Azole-resistant Aspergillus fumigatus with the environmental TR 46 / Y 121 F / T 289 A mutation in India

Sir, Triazole antifungals are the mainstay of therapy for patients with aspergillosis. Notably, the mortality associated with aspergillosis is high and the rate of treatment failure is much higher if patients are infected with multiple-triazole-resistant (MTR) Aspergillus fumigatus. MTR A. fumigatus strains with the mutation TR34/ L98H occur both in azole-treated as well as in azole-naive patients and have increasingly been reported from Dutch patients and their environment and from other European and Asian countries. – 7 Molecular studies from Europe and India suggested that use of azole fungicides in the environment selects MTR A. fumigatus TR34/L98H strains. 7,8 This issue is further complicated by the emergence of a new resistance mechanism, TR46/Y121F/T289A in the cyp51A gene responsible for voriconazole resistance in A. fumigatus, which was detected in 2009 in a Dutch patient and has been recently reported in series of patients from the Netherlands. We report the occurrence of the same TR46/Y121F/T289A mutations in environmental A. fumigatus strains in India, which were also cross-resistant to commonly used azole fungicides. A total of 105 soil samples from the agricultural fields of the river Yamuna bank, Delhi (n1⁄463) and Varanasi, Uttar Pradesh (n1⁄442), located 800 km apart, were investigated during 2012–13. Samples were processed as described previously. A total of 37 (35%) samples (Delhi, n1⁄427; Varanasi, n1⁄410) yielded 126 A. fumigatus strains, including 88 from Delhi and 38 from Varanasi, on Sabouraud dextrose agar (SDA) plates. All A. fumigatus strains were screened for resistance on SDA plates supplemented with 4 mg/L itraconazole and 1 mg/L voriconazole. Six A. fumigatus strains grew on voriconazole plates and eight grew on itraconazole plates. Voriconazoleresistant A. fumigatus strains originated from Varanasi, five from a potato (Solanum tuberosum) field and one from a fenugreek (Trigonella foenum-graecum) field, which were located 2 km apart from each other. Four of the eight itraconazole-resistant A. fumigatus strains originated from rose (Rosa species) bed soil and red chilli (Capsicum annuum) fields, Delhi. Of the remaining four itraconazole-resistant A. fumigatus strains, one each originated from fields of aubergine (Solanum melongena), mustard (Brassica juncea), potato and fenugreek in Varanasi. Notably, 5.7% of the soil samples harboured voriconazole-resistant A. fumigatus and 7.6% harboured itraconazole-resistant A. fumigatus. The overall isolation rate of both itraconazoleand voriconazole-resistant A. fumigatus was found to be higher in Varanasi (26.3%; 10/38) than in Delhi (4.5%; 4/88). Identification of resistant A. fumigatus strains was confirmed by sequencing of the internal transcribed spacer region, b-tubulin and the calmodulin gene. Further, A. fumigatus strains were screened for triazole resistance mutations using a mixed-format real-time PCR. All six voriconazoleresistant A. fumigatus strains exhibited the TR46/Y121F/T289A mutation and the eight itraconazole-resistant strains showed the TR34/ L98H mutation. Microsatellite genotyping of TR46/Y121F/T289A A. fumigatus strains was performed with a panel of nine short tandem repeats. For phylogenetic analysis, 32 resistant strains of A. fumigatus from the Netherlands (TR46, 14 clinical; TR34, 2 clinical and 3 environmental), India (TR46, 6 environmental; TR34, 2 clinical and 3 environmental), France (TR34, 1 clinical) and Germany (TR34, 1 clinical) were included along with 30 wild-type strains from India (8 clinical and 14 environmental) and the Netherlands (n1⁄48). The Indian environmental TR46/Y121F/T289A strains were similar toDutch clinicalTR46/Y121F/T289A strains and clustered separately from TR34/L98H A. fumigatus strains (Figure 1). The in vitro activity of azole antifungals and fungicides against 6 TR46/Y121F/T289A and 14 wild-type environmental A. fumigatus strains obtained from the same geographical locality were determined using the CLSI M38-A2 broth microdilution method. The azole fungicides tested were bromuconazole, cyproconazole, difenoconazole, epoxiconazole, penconazole, tebuconazole, triadimefon, metconazole (gifted by Dr P. E. Verweij, The Netherlands), hexaconazole (Rallis India, Mumbai, India) and tricyclazole (Cheminova India, Mumbai, India). All six TR46/Y121F/T289A isolates showed high MICs of voriconazole (.16 mg/L) and isavuconazole (8 mg/L), reducedsusceptibility to itraconazole (range,1–2 mg/L)and posaconazole (range, 0.25–0.5 mg/L) and cross-resistance to all the fungicides tested (range, 4 to .32 mg/L). However, all the wild-type isolates revealed low MICs of the azole antifungals (range, 0.06– 0.5 mg/L) and fungicides (range, 0.125–8 mg/L) tested. The emergence of a new azole resistance mechanism in environmental A. fumigatus strains in India raises concern, as this mechanism, TR46/Y121F/T289A, has been associated with invasive infections and therapeutic failure with voriconazole. So far, this