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. 2015 Nov 2;60(1):387-92.
doi: 10.1128/AAC.02326-15. Print 2016 Jan.

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

Faezeh Mohammadi  1 , Seyed Jamal Hashemi  1 , Jan Zoll  2 , Willem J G Melchers  2 , Haleh Rafati  3 , Parvin Dehghan  4 , Sasan Rezaie  1 , Ali Tolooe  5 , Yalda Tamadon  5 , Henrich A van der Lee  2 , Paul E Verweij  2 , Seyedmojtaba Seyedmousavi  6
Affiliations

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

Faezeh Mohammadi et al. Antimicrob Agents Chemother. .

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.

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Figures

FIG 1
FIG 1
Endpoint fluorescence plot of single-nucleotide variance for detection of the L98H mutation in clinical Aspergillus fumigatus isolates using a quantitative PCR assay. Relative L98 (6-carboxyfluorescein [6FAM]) and L98H (cyanine 5 [Cy5]) fluorescence levels are plotted on the y and x axes, respectively. Blue diamonds, control isolates; purple circles, clinical A. fumigatus isolates without a mutation in the cyp51A gene at L98; black circles, clinical A. fumigatus isolates harboring the L98H substitution in the cyp51A gene; X, nuclease-free water, which was used as a negative control.
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