Objective: Invasive aspergillosis (IA), mainly caused by Aspergillus fumigatus, has emerged as a life-threatening infection in immunocompromised patients. Triazoles are the most effective drugs for treatment of IA. Triazole-resistant A. fumigatus strains have been isolated from both clinical specimens and environmental sources and the dominant resistance mechanism involves TR34/L98H mutations in cyp51A. This study developed low-cost PCR and PCR-RFLP assays targeting the promoter and codon 98 regions for rapid detection of triazole-resistant A. fumigatus isolates carrying TR34/L98H mutations for resource-poor settings. Methods: Reference A. fumigatus strains carrying wild-type and mutant sequences in promoter region and codon 98 in cyp51A (cyp51A98) and 40 itraconazole-susceptible and 35 itraconazole-resistant clinical and environmental isolates were used. Drug susceptibility testing of A. fumigatus isolates to triazoles was determined by Etest. PCR assay targeting TR34 region was developed to yield varying amplicons that could be easily resolved by 2.5% agarose gels. The L98H mutation was detected by PCR amplification of cyp51A98 region followed by restriction digestion of amplicons with AluI to yield different RFLP patterns. Results were confirmed by direct DNA sequencing of respective gene fragments from selected isolates. Results: PCR amplification of promoter region yielded distinct 105 bp and 139 bp amplicons in agarose gels from isolates containing wild-type sequences and tandem-repeats (TR34), respectively. PCR-RFLP assay from all 40 itraconazole-susceptible isolates yielded three (71 bp, 90 bp and 189 bp) fragments indicating wild-type sequence at cyp51A98. PCR-RFLP assay from 32 itraconazole-resistant isolates yielded two (71 bp and 279 bp) fragments indicating L98H mutation while 3 isolates yielded wild-type pattern at cyp51A98. The latter 3 isolates contained a mutation either at codon 54 or codon 220. All 32 itraconazole-resistant isolates with L98H mutation at cyp51A98 yielded 139 bp amplicons indicating the presence of TR34 while the remaining 3 itraconazole-resistant and all 40 itraconazole-susceptible isolates yielded 105 bp amplicons indicating wild-type sequence in the promoter region. The results were confirmed by direct DNA sequencing of respective gene fragments from selected isolates. Conclusions: Simple, low-cost methods for molecular detection of TR34/L98H mutations in cyp51A gene have been developed for rapid identification of triazole-resistant A. fumigatus isolates in resource-poor settings. The methods will help in determining the prevalence of TR34/L98H mutations in cyp51A gene in clinical and environmental A. fumigatus isolates and may also help in proper management of patients with IA. Supported by KURS grant MI 01/09.
- ECCMID 24th (2014)