CLINICAL ISSUES :: MOLECULAR DIAGNOSTICS FOR DERMATOMYCOSIS Method Advantages Microscopy


• Low cost of materials • Fast


• Well-established standard method


Culture


• Low cost of materials • Well-established standard method


Molecular


• Early pathogen-specific treatment possible


• Quick identification of human and ani- mal carriers (avoiding epidemic spread)


• High Sensitivity


• Species identification also possible after therapy start


• Less false-negative results at multiple infections


• Less false-negative results for nail material


Disadvantages • High false-negative rate (30%)


• False-positive results possible due to contaminations / artifacts


• No species differentiation possible (only general fungi detection)


• Manual / subjective analysis


• Experienced labora- tory staff needed


• Long growth time (at least 2 weeks)


• False-negative results possible, since some fungal species do not grow


- Often after therapy starts (e.g. with topic antimycotics)


• Multiple infections will eventually / partially not be detected (fungal overgrowth)


• Very experienced laboratory staff needed for microscopic and macroscopic evaluation


• Manual / subjective analysis


• When microscopic and macroscopic evaluation eventually not clear a bio- chemical differentiation necessary


Table 1. Laboratory diagnostic methods for Dermatomycosis10,14,18,20,26


Primers target components of a fungi’s genome, including the nontranscribed spacer (NTS) regions, metalloprotease gene, chitin synthase (CHS) gene, tubulin gene, promoter region within ribosomal intergenic spacer, transcription elongation factor 1, actin gene, and calmodulin gene.9


Notably, PCR is 20–30% more


specific than culture and is capable of detecting dermatophytes that are difficult to grow in culture.11 Nested PCR aims to increase sensitivity or specificity by using


two sets of primers in two sequential amplification reactions; the product from the first reaction serves as the template for the second run.16


Prior work has found improved sensitivity for


dermatophyte detection with nested PCR using CHS primers compared to conventional PCR.17 Real-time PCR: Real-time PCR provides rapid quantitative results for the identification of DNA or RNA, allowing estimation of a sample’s fungal load.18


Previous work has found that real-time


PCR using a pan-dermatophyte primer targeting CHS is more sensitive (93%) than nested PCR (73.3%) targeting the same gene.19 Additionally, real-time PCR demonstrated higher sensitivity and specificity compared to microscopy with KOH. Real-time PCR showed a sensitivity of 93.3% and specificity of 40%, whereas KOH microscopy had a sensitivity of 80% and specificity of 16%.19 DNA microarray direct detection: PCR-based detection by


DNA microarray enables direct, species-specific detection of fungal pathogens. DNA microarray methodology allows for multiplexing, enabling simultaneous detection of different species. While traditional PCR usually identifies 1 to 6 fungal species at a time,20


sensitivities. While combination of culture and direct microscopy resulted in fairly low sensitivities of 79% and 59% for OM and TP respectively, the combination of culture and DNA microarray increased sensitivity to 87% for OM and 94% for TP. Additionally, combining direct microscopy and DNA microarray achieved sensi- tivity of 92% for OM and 96% for TP.22


Overall, the combination of


PCR-based DNA microarray methodology with direct microscopy yields sensitivity nearing 100% with the added benefits of species identification and a short turnaround time for results. Next-generation sequencing: Next-generation sequencing (NGS) compares nucleotides in a sample against a catalogue library. NGS allows high throughput, rapid turnaround time, and accurate de- tection. Additionally, previous work has found that NGS is signifi- cantly more sensitive than culture for the detection of bacterial and fungal infections (95% versus 60%).23


of several species are available, including T. rubrum, Arthroderma vanbreuseghemii, A. benhamiae, and T. verrucosum.24,25


Advantages of molecular versus conventional methods


one commercially available PCR-based DNA


microarray kit provides direct detection of 50 dermatophytes, with 23 species-specific dermatophytes in addition to six yeasts and molds within approximately three hours.21 Previous work comparing detection methods for OM and tinea pedis (TP) fungal infections found that the highest sensi- tivity among methodologies was PCR-based DNA microarray technology, with a sensitivity of 79% for OM and 91% for TP.22 In contrast, direct microscopy showed sensitivities of 68% for OM and 37% for TP. Fungal culture had the lowest sensitivities, 29% for OM and 44% for TP.22 The same study found that combining conventional diagnostic methods with PCR-based DNA microarray yielded the higher


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Species identification is crucial to appropriately treat patients with dermatomycosis. Differential diagnosis from noninfectious disorders is critical because they require different treatments.26 Additionally, dermatophyte or non-dermatophyte species iden- tification is also essential for determining the optimal treatment and its duration.27,28


Providing an inappropriate treatment can


lead to longer treatments with potential side effects. Furthermore, identification of the fungal species and antifungal susceptibility testing lowers the chance of resistance development.27,29,30 Direct detection with molecular methods enables rapid iden- tification of fungal species, including those that are difficult to grow in culture. Molecular testing offers a faster turnaround time from collection to results, allowing for more expedient initiation of treatment. Molecular methods also have the advantage of improved


sensitivity compared to conventional diagnostics. A recent study by Pospischil, et al. 202226


examined specimens with a positive result by direct microscopy but a negative or contaminated


because NGS requires a genomic library, false negative results can occur if the library does not include the causative pathogen.


• Specific equipment needed • Higher cost than conventional methods


The genome sequences However,


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