Long Amplification PCR (LA-PCR) Detection of Azole Resistant Trichophyton indotineae

Trichphyton indotineae, a species newly designated in 2020 independent of T. interdigitale, comprises highly terbinafine (TRF)-resistant dermatophytosis that is epidemic in North India and spreding to worldwide. Some clinical isolates of T. indotineae have been resistance both TRF and azoles that might be caused the treatment failure. To detect the azole resistance strains, we developed a long amplification PCR (LA-PCR) detection method for the tandem repeat of the CYP51B (encoding sterol 14a-demethylase gene) in T. indotineae. Contrasting the drug susceptibility test results with the LA-PCR results confirmed a trend toward low susceptibility to azole antifungal agents in strains with amplifications of 9.5 kbp or greater (3 or more copies of CYP51B). Our results suggest that the method could be detected rapidly of low-susceptibility strains to azole antifungal agents.

Antifungal susceptibility testing such as the Clinical and Laboratory Standards Institute (CLSI) protocol M38 microdilution technique has been performed on clinical isolates to detect drug-resistant strains as a conventional method [6].However, it takes at least 4 weeks including pure culture on potato dextrose agar and incubation in 96-well plates to obtain results.Therefore, it is necessary to detect drug-resistant strains using a simple and quick method.
In previous study, we sequenced the genomes of azole-sensitive and resistant strains by next-generation sequencing analysis and indicated the azole resistance mechanism by overexpression of CYP51B (encoding sterol 14a-demethylase gene) [7].In Aspergillus fumigatus and Candida albicans, resistance to azole antifungal agents has been reported to be due to reduced drug binding ability caused by mutations in the Cyp51 genes and to overexpression of the Cyp51 genes by mutations in the transporter region [7].However, Yamada et al. [7] found that T. indotinea has acquired azole resistance by a unique method that has not been reported in other fungi.They found that its overexpression was derived from a tandem repeat of the CYP51B in their genomes [7].We carried out for detecting these tandem repeats by genom sequencing analysis, but we felt that a simpler method was needed for clinical purposes.
In the present study, we improved the rapid and simple molecular detection method for the tandem repeat of the CYP51B in azole-sensitive and resistant strains of T. indotineae.
The 7 strains of T. indotineae examined in this study are listed in Table 1.To assess azole susceptibility in these isolates, the broth microdilution assay was performed based on the CLSI M38 guidelines with modifications as previously described [6].The number of tandem repeats of CYB51B in the each strain was analyzed by the genom sequence analysis reported by previous our report [7].
Genomic DNA was extracted according to the method of Girardin and Latge [8], with several minor modificartions.The growing mycelia from each dermatophyte strain were collected after incubation on Saboraud's dextrose agar (1% of peptone, 2% of glucose and 2% of agar) for 3 to 4 days at 28 °C, frozen and gournd twice and cooled on liquid nitrogen with Multi-Beads shocker (Yasui Kikai Corporation, Osaka, Japan) at 2000 r.p.m for 10 s.Genomic DNA (approximatory100 ng) from isolates was amplified by long analysis PCR (LA-PCR) in a volume of 25 lL, using a 2.5 lL of 10 9 reaction mixture, 5 lL of 5 9 PCR enhancer, 1.5 lL of 2.5 mM of each deoxynucleoside triphosphate, 0.75 U of long range PCR enzyme mix (biotechrabbit GmbH, Berlin, Germany), and 0.3 lM of primer pairs.The reaction mixture was heated for template denaturation (2 min, 95 °C).Then thirty cycles of PCR amplification were performed with the following conditions: denaturation for 30 s at 95 °C, primer annealing and extension for 25 min at 68 °C.The resulting amplified DNA fragments were electrophoresed on a 0.6% (w/v) agarose gel with 1 9 TAE buffer and visualized by ethidium bromide staining.123 The electrophoresis image showed a amplicon of approximately 4.7 kbp in the strain thought to harbor one copy of CYP51B, ammplicons of approximately 4.7, 7.1 and 9.5 kbps in the strain thought to harbor 3 copies of the genes, and amolocons of approximately 4.7, 7.1, 9.5, 12 and 14.3 kbp in the strain thought to harbor more 5 copies of the genes (Fig. 2).However, amplified products of greater length than 15 kbp could not be clearly separated by our agarose (0.6%) electrophoresis method.Contrasting the drug susceptibility test results with the LA-PCR results confirmed a trend toward low susceptibility to azole antifungal agents in strains with amplifications of 9.5 kbp or greater (3 or more copies of CYP51B) (Table 1).In this study, we developed a LA-PCR detection method for the tandem repeat of the CYP51B in T. indotineae.Our results suggest that the method could be detected rapidly of low-susceptibility strains to azole antifungal agents without next-generation sequencing analysis of CYP51B.
Currently, multi-drug resistant T. indotoneae have been isolated in many parts of the world.We believe that our developed method can contribute to the treatment of this intractable dermatophytosis.In the future, we plan to perform this LA-PCR analysis on more T. indotineae isolates to evaluate their clinical usefulness.

Fig. 1
Fig. 1 Schema of the primer positions and estimated PCR amplicon lengths of T. indotineae CYP51B.The expected amplified gene lengths per copy number of CYP51B were