Translation elongation factor 1-alpha gene as a marker for diagnosing of candidal onychomycosis

Document Type : Original Articles

Authors

1 Department of Medical Mycology and Parasitology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

2 Basic Sciences in Infectious Diseases Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

3 Department of Medical Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

4 Medical mycology laboratory, Razi hospital, Tehran, Iran

5 Department of Medical Parasitology and Mycology, School of Medicine, Shiraz University of Medical

Abstract

Background and Purpose: Culture-based identification methods have been the gold standard for the diagnosis of candidal onychomycosis. Molecular technologies, such as polymerase chain reaction (PCR) assays, can provide an alternative for the rapid detection of Candida species. The present study was conducted to investigate a pan-Candida PCR assay based on the translation elongation factor 1-alpha (TEF-1α) gene for the detection of the most prevalent pathogenic Candida species.
Materials and Methods: For the purpose of the study, an optimized pan-Candida PCR primer pair was designed, and the target was amplified and sequenced. The analytical and clinical diagnostic performance of the designed primers was tested using 17 reference strains, 137 nail scrapings suspected of onychomycosis, and 10 healthy nail specimens.
Results: The use of the universal pan-Candida primers designed on TEF-1α gene resulted in the successful amplification of a 270-base pair fragment in all Candida species tested, except for C. glabrata, and reacted neither with other fungi nor with E. coli. The sequence difference count matrix showed poor insertion/deletion differences (0-2 nt) among Candida species. Among 137 nail specimens, 35% (n=48), 30.7% (n=42), and 40.1% (n=55) of the samples were found to be positive by direct microscopy, culture, and pan-Candida PCR, respectively.
Conclusion: Based on the findings, the PCR-based detection targeting the DNA TEF-1α gene is a rapid and simple procedure for the diagnosis of candidal onychomycosis directly from nail sample.

Keywords


1. Xiang H, Xiong L, Liu X, Tu Z. Rapid simultaneous detection and identification of six species Candida using polymerase chain reaction and reverse line hybridization assay. J Microbiol Methods. 2007; 69(2):282-7.
2. Low CY, Rotstein C. Emerging fungal infections in immunocompromised patients. F1000 Med Rep. 2011; 3:14.
3. Jesudanam TM, Rao GR, Lakshmi DJ, Kumari GR. Onychomycosis: a significant medical problem. Indian J Dermatol Venereol Leprol. 2002; 68(6):326-9.
4. Dubljanin E, Dzamic A, Mitrovic S, Arsenijevic VA, Calovski IC. Onychomycosis: clinical findings, etiological agents and evaluation of laboratory methods. Arch Biol Sci. 2014; 66(2):587-94.
5. Selvarangan R, Bui U, Limaye AP, Cookson BT. Rapid identification of commonly encountered Candida species directly from blood culture bottles. J Clin Microbiol. 2003; 41(12):5660-4.
6. Klingspor L, Jalal S. Molecular detection and identification of Candida and Aspergillus spp. from clinical samples using real‐time PCR. Clin Microbiol Infect. 2006; 12(8):745-53.
7. Taverna CG, Bosco-Borgeat ME, Murisengo OA, Davel G, Boite MC, Cupolillo E, et al. Comparative analyses of classical phenotypic method and ribosomal RNA gene sequencing for identification of medically relevant Candida species. Mem Inst Oswaldo Cruz. 2013; 108(2):178-85.
8. Barbedo LS, Figueiredo-Carvalho MH, Muniz Mde M, Zancopé-Oliveira RM. The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA. Mem Inst Oswaldo Cruz. 2016; 111(4):267-70.
9. Kristensen R, Torp M, Kosiak B, Holst-Jensen A. Phylogeny and toxigenic potential is correlated in Fusarium species as revealed by partial translation elongation factor 1 alpha gene sequences. Mycol Res. 2005; 109(Pt 2):173-86.
10. Silva FP, Vechiato MH, Harakava R. EF-1α gene and IGS rDNA sequencing of Fusarium oxysporum f. sp. vasinfectum and F. oxysporum f. sp. phaseoli reveals polyphyletic origin of strains. Trop Plant Pathol. 2014; 39(1):64-73.
11. Voigt K, WoÈstemeyer J. Phylogeny and origin of 82 zygomycetes from all 54 genera of the Mucorales and Mortierellales based on combined analysis of actin and translation elongation factor EF-1α genes. Gene. 2001; 270(1-2):113-20.
12. Mirhendi H, Makimura K, de Hoog GS, Rezaei-Matehkolaei A, Najafzadeh MJ, Umeda Y, et al. Translation elongation factor 1-α gene as a potential taxonomic and identification marker in dermatophytes. Med Mycol. 2014; 53(3):215-24.
13. Carberry S, Neville CM, Kavanagh KA, Doyle S. Analysis of major intracellular proteins of Aspergillus fumigatus by MALDI mass spectrometry: identification and characterisation of an elongation factor 1B protein with glutathione transferase activity. Biochem Biophys Res Commun. 2006; 341(4):1096-104.
14. Ignjatovic VA, Stevanovic MT, Durdevic VS, Petrovic MM, Stanucevic MP, Dzamic AM, et al. Onychomycosis: sampling, diagnosing as efficiant part of hospital pharmacology. Hosp Pharmacol Int Multidisciplinary J. 2014; 1(3):130-7.
15. Abd-Elsalam KA. Bioinformatic tools and guideline for PCR
primer design. Afr J Biotechnol. 2003; 2(5):91-5.
16. Silva GA, Bernardi TL, Schaker PD, Menegotto M, Valente P. Rapid yeast DNA extraction by boiling and freeze-thawing without using chemical reagents and DNA purification. Braz Arch Biol Technol. 2012; 55(2):319-27.
17. Petinataud D, Berger S, Ferdynus C, Debourgogne A, Contet‐Audonneau N, Machouart M. Optimising the diagnostic strategy for onychomycosis from sample collection to fungal identification evaluation of a diagnostic kit for real‐time PCR. Mycoses. 2016; 59(5):304-11.
18. Barbedo LS, Figueiredo-Carvalho MH, Muniz MN, Zancope-Oliveira RM. Comparison of four molecular approaches to identify Candida parapsilosis complex species. Mem Inst Oswaldo Cruz. 2017; 112(3):214-9.
19. Kanbe T, Horii T, Arishima T, Ozeki M, Kikuchi A. PCR‐based identification of pathogenic Candida species using primer mixes specific to Candida DNA topoisomerase II genes. Yeast. 2002; 19(11):973-89.
20. Sundstrom P, Smith D, Sypherd PS. Sequence analysis and expression of the two genes for elongation factor 1 alpha from the dimorphic yeast Candida albicans. J Bacteriol. 1990; 172(4):2036-45.
21. Khan ZU, Mustafa AS. Detection of Candida species by polymerase chain reaction (PCR) in blood samples of experimentally infected mice and patients with suspected candidemia. Microbiol Insights. 2001; 156(1):95-102.
22. Zhang J, Hung GC, Nagamine K, Li B, Tsai S, Lo SC. Development of Candida-specific real-time PCR assays for the detection and identification of eight medically important Candida species. Microbiol Insights. 2016; 9:21-8.
Volume 6, Issue 1
March 2020
Pages 15-21
  • Receive Date: 05 September 2019
  • Accept Date: 10 February 2020
  • First Publish Date: 01 March 2020
  • Publish Date: 01 March 2020