Volume 19, Number 5—May 2013
Azole-Resistant Aspergillus fumigatus, Iran
To the Editor: Aspergillus fumigatus causes a variety of diseases in humans. The drugs recommended for treatment of Aspergillus diseases are the mold-active azole antifungal drugs (1). However, a wide range of mutations in A. fumigatus confer azole resistance, which commonly involves modifications in the cyp51A gene (2), the target for azole antifungal drugs.
Azole resistance is thought to be selected for as a result of patient therapy or exposure to azole compounds in the environment; resistance in clinical A. fumigatus isolates has been increasingly reported in several European countries, Asia, and the United States (3–7). The most frequently reported resistance mechanism is a 34-bp tandem repeat (TR34) in combination with a substitution at codon 98 (TR34/L98H) (4); this mechanism is believed to have been selected for through environmental exposure to azole fungicides.
Because routine in vitro susceptibility testing of clinical Aspergillus isolates is not common in many centers worldwide, the prevalence of azole resistance might be underestimated. We investigated the prevalence of azole resistance in clinical A. fumigatus isolates stored for 6 years (2003–2009) at Tehran University Mycology Reference Centre and Islamic Azad University, Ardabil Branch, Iran.
We investigated 124 clinical A. fumigatus isolates obtained from patients with Aspergillus diseases (Technical Appendix Table 1 [PDF - 160 KB - 4 pages]). We conducted strain identification, in vitro antifungal susceptibility testing, and sequence-based analysis of the Cyp51A gene, as described (4). We performed microsatellite genotyping of all A. fumigatus isolates for which the MIC of itraconazole was ≥16 mg/L (8) by using a short tandem repeat A. fumigatus assay, and we compared the results with those reported for the Netherlands (20 isolates) and other European countries (24 isolates) (Technical Appendix Figure [PDF - 160 KB - 4 pages]).
The distribution of azole-resistant and wild-type A. fumigatus isolates examined in this study, according to year of isolation, is shown in online Technical Appendix Table 1. Of 124 A. fumigatus isolates, 4 grew on the wells containing itraconazole and voriconazole, indicating a multidrug-resistant phenotype. Of these resistant isolates, 3 were from patients with chronic pulmonary aspergillosis and 1 was from a patient with allergic bronchopulmonary aspergillosis (Table).
Sequence analysis of the CYP51A gene indicated the presence of TR34/L98H in 3 isolates and no mutations in the other isolate (Table). The first TR34/L98H isolate had been recovered in 2005, which is relatively early compared with reported isolations in other countries (Technical Appendix Table 2 [PDF - 160 KB - 4 pages]). Microsatellite typing of 6 short tandem repeat loci demonstrated identical patterns for 2 of the 3 azole-resistant isolates from Iran, but the TR34/L98H isolates from Iran did not cluster with those from the Netherlands and other European countries, indicating no close genetic relatedness (Technical Appendix Figure [PDF - 160 KB - 4 pages]).
The TR34/L98H azole resistance mechanism was first described in 1998 in the Netherlands; since then, its presence in clinical and environmental A. fumigatus isolates in multiple European countries and recently in Asia has been increasingly reported (Technical Appendix Table 2 [PDF - 160 KB - 4 pages]) (3–7). In the study reported here, prevalence of azole resistance in clinical A. fumigatus isolates obtained from patients in Iran was 3.2%; most isolates exhibited the TR34/L98H resistance mechanism. The fact that the first TR34/L98H isolate was found relatively early, in 2005, underscores the possibility that prevalence of azole resistance might be underestimated in many countries because in vitro susceptibility testing of A. fumigatus is not routinely performed.
Microsatellite genotypic analysis of A. fumigatus isolates from the Netherlands and various European countries showed that the genetic diversity of TR34/L98H isolates is lower than that of wild-type controls (8). It has been suggested that TR34/L98H isolates might have a common ancestor that developed locally and subsequently migrated across Europe. In contrast, genotyping of TR34/L98H originating from India suggested a different dynamic; all environmental and clinical TR34/L98H isolates from India shared the same multilocus microsatellite genotype not found in any other analyzed samples, from within India or from the Netherlands, France, Germany, or the People’s Republic of China (9). The molecular epidemiology of the TR34/L98H isolates from Iran suggests that they cluster apart from the European isolates, indicating that migration from Europe to Iran, or vice versa, is unlikely. Genotyping of more TR34/L98H isolates from the Middle East and comparison with those from India would enhance understanding of the origin and geographic spread of TR34/L98H.
Our study indicates that TR34/L98H was in Iran in 2005; this finding adds to the growing list of regions where acquired resistance in A. fumigatus of environmental origin is documented. From a global perspective, fungicide use is second highest in the Asia–Pacific regions (24%), preceded only by western Europe (37%) (10). For a bettering understand of the scale of this emerging public health problem and for insight into the dynamics of geographic migration, surveys of fungal culture collections for TR34/L98H and molecular typing studies are warranted. These data would be useful not only for clinical management of Aspergillus diseases but also for enabling policy makers to develop strategies that prevent resistance selection by the environmental route.
We thank Jacques F. Meis and Ferry Hagen for performing additional real-time PCRs to screen mutations in 4 azole-resistant isolates.
This work was supported in part by a research grant from the Faculty of Medicine and Medical Sciences, Islamic Azad University, Ardabil Branch, Iran, and the School of Hygiene & Institute of Public Health Research, Tehran University of Medical Sciences, Tehran, Iran.
S.J.M., E.Z., M.T.H., J.Z., and W.J.G.M. have no conflicts of interest. S.S received a research grant from the Faculty of Medicine and Medical Sciences, Islamic Azad University, Ardabil Branch, Iran. J.W.M. and P.E.V. have served as consultants to and have received research grants from Astellas, Basilea, Gilead Sciences, Merck, and Pfizer.
- Walsh TJ, Anaissie EJ, Denning DW, Herbrecht R, Kontoyiannis DP, Marr KA, Treatment of aspergillosis: clinical practice guidelines of the Infectious Diseases Society of America. Clin Infect Dis. 2008;46:327–60 .
- Verweij PE, Howard SJ, Melchers WJ, Denning DW. Azole-resistance in Aspergillus: proposed nomenclature and breakpoints. Drug Resist Updat. 2009;12:141–7.
- Howard SJ, Cerar D, Anderson MJ, Albarrag A, Fisher MC, Pasqualotto AC, Frequency and evolution of azole resistance in Aspergillus fumigatus associated with treatment failure. Emerg Infect Dis. 2009;15:1068–76.
- Snelders E, van der Lee HA, Kuijpers J, Rijs AJ, Varga J, Samson RA, Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism. PLoS Med. 2008;5:e219.
- Mellado E, De La Camara R, Buendia B, Rodriguez-Tudela JL, Cuenca-Estrella M. Breakthrough pulmonary Aspergillus fumigatus infection with multiple triazole resistance in a Spanish patient with chronic myeloid leukemia. Rev Iberoam Micol. 2013;30:64–8.
- Lockhart SR, Frade JP, Etienne KA, Pfaller MA, Diekema DJ, Balajee SA. Azole resistance in Aspergillus fumigatus isolates from the ARTEMIS global surveillance study is primarily due to the TR/L98H mutation in the cyp51A gene. Antimicrob Agents Chemother. 2011;55:4465–8 .
- Rath PM, Buchheidt D, Spiess B, Arfanis E, Buer J, Steinmann J. First reported case of azole-resistant Aspergillus fumigatus due to the TR/L98H mutation in Germany. Antimicrob Agents Chemother. 2012;56:6060–1.
- Camps SM, Rijs AJ, Klaassen CH, Meis JF, O'Gorman CM, Dyer PS, Molecular epidemiology of Aspergillus fumigatus isolates harboring the TR34/L98H azole resistance mechanism. J Clin Microbiol. 2012;50:2674–80.
- Chowdhary A, Kathuria S, Xu J, Sharma C, Sundar G, Singh PK, Clonal expansion and emergence of environmental multiple-triazole-resistant Aspergillus fumigatus strains carrying the TR(34)/L98H mutations in the cyp51A gene in India. PLoS ONE. 2012;7:e52871.
- Stensvold CR, Jørgensen LN, Arendrup MC. Azole-resistant invasive aspergillosis: relationship to agriculture. Current Fungal Infection Reports. 2012;6:178–91.
Suggested citation for this article: Seyedmousavi S, Hashemi SJ, Zibafar E, Zoll J, Hedayati MT, Mouton JW, et al. Azole-resistant Aspergillus fumigatus, Iran [letter]. Emerg Infect Dis [Internet]. 2013 May [date cited]. http://dx.doi.org/10.3201/eid1905.130075