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 (). However, a wide range of mutations in A. fumigatus confer azole resistance, which commonly involves modifications in the cyp51A gene (), 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 (–). The most frequently reported resistance mechanism is a 34-bp tandem repeat (TR34) in combination with a substitution at codon 98 (TR34/L98H) (); 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). We conducted strain identification, in vitro antifungal susceptibility testing, and sequence-based analysis of the Cyp51A gene, as described (). We performed microsatellite genotyping of all A. fumigatus isolates for which the MIC of itraconazole was ≥16 mg/L () 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).

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).

Table

Characteristics of 4 azole-resistant clinical Aspergillus fumigatus isolates, Iran*

Isolate	Underlying disease	Previous azole exposure	34-bp tandem repeat†	Amino acid substitution in cyp51A gene‡
					MIC, mg/L
					Amphotericin B	Itraconazole	Voriconazole	Posaconazole
T-IR-AF 12	CPA	Yes	Positive	L98H	0.5	≥16	4.0	0.5
T-IR-AF 17	CPA	No	Positive	L98H	0.5	≥16	4.0	0.5
T-IR-AF 433	CPA	Yes	Negative	ND	0.5	≥16	8.0	0.5
T-IR-AF 890	ABPA	No	Positive	L98H	0.5	≥16	8.0	0.25

*CPA, chronic pulmonary aspergillosis; ND, not detected; ABPA, allergic bronchopulmonary aspergillosis.
†34-bp tandem repeat in the promoter region of CYP51A gene.
‡The numbers indicate the position at which an amino acid change occurs. Nucleotides are numbered from the translation start codon ATG of cyp51A.

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). 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).

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) (–). 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 (). 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 (). 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%) (). 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.

Technical Appendix

Distribution of azole-resistant and azole-susceptible Aspergillus fumigatus isolates, Iran, 2003–2009; first reports of multiple-triazole-resistant A. fumigatus isolate(s) carrying the TR34/L98H mutations in the CYP51A gene, by country; and minimum spanning tree comparing genotypic relatedness of clinical azole-resistant A. fumigatus isolates carrying TR34/L98H alteration in the CYP51A gene from Iran with those reported from European countries. TR, tandem repeat.