Simple and rapid detection of TR34/L98H mutations in the Cyp51A gene of triazole-resistant Aspergillus fumigatus by tetra-primer ARMS-PCR technique
Sadegh Khodavaisy1, Shahram Mahmoudi1, Hamid Badali2, Mohammad kord1, Assad Azarnejad3,4
1 Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Medical Mycology and Parasitology, Invasive Fungi Research Center, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
3 Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
4 Cellular and Molecular Research Center, School of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Kurdistan, Iran

Introduction: Single nucleotide polymorphism (SNP) detection has been used extensively for genetic association studies. Alteration of the drug target (Cyp51A) is the principal mechanism of triazole resistance among Aspergillus fumigatus isolates. The most frequently characterized (hotspot) mutation in Cyp51A gene is at codons L98 accompanied by a tandem repeat of 34 base pairs in the 5´upstream region of cyp51A (TR34/L98H). Rapid identification of triazole-resistant point mutations is important for management of aspergillosis. Amplification-Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR) is an inexpensive, reliable and rapid way to investigation and identification of point mutations. In this study we evaluated detection of TR34/L98H mutations in the Cyp51A gene of triazole-resistant A. fumigatus by tetra-primer ARMS-PCR technique.
Materials and Methods: Reference A. fumigatus strains (10042001/02 and 10042003/04) carrying wild-type and mutant (TR34/L98H) were used for the establishment of ARMS-PCR assays. Optimization of ARMS-PCR was carried out in a step by step manner. In this technique, four primers in one reaction were done for amplification of indicative amplicons in wild-type and triazole-resistant A. fumigatus carrying TR34/L98H mutations. The assays were evaluated using 5 susceptible and 10 triazole resistant isolates.
Results: ARMS-PCR assay from reference triazole-resistant A. fumigatus isolate containing TR34/ L98H mutations at cyp51A yielded 942 bp & 212 bp DNA fragments.  PCR amplification from reference A. fumigatus isolates containing wild-type sequence yielded 904 bp & 741 bp DNA fragments. The DNA sequencing data confirmed the results of ARMS-PCR assays for all the isolates analyzed in this study. None of the A. fumigatus isolates lacking TR34/L98H mutations yielded false-positive results by ARMS-PCR assays.
Conclusion: ARMS-PCR assays developed in this study is a fast, easy, low cast, and user friendly method that may also help in rapid identification of azole resistant A. fumigatus carrying TR34/L98H mutations for proper management of patients with invasive aspergillosis in developing countries.
Key Words: A. fumigatus, Triazole-resistant, TR34/L98H, ARMS-PCR