A Simple Method To Detect Point Mutations in Aspergillus fumigatus
            <i>cyp51A</i>
            Gene Using a Surveyor Nuclease Assay

Arai, Teppei; Majima, Hidetaka; Watanabe, Akira; Kamei, Katsuhiko

doi:10.1128/aac.02271-19

Cited by 12 publications

(13 citation statements)

References 26 publications

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“…The advantage of pyrosequencing over newer sequencing approaches is the determination of definitive sequence information rather than a reliance on the restriction pattern differences generated by surveyor nucleases and detected by gel electrophoresis. 54 …”

Section: Discussionmentioning

confidence: 99%

Deciphering Aspergillus fumigatus cyp51A-mediated triazole resistance by pyrosequencing of respiratory specimens

Frazer

Anees-Hill

Hassan

et al. 2020

Journal of Antimicrobial Chemotherapy

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Background Infections caused by triazole drug-resistant Aspergillus fumigatus are an increasing problem. The sensitivity of standard culture is poor, abrogating susceptibility testing. Early detection of resistance can improve patient outcomes, yet tools for this purpose are limited. Objectives To develop and validate a pyrosequencing technique to detect resistance-conferring cyp51A polymorphisms from clinical respiratory specimens and A. fumigatus isolates. Methods Method validation was performed by Sanger sequencing and pyrosequencing of 50 A. fumigatus isolates with a spectrum of triazole susceptibility patterns. Then, 326 Aspergillus quantitative PCR (qPCR)-positive respiratory samples collected over a 27 month period (January 2017–March 2019) from 160 patients at the UK National Aspergillosis Centre were assessed by cyp51A pyrosequencing. The Sanger sequencing and pyrosequencing results were compared with those from high-volume culture and standard susceptibility testing. Results The cyp51A genotypes of the 50 isolates analysed by pyrosequencing and Sanger sequencing matched. Of the 326 Aspergillus qPCR-positive respiratory specimens, 71.2% were reported with no A. fumigatus growth. Of these, 56.9% (132/232) demonstrated a WT cyp51A genotype and 31.5% (73/232) a resistant genotype by pyrosequencing. Pyrosequencing identified the environmental TR34/L98H mutation in 18.7% (61/326) of the samples in contrast to 6.4% (21/326) pan-azole resistance detected by culture. Importantly, pyrosequencing detected resistance earlier than culture in 23.3% of specimens. Conclusions The pyrosequencing assay described could detect a wide range of cyp51A polymorphisms associated with triazole resistance, including those not identified by commercial assays. This method allowed prompt recognition of resistance and the selection of appropriate antifungal treatment when culture was negative.

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Section: Discussionmentioning

confidence: 99%

Deciphering Aspergillus fumigatus cyp51A-mediated triazole resistance by pyrosequencing of respiratory specimens

Frazer

Anees-Hill

Hassan

et al. 2020

Journal of Antimicrobial Chemotherapy

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“…Quantitative PCRs with molecular beacons were also widely used to detect resistance to itraconazole and other triazoles (voriconazole, posaconazole and ravuconazole) by targeting the codons 54, 98, 138 and 220 (Balashov et al, 2005;Garcia-Effron et al, 2008). Recently, a surveyor nuclease assay was also developed to rapidly detect CYP51A point mutations in resistant A. fumigatus isolates (Arai et al, 2020). Interestingly, detection of mutations linked to azole resistance can be performed either on Aspergillus cultures or directly from clinical samples.…”

Section: Molecular Methods For Susceptibility Testing In Molds Azole Resistancementioning

confidence: 99%

“…Real-time PCR methods have been developed for that purpose, and for instance, a TR34/L98H mutation in A. fumigatus was found within a brain biopsy and sputum samples (van der Linden et al, 2010;Zhao et al, 2016a). Techniques based on pyrosequencing of A. fumigatus DNA were also developed to assess azole resistance (van der Linden et al, 2010;Arai et al, 2020), and were optimized recently to be used directly on clinical respiratory samples (Chong et al, 2015;Dannaoui et al, 2017).…”

Section: Molecular Methods For Susceptibility Testing In Molds Azole Resistancementioning

confidence: 99%

Can We Improve Antifungal Susceptibility Testing?

Durand

Maubon

Cornet

et al. 2021

Front. Cell. Infect. Microbiol.

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Systemic antifungal agents are increasingly used for prevention or treatment of invasive fungal infections, whose prognosis remains poor. At the same time, emergence of resistant or even multi-resistant strains is of concern as the antifungal arsenal is limited. Antifungal susceptibility testing (AFST) is therefore of key importance for patient management and antifungal stewardship. Current AFST methods, including reference and commercial types, are based on growth inhibition in the presence of an antifungal, in liquid or solid media. They usually enable Minimal Inhibitory Concentrations (MIC) to be determined with direct clinical application. However, they are limited by a high turnaround time (TAT). Several innovative methods are currently under development to improve AFST. Techniques based on MALDI-TOF are promising with short TAT, but still need extensive clinical validation. Flow cytometry and computed imaging techniques detecting cellular responses to antifungal stress other than growth inhibition are also of interest. Finally, molecular detection of mutations associated with antifungal resistance is an intriguing alternative to standard AFST, already used in routine microbiology labs for detection of azole resistance in Aspergillus and even directly from samples. It is still restricted to known mutations. The development of Next Generation Sequencing (NGS) and whole-genome approaches may overcome this limitation in the near future. While promising approaches are under development, they are not perfect and the ideal AFST technique (user-friendly, reproducible, low-cost, fast and accurate) still needs to be set up routinely in clinical laboratories.

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“…We determined MICs by the Clinical and Laboratory Institute (CLSI) M38‐E3 broth microdilution method, 25 with slight modifications as described previously 26 . In brief, we measured MICs against itraconazole (ITCZ) and voriconazole (VRCZ) in RPMI 1640 medium (pH 7.0) at 35°C using EIKEN plates (Eiken Chemicals).…”

Section: Methodsmentioning

confidence: 99%

Genetic differences between Japan and other countries in cyp51A polymorphisms of Aspergillus fumigatus

Majima

Arai

Kusuya

et al. 2021

Mycoses

Self Cite

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Background: Mutations in cyp51A gene are known as main mechanisms of azole resistance in Aspergillus fumigatus, whereas azole-susceptible strains also carry cyp51A mutations (polymorphisms). The polymorphisms found in Europe mainly consist of two combinations of mutations, that is combinations of five single-nucleotide polymorphisms (SNPs) of cyp51A, referred to as cyp51A-5SNPs, and combinations of three SNPs of cyp51A, referred to as cyp51A-3SNPs. Few studies have compared the distributions of cyp51A polymorphisms between different regions. Objectives:The aim of this study was to investigate the regional differences of cyp51A polymorphisms. Methods:We compared the proportions of cyp51A polymorphisms in clinical and environmental strains isolated in various countries, and analysed the strains phylogenetically using short tandem repeats (STRs) and whole-genome sequence (WGS).Results: Among the Japanese strains, 15 out of 98 (15.3%) clinical strains and 8 out of 95 (8.4%) environmental strains had cyp51A polymorphisms. A mutation of cyp-51A N248K was the most prevalent polymorphism in both clinical (n = 14, 14.3%) and environmental strains (n = 3, 3.2%). Only one environmental strain harboured cyp51A-5SNPs, which was reported to be the most prevalent in Europe. For phylogenetic analyses using STRs and WGS, 183 and 134 strains, respectively, were employed.They showed that most of the strains with cyp51A N248K clustered in the clades different from those of the strains with cyp51A-5SNPs and cyp51A-3SNPs as well as from those with TR 34 /L98H mutations.Conclusions: This study suggests that there are genetic differences between cyp51A polymorphisms of A. fumigatus in Japan and Europe.

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A Simple Method To Detect Point Mutations in Aspergillus fumigatus cyp51A Gene Using a Surveyor Nuclease Assay

Cited by 12 publications

References 26 publications

Deciphering Aspergillus fumigatus cyp51A-mediated triazole resistance by pyrosequencing of respiratory specimens

Deciphering Aspergillus fumigatus cyp51A-mediated triazole resistance by pyrosequencing of respiratory specimens

Can We Improve Antifungal Susceptibility Testing?

Genetic differences between Japan and other countries in cyp51A polymorphisms of Aspergillus fumigatus

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