SLR - November 2017 - David Liou

Molecular Diagnostic Techniques for Onychomycosis: Validity and Potential Application

Reference: Watanabe S, Ishida, K. Molecular Diagnostic Techniques for Onychomycosis: Validity and Potential Application. Am J Clin Dermatol. 2017 Apr 18: 281.

Scientific Literature Review

Reviewed By: David Liou, DPM
Residency Program: Cambridge Health Alliance, Cambridge, MA

Podiatric Relevance: Onychomycosis is a common pathology seen in the podiatric clinic. For definitive treatment of onychomycosis, diagnosis is required through positive findings by direct microscopy and fungal culture. Despite fungal culture being the gold standard, it is slow and difficult, with low yields. Recent advancement in molecular identification of fungal pathogens through polymerase chain reaction (PCR), and decrease in cost of this method, have allowed for more effective identification of the pathologic organism. This study compares the PCR method with conventional fungal culture for identification of fungi in 149 specimens with onychomycosis diagnosed by direct microscopy. This study aims to validate the CPR method.

Method: Three hundred nail specimens were collected from February 21 to July 6, 2011; 149 were positive by direct microscopy using potassium hydroxide. These specimens were then subjected to the modified real-time PRC assay of Miyajima et al. and fungal culture. Fungal culture involved inoculation onto Sabouraud dextrose CG agar containing chloramphenicol and gentamicin and intubation at 27 oC. PCR of specimens involved extraction of DNA using the QIAamp DNA Micro Kit and real-time PRC using StepOnePlus system with custom primers. In both methods, fungi were identified at the genus level. This study further compares the time to obtain definite results and whether there is co-infection of a single specimen with two different species of fungi.

Of the 149 specimens, 142 (95.3 percent) were positive for Trichophyton rubrum or Trichophyton mentagrophytes (including Trichophyton interdigitales) by PCR. Compared to conventional fungal culture, only 104 (69.8 percent) were positive. No specimen was negative by PCR but positive by culture. The PCR method was able to identify T. rubrum in 11 of the 17 cultured specimens that fungal culture was not able to identify either T. rubrum or T. mentagrophytes. Furthermore, real-time PCR allows for definitive result with identification of the fungal pathology in less than two days, compared to fungal culture, which requires up to 28 days. No specimen was positive for both T. rubrum and T. mentagrophytes by either PRC or culture.

Conclusion: Despite recent advancements in the treatment of onychomycosis, definitive diagnosis of onychomycosis remains basic and unreliable. Real-time PCR achieved higher sensitivity and higher dermatophyte identification rate. This advantage is attributed to PCR requiring a small amount and even low viability of the specimen. PCR also allows for more rapid identification of causative fungi (<2 days vs. 28 days). Despite the advantages of the PCR technique, the cost of PCR has been a major hindrance. At present, the PCR test is $100/sample. However, with maturation of this technology, the cost is likely to reduce to around $50/sample. At this price point, PCR will be comparable to fungal culture in terms of cost and will offer better diagnosis of onychomycosis. This will allow for more accurate and rapid diagnosis of onychomycosis and will improve the treatment of this pathology.

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