes of these infections include dermatophytes, nondermatophytic moulds [NDMs], and yeasts. All dermatophytes belong to three genera Trichophyton, Microsporum, and Epidermophyton. Based on the natural habitat, these fungi can be differentiated into geophilic, zoophilic, and anthropophilic species that are acquired from soil, animals, and human, respectively.[2] The various clinical manifestation of dermatophytosis has been described. Although the disease can be diagnosed based on typical clinical presentation, laboratory confirmation may be essential for those with rare presentations. In addition, in view of the present problem of relapse or recurrences in India, laboratory diagnosis in terms of isolation, identification and in vitro susceptibility testing is gaining importance. For optimal management of the onychomycosis, identification of causative agent plays an important role as the treatment with specific antifungal agents varies for the dermatophytes and NDMs or yeast.

The efficiency of the laboratory diagnosis of dermatophytosis depends on the quality of samples collected. Few important attributes that should be considered while collecting the samples are [i] cleaning the lesion site with 70% alcohol to remove the contaminants and [ii] sample collection before initiating systemic treatment and if already applying topical antifungals or on oral antifungals, abstinence for at least 15 days or 3 months respectively is essential before sample collection.[3] Samples from skin should include epidermal scales usually collected from the advancing edge of the lesions using sterile dermal blunt curette [Brocq's curette], scalpel blades, or vaccinostyles. Whereas, nail samples are collected near the area of nail bed where viable fungal hyphae are usually present. While collecting infected hair for diagnosis, it is important to include the hair follicle to increase the chance of detection and isolation. Hence, hairs should be plucked from the basal part using sterile forceps and not clipped.[4]

Examination of the clinical specimen using microscope is one of the simple and rapid screening techniques that allow the clinician to initiate antifungal therapy for dermatophytosis. Direct microscopic visualization requires the addition of clearing agent to digest keratin from the sample. A variety of clearing agents such as 10% or 20% potassium hydroxide [KOH] with or without dimethyl sulfoxide, 10% sodium hydroxide, Amann's chloral lactophenol, and detergents such as sodium dodecyl sulfate [SDS] can be used.[3] KOH is a simplest and low-cost method for keratin dissociation used in every mycology laboratory. As the KOH do not stain the fungi and can be visualized only based on the difference in the refraction, detection of fungi in this preparation is difficult for the beginners and requires some amount of expertise. To provide the contrast, and increase the chance of detection and sensitivity of direct microscopy, various stains such as cotton blue C4B [Bacti-Lab Inc., RAL or Bio-Rad, associated with lactic acid and phenol] or BlueBlack Ink permanent [Parker Quink], or chlorazol black E [CBE; Sigma-Aldrich] stain which imparts deep blue or black color to fungal element are used.[4] In addition, other stains such as Periodic acid-Schiff [PAS] which stains polysaccharide glycosaminoglycans, Congo red which stains ß-D-glucans and fluorochromes such as calcofluor white [CW; Fluorescent brightener 28, Sigma-Aldrich], BlankophorPFlussig [Bayer] or Uvitex 2B [Fungiqual A, Ciba Corning] which binds to chitin of the fungal cell wall have been evaluated and found to be beneficial.[3] Mycetocolor® and Mycetfluo® are commercial reagents that contain Congo red and calcofluor respectively along with SDS as a clearing agent. Recently, Pihet et al. evaluated four different staining techniques, KOH with chlorazol black, charcoal-lactophenol, Mycetocolor® and Mycetfluo® and showed that performance of Mycetfluo® was better [57%] and statistically significant when compared to other three staining techniques [42%] for detecting fungal elements in skin, hair, and nail samples.[5] Histological examination using stains like PAS or Gomoris methenamine silver on which the fungi appears red or gray black respectively also helps in the diagnosis especially for the examination of the nail specimen. Cyanoacrylate surface skin scrapping is a simple strip biopsy test performed by removing the uniform thickness of stratum corneum with the adhesive. This test has been evaluated for the detection of dermatophytes from the skin specimen and has shown the sensitivity of 62% with high negative predictive value of 92%.[6] However, the main disadvantage of this technique is that it cannot be applied for hair or nail samples.

Isolation and identification of the etiological agent responsible for dermatophytosis are essential as the treatment regimens may differ for different dermatophyte species for example - Trichophyton tonsurans in tinea capitis tends to require shorter duration of therapy than that caused by Microsporum canis; onychomycosis due to nondermatophyte mold may not respond to the standard therapy used for dermatophytosis. Sabouraud's dextrose agar or potato dextrose agar supplemented with antibiotics [chloramphenicol ± gentamicin], and cycloheximide are generally used for the primary isolation of fungi from a clinical specimen. Cultures are usually incubated at 20°C25°C for 46 weeks, but higher incubation temperatures of 30°C32°C may be required if Trichophyton verrucosum is suspected.[3] Dermatophyte test medium was developed by Taplin as a selective and differential medium for detection and identification of dermatophytes. The growth of dermatophytes on this media may be presumptively identified based on gross morphology and the production of alkaline metabolites, which raise the pH and cause the phenol red indicator to change the color of the medium from yellow to pink-red.[7] In addition, specialized isolation media like casamino acids-erythritol albumin agar may help for isolation of the dermatophytes especially when the sample is contaminated with bacteria or yeast. Bromocresol purple casein yeast extract agar may be used for the rapid identification of T. verrucosum.[4] Spores are essential for the morphological identification of dermatophytes. Most of the dermatophytes take a longer time to sporulate or sometimes do not sporulate on primary isolation media, hence, it is essential to induce spores on the sporulation media to proceed for morphological identification. Many sporulation media such as potato dextrose agar, Borelli lactrimel agar, pablum cereal, brain heart infusion agar, Baxter's medium, Takasio medium, malt agar, or water agar which stimulate conidiation and pigment production of dermatophytes may be used. Apart from that, special culture media that can differentiate different species and genus of dermatophytes are bromocresol purple milk solid glucose agar [turns violet color in the presence of Trichophyton interdigitale, differentiating it from Trichophyton rubrum and Microsporum persicolor]; polished rice grains that can differentiate Microsporum audouinii [brownish pigment] from M. canis [yellow pigment];[4],[8] urea indole broth or Christensen's urea agar medium that can differentiate Trichophyton mentagrophytes from T. rubrum and Trichophyton soudanense from urease negative organism.In vitro hair perforation test can differentiate T. rubrum [no perforating organs] from T. mentagrophytes and M. canis [positive test] from M. audouinii or M. equinum [negative test].[4] Comparative evaluation of direct microscopy and culture from the clinical samples is summarized in [Table 1].

The rate of isolation of the fungi from the clinical specimen has been found to be lesser than the positivity rate of direct microscopic examination. These discrepancies in the result of direct microscopy and culture has been attributed to several factors such as inadequate quantity of sample, presence of nonviable fungal hyphae, and antifungal treatment before sample collection. The presence of antifungals in the epidermal layer of skin inhibits the growth of the fungi. A medium containing lecithin and polysorbate 80 has been developed and being used to minimize the carryover effect of antifungals.[3],[9] Direct microscopy has a limitation that it only detects the presence or absence of fungal element but cannot help in the differentiation of the different species. Even after successful isolation of the dermatophytes from the clinical specimen, identification is difficult due to morphological similarities shared between species necessitating molecular techniques.[10]

Isolation and identification of dermatophytes from the clinical samples are considered as a gold standard method for diagnosis of dermatophytosis. However, the dermatophyte usually takes long time to grow in culture and sporulate leading to delayed diagnosis. Rapid diagnosis and accurate identification of dermatophytes helps in successful management.[11] Nucleic acid-based molecular methods are increasingly being employed in the clinical microbiological laboratory for rapid and specific identification of the fungi as well as for the detection of etiological agent directly from the clinical specimen.[12]

Extraction of the DNA from the clinical specimens can be done by phenolchloroform method or using commercially available DNA extraction kit. Before DNA extraction step, keratin from the clinical sample should be disrupted which can be achieved by mechanical method or enzymatic digestion of keratin with proteinase K or nonenzymatic disruption by Na2S solution.[13] As very low quantity of the fungal DNA is expected from clinical samples, different approaches have been used to increase the yield of DNA. The sensitivity of detection depends on selection of the target DNA for amplification, and application of techniques that could detect the specific amplicon. Molecular diagnoses for rapid detection of etiological agent from the clinical specimens may be done either by conventional polymerase chain reaction [PCR] or by real-time PCR.[14]

Conventional PCR is one of the widely used, simple and inexpensive tool for detection of a particular species with specific primers and its interpretation is mainly based on amplicon size in agarose gel.[15] The identification of an organism is achieved using variety of primers such as species specific primer [amplify only the species of interest], pan dermatophyte primer [amplifies the DNA from all dermatophytes], or pan fungal primer [amplifies the DNA of any fungal agent which usually targets internal transcribed region [ITS] or 28S region of ribosomal DNA sequence or gene encoding topoisomerase II, or chitin synthase I [CHS I]].[12],[16] Brillowska-Dabrowska et al. developed the PCR test using ITS primers designed to amplify 302 bp amplicon of Trichophyton species and 279 bp amplicon of Microsporum species. Of the 58 isolates tested, they could successfully identify 42 isolates including 35/35 - Trichophyton isolates belonging to ten different species and 3/3 - M. canis and 4/4 - M. audouinii samples. They could not identify 2 - Epidermophyton floccosum, 11 - Microsporum gypseum, and 3 - M. persicolor.[17] The same group further developed multiplex PCR test for detection of dermatophytes from tinea unguium cases by adding species specific primers.[18] Multiplex PCR based on CHS I and ITS region for identification of T. rubrum and T. mentagrophytes showed a sensitivity of 97% which was higher [81.1%] than the conventional method.[12] In many countries, duplex PCR has been used for detection of T. rubrum from onychomycosis and tinea pedis cases.[19],[20],[21],[22] Kondori et al. evaluated the duplex PCR and reported positivity rate of 44% for dermatophytes compared to 34% by culture, the sensitivity and specificity was 94% and 85%, respectively.[19] Garg et al. performed pan-dermatophyte nested PCR targeting CHS I and ITS and showed a sensitivity of 83.8% which was far higher than the KOH wet mount examination. They concluded that this method should be considered as the gold standard method for the diagnosis of onychomycosis.[23],[24] Although nested PCR has high sensitivity, this test cannot be recommended for clinical diagnosis because of increased chance of contamination due to two successive amplifications steps involved in the procedure.[25]

Real-time PCR is a rapid and sensitive approach to detect an organism directly from clinical samples. As real-time PCR assay is conducted in a closed tube system, it limits the risk of contamination and helps in detection of multiple species of dermatophytes using different species-specific probes.[14] Arabatzis et al. detected and identified different dermatophytes from the clinical samples by real-time PCR using ITS primers.[26] Alexander et al., mainly targeted T. rubrum using T. rubrum specific primers and a probe whereas Bergmans et al., could differentiate 11 dermatophytes within 72 h in a single real-time PCR reaction with melt curve analysis.[20],[27] Real-time PCR could detect both dermatophytes and nondermatophytes with a sensitivity of 97% when compared with culture. The concordance between the culture and the real-time PCR in identifying the dermatophytes to the species and genus level was 94.3% and 97.4%, respectively.[28] These results suggests that real-time PCR technique can be applied successfully for the diagnosis of dermatophytosis as an alternative to the classical diagnostic methods.[29],[30]

Post-PCR techniques like PCR-ELISA has been reported to be highly sensitive for detection of dermatophytes to the species level from the clinical specimens.[21] Five common species of dermatophytes including T. rubrum, T. interdigitale, T. violaceum, M. canis, and Epidermophyton floccosum was directly detected from the clinical samples by PCR ELISA by amplifying topoisomerase II gene and detection by hybridization using digoxigenin labeled probes.[21] PCR ELISA from onychomycosis cases showed sensitivity of 79.0% and specificity of 85.5% in comparison with conventional method.[12],[21] Sato et al. developed a simple PCR-based DNA microarray [PCR DM] technique to identify 26 reference strains of clinically important fungi. They found that 92% of 106 microscopic positive onychomycosis cases could be identified by PCR DM.[22] Further, PCR reverse line blot assay provide rapid detection and identification of dermatophytes within 24 h from clinical samples.[31] PCR terminal restriction fragment length polymorphism is simple and reliable for routine diagnosis of etiological agent identification from onychomycosis. This approach could detect fungi in 74% [162/219] of culture negative samples and can be performed with the minimum amount of nail sample [20100 mg].[32]

In view of the morphological similarities or due to less or rare production of macroconidia and/or microconidia most of the dermatophytes remains unidentified by classical conventional methods. [Table 2] summarizes the complete comparison of molecular diagnostic technique with conventional diagnostic methods. Yüksel and Ilkit, evaluated 64 dermatophyte isolates, including 35 isolates which rarely produce macroconidia, by real-time PCR and showed accurate identification of ten taxonomically distinct dermatophytes with 100% sensitivity.[33] At present, sequencing of the ITS region of rDNA is considered as a gold standard for the identification of the dermatophytes[28],[29],[30],[34] However, ITS-rDNA sequence based identification is limited as it has certain limitation such as small number of nucleotide differences observed in several ecologically and phenotypically separated Trichophyton species.[35],[36],[37],[38] Hence, other genetic markers such as partial beta tubulin, 60S ribosomal protein L10, calmodulin are being attempted to discriminate between closely related species of dermatophytes.[36] Sequencing method also helps in detection of novel species [e.g., Trichophyton bullosum and Microsporum mirabile was described based on sequencing][14]

Matrix-assisted laser desorption ionization time of flight [MALDI-TOF] mass spectrometry is relatively a new technique that is being used in the microbiology laboratory for the rapid identification of the microorganisms. This technique has been evaluated for the identification of dermatophytes and the results have revealed that this technique is accurate and comparable to the sequencing technique. The recent review on MALDI-TOF-based identification of dermatophytosis recommends the use of formic acid extraction step instead of direct mounting to improve the quality of the peaks obtained and its analysis. The major limitation of this technique is inadequate representation of dermatophyte species in reference spectrum libraries of different commercial systems. This limitation can be overcome by the laboratories by developing an in-house reference library for inter- and intra-specific dermatophyte diversity.[16],[39],[66]

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Conflicts of interest

There are no conflicts of interest.

[Table 1], [Table 2]