title: Quantitative Analysis of Single-Nucleotide Polymorphism for Rapid Detection of TR34/L98H- and TR46/Y121F/T289A-Positive Aspergillus fumigatus Isolates Obtained from Patients in Iran from 2010 to 2014.
abstract: We employed an endpoint genotyping method to update the prevalence rate of positivity for the TR34/L98H mutation (a 34-bp tandem repeat mutation in the promoter region of the cyp51A gene in combination with a substitution at codon L98) and the TR46/Y121F/T289A mutation (a 46-bp tandem repeat mutation in the promoter region of the cyp51A gene in combination with substitutions at codons Y121 and T289) among clinical Aspergillus fumigatus isolates obtained from different regions of Iran over a recent 5-year period (2010 to 2014). The antifungal activities of itraconazole, voriconazole, and posaconazole against 172 clinical A. fumigatus isolates were investigated using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. For the isolates with an azole resistance phenotype, the cyp51A gene and its promoter were amplified and sequenced. In addition, using a LightCycler 480 real-time PCR system, a novel endpoint genotyping analysis method targeting single-nucleotide polymorphisms was evaluated to detect the L98H and Y121F mutations in the cyp51A gene of all isolates. Of the 172 A. fumigatus isolates tested, the MIC values of itraconazole (≥16 mg/liter) and voriconazole (>4 mg/liter) were high for 6 (3.5%). Quantitative analysis of single-nucleotide polymorphisms showed the TR34/L98H mutation in the cyp51A genes of six isolates. No isolates harboring the TR46/Y121F/T289A mutation were detected. DNA sequencing of the cyp51A gene confirmed the results of the novel endpoint genotyping method. By microsatellite typing, all of the azole-resistant isolates had genotypes different from those previously recovered from Iran and from the Dutch TR34/L98H controls. In conclusion, there was not a significant increase in the prevalence of azole-resistant A. fumigatus isolates harboring the TR34/L98H resistance mechanism among isolates recovered over a recent 5-year period (2010 to 2014) in Iran. A quantitative assay detecting a single-nucleotide polymorphism in the cyp51A gene of A. fumigatus is a reliable tool for the rapid screening and monitoring of TR34/L98H- and TR46/Y121F/T289A-positive isolates and can easily be incorporated into clinical mycology algorithms.
authors.affiliation: Department of Medical Mycology and Parasitology, School of Hygiene & Institute of Public Health Research, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Faezeh
authors.initials: F
authors.lastname: Mohammadi
authors.affiliation: Department of Medical Mycology and Parasitology, School of Hygiene & Institute of Public Health Research, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Seyed Jamal
authors.initials: SJ
authors.lastname: Hashemi
authors.affiliation: Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, the Netherlands.
authors.forename: Jan
authors.initials: J
authors.lastname: Zoll
authors.affiliation: Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, the Netherlands.
authors.forename: Willem J G
authors.initials: WJ
authors.lastname: Melchers
authors.affiliation: Department of Biochemistry, Erasmus University Medical Center, Rotterdam, the Netherlands.
authors.forename: Haleh
authors.initials: H
authors.lastname: Rafati
authors.affiliation: Department of Medical Mycology and Parasitology, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Parvin
authors.initials: P
authors.lastname: Dehghan
authors.affiliation: Department of Medical Mycology and Parasitology, School of Hygiene & Institute of Public Health Research, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Sasan
authors.initials: S
authors.lastname: Rezaie
authors.affiliation: Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Tolooe
authors.affiliation: Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
authors.forename: Yalda
authors.initials: Y
authors.lastname: Tamadon
authors.affiliation: Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, the Netherlands.
authors.forename: Henrich A
authors.initials: HA
authors.lastname: van der Lee
authors.affiliation: Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, the Netherlands.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Verweij
authors.affiliation: Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, the Netherlands Invasive Fungi Research Center, Mazandaran University of Medical Sciences, Sari, Iran Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center, Rotterdam, the Netherlands S.Seyedmousavi@gmail.com.
authors.forename: Seyedmojtaba
authors.identifier: http://orcid.org/0000-0002-6194-7447
authors.initials: S
authors.lastname: Seyedmousavi
mesh_terms: D000935:Antifungal Agents / Q000627:therapeutic use*; D001228:Aspergillosis / Q000188:drug therapy / Q000453:epidemiology / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics; D025141:Drug Resistance, Fungal / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007492:Iran / Q000453:epidemiology; D017964:Itraconazole / Q000627:therapeutic use; D008826:Microbial Sensitivity Tests; D018895:Microsatellite Repeats; D016533:Mycological Typing Techniques; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D012189:Retrospective Studies; D017422:Sequence Analysis, DNA; D014230:Triazoles / Q000627:therapeutic use; D065819:Voriconazole / Q000627:therapeutic use
chemical_list: D000935:Antifungal Agents; D004271:DNA, Fungal; D005656:Fungal Proteins; D014230:Triazoles; D017964:Itraconazole; C101425:posaconazole; D003577:Cytochrome P-450 Enzyme System; C465526:cytochrome P-450 CYP51A, Aspergillus; D065819:Voriconazole