Molecular Profile of Trichophyton mentagrophytes and Microsporum canis based on PCR-RFLP of Internal Transcribed Spacer

Dwi Endrawati, Eni Kusumaningtyas

Abstract

Trichophyton mentagrophytes and Microsporum canis are dermatophytes fungi which commonly infect animal and human. Conventional and molecular methods were used for identification of the fungus. The region of internal transcribed spacer (ITS) has a high probability for fungal identification. PCR-RFLP was reported as a useful method to differentiate dermatophytes fungi. The objective of the study was to compare molecular profile of T. mentagrophytes and M. canis based on the result of ITS fragment digestion using Dde I, Hinf I and Mva I. The molds were isolated from skin scrapping of 18 animals which showed dermatophytosis lesion. The isolated molds were grown on agar plate for 14 days of incubation at 37oC and then identified based on macro and microscopic morphologies. Amplification of chitin synthase gene was used for confirmation and separation of dermatophytes from other fungi. ITS fragment was amplified and then digested using restriction enzymes Dde I, Hinf I and Mva I. The result showed that digestion products from ITS fragment of T. mentagrophytes and M. canis were different. The fragment 159 bp from Dde I, 374 bp from Hinf I and 89 bp from Mva I were present in T. mentagrophytes but absent in M. canis.   Based on these results, specific RFLP profile of digestion ITS region by Dde I, Hinf I and Mva I can be used as a specific marker for species of dermatophytes fungi.

Keywords

Dermatophytes, Internal transcribed spacer, PCR RFL

References

Aala F. 2012. Conventional and molecular characterization of Trichophyton rubrum. African J Microbiol Res. 6:6502–6516.

Abdel-Fatah B, Ahmad M, Moharam M, El-Din A, Moubasher AH A-RM. 2013. Genetic relationships and isozyme profile of dermatophytes and Candida strain from Egypt and Libya. Am J Biochem Mol Biol. 3:271–292.

Aneke CI, Otranto D, Cafarchia C. 2018. Therapy and antifungal susceptibility profile of Microsporum canis. J Fungi. 4:1–14.

Begerow D, Nilsson H, Unterseher M MW. 2010. Current state and perspectives of fungal DNA barcoding and rapid identification procedures. Appl Microbiol Biotechnol. 87:99–108.

Brillowska-Dabrowska A, MichaŁek E, Saunte DML, Sogaard Nielsen S, Arendrup MC. 2013. PCR test for Microsporum canis identification. Med Mycol. 51:576–579.

Cafarchia C, Gasser RB, Figueredo LA, Weigl S, Danesi P, Capelli G, Otranto D. 2013. An improved molecular diagnostic assay for canine and feline dermatophytosis. Med Mycol. 51:136–143.

Dhieb C, Essghaier B, El Euch D, Sadfi-Zouaoui N. 2014. Phenotypical and molecular characterization of Microsporum canis strains in North-Tunisia. Polish J Microbiol. 63:307–315.

Elavarashi E, Kindo AJ, Kalyani J. 2013. Ooptimization of PCR-RFLP directly from the skin and nails in cases of dermatophy-tosis, targeting the ITS and the 18s ribosomal DNA regions. J Clin Diagnostic Res. 7:646–651.

Emam SM, Abd El-salam OH. 2016. Real-time PCR: A rapid and sensitive method for diagnosis of dermatophyte induced onychomycosis, a comparative study. Alexandria J Med. 52:83–90.

Ferrer C, Colom F, Frasés S, Mulet E, Abad JL, Alió JL. 2001. Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA typing in ocular infections. J Clin Microbiol. 39:2873–2879.

Hryncewicz-Gwóźdź A, Beck-Jendroschec V, Brasch J, Kalinowska K, Jagielski T. 2011. Tinea capitis and Tinea corporis with a severe inflammatory response due to Trichophyton tonsurans. Acta Derm Venereol. 91:708–710.

Ilhan Z, Karaca M, Ekin IH, Solmaz H, Akkan HA, Tutuncu M. 2016. Detection of seasonal asymptomatic dermatophytes in Van cats. Brazilian J Microbiol. 47:225–230.

Jung HJ, Kim SY, Jung JW, Park HJ, Lee YW, Choe YB, Ahn KJ. 2014. Identification of dermatophytes by polymerase chain reaction-restriction fragment length polymorphism analysis of metalloproteinase-1. Ann Dermatol. 26:338–342.

Katiraee F, Asharafi Helan J, Teifoori F. 2016. Multiple Cases of Feline Dermatophytosis Due to Microsporum anis Transmitted to Their Owners. J Zoonotic Drsease. 1:24–27.

Mirzahoseini H, Omidinia E, Shams-Ghahfarokhi M, Sadeghi G, Razzaghi-Abyaneh M. 2009. Application of PCR-RFLP to rapid identification of the main pathogenic dermatophytes from clinical specimens. Iran J Public Health. 38:18–24.

Mohammadi R, Abastabar M, Mirhendi H, Badali H, Shadzi S, Chadeganipour M, Pourfathi P, Jalalizand N, Haghani I. 2015. Use of restriction fragment length polymorphism to rapidly identify dermatophyte species related to dermatophytosis. Jundishapur J Microbiol. 8:4–9.

Pal M, Dave P. 2013. Ringworm in cattle and man caused by Microsporum canis: Transmission from dog. Int J Livest Res. 3:100.

Pasquetti M, Min ARM, Scacchetti S, Dogliero A, Peano A. 2017. Infection by Microsporum canis in paediatric patients: A veterinary perspective. Vet Sci. 4:2–7.

Pham T, Wimalasena T, Box WG, Koivuranta K, Storgårds E, Smart KA, Gibson BR. 2011. Evaluation of ITS PCR and RFLP for differentiation and identification of brewing yeast and brewery “wild” yeast contaminants. J Inst Brew. 117:556–568.

Putty K, Shiva Jyothi J, Sharanya M, Srikanth Reddy M, Sai Ram Sandeep G, Abhilash M, Venkatesh Yadav J, Purushotham P, Kesavulu Naidu I, Uma Chowdhary A, et al. 2018. PCR as a rapid diagnostic tool for detection of dermatophytes. Int J Curr Microbiol Appl Sci. 7:2021–2025.

Rezaei-Matehkolaei A, Makimura K, Sybren de Hoog G, Shidfar MR, Satoh K, Najafzadeh MJ, Mirhendi H. 2012. Multilocus differentiation of the related dermatophytes Microsporum canis, Microsporum ferrugineum and Microsporum audouinii. J Med Microbiol. 61:57–63.

Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W, Bolchacova E, Voigt K, Crous PW, et al. 2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci U S A. 109:6241–6246.

Sharma R, Gupta S, Asati DP, Karuna T, Purwar S BD. 2017. A pilot study for the evaluation of PCR as a diagnostic tool in patients with suspected dermatophytosis. Indian Dermatol Online J. 8:176–180.

Tartor YH, El Damaty HM MY. 2016. Diagnostic performance of molecular and conventional methods for identification of dermatophyte species from clinically infected Arabian horse in Egypt. Vet Dermatol. 27:401-e102.

Torres-Guerrero E, González de Cossío AC, Segundo ZC, Cervantes ORA, Ruiz- Esmenjaud J, Arenas R. 2016. Microsporum canis and other dermatophytes isolated from humans, dogs and cats in mexico city. Glob Dermatology. 3:275–278.

White TJ, Bruns TD, Lee SB, Taylor JWWhite TJ, Bruns TD, Lee SB TJ. 1990. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. In: PCR Protoc A Guid to Methods Appl. New York (US): Academic Press; p. 315-322.

Zhang F, Tan C, Xu Y, Yang G. 2019. FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis. Int J Mol Med. 44:2047–2056.

Zhang R, Ran Y, Day Y, Zhang H LY. 2011. A case of kerion celsi caused by Microsporum gypseum in a boy following dermatoplasty for a scalp wound from a road accident. Med Myco. 49:90–93.

Refbacks

  • There are currently no refbacks.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.