Use of restriction fragment length polymorphism to identify Candida species, related to onychomycosis


1 Department of Medical Parasitology and Mycology, Isfahan University of Medical Sciences, Isfahan, Iran

2 Prof. Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

3 Department of Medical Mycology and Parasitology, Invasive Fungi Research Center (IFRC), School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran


Background: Onychomycosis is one of the most common clinical forms of fungal infections due to both filamentous fungi and yeasts. The genus of Candida is one of the most prominent causes of onychomycosis in all around the world. Although Candida albicans is still the most frequent cause of nail infections, use of broad-spectrum antifungal agents has led to a shift in the etiology of C. albicans to non-albicans species. The aim of the present study is rapid and precise identification of candida species isolated from nail infection by using of PCR-RFLP technique.
Materials and Methods: A total of 360 clinical yeast strains were collected from nail infections in Iran. Genomic DNA was extracted using FTA; cards. ITS1-5.8SrDNA-ITS2 region was amplified using universal primers and subsequently products were digested with the restriction enzyme MspI. For identification of newly described species (C. parapsilosis complex), the SADH gene was amplified, followed by digestion with Nla III restriction enzyme.
Results: Candida albicans was the most commonly isolated species (41.1%), followed by C. parapsilosis (21.4%), C. tropicalis (12.8%), C. kefyr (9.4%), C. krusei (5.5%), C. orthopsilosis (4.1%), C. glabrata (2.8%), C. guilliermondii (1.4%), C. rugosa (0.8%), and C. lusitaniae (0.5%). Patients in the age groups of 51-60 and 81-90 years had the highest and lowest distribution of positive specimens, respectively.
Conclusion: Rapid and precise identification of Candida species from clinical specimens lead to appropriate therapeutic plans.


Baran R, Hay RJ, Tosti A, Haneke E. A new classification of onychomycosis. Br J Dermatol 1998;139:567-71.  Back to cited text no. 1
Scher RK. Onychomycosis: A significant medical disorder. J Am Acad Dermatol 1996;35:S2-5.  Back to cited text no. 2
Drake LA, Dinehart SM, Farmer ER, Goltz RW, Graham GF, Hordinsky MK, et al.; Guidelines/Outcomes Committee. Guidelines of care for superficial mycotic infections of the skin: Onychomycosis. J Am Acad Dermatol 1996;34:116-21.  Back to cited text no. 3
Elewski BE, Charif MA. Prevalence of onychomycosis in patients attending a dermatology clinic in northeastern Ohio for other conditions. Arch Dermatol 1997;133:1172-3.  Back to cited text no. 4
Lohoué Petmy J, Lando AJ, Kaptue L, Tchinda V, Folefack M. Superficial mycoses and HIV infection in Yaounde. J Eur Acad Deramtol Venereol 2004;18:301-4.  Back to cited text no. 5
Zomorodian K, Rahimi MJ, Pakshir K, Motamedi M, Ghiasi MR, Rezashah H. Determination of antifungal susceptibility patterns among the clinical isolates of Candida species. J Glob Infect Dis 2011;3:357-60.  Back to cited text no. 6
André J, Achten G. Onychomycosis. Int J Dermatol 1987;26:481-90.  Back to cited text no. 7
Borst A, Theelen B, Reinders E, Boekhout T, Fluit AC, Savelkoul PH. Use of amplified fragment length polymorphism analysis to identify medically important Candida spp., including C. dubliniensis. J Clin Microbiol 2003;41:1357-62.  Back to cited text no. 8
Soll DR. The ins and outs of DNA fingerprinting the infectious fungi. Clin Microbiol Rev 2000;13:332-70.  Back to cited text no. 9
Enger L, Joly S, Pujol C, Simonson P, Pfaller M, Soll DR. Cloning and characterization of a complex DNA fingerprinting probe for Candida parapsilosis. J Clin Microbiol 2001;39:658-69.  Back to cited text no. 10
Borman AM, Linton CJ, Miles SJ, Campbell CK, Johnson EM. Ultra-rapid preparation of total genomic DNA from isolates of yeast and mould using Whatman FTA filter paper technology-a reusable DNA archiving system. Med Mycol 2006;44:389-98.  Back to cited text no. 11
White TJ, Bruns T, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR Protocols: A Guide to Methods and Applications. London, UK: Academic Press; 1990. p. 315-22.  Back to cited text no. 12
Mirhendi H, Makimura K, Khoramizadeh M, Yamaguchi H. A one-enzyme PCR-RFLP assay for identification of six medically important Candida species. Nihon Ishinkin Gakkai Zasshi 2006;47:225-9.  Back to cited text no. 13
Tavanti A, Davidson AD, Gow NA, Maiden MC, Odds FC. Candida orthopsilosis and Candida metapsilosis spp. nov. to replace Candida parapsilosis groups II and III. J Clin Microbiol 2005;43:284-92.  Back to cited text no. 14
Mirhendi H, Bruun B, Schønheyder HC, Christensen JJ, Fuursted K, Gahrn-Hansen B, et al. Molecular screening for Candida orthopsilosis and Candida metapsilosis among Danish Candida parapsilosis group blood culture isolates: Proposal of a new RFLP profile for differentiation. J Med Microbiol 2010;59:414-20.  Back to cited text no. 15
Mohammadi R, Mirhendi H, Yadegari MH, Ghahri M, Shadzi S, Jalalizand N, et al. Evaluation of prevalence of the new Candida Species (C. Orthopsilosis and C. Metapsilosis) among C. Parapsilosis Group isolated from patients by PCR-RFLP of SADH Gene in Iran. J Isfahan Med Sch 2011;29:1-10.  Back to cited text no. 16
Odds FC, Bernaerts R. CHROMagar Candida, a new differential isolation medium for presumptive identification of clinically important Candida species. J Clin Microbiol 1994;32:1923-9.  Back to cited text no. 17
Daef E, Moharram A, Eldin SS, Elsherbiny N, Mohammed M. Evaluation of chromogenic media and seminested PCR in the identification of Candida species. Braz J Microbiol 2014;45:255-62.  Back to cited text no. 18
Weitzman I, Summerbell RC. The dermatophytes. Clin Microbiol Rev 1995;8:240-59.  Back to cited text no. 19
Zaias N, Glick B, Rebell G. Diagnosing and treating onychomycosis. J Fam Pract 1996;42:513-8.  Back to cited text no. 20
Nucci M, Marr KA. Emerging fungal diseases. Clin Infect Dis 2005;41:521-6.  Back to cited text no. 21
Gumbo T, Isada CM, Hall G, Karafa MT, Gordon SM. Candida glabrata Fungemia. Clinical features of 139 patients. Medicine (Baltimore) 1999;78:220-7.  Back to cited text no. 22
Kaur R, Kashyap B, Bhalla P. Onychomycosis--epidemiology, diagnosis and management. Indian J Med Microbiol 2008;26:108-16.  Back to cited text no. 23
[PUBMED]  Medknow Journal  
Tainwala R, Sharma YK. Current clinico-mycological trends of onychomycosis in Pune. Indian J Dermatol 2012;57:242-3.  Back to cited text no. 24
[PUBMED]  Medknow Journal  
Bramono K, Budimulja U. Epidemiology of onychomycosis in Indonesia: Data obtained from three individual studies. Nihon Ishinkin Gakkai Zasshi 2005;46:171-6.  Back to cited text no. 25
Banerjee U, Sethi M, Pasricha JS. Study of onychomycosis in India. Mycoses 1990;33:411-5.  Back to cited text no. 26
Haneke E. Fungal infections of the nail. Semin Dermatol 1991;10:41-53.  Back to cited text no. 27
Williams HC. The epidemiology of onychomycosis in Britain. Br J Dermatol 1993;129:101-9.  Back to cited text no. 28
Mohammadi R, Mirhendi H, Rezaei-Matehkolaei H, Ghahri M, Shidfar MR, Jalalizand N, et al. Molecular identification and distribution profile of Candida species isolated from Iranian patients. Med Mycol 2013;51:657-63.  Back to cited text no. 29
Feng X, Ling B, Yang G, Yu X, Ren D, Yao Z. Prevalence and distribution profiles of Candida parapsilosisCandida orthopsilosis and Candida metapsilosis responsible for superficial candidiasis in a Chinese university hospital. Mycopathologia 2012;173:229-34.  Back to cited text no. 30
Silva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J. Candida glabrataCandida parapsilosis and Candida tropicalis: Biology, epidemiology, pathogenicity and antifungal resistance. FEMS Microbiol Rev 2012;36:288-305.  Back to cited text no. 31
Alcoba-Flórez J, Méndez-Alvarez S, Cano J, Guarro J, Pérez-Roth E, del Pilar Arévalo M. Phenotypic and molecular characterization of Candida nivariensis sp. nov., a possible new opportunistic fungus. J Clin Microbiol 2005;43:4107-11.  Back to cited text no. 32
Correia A, Sampaio P, James S, Pais C. Candida bracarensis sp. nov., a novel anamorphic yeast species phenotypically similar to Candida glabrata. Int J Syst Evol Microbiol 2006;56:313-7.  Back to cited text no. 33
Mirhendi H, Bruun B, Schønheyder HC, Christensen JJ, Fuursted K, Gahrn-Hansen B, et al. Differentiation of Candida glabrataC. nivariensis and C. bracarensis based on fragment length polymorphism of ITS1 and ITS2 and restriction fragment length polymorphism of ITS and D1/D2 regions in rDNA. Eur J Clin Microbiol Infect Dis 2011;30:1409-16.  Back to cited text no. 34
Chai LY, Denning DW, Warn P. Candida tropicalis in human disease. Crit Rev Microbiol 2010;36:282-98.  Back to cited text no. 35
Chadeganipour M, Nilipour S, Ahmadi G. Study of onychomycosis in Isfahan, Iran. Mycoses 2010;53:153-7.  Back to cited text no. 36
Khosravi AR, Mansouri P. Onychomycosis in Tehran, Iran: Prevailing fungi and treatment with itraconazole. Mycopathologia 2006;150:9-13.  Back to cited text no. 37
Hashemi SJ, Gerami M, Zibafar E, Daei M, Moazeni M, Nasrollahi A. Onychomycosis in Tehran: Mycological study of 504 patients. Mycoses 2010;53:251-5.  Back to cited text no. 38