Detecting Azole-Antifungal Resistance in Aspergillus fumigatus by Pyrosequencing

Torre, Mireille H van der; Frazer, Lilyann Novak; Rautemaa‐Richardson, Riina

doi:10.3390/jof6010012

Cited by 23 publications

(21 citation statements)

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“…Pyrosequencing is an alternative method, with the potential to screen for all known mutations within specific target genes and has been successfully applied to the testing of respiratory samples from patients with chronic pulmonary aspergillosis [ 28 , 29 ]. A surveyor nuclease assay has also been designed to detect point mutations associated with azole resistance in the cyp51A gene of A. fumgiatus [ 30 ].…”

Section: Advances In Molecular Diagnosticsmentioning

confidence: 99%

Recent Advances and Novel Approaches in Laboratory-Based Diagnostic Mycology

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2021

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What was once just culture and microscopy the field of diagnostic mycology has significantly advanced in recent years and continues to incorporate novel assays and strategies to meet the changes in clinical demand. The emergence of widespread resistance to antifungal therapy has led to the development of a range of molecular tests that target mutations associated with phenotypic resistance, to complement classical susceptibility testing and initial applications of next-generation sequencing are being described. Lateral flow assays provide rapid results, with simplicity allowing the test to be performed outside specialist centres, potentially as point-of-care tests. Mycology has responded positively to an ever-diversifying patient population by rapidly identifying risk and developing diagnostic strategies to improve patient management. Nowadays, the diagnostic repertoire of the mycology laboratory employs classical, molecular and serological tests and should be keen to embrace diagnostic advancements that can improve diagnosis in this notoriously difficult field.

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Section: Advances In Molecular Diagnosticsmentioning

confidence: 99%

Recent Advances and Novel Approaches in Laboratory-Based Diagnostic Mycology

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2021

JoF

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“…However, resistance acquisition in A. fumigatus sensu stricto is emerging due to selective pressure caused by the prolonged azole treatment of chronic aspergillosis patients (that can range from several weeks to years, or even a patient’s full lifetime) or due to environmental selective pressure. The mechanisms of azole resistance are often associated with mutations in genes involved in the A. fumigatus ergosterol pathway [ 25 ], particularly in the cyp51A gene which encodes the cytochrome P450 14-α-lanosterol demethylase, the main target of azole antifungals [ 26 ]. Point mutations within the cyp51A gene (G54, M220) are more frequently associated with prolonged azole prophylaxis/therapy [ 27 , 28 ].…”

Section: Introductionmentioning

confidence: 99%

“…Point mutations within the cyp51A gene (G54, M220) are more frequently associated with prolonged azole prophylaxis/therapy [ 27 , 28 ]. The most common pan-azole resistance mutation is a combination of a 34-bp long tandem repeat in the promoter region and a leucine-to-histidine substitution in codon 98, TR 34 /L98H [ 25 , 29 , 30 ]. This mutation, firstly described in Dutch A. fumigatus isolates, is now spread worldwide [ 31 ] due to extensive use of azole fungicides in animal, agricultural, and processing industries.…”

Section: Introductionmentioning

confidence: 99%

“…After infection of azole-naïve individuals with these resistant strains, subsequent treatment failure with triazole therapy (the first choice for treatment and prophylaxis of aspergillosis) may occur [ 34 ]. Consequently, higher morbidity and mortality rates associated with azole resistance are likely to become a major public health concern [ 25 , 29 , 34 , 35 ].…”

Section: Introductionmentioning

confidence: 99%

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Azole-Resistant Aspergillus fumigatus Harboring the TR34/L98H Mutation: First Report in Portugal in Environmental Samples

et al. 2020

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Introduction: The frequency in detection of azole-resistant Aspergillus fumigatus isolates has increased since 2010. In Portugal, the section Fumigati is one of the most frequent, and resistant strains to have been found in clinical and environmental contexts. Although several cryptic species within the Fumigati section show intrinsic resistance to azoles, one factor driving (acquired) resistance is selective pressure deriving from the extensive use of azoles. This is particularly problematic in occupational environments where high fungal loads are expected, and where there is an increased risk of human exposure and infection, with impact on treatment success and disease outcome. The mechanisms of resistance are diverse, but mainly associated with mutations in the cyp51A gene. Despite TR34/L98H being the most frequent mutation described, it has only been detected in clinical specimens in Portugal. Methods: We analyzed 99 A. fumigatus isolates from indoor environments (healthcare facilities, spas, one dairy and one waste sorting unit) collected from January 2018 to February 2019 in different regions of Portugal. Isolates were screened for resistance to itraconazole, voriconazole and posaconazole by culture, and resistance was confirmed by broth microdilution. Sequencing of the cyp51A gene and its promoter was performed to detect mutations associated with resistance. Results: Overall, 8.1% of isolates were able to grow in the presence of at least one azole, and 3% (isolated from the air in a dairy and from filtering respiratory protective devices in a waste sorting industry) were pan-azole-resistant, bearing the TR34/L98H mutation. Conclusion: For the first time in Portugal, we report environmental isolates bearing the TR34/L98H mutation, isolated from occupational environments. Environmental surveillance of the emergence of azole-resistant A. fumigatus sensu stricto strains is needed, to ensure proper and timely implementation of control policies that may have a positive impact on public and occupational health.

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“…We report the design and validation of a pyrosequencing method to determine resistance-conferring polymorphisms within the cyp51A region of A. fumigatus 38 from isolates and clinical respiratory samples. This assay was then used to screen respiratory samples from patients attending the UK National Aspergillosis Centre (NAC) to determine its utility to detect triazole resistance compared with high-volume culture (HVC) 29 and routine susceptibility testing.…”

Section: Introductionmentioning

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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Detecting Azole-Antifungal Resistance in Aspergillus fumigatus by Pyrosequencing

Cited by 23 publications

References 145 publications

Recent Advances and Novel Approaches in Laboratory-Based Diagnostic Mycology

Recent Advances and Novel Approaches in Laboratory-Based Diagnostic Mycology

Azole-Resistant Aspergillus fumigatus Harboring the TR34/L98H Mutation: First Report in Portugal in Environmental Samples

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

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