Detection of Microsporum Canis and Trichophyton mentagrophytes PDF

Received: 8 November 2021 | Accepted: 16 June 2022 DOI: 10.1111/vde.13111 ORIGINAL ARTICLE Detection of Microsporum canis and Trichophyton mentagrophytes by loop-­mediated isothermal amplification (LAMP) and real-­time quantitative PCR (qPCR) methods Hamit Kaan Müştak...

Received: 8 November 2021 | Accepted: 16 June 2022 DOI: 10.1111/vde.13111 ORIGINAL ARTICLE Detection of Microsporum canis and Trichophyton mentagrophytes by loop-­mediated isothermal amplification (LAMP) and real-­time quantitative PCR (qPCR) methods Hamit Kaan Müştak | Gültekin Ünal | İnci Başak Müştak Ankara University Faculty of Veterinary Medicine, Department of Microbiology, Abstract Ankara, Turkey Background: Dermatophytes are infectious zoonotic fungal agents that are Correspondence common in animals worldwide. A new loop-­mediated isothermal amplifi- İnci Başak Müştak, Ankara University Faculty of Veterinary Medicine, cation (LAMP) method and quantitative (q)PCR can be used for identifying Department of Microbiology, Dışkapı, Ankara 06110, Turkey. these agents. Both methods have high specificity and sensitivity, and are Email: [email protected] simple and quick to use. Funding information Hypothesis/Objectives: To develop a LAMP and a rapid multiplex qPCR Ankara University Scientific Research method for detecting Microsporum canis and Trichophyton mentagrophytes, Projects Coordination Unit, Grant/Award Number: 19B0239001 which are the most common fungal species isolated from cats and dogs. Material and Methods: Both methods targeted the CHS-­1 gene. Their spec- ificity and sensitivity were tested using 64 M. canis and 44 T. mentagrophytes field strains. The validation of the methods was performed using 250 clinical fungal-­positive hair samples. Results: The specificity value was 100% for both methods. For LAMP, the sensitivity value was 96.9% for M. canis and 93.2% for T. mentagrophytes. For qPCR, the sensitivity values were 98.4% for M. canis and 97.7% for T. mentagrophytes. Similar specificity and sensitivity results were obtained from the validation study using 250 clinical hair samples. LAMP and multiplex qPCR took 30 and 45 min (respectively) for both targets. The limit of detec- tion (LOD) assays for both targets were 10 and 1 spore/mL for LAMP and multiplex qPCR, respectively. Conclusion: These findings demonstrate that the LAMP and multiplex qPCR methods targeting CHS-­1 gene developed in this study can be used both for point-­of-­care testing and in the laboratory for detecting M. canis and T. men- tagrophytes with high specificity and sensitivity with an internal control. INTRODUCTION species most frequently isolated from animals are M. canis (usually cat, dog), Nannizzia gypsea (dog), Dermatophytes (phylum Ascomycota, class Trichophyton verrucosum (cattle, small ruminant), Euascomycetes, order Onygenales and family M. equinum (equidae), T. mentagrophytes (cat, dog) Arthrodermataceae) are divided into Microsporum, and T. mentagrophytes var. erinacei (hunting dogs). Trichophyton and Epidermophyton genera accord- Microsporum canis 2,3,4 and Trichophyton menta- ing to their asexual characteristics. These agents grophytes 2,5,6 are the most commonly isolated infect body parts such as skin, hair, feathers, dermatophytes from cats and dogs with zoonotic horns, hooves, nails and claws.1 The dermatophyte importance.1– ­3 516 | © 2022 ESVD and ACVD. wileyonlinelibrary.com/journal/vde Veterinary Dermatology. 2022;33:516–522. DETECTION OF DERMATOPHYTES USING LAMP AND qPCR Veterinary Dermatology    | 517 Clinical diagnosis of T. mentagrophytes and M. canis Escherichia coli, Salmonella Enteritidis, Pasteurella is performed from skin lesions while laboratory diagno- multocida, Listeria monocytogenes, Staphylococcus sis is performed using direct microscopic hair examina- aureus and Trueperella pyogenes. tion with 10%–­20% potassium hydroxide (KOH) and For the validation studies, the following 250 fungal-­ mycological culture.2 Classical mycological culturing, positive hair samples were used: 49 M. canis, 33 which is accepted as the reference method for identi- T. mentagrophytes, 48 Aspergillus spp., 27 Penicillium fication, has five main disadvantages: (i) some derma- spp., 21 Alternaria spp., 19 Mucor spp., 16 Candida tophytes do not grow on media; (ii) diagnosis relies on spp., nine Fusarium spp., nine Rhizopus spp., eight subjective macroscopic and microscopic evaluation; (iii) Blastomyces spp., six Microsporum spp. and five diagnosis requires well-­trained personnel; (iv) misdiag- Sporotrix spp. All strains were initially isolated from cats nosis is possible while evaluating certain transitional and dogs, identified by mycological culture method and forms in fungi; and (v) most importantly, diagnosis takes confirmed by ITS region sequencing. 10–­14 days.7 Given these disadvantages, numerous studies have investigated the use of molecular methods for rapid DNA extraction detection of dermatophytes. These include conven- tional PCR,8 quantitative (q)PCR,9 nested-­PCR10 and DNA extraction of the reference fungal strains was per- sequence analysis of the internal transcribed spacer formed using Quick-­DNA Fungal/Bacterial Miniprep kit (ITS) region.11 Molecular methods can identify fungal (catalogue no. D6005, Zymo Research Corp.). Genomic species as well as detect virulence and antifungal resis- DNA extraction of bacterial strains was performed using tance genes.12 theBiospeedy High Concentration DNA Isolation Kit Many gene regions varying in specificity and sen- from Bacterial Cultures (Bioeksen R&D Technologies sitivity have been used in the molecular diagnosis of Ltd.). All procedures were performed according to the T. mentagrophytes and M. canis, most frequently 18S, respective manufacturer's instructions. 5.8S, 28S rDNA, ITS1, ITS2 and the intergenic spacer To extract DNA from hair samples, a modified method (IGS) gene regions.13 Other regions include partial α-­ based on Kolukırık et al.18 and Zhang et al.19 was used. tubulin II (TUB2), γ-­actin (ACT ), translation elongation At least five hair samples were added to 200 μl solution factor 1-­α (TEF1α), RNA polymerase II (partial RPB2), (6 mg/mL BSA, 20 mg/mL PEG 400, 0.25% Tween 20, fungal-­specific translation elongation factor 3 (TEF3), 15 mM peptide complex, 20 mM Tris–­HCl pH 8.0), vor- small ribosomal protein required for t-­ RNA binding, texed for 10 min, then incubated at 85°C for 15 min. DNA topoisomerase I (TOPI), phosphoglycerate kinase The purity (260/280 nm) and concentration of the (PGK ), hypothetical protein (LNS2) and the alternative extracted DNA were measured by Nanodrop ND-­1000 region of TEF1α.14 Chitin synthases (CHSs) are enzymes (Thermo Fisher Scientific) and stored at −20°C until used. used in the biosynthesis of chitin, an important struc- tural component of fungal cell walls. CHS-­1 gene is a member of the CHS gene family which is conserved Oligonucleotides among fungal species and that best discriminates be- tween T. mentagrophytes and M. canis.15–­17 The chitin synthase-­1 (CHS-­1) gene, a conserved gene In this study, we developed two methods for detect- region of 3592 bp encoding a cellulose-­like polysaccha- ing T. mentagrophytes and M. canis: a LAMP method ride in the cell wall, was used as a target gene to de- for on-­site diagnosis and a multiplex qPCR method for tect M. canis and T. mentagrophytes. Sequences of the laboratory conditions. CHS-­1 gene of each target organism were obtained from GenBank (NCBI). The resulting sequences were aligned with Clustal-­Omega, and conserved regions found for MATERIALS AND METHODS each species were identified in the aligned sequences. Suitable primers for LAMP, and primers and Samples and reference strains probes for multiplex qPCR assays were designed with PrimerExplorer v5 and Primer3 v0.4.0 software, Microsporum canis (CBS 566.80) and T. mentagro- respectively. phytes (CBS 106.67) reference strains were used as Amplification of the target gene was observed in the positive controls for the optimization and validation FAM, ROX and HEX channels for M. canis, T. mentag- studies of the developed methods. rophytes and the internal control, respectively. For in- To demonstrate that the developed methods can ternal control of the multiplex qPCR method, primers only detect target organisms, selectivity studies were and a probe amplifying the glyceraldehyde 3-­phosphate performed with the following fungal and bacterial dehydrogenase (GAPDH) gene were used according to strains: for fungi, Trichophyton tonsurans (CBS 112818), Radonic et al.20 Trichophyton rubrum (CBS 118892), Nannizzia gypseum (CBS 118893), Aspergillus niger (CBS 513.88), Candida albicans (CBS 562), Rhizopus oryzae (CBS 257.28), Optimization of the LAMP method Histoplasma capsulatum (CBS 136.72), Fusarium sola- nii (CBS 490.63), Alternaria alternata (CBS 916.96) and Bst DNA polymerase 2.0 Warmstart (New England Malessezia pachydermatitis (CBS 1879); for bacteria, BioLabs Japan Inc.) enzyme was used for optimization 518 |    Veterinary Dermatology MÜŞTAK et al. and hydroxy naphthol blue (HNB) (Sigma-­Aldrich Corp.) (Merck Millipore) filters so that only hyphae remained was used to visualize the DNA product.21 in the filter and a mixture consisting only of spores Reaction content, primer concentrations and analy- was prepared. The suspension then was transferred sis duration were decided based on the Bst polymerase to Sabouraud Dextrose Broth (CM0147; Oxoid) sup- enzyme, as reported by New England BioLabs (https:// plemented with chloramphenicol and incubated over- inter​natio​nal.neb.com). The LAMP reaction mix of night at 30°C. Concentrations were adjusted to 1 × 10 6 25 μL consisted of 6 mg/mL BSA, 20 mg/mL PEG 400, spores/mL according to the McFarland turbidimetric 0.25% Tween 20, 15 mM peptide complex, 20 mM method. Ten-­fold serial dilutions were prepared from Tris–­HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM these samples. Hair samples known to be negative for dNTP mix, 0.1 U Bst DNA polymerase, 120 μM HNB, M. canis (n = 7) and T. mentagrophytes (n = 7) were primers at different concentrations (40 μM FIP, 40 μM spiked with each dilution of these spore samples and BIP, 5 μM F3, 5 μM B3, 10 μM Loop-­F, 10 μM Loop-­B1) DNA extraction was performed. The limit of detec- and 10 μl (50 ng/μL) of template nucleic acid to final tion studies for both LAMP and multiplex qPCR tested volume. The amplification conditions of the primer sets with duplicates on different days. Nuclease-­free water were optimized at 65°C for 60 min in line with the en- (DEPC-­treated, molecular biology grade) was used as zyme manufacturer's instructions. the negative control in all reactions. The results were evaluated in 60 min using a QuantStudio 5 instrument (Applied Biosystems/ Thermo Fisher Scientific) by selecting the FAM chan- Specificity and sensitivity of the assays nel. The results were recorded as positive if there was a sigmoidal curve and a colour change in the reac- Specificity and sensitivity were tested in a blinded man- tion mix from purple to sky blue, detectable with the ner using DNA samples of 64 M. canis and 44 T. men- naked eye; results were recorded as negative if there tagrophytes field strains isolated from cats and dogs. was no sigmoidal curve or detectable colour change. The conventional mycological method was used as Nuclease-­free water (DEPC-­treated, molecular biology the ‘gold standard’ for comparing the developed meth- grade) was used as the negative control in all reactions. ods. Sensitivity and specificity were calculated using the following formulas, respectively: a/(a + c) × 100; d/ (b + d) × 100. Optimization of the multiplex qPCR method RESULTS The multiplex qPCR mix of 20 μL consisted of 6 mg/ mL bovine serum albumin (BSA), 11 mg/mL betaine, LAMP and multiplex qPCR 20 mg/mL PEG 400, 0.25% Tween 20, 20 mM Tris–­ oligonucleotides HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTP mix, 1 U Biospeedy Hot-­Start Mutant Taq DNA Oligonucleotides for the LAMP assay were designed Polymerase (Bioeksen R&D Technologies Ltd.), 0.1 μM by selecting specific regions within the CHS-­1 gene for of each primer (M. canis, T. mentagrophytes and inter- M. canis and T. mentagrophytes with lengths of 619 nal control) and 5 μL (50 ng/μL) of template nucleic acid and 504 bp, respectively (Table 1). The primers and to final volume. The amplification conditions for the multiplex qPCR were 95°C for 5 min followed by 40 cy- TA B L E 1 Primer sequences used in loop-­mediated isothermal cles of 95°C for 10 s and finally 55°C for 50 s. The mul- amplification (LAMP) for identifying Microsporum canis (MC) and tiplex qPCR assay was performed on a QuantStudio 5 Trichophyton mentagrophytes (TM) instrument (Applied Biosystems). Amplification of the Primer Sequence (5′–­3′) target gene was observed in the FAM, ROX and HEX channels for M. canis, T. mentagrophytes and internal MC F3 GCATTGCCAAACAGCAGGT control, respectively. The results were recorded as B3 AGGATGCGGCCGAAGG negative if there was no sigmoidal curve and Cq > 35, FIP GCCCTTGACCTCCATGCCGATTTTAAGG​ and positive if there was sigmoidal curve with Cq < 35. ACGTCACCGCCCA BIP GCCCGTCCAGCTCCTCTTCTTTTACCGG​ TGCGAGTTGATCT Limit of detection (LOD) in artificially L-­F CTGGGTGGTGTACTCGTAGAT spiked samples L-­B TGCCTCAAGGAGAAGAACCAG TM F3 AGCAGCAAGACATGGGGTA To detect M. canis CBS 566.80 and T. mentagrophytes B3 TAACCTGGGTGCCCTTGA CBS 106.67 standard strains, the methods based on FIP GAACAGCTCTCGTACGCGGATTTTTTAG​ De Hoog et al.22 and Aberkane et al.23 were revised. CCTGGAAGAAGATTGTCG For this purpose, 2–­4 cm3 of the reference strains colo- BIP GATGGCATTGCCAAACAGCAGGTTTTCC​ nies cultured on Sabouraud Dextrose Agar (CM0041B, ATGCCTATCTGGGTGGTA Oxoid) were taken and mixed in a sterile 1.5-­ mL L-­F CGACCGTCTGAGACGATACAAA Eppendorf tube containing phosphate-­buffered saline L-­B TCAACGGTAAAGACGTCACTGC (PBS). This suspension was passed through 11 μm DETECTION OF DERMATOPHYTES USING LAMP AND qPCR Veterinary Dermatology    | 519 probes for the multiplex qPCR assay were designed by Optimization of the assays selecting 421 and 391 bp regions of the CHS-­1 gene specific for M. canis and T. mentagrophytes, respec- Table 3 presents the optimized parameters for LAMP tively (Table 2). and multiplex qPCR. The designed oligonucleotides of the LAMP test were positive at 65°C at the end of 30 min for both tar- gets. Figure 1 shows the results of the multiplex qPCR TA B L E 2 Primer and probe sequences used in multiplex assay for all targets. As expected, the animal GAPDH quantitative PCR for identifying Microsporum canis (qMC) and DNA, which was used as the internal control, showed Trichophyton mentagrophytes (qTM) a sigmoidal curve in its channel for all targets. That is, Primers Sequence (5′–­3′) there was no inhibition of the reaction. qMC F GCATTGCCAAACAGCAGGT R AGGATGCGGCCGAAGG LOD, specificity and Probe TGGAGGTCAAGGGCACCCAG sensitivity of the assays qTM F CCAGGTTATTCTCAAGCCGC R GGTATATACTTCGGCCGCCT The multiplex qPCR assay LODs for M. canis and T. men- Probe GGTCCAGCTCCTCTTCTGTC tagrophytes were 58.1 ng/μL and 60.44 ng/μL DNA (re- spectively) from the first dilution (1 × 10 6 spores/mL) of the reference strains. The LOD of the LAMP test was 10 spores/mL for both agents (Figures 2 and 3). TA B L E 3 Reaction content in loop-­mediated isothermal After applying multiplex qPCR to all dilutions in amplification (LAMP) and multiplex quantitative (q)PCR for duplicate, starting at 10,4 a gradual decrease in the Microsporum canis and Trichophyton mentagrophytes Cq values was obtained for each target as DNA con- Volume (μL) centrations decreased (Figures 4 and 5). The LOD for the multiplex qPCR assay was 1 spore/mL for both Reagent LAMP Multiplex qPCR targets. 2× master mix -­ 10 The specificities of multiplex qPCR and LAMP 10× primer mix 2.5 3 were 100% for both targets while the sensitivities of LAMP and multiplex qPCR were 96.9% (62 of 64) and 10× buffer 2.5 -­ 98.4%(63 of 64) for M. canis, and 93.2% (41 of 44) and BST 2.0 (NEB) 1 -­ 97.7% (43 of 44) for T. mentagrophytes, respectively. 100 nM MgSO 4 1.5 -­ 10 nM dNTP 3.5 -­ HNB 1 -­ Validation of the assays with Water 3 2 clinical samples DNA 10 5 Of the 250 hair samples used in a blinded manner, Total volume 25 20 168 hair samples negative for M. canis and T. menta- Abbreviations: HNB, hydroxy naphthol blue; NEB, New England BioLabs. grophytes, also were negative for both targets using F I G U R E 1 Microsporum canis (purple curve) and Trichophyton mentagrophytes (red curve) multiplex quantitative PCR results, showing positive results for both targets (Cq < 35). The green sigmoidal curve refers to the internal control 520 |    Veterinary Dermatology MÜŞTAK et al. F I G U R E 2 Limit of detection (LOD) result of Microsporum canis loop-­mediated isothermal amplification (LAMP) method. 1–­7, DNA dilutions of 10 6 –­10 0 (respectively) of M. canis. Colours: Positive result, sky blue; negative result, purple. NTC, negative control F I G U R E 3 Limit of detection (LOD) result of Trichophyton mentagrophytes loop-­mediated isothermal amplification (LAMP) method. 1–­ 7, DNA dilutions of 10 6 –­10 0 (respectively) of Trichophyton mentagrophytes. Colours: Positive result, sky blue; negative result, purple. NTC, negative control F I G U R E 4 Sigmoidal curves of Microsporum canis spore dilutions tested in the FAM channel. Curve with the highest Cq value (top curve) has a dilution rate of 10 4 and curve with the lowest Cq value (bottom curve) has a dilution rate of 10 0 both LAMP and multiplex qPCR. Neither method gave DISCUSSION false-­ positive results for nontarget fungi samples. However, LAMP failed to detect two of 49 M. canis Microsporum canis and T. mentagrophytes are the fun- and two of 33 T. mentagrophytes samples. Thus, gal dermatophytes most frequently isolated from cats the selectivities of the LAMP method were 95.9% and dogs. Because they can cause serious zoonotic in- and 93.9% for M. canis and T. mentagrophytes, fections when spreading to humans, they have implica- respectively. tions for public health. In this study, we developed the By contrast, multiplex qPCR correctly identified all first LAMP method for quickly identifying M. canis and 49 M. canis and 32 of 33 T. mentagrophytes samples. T. mentagrophytes, and a multiplex qPCR method to Thus, the selectivities of the multiplex qPCR method detect both agents simultaneously. were 100% and 96.9% for M. canis and T. mentagro- As for other fungal agents, mycological culturing is phytes, respectively. the gold standard method for detecting M. canis and DETECTION OF DERMATOPHYTES USING LAMP AND qPCR Veterinary Dermatology    | 521 F I G U R E 5 Sigmoidal curves of Trichophyton mentagrophytes spore dilutions tested in the ROX channel. Curve with the highest Cq value (top curve) has a dilution rate of 10 4 and curve with the lowest Cq value (bottom curve) has a dilution rate of 10 0 T. mentagrophytes. However, this method takes about AU T H O R C O N T R I B U T I O N S seven to 21 days to produce results. The two new Hamit Kaan Müştak: Conceptualization; data curation; methods developed here offer significant time savings formal analysis; investigation; methodology; project while providing objective and accurate results: 30 min administration; writing –­original draft; writing –­review for both targets using LAMP and 45 min using multi- and editing. Gültekin Ünal: Data curation; formal anal- plex qPCR. ysis; investigation; methodology; writing –­review and In comparison with the gold standard method, editing. İnci Başak Müştak: Data curation; supervi- both new methods had 100% specificity for both tar- sion; writing –­review and editing. gets. Ohst et al.24 showed that a modular singleplex qRT-­PCR assay for detecting the most common der- F U N D I N G I N F O R M AT I O N matophytes had greater specificity (88.2%) and sen- This research was supported by Ankara University sitivity (60.7%) than both microscopy and cultures. In Scientific Research Projects Coordination Unit, project our study, the sensitivity results were >97% for both no. 19B0239001, 2020. targets using qPCR and > 93% for LAMP. These results were further supported by the validation study using CONFLICT OF INTEREST field samples, which produced similar specificity and None declared. sensitivity values. Garg et al.25 reported that nested PCR targeting ORCID the CHS1 gene may be considered the gold standard Hamit Kaan Müştak https://orcid. for detection of dermatophytes in patients. They also org/0000-0002-3694-1959 concluded that the nested-­PCR technique is not only Gültekin Ünal https://orcid. rapid, but also simple and cheap in comparison with org/0000-0002-8996-7028 other molecular methods for detection of dermato- I˙nci Başak Müştak https://orcid. phytes. However, both methods developed in our study org/0000-0001-9180-5768 are cheaper and more rapid than the other PCR-­based techniques. REFERENCES 1. Samanta I. 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Evolution Müştak İB Detection of Microsporum canis and and functional insights of different ancestral orthologous Trichophyton mentagrophytes by loop-mediated clades of chitin synthase genes in the fungal tree of life. Front isothermal amplification (LAMP) and real-time Plant Sci. 2016;7:37. quantitative PCR (qPCR) methods. Vet Dermatol. 17. Emam SM, El-­ salam OHA. Real-­ time PCR: a rapid and 2022;33:516–­522. https://doi.org/10.1111/ sensitive method for diagnosis of dermatophyte induced onychomycosis, a comparative study. Alexandria J Med. vde.13111 2016;52:83– ­9 0. Résumé Contexte: Les dermatophytes sont des agents fongiques zoonotiques infectieux qui sont courants chez l’animal dans le monde entier. Une nouvelle méthode d'amplification isotherme médiée par les boucles (LAMP) et une PCR(q) quantitative peuvent être utilisées pour identifier ces agents. Les deux méthodes ont une spécificité et une sensibilité élevées, et sont simples et rapides à utiliser. Hypothèses/Objectifs: Développer une LAMP et une méthode PCRq multiplexe rapide pour détecter Microsporum canis et Trichophyton mentagrophytes, qui sont les espèces fongiques les plus courantes isolées chez les chats et les chiens. Matériel et méthodes: Les deux méthodes ciblaient le gène CHS-­1. Leur spécificité et leur sensibilité ont été testées à l'aide de 64 souches de M. canis et 44 de T. mentagrophytes. La validation des méthodes a été réalisée à l'aide de 250 échantillons cliniques de poils fongiques positifs. Résultats: La valeur de spécificité était de 100 % pour les deux méthodes. Pour LAMP, la valeur de sensibilité était de 96,9 % pour M. canis et de 93,2 % pour T. mentagrophytes. Pour la PCRq, les valeurs de sensibilité étaient de 98,4 % pour M. canis et de 97,7 % pour T. mentagrophytes. Des résultats de spécificité et de sensibilité similaires ont été obtenus à partir de l'étude de validation utilisant 250 échantillons cliniques de poils. LAMP et PCRq mul- tiplex ont pris 30 min et 45 min (respectivement) pour les deux cibles. La limite de détection (LOD) pour les deux cibles était respectivement de 10 spores/mL et 1 spore/mL pour LAMP et multiplex PCRq. Conclusion: Ces résultats démontrent que les méthodes LAMP et PCRq multiplex ciblant le gène CHS-­1 dével- oppées dans cette étude peuvent être utilisées à la fois pour les tests au chevet du patient et en laboratoire pour détecter M. canis et T. mentagrophytes avec une spécificité et une sensibilité élevées avec un contrôle interne. Resumen Introducción: los dermatofitos son agentes fúngicos zoonóticos infecciosos que son comunes en los animales de todo el mundo. Se puede utilizar un nuevo método de amplificación isotérmica mediada por bucle (LAMP) y PCR cuantitativa (q)PCR para identificar estos agentes. Ambos métodos tienen alta especificidad y sensibilidad, y son simples y rápidos de usar. Hipótesis/Objetivos: Desarrollar un método LAMP y un método qPCR multiplex rápido para la detección de Microsporum canis y Trichophyton mentagrophytes, que son las especies fúngicas más comunes aisladas de gatos y perros. Material y Métodos: Ambos métodos se dirigieron al gen CHS-­1. Su especificidad y sensibilidad se probaron utilizando 64 cepas de campo de M. canis y 44 de T. mentagrophytes. La validación de los métodos se realizó utilizando 250 muestras clínicas de pelos positivas para hongos. Resultados: La especificidad fue del 100% para ambos métodos. Para LAMP, el valor de sensibilidad fue del 96,9 % para M. canis y del 93,2 % para T. mentagrophytes. Para qPCR, los valores de sensibilidad fueron del 98,4 % para M. canis y del 97,7 % para T. mentagrophytes. Se obtuvieron resultados similares de especificidad y sensibi- lidad del estudio de validación utilizando 250 muestras clínicas de pelol. LAMP y multiplex qPCR tardaron 30 min y 45 min (respectivamente) para ambos objetivos. Los ensayos de límite de detección (LOD) para ambos objetivos fueron 10 esporas/mL y 1 espora/mL para LAMP y multiplex qPCR, respectivamente. Conclusión: estos hallazgos demuestran que los métodos LAMP y multiplex qPCR dirigidos al gen CHS-­1 desar- rollados en este estudio pueden usarse tanto para pruebas en primer punto de atención como en el laboratorio para detectar M. canis y T. mentagrophytes con alta especificidad y sensibilidad con un control interno. Zusammenfassung Hintergrund: Dermatophyten sind infektiöse zoonotische Pilzelemente, die bei Tieren weltweit häufig vorkom- men. Eine neue Loop-­mediierte isothermale Amplifizierungsmethode (LAMP) sowie eine quantitative (q) PCR kön- nen bei der Identifizierung dieser Elemente eingesetzt werden. Beide Methoden haben eine hohe Spezifität und Sensitivität und sind einfach und rasch anzuwenden. Hypothese/Ziele: Die Entwicklung einer LAMP und einer rapiden Multiplex qPCR Methode, um Microsporum canis und Trichophyton mentagrophytes zu finden, die die am häufigsten bei Katzen und Hunden isolierte Pilzspezies darstellen. Material und Methoden: Beide Methoden zielten auf das CHS-­1 Gen ab. Ihre Spezifität und Sensibilität wurden mittels 64 M. canis und 44 T. mentagrophytes Feldstämmen getestet. Die Validierung dieser Methoden wurde anhand von 250 klinischen Pilz-­positiven Haarproben durchgeführt. Ergebnisse: Die Spezifität lag bei beiden Methoden bei 100%. Für LAMP lag die Sensitivität bei 96,9% für M. canis und 93,2% für T. mentagrophytes. Für die qPCR lagen die Sensibilitätswerte für M. canis bei 98,4% und für T. mentagrophytes bei 97,7%. Ähnliche Ergebnisse in Bezug auf Spezifität und Sensibilität wurden bei der Validierungsstudie anhand der 250 klinischen Haarproben gefunden. LAMP und Multiplex qPCR benötigten 30 min beziehungsweise 45 min für beide Targets. Die niedrigste Nachweisgrenze (LOD) lag bei beiden Targets bei 10 Sporen/mL und 1 Spore/mL für LAMP beziehungsweise für multiplex qPCR. Schlussfolgerung: Diese Ergebnisse zeigen, dass die LAMP und die Multiplex qPCR Methoden, die auf das CHS-­ 1 Gen abzielen, welche in dieser Studie entwickelt wurden, sowohl für Point-­of care Untersuchungen sowie im Labor zur Identifizierung von M. canis und T. mentagrophytes mit einer hohen Spezifität und Sensitivität mit einer internen Kontrolle eingesetzt werden können. 要約 背景: 皮膚糸状菌は人獣共通感染症の原因となる真菌であり，世界中の動物に広く感染している。皮膚糸状菌の同定には， 新しいLAMP法および定量PCR法を用いることができる。両手法とも高い特異度および感度を有し、簡便かつ迅速に使用 できる。 仮説/目的: 本研究の目的は、犬猫から最も多く分離される真菌であるMicrosporum canisとTrichophyton mentagro- phytes​を検出するためのLAMP法および迅速なmultiplex qPCR法を開発することであった。 材料と方法: 両手法ともCHS-­1遺伝子を標的とした。M. canis 64株およびT. mentagrophytes 44株の野外株を用いて， 両者の特異度および感度を検証した。また、臨床真菌陽性毛250検体を用いて、両手法の妥当性を検証した。 結果: 特異度は両法とも100%であった。LAMP法では、M. canisに対して96.9%の感度、T. mentagrophytesに対し て93.2%の感度を示した。また、qPCR法では、M. canisに対して98.4%、T. mentagrophytesに対して97.7%の感度 が得られた。また，臨床毛250検体を用いた検証試験においても、同様の特異度、感度が得られた。LAMP法では30 分、multiplex qPCR法では45分という短い時間で両ターゲットの検出が可能であった。また、検出限界はLAMP法で10 spore/mL、multiplex法では1 spore/mLであった。 結論: 本研究で開発したCHS-­1遺伝子を標的としたLAMP法およびmultiplex qPCR法は、内部コントロールによる高い 特異性および感度でM. canisおよびT. mentagrophytesを検出するため、ポイントオブケア検査およびラボの両方で使用 できることが示された。 摘要 背景: 皮肤癣菌是世界范围内动物常见的传染性人畜共患真菌。一种新的环介导等温扩增 (LAMP) 方法和定量 (q)PCR 可用于鉴定这些试剂。两种方法均具有较高的特异性和敏感性，且使用简单、快捷。 假设/目的: 开发用于检测犬小孢子菌和须毛癣菌(从猫和犬中分离的最常见真菌菌种)的 LAMP 和快速多重 qPCR 方法。 材料和方法: 两种方法均靶向 CHS-­1 基因。采用64株犬小孢子菌和44株须癣毛癣菌田间菌株检测其特异性和敏感性。 采用250例临床真菌阳性毛发样本进行方法的验证。 结果: 两种方法的特异性值均为100%。对于LAMP，犬小孢子菌的灵敏度值为96.9%，须癣毛癣菌的灵敏度值为93.2% 。对于qPCR，犬小孢子菌的灵敏度值为98.4%，须毛癣菌的灵敏度值为97.7%。使用250份临床毛发样本的验证研究获 得了相似的特异性和灵敏度结果。两个靶标的 LAMP 和多重 qPCR 分别需要 30 min 和45 min。对于 LAMP 和多重 qPCR，两种靶标的检测限 (LOD) 试验分别为10个孢子/mL 和1个孢子/mL。 结论: 这些发现证明，有内部对照组的情况下，本研究开发的靶向 CHS-­1 基因的 LAMP 和多重 qPCR 方法既可用于临床 检测，也可用于实验室检测犬小孢子菌和须癣毛癣菌，具有较高的特异性和敏感性。 Resumo Contexto: Dermatófitos são fungos causadores de infecções zoonóticas comuns em animais mundialmente. Um novo método de amplificação isotérmica mediada por loop (LAMP) e PCR quantitativa (qPCR) podem ser utilizados para identificar esses agentes. Ambos os métodos têm alta especificidade e sensibilidade, e são simples e rápidos para utilizar. Hipótese/Objetivos: Desenvolver um método de LAMP e qPCR multiplex rápida para detectar Microsporum canis e Trichophyton mentagrophytes, que são as espécies fúngicas mais comumente isoladas de cães e gatos. Material e métodos: Ambos os métodos foram focados no gene CHS-­1. A sua sensibilidade e especificidade foram testadas utilizando 64 amostras de campo de M. canis e 44 de T. mentagrophytes. A validação foi realizada usando 250 amostras de pelo positivas para fungos. Resultados: O valor da especificidade foi 100% para ambos os métodos. Para o LAMP, o valor da sensibilidade foi 96,9% para M. canis e 93,25 para T. mentagrophytes. Para o qPCR, o valor de sensibilidade foi de 98,4% para M.canis e de 97,7% para T. mentagrophytes. Valores de sensibilidade e especificidade similares foram obtidos no estudo de validação utilizando 250 amostras de pelo positivas para fungos. LAMP e qPCR multiplex levaram 30 min e 45 min (respectivamente) para ambos os alvos. Os ensaios de limite de detecção (LOD) para ambos os alvos foram 10 esporos/mL e 1 esporo/mL para LAMP e qPCR multiplex, respectivamente. Conclusão: Esses achados demonstram que os métodos LAMP e qPCR multiplex visando o gene CHS-­1 desen- volvidos neste estudo podem ser usados tanto para testes no local do atendimento quanto no laboratório para detectar M. canis e T. mentagrophytes com alta especificidade e sensibilidade com controle interno. Copyright of Veterinary Dermatology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.