Terbinafine resistance of dermatophytes in India and Germany
Resistance of dermatophytes against antifungal agents was not perceived as a problem for decades. Therapy failure in onychomycosis or tinea capitis was never related to a reduced in vitro susceptibility of the causative species of dermatophytes. The situation has changed dramatically. The starting point, was, most likely, India. An incredible increase in chronic recalcitrant dermatophytoses over the past few years has been noted in India. The main causative pathogen is the formerly zoophilic, but nowadays anthropophilic dermatophyte Trichophyton (T.) mentagrophytes of the ITS genotype VIII. Due to travelling and migration, spread of the mostly terbinafine resistant strains of T. mentagrophytes Type VIII from India to other parts of the world, e.g. Germany, is ongoing.
Patients and methods/India: Three epidemiologic exercises, for the purpose of speciation and also to look into the possibility of antifungal resistance were undertaken with cooperation from eight Indian centers with German and Swiss mycologists. A total of 402 Indian patients were included in the epidemiological study. Terbinafine antifungal susceptibility testing (breakpoint method) and genetic point mutation analysis of the squalene epoxidase (SQLE) gene was done. Itraconazole and voriconazole minimal inhibitory concentrations (MICs) were determined.
Patients/Germany: From September 2016 until March 2020, altogether 29 strains of T. mentagrophytes Type VIII India were isolated in Germany.
Results/India: All 402 samples were evaluated for growth on culture media. From 289 (71.9%) samples, T. mentagrophytes was isolated. T. rubrum was cultivated from 19 (4.7%) samples, while 94 (23.4%) samples yielded no dermatophyte growing on media. 265 samples were screened for presence of dermatophytes by PCR-ELISA. In 235 (88.7 %) samples, species belonging to the T. interdigitale/T. mentagrophytes complex were found. 11 (4.1 %) samples harboured T. rubrum and in 19 (7.2 %) samples, no dermatophyte DNA was detected. 71 % of isolates were resistant to terbinafine. The amino acid substitution Phe397Leu in the squalene epoxidase of resistant T. mentagrophytes was highly prevalent (91 %). Two novel substitutions in resistant Trichophyton strains, Ser395Pro and Ser443Pro, were discovered. The missense substitution Ala448Thr was found in terbinafine sensitive and resistant isolates but was associated with increased MICs of itraconazole and voriconazole.
Results/Germany: All patients were residents in Germany, 12 females, 15 males, the gender of the remaining 2 was not assignable. Patients originated from India (11), from Pakistan (2), Bangladesh (2), Iraq (2), Bahrain (1), and Libya (1), or from not specified countries (10). At least, 2 patients were German born residents. Most isolates (21) were found 2019 and 2020. 29 T. mentagrophytes isolates were sequenced (“internal transcribed spacer”, ITS) and TEF1-α. All 29 strains belonged to genotype VIII (India) of T. mentagrophytes. In vitro resistance testing revealed that 13 strains (45 %) out of 29 were terbinafine resistant with breakpoints >0.2 µg/ml. The remaining 16 strains (55 %) were terbinafine sensitive. Point mutation analysis revealed, that among 13 resistant strains, 10 exhibited Phe397Leu amino acid substitution of the squalene epoxidase (SQLE) indicative for in vitro resistance to terbinafine. Two resistant strains showed combined Phe397Leu and Ala448Thr amino acid substitutions, and one stain single Leu393Phe amino acid substitution. Out of 16 terbinafine sensitive strains, in 8 Ala448Thr, and in one Ala448Thr + new Val444 Ile amino acid substitutions were detected. Treatment includes topical ciclopirox olamine plus miconazole or sertaconazole. Appropriate oral antifungal agent is itraconazole 200 mg twice daily for at least 4 to 8 weeks.
Conclusion: The dramatic increase in terbinafine resistant T. mentagrophytes ITS VIII from all over India within such a short period of time underscores the issue of development of resistance in patients with chronic dermatophytoses. A strong association was found between in vitro terbinafine resistance of T. mentagrophytes and the occurrence of single point mutations of the SQLE gene and distinct amino acid substitutions, respectively. Alterations at position Phe397Leu, Leu393Ser, Leu393Phe, Gln408Leu, His440Tyr, of the SQLE were associated with terbinafine resistance. Increasing number of patients in Europe (Germany, Finland, Estonia) and from United States have been seen to be presenting with chronic recalcitrant dermatophytosis due to terbinafine-resistants T. mentagrophyte VIII from India. Transmission of the Indian T. mentagrophytes VIII to other countries due to travel, migration and in general, globalization, appears to be a serious issue even from a public health perspective. The here shown 29 Indian strains of T. mentagrophytes ITS VIII isolated from patients with dermatophytoses residing in Germany represent probably “The tip of the iceberg”. Further studies are the need of the day to understand this phenomenon better and find answers to combat it.