Novel Genotyping and Quantitative Analysis of Neuraminidase Inhibitor Resistance-Associated Mutations in Influenza A Viruses by Single-Nucleotide Polymorphism Analysis

Duan, Susu; Boltz, David A.; Li, Jiang; Oshansky, Christine M.; Marjuki, Henju; Barman, Subrata; Webby, Richard J.; Webster, Robert G.; Govorkova, Elena A.

doi:10.1128/aac.00316-11

Cited by 22 publications

(25 citation statements)

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“…In the current study, we employed for the first time a rapid and simple one-step endpoint genotyping quantitative PCR assay (11)(12)(13)(14)(15)(16)(17)(18)(19)(20) to detect the L98H and Y121F mutations in TR 34 /L98H-and TR 46 /Y121F/T289A-positive azole-resistant A. fumigatus isolates. Interestingly, all of the A. fumigatus isolates in which the L98H mutation was confirmed by PCR sequencing of the cyp51A gene were correctly found to be mutated from the endpoint fluorescence plots (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…1). The quantitative SNP assay used in the current study is based on the competition between probes detecting the wild type and the mutants (11,(16)(17)(18)44). Endpoint measurements of the fluorescent signal for the mutant probe versus that for the wild-type probe were used for target detection and SNP discrimination (16)(17)(18)(19)(20).…”

Section: Discussionmentioning

confidence: 99%

“…In this assay, an extension probe can be simply designed to anneal to the template in a posi-tion that places the mutation site immediately adjacent to the 3= end of the probe, and the use of dideoxynucleoside triphosphates (ddNTPs) allows the extension of only 1 nucleotide from the 3= end of the probe. Labeling of each ddNTP with a different fluorescent dye allows the differentiation of the genotype at the SNP by the color of the extended probes (11)(12)(13)(14)(15)(16)(17)(18)(19)(20).…”

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confidence: 99%

“…The quantitative analysis of SNPs has been a reliable method in diagnostic microbiology for identification of a single nucleotide in the genomes of humans (11)(12)(13)(14)(15), viruses (16)(17)(18)(19)(20), and bacteria (18). In this assay, an extension probe can be simply designed to anneal to the template in a posi-tion that places the mutation site immediately adjacent to the 3= end of the probe, and the use of dideoxynucleoside triphosphates (ddNTPs) allows the extension of only 1 nucleotide from the 3= end of the probe.…”

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Quantitative Analysis of Single-Nucleotide Polymorphism for Rapid Detection of TR ₃₄ /L98H- and TR ₄₆ /Y121F/T289A-Positive Aspergillus fumigatus Isolates Obtained from Patients in Iran from 2010 to 2014

Mohammadi

Hashemi

Zoll

et al. 2016

Antimicrob Agents Chemother

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gWe employed an endpoint genotyping method to update the prevalence rate of positivity for the TR 34 /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 TR 46 A zole resistance in Aspergillus fumigatus is a global and evolving public health threat which translates into treatment failure (1). Surveillance studies indicate that the incidence of azole resistance is increasing (2-6), with the TR 34 /L98H mutation (a 34-bp tandem repeat mutation in the promoter region of the cyp51A gene in combination with a substitution at codon L98) emerging in multiple European countries and in the Middle East, Asia, and Africa and with a new resistance mechanism, the TR 46 /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), emerging more recently in Europe and India (2-6). We also previously reported the occurrence of the TR 34 /L98H mutation in 3.2% of clinical Aspergillus fumigatus isolates obtained from patients in Iran to the end of 2009 (5).The trend of increases in the rates of azole resistance among A. fumigatus isolates in different regions and patient groups exemplifies the fact that knowledge of the (local) epidemiology of azoleresistant Aspergillus diseases is important for clinical mycology/ microbiology reference laboratories (7-9). Moreover, rapid and specific molecular methods for the identification of the recently identified azole-resistant A. fumigatus strains can significantly influence a timely decision on patient management (10).In our search for a novel, rapid, sensitive, accurate, and highthroughput method for detection and screening of azole resistance in A. fumigatus, we found that endpoint genotyping targeting a single-nucleotide polymorphism (SNP) in the cyp51A gene could provide an option. The quantitative analysis of SNPs has been a reliable method in diagnostic microbiology for identification of a single nucleotide in the genomes of humans (11-15), viruses (16-20), and bacteria (18). In this assay, an extension probe can be simply designed to anneal to the template in a posi-

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Quantitative Analysis of Single-Nucleotide Polymorphism for Rapid Detection of TR ₃₄ /L98H- and TR ₄₆ /Y121F/T289A-Positive Aspergillus fumigatus Isolates Obtained from Patients in Iran from 2010 to 2014

Mohammadi

Hashemi

Zoll

et al. 2016

Antimicrob Agents Chemother

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“…Baby Hamster Kidney (BHK) cells were kindly provided by Dr. Charles Russell and maintained as described previously (137). The Normal Human Bronchial Epithelial (NHBE) cells (Lonza, Basel, Switzerland) were cultured on an air-liquid interface in 12-well plates and the infection with influenza viruses was described as previously (138). For growth curve, the inoculums were added to the apical surface of cells and were removed after 1 h of incubation at 37 C. Apical supernatants containing progeny viruses were collected by adding fresh medium into apical compartment 30 mins prior to the indicated time point and stored at -70°C for further titration in MDCK cells All the cell lines were maintained in a humidified atmosphere of 5% CO 2 at 37 ºC.…”

Section: Cellsmentioning

confidence: 99%

The Molecular Basis of Fitness and Transmissibility of Neuraminidase Inhibitor Resistant Influenza A Viruses

Duan¹

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DEDICATIONThis dissertation is dedicated to my parents and my own family who have been always giving me unconditional love, indissoluble strength, endless inspiration. iv ACKNOWLEDGEMENTSAll of this would not have been possible had Dr. Robert G. Webster not taken me as his graduate student. I will always remember my first conversation with Dr. Webster about my rotating his lab and the first conversation with Dr. Webster at the day I officially joined the lab: "Work hard, young people." I have been doing nothing but following this simple instruction through all my studies during these years. I will be eternally grateful for Dr. Webster for having faith in me to take me as his student and mentoring my Ph.D study. I thank Dr. Webster for being my mentor in every way, for always giving me the biggest vision about the research and career, for teaching me to start strong and end strong a scientific story, for enlightening me with the most important and critical questions for now and for future in the world of influenza virology.This dissertation would not have been completed in its present way without Dr. Elena G. Govorkova's supervision. The greatest appreciation is to Elena as my supervisor in all kinds of trainings: from introducing me the knowledge of the antiviral field, to helping me experiments in person, to supporting me to develop different projects, to teaching me to write manuscripts, to educating me to be a philosopher, and always reminding me to look pretty as a woman. I will always value all these precious training and experience.Special deep thanks to Dr. Richard J. Webby for providing me different training opportunities in other aspects of influenza virology, for giving me different visions to see different questions. Especially many thanks for his biggest push to greatly move forward the second study in this thesis by adding new angles and much more depth.Special thanks to my committee members, Dr. Paul Thomas, Dr. Michael Whitt and Dr. Tony Marion for giving me different perspectives to look the virology field, for always teaching me to think questions harder, deeper and more precisely.Special thanks to Scott Krauss for his general support for all studies.Special thanks to Dr. Subrata Barman and Dr. Charles Russell for kindly providing related experimental materials and working instructions.Many thanks to a former postdoctor fellow in our lab, Dr. David Boltz, for being a good teacher and good friend to me, for introducing me the influenza genome analysis, for helping me to develop the methodology project and for giving me encouragement to through the ups and downs of the studies. Many thanks to another former postdoctor fellow, Dr. Hui-ling Yen, for also being a great teacher and friend to me, for her unique comments and inspiring discussion on many influenza questions and for her dear encouragement for me all the time. Many thanks to other current and former postdoctor fellows in our flu group for helping me with their respective expertise and those include Dr. Hassan, Zarakat, Dr. Henju Marjuki, D...

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Susceptibility Test Methods: Viruses

Pinsky

2023

ClinMicroNow

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Testing for viral susceptibility, primarily via genotypic drug resistance mutation testing, is now standard practice for the management of viral infections for optimum patient care. Antiviral resistance is expressed as a drop in the efficacy of a drug to inhibit viral replication. The virus being treated is inherently resistant to the drug(s) being given. Alternatively, host biological and socio‐behavioral factors or inadequate dosing may influence therapeutic success. Host physiologic factors can interfere with a successful clinical response. Viral susceptibility testing can identify resistance and guide patient management strategies. The clinical category and status of the patient are important in determining whether antiviral resistance testing is indicated. Genotypic viral susceptibility testing requires knowledge of the mutations that confer antiviral resistance. FDA‐cleared genotypic antiviral resistance tests include software to analyze the raw sequence and render a resistance interpretation. The pharmaceutical industry is actively investigating and developing new antiviral drugs.

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Novel Genotyping and Quantitative Analysis of Neuraminidase Inhibitor Resistance-Associated Mutations in Influenza A Viruses by Single-Nucleotide Polymorphism Analysis

Cited by 22 publications

References 39 publications

Quantitative Analysis of Single-Nucleotide Polymorphism for Rapid Detection of TR ₃₄ /L98H- and TR ₄₆ /Y121F/T289A-Positive Aspergillus fumigatus Isolates Obtained from Patients in Iran from 2010 to 2014

Quantitative Analysis of Single-Nucleotide Polymorphism for Rapid Detection of TR ₃₄ /L98H- and TR ₄₆ /Y121F/T289A-Positive Aspergillus fumigatus Isolates Obtained from Patients in Iran from 2010 to 2014

The Molecular Basis of Fitness and Transmissibility of Neuraminidase Inhibitor Resistant Influenza A Viruses

Susceptibility Test Methods: Viruses

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Novel Genotyping and Quantitative Analysis of Neuraminidase Inhibitor Resistance-Associated Mutations in Influenza A Viruses by Single-Nucleotide Polymorphism Analysis

Cited by 22 publications

References 39 publications

Quantitative Analysis of Single-Nucleotide Polymorphism for Rapid Detection of TR 34 /L98H- and TR 46 /Y121F/T289A-Positive Aspergillus fumigatus Isolates Obtained from Patients in Iran from 2010 to 2014

Quantitative Analysis of Single-Nucleotide Polymorphism for Rapid Detection of TR 34 /L98H- and TR 46 /Y121F/T289A-Positive Aspergillus fumigatus Isolates Obtained from Patients in Iran from 2010 to 2014

The Molecular Basis of Fitness and Transmissibility of Neuraminidase Inhibitor Resistant Influenza A Viruses

Susceptibility Test Methods: Viruses

Contact Info

Product

Resources

About

Quantitative Analysis of Single-Nucleotide Polymorphism for Rapid Detection of TR ₃₄ /L98H- and TR ₄₆ /Y121F/T289A-Positive Aspergillus fumigatus Isolates Obtained from Patients in Iran from 2010 to 2014

Quantitative Analysis of Single-Nucleotide Polymorphism for Rapid Detection of TR ₃₄ /L98H- and TR ₄₆ /Y121F/T289A-Positive Aspergillus fumigatus Isolates Obtained from Patients in Iran from 2010 to 2014