In the present study, we examined itraconazole, voriconazole and posaconazole susceptibility in a spectrum of clinically significant Aspergillus spp. A. flavus was found to be the most common clinically significant Aspergillus spp. in this study followed by A. fumigatus, A. niger and A. terreus, respectively. We screened 114 isolates for azole resistance but we could not detect any triazole resistant Aspergillus isolate. MICs of representative isolates were tested by broth microdilution and all of them were found to be triazole susceptible. Hence, triazole resistance could not be detected in clinical Aspergillus isolates from a single laboratory in our country over the last four years. Our data can nevertheless serve as baseline for future surveillance of triazole susceptibility in clinically significant Aspergillus spp. in our region, as such data in either clinical or environmental Aspergillus isolates is currently lacking from Pakistan.
Resistant isolates harboring either TR34/L98H or TR46/Y121F/T289A mutations have been found in environmental and clinical samples from several countries including the USA, United Kingdom, Ireland, most countries in Europe, Tanzania, and Australia (22). Arendrup et al. screened a total of 3,788 Aspergillus isolates for azole resistance from a similar clinical spectrum to our study, but as a multicenter international surveillance during January 2009–January 2011. A. fumigatus species complex constituted 77.6% of their isolates, while in our study, A. flavus was the most common species (56.1%), and A. fumigatus was only 33.3% of the isolates. Acquired azole resistance in A. fumigatus was detected in 11 of 17 European centers in nine countries, with an overall 3.2% prevalence of azole resistance. The median MICs of itraconazole, voriconazole and posaconazole for the resistant isolates were >8, 2 and 1 mg/L respectively. TR34/L98H was the predominant mechanism of resistance. Our isolates showed lower MICs. The MIC90 of itraconazole, voriconazole and posaconazole of our A .fumigatus isolates were 0.5, 1 and 0.5 mg/L respectively (table 2) (23, 24).
Data on azole resistant Aspergillus spp. from clinical and environmental isolates is also available from Far East: Taiwan, Thailand, Japan; and our neighboring countries, India, China, and Iran. Tashiro et al. confirmed that long-term itraconazole therapy induced azole resistance in A. fumigatus isolates with itraconazole MICs ≥4mg/L is isolates that were exposed to itraconazole for >115 days (24). Five of our isolates were previously exposed to azoles, as described above. However, we could not detect any triazole resistance in our isolates. One of these had an exposure of 197 days to itraconazole, but its MIC was 0.25 mg/L. Liu et al, from China identiﬁed four azole resistant A. fumigatus strains out of 72 clinical isolates, based on mutations of cyp51A. Three strains were highly resistant to itraconazole (MIC 16 mg/L), two of which exhibited the TR34/L98H/S297T/F495I mutation, while one carried only the TR34/L98H mutation. The fourth multiazaole-resistant isolate (itaconazole 4mg/L, voriconazole 2mg/L) carried a new G432A mutation (25). Chowdhary et al. found azole resistance in (12) 1.7% A. fumigatus clinical isolates during four years in a referral Chest Hospital in Delhi, India. These isolates harbored TR34/L98H mutation in 83.3% isolates with a pan-azole resistant phenotype, linked to the use of fungicide azoles in agricultural practices. Of the 12 resistant A. fumigatus, 11 showed a pan-azole resistant phenotype exhibiting high MIC of all the triazoles, itraconazole [geometric mean (GM) MIC=16 mg/L], voriconazole (GM MIC=8 mg/L), and posaconazole (GM MIC=2.82 mg/L). In contrast, a solitary A. fumigatus isolate exhibited high MIC (>16 mg/L) against itraconazole only (8). Chowdhary et al. also reported 7 % triazole resistance in A. fumigatus isolates from 24 environmental samples in India, which shared the same TR34/L98H mutation in the cyp51 gene and showed cross-resistance to itraconazole, voriconazole and posaconazole, and to six triazole fungicides used extensively in agriculture. The mutated environmental strains showed high MICs ( itraconazole GM MIC=16 mg/L], voriconazole (GM MIC=8.7 mg/L), and posaconazole (GM MIC=1.03 mg/L), and the mutated clinical isolates showed the following MICs ( itraconazole GM MIC=16 mg/L], voriconazole (GM MIC=5.9 mg/L), and posaconazole (GM MIC=3.2 mg/L)(26) (26). Nabili et al in a three-year study, screened 513 samples (213 clinical and 300 environmental samples) from ten provinces of Iran for azole resistance, and found a 6.6% prevalence of azole-resistant A. fumigatus in Iran ([clinical and environmental A. fumigatus isolates with decreased drug susceptibility and mutations: itraconazole MIC range 4 to >16mg/L, voriconazole MIC range 0.25 to >16mg /L posaconazole MIC range 0.016 to 4mg/L]. Among resistant isolates, TR34/L98H mutations in the CYP51A gene were the most prevalent (80 %) (27). In comparison to these studies, our isolates showed comparatively lower MICs.
J.F. Meis and, A. Chowdhary et al. recently sequenced and analysed 24 genomes of A. fumigatus from across the world. These isolates were rationally chosen to include an informative selection of clinical and environmental isolates that were wild-type, or known to carry the TR34/L98H allele. This population genomic analysis showed that A. fumigatus was broadly panmictic, with as much genetic diversity found within a country as is found between continents (28).
The occurrence of azoles resistant isolates of A. fumigatus varies worldwide, from 2.1–20% in the UK, 10–12% in the Europe, 10% in Asia, Africa, America and Australia to 1.75% in India, probably due to varying usage of azole fungicides that may select for resistance (29).
Amongst our isolates, A. flavus was the most common one. At present, the development of azole resistance is mainly associated with A. fumigatus and less so with A. flavus and A. terreus; however, further surveillance is warranted. A. flavus is the cause of a broad spectrum of human diseases predominantly in Asia, the Middle East, and Africa possibly due to its ability to survive better in hot and arid climatic conditions compared to other Aspergillus spp (30). In a collection of 590 clinical isolates, from five centers in USA and Europe, the rate of voriconazole resistance in A. flavus was estimated at ~2% using an ECV of >1 μg/mL (31).
There is limited data from Pakistan on the mold antifungal surveillance. A study was conducted in the Armed Forces Institute of Pathology, Rawalpindi from January through December, 2016. 110 isolates; 45 (40.9%) A. fumigatus, 40 (36.3%) Alternaria alternata, and 25 (22.7%) Cladosporium sphaerospermum were tested against amphotericin B, fluconazole and voriconazole by broth microdilution method. The overall voriconazole and amphotericin B susceptibility rates were 82.2% and 84.5%, respectively. Voriconazole resistance was seen in only 1 (2.5%) A. fumigatus isolate, which was not confirmed by genotyping (32). Due to our limited resources, our study also lacks the detection of molecular mechanisms of azole resistance in our clinical isolates.
The limitations of our study are the small sample size, and single centre which couldn’t find any triazole resistant Aspergillus isolate. However, we feel the bench experience will help introduce the screening and MICs into routine clinical practice, though Azole resistant Aspergillus disease is a rare entity and difficult to diagnose(10). Unfortunately, the susceptibilities could not be performed in real-time but in batches, due to logistic issues which reduced the clinical utility for some patients.
Future directions necessitate surveillance of our environmental and clinical isolates, to determine any rising MICs, and change in our local epidemiology. This MIC data should be linked to clinical antifungal usage and therapeutic drug monitoring in patients who are on long-term azole therapy, as in CPA cases. Another concern is the missing data on the quantity and the types of antifungals used in agriculture, as Pakistan is an agricultural economy and there is high risk of excessive use.