Molecular identification of yeast communities isolated from nail specimens by PCR-RFLP and PCR-FSP methods

Abstract

Background and purpose: Onychomycosis is a common fungal infection that affects the nails, caused by various fungal agents. Moreover, yeast onychomycosis has increased in recent years. Yeast isolates might not be identified at the species level by conventional methods, whereas molecular methods can identify yeast isolates more accurately. This study aimed to identify yeast communities isolated from nail specimens by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and PCR- fragment size polymorphism (FSP) methods.

Materials and methods: This experimental study was conducted on archival yeast isolates obtained from 269 patients suspected of onychomycosis who referred to the Medical Mycology Laboratory at Shiraz University of Medical Sciences in Shiraz, Iran, between April 2022 and March 2023. Onychomycosis was confirmed through direct examination and culture of nail specimens. The PCR-RFLP and PCR-FSP methods were used to identify yeast isolates.

Results: In total, 78 (28.99%) yeast strains were identified. Candida albicans was the most common species, followed by Candida parapsilosis complex and Candida tropicalis. Uncommon species of yeasts, such as Candida utilis, Candida pararugosa, Candida nivariensis, and Rhodotorula rubra were identified by molecular methods. The PCR-FSP method showed a strong agreement with the PCR-RFLP method in the identification of common yeast agents causing onychomycosis (κ=0.84).

Conclusion: It seems necessary to use molecular diagnostic tools in addition to conventional methods to identify yeast isolates in clinical laboratories. The rapid and accurate identification of fungal agents causing onychomycosis is useful for the selection of an appropriate treatment strategy.

Keywords: Molecular identification; Onychomycosis; PCR-FSP; PCR-RFLP; Yeast communities.

Figure 1

A) Agarose gel electrophoretic band pattern of polymerase chain reaction-restriction fragment length polymorphism products of yeast isolates; Lane M: 100-bp DNA ladder; Lanes 1 and 8: C. kefyr (720 bp); Lanes 2, 5, and 11: C. albicans (239, 298 bp); Lanes 3, 4, 6, and 9: C. glabrata (320, 561 bp); Lanes 7 and 10: C. tropicalis (186, 340 bp); Lane N: negative control. B) Agarose gel electrophoresis pattern of PCR-FSP products of yeast isolates; Lane M: 100-bp DNA ladder; Lane 1: C. albicans (219, 338 bp); Lane 2: C. glabrata (419, 482 bp); Lane 3: C. parapsilosis (220, 310 bp); Lane 4: C. krusei (182, 347 bp); Lane 5: C. tropicalis (218, 327 bp); Lane 6: C. pararugosa (164, 271 bp); Lane 7: C. orthopsilosis (220, 311 bp); Lane N: negative control; Lane 8: C. pararugosa (164, 271 bp); Lane 9: C. utilis (220, 364 bp); Lane 10: (mix and unidentified isolates); Lane 11: C. utilis(220, 364 bp); Lane 12: Rhodotorula rubra (232, 404 bp); Lane 13: C. orthopsilosis (220, 311 bp); and Lane 14: C. kefyr (309, 432 bp).