Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus

ABSTRACT The global emergence of azole-resistant Aspergillus fumigatus strains is a growing public health concern. Different patterns of azole resistance are linked to mutations in cyp51A. Therefore, accurate characterization of the mechanisms underlying azole resistance is critical to guide selection of the most appropriate antifungal agent for patients with aspergillosis. This study describes a new sequencing-free molecular screening tool for early detection of the most frequent mutations known to be associated with azole resistance in A. fumigatus. PCRs targeting cyp51A mutations at positions G54, Y121, G448, and M220 and targeting different tandem repeats (TRs) in the promoter region were designed. All PCRs were performed simultaneously, using the same cycling conditions. Amplicons were then distinguished using a high-resolution melting assay. For standardization, 30 well-characterized azole-resistant A. fumigatus strains were used, yielding melting curve clusters for different resistance mechanisms for each target and allowing detection of the most frequent azole resistance mutations, i.e., G54E, G54V, G54R, G54W, Y121F, M220V, M220I, M220T, M220K, and G448S, and the tandem repeats TR34, TR46, and TR53. Validation of the method was performed using a blind panel of 80 A. fumigatus azole-susceptible or azole-resistant strains. All strains included in the blind panel were properly classified as susceptible or resistant with the developed method. The implementation of this screening method can reduce the time needed for the detection of azole-resistant A. fumigatus isolates and therefore facilitate selection of the best antifungal therapy in patients with aspergillosis.

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Single-Center Evaluation of an Agar-Based Screening for Azole Resistance in Aspergillus fumigatus by Using VIPcheck

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01250-17 ◽

2017 ◽

Vol 61 (12) ◽

Cited By ~ 19

Author(s):

J. B. Buil ◽

H. A. L. van der Lee ◽

A. J. M. M. Rijs ◽

J. Zoll ◽

J. A. M. F. Hovestadt ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Sensitivity And Specificity ◽

Antifungal Susceptibility ◽

Screening Method ◽

Point Mutations ◽

Azole Resistance ◽

Minimal Growth ◽

Content Type ◽

Mean Sensitivity ◽

Selection Of

ABSTRACT Antifungal susceptibility testing is an essential tool for guiding therapy, although EUCAST and CLSI reference methods are often available only in specialized centers. We studied the performance of an agar-based screening method for the detection of azole resistance in Aspergillus fumigatus cultures. The VIPcheck consists of four wells containing voriconazole, itraconazole, posaconazole, or a growth control. Ninety-six A. fumigatus isolates were used. Thirty-three isolates harbored a known resistance mechanism: TR34/L98H (11 isolates), TR46/Y121F/T289A (6 isolates), TR53 (2 isolates), and 14 isolates with other cyp51A gene point mutations. Eighteen resistant isolates had no cyp51A-mediated azole resistance. Forty-five isolates had a wild-type (WT) azole phenotype. Four technicians and two inexperienced interns, blinded to the genotype/phenotype, read the plates visually after 24 h and 48 h and documented minimal growth, uninhibited growth, and no growth. The performance was compared to the EUCAST method. After 24 h of incubation, the mean sensitivity and specificity were 0.54 and 1.00, respectively, with uninhibited growth as the threshold. After 48 h of incubation, the performance mean sensitivity and specificity were 0.98 and 0.93, respectively, with minimal growth. The performance was not affected by observer experience in mycology. The interclass correlation coefficient was 0.87 after 24 h and 0.85 after 48 h. VIPcheck enabled the selection of azole-resistant A. fumigatus colonies, with a mean sensitivity and specificity of 0.98 and 0.93, respectively. Uninhibited growth on any azole-containing well after 24 h and minimal growth after 48 h were indicative of resistance. These results indicate that the VIPcheck is an easy-to-use tool for azole resistance screening and the selection of colonies that require MIC testing.

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Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae

Journal of Clinical Microbiology ◽

10.1128/jcm.03354-15 ◽

2016 ◽

Vol 54 (8) ◽

pp. 2074-2081 ◽

Cited By ~ 19

Author(s):

Valentina Donà ◽

Sara Kasraian ◽

Agnese Lupo ◽

Yuvia N. Guilarte ◽

Christoph Hauser ◽

...

Keyword(s):

High Resolution ◽

Antimicrobial Resistance ◽

Neisseria Gonorrhoeae ◽

Real Time ◽

Sensitivity And Specificity ◽

Real Time Pcr ◽

High Resolution Melting ◽

Health Concern ◽

Content Type ◽

Resistance To Antibiotics

Resistance to antibiotics used againstNeisseria gonorrhoeaeinfections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences forN. gonorrhoeaeidentification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterizedN. gonorrhoeaestrains, 19 commensalNeisseriaspecies strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identifiedN. gonorrhoeaeand the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcalNeisseriaspecies, and the detection limit was 103to 104genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing.

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Nonrandom Distribution of Azole Resistance across the Global Population of Aspergillus fumigatus

mBio ◽

10.1128/mbio.00392-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 22

Author(s):

Thomas R. Sewell ◽

Jianing Zhu ◽

Johanna Rhodes ◽

Ferry Hagen ◽

Jacques F. Meis ◽

...

Keyword(s):

Genetic Diversity ◽

Drug Resistance ◽

Aspergillus Fumigatus ◽

Pathogenic Fungus ◽

Crop Protection ◽

Resistance Mechanisms ◽

Health Concern ◽

Azole Resistance ◽

Content Type ◽

Global Population

ABSTRACT The emergence of azole resistance in the pathogenic fungus Aspergillus fumigatus has continued to increase, with the dominant resistance mechanisms, consisting of a 34-nucleotide tandem repeat (TR34)/L98H and TR46/Y121F/T289A, now showing a structured global distribution. Using hierarchical clustering and multivariate analysis of 4,049 A. fumigatus isolates collected worldwide and genotyped at nine microsatellite loci using analysis of short tandem repeats of A. fumigatus (STRAf), we show that A. fumigatus can be subdivided into two broad clades and that cyp51A alleles TR34/L98H and TR46/Y121F/T289A are unevenly distributed across these two populations. Diversity indices show that azole-resistant isolates are genetically depauperate compared to their wild-type counterparts, compatible with selective sweeps accompanying the selection of beneficial mutations. Strikingly, we found that azole-resistant clones with identical microsatellite profiles were globally distributed and sourced from both clinical and environmental locations, confirming that azole resistance is an international public health concern. Our work provides a framework for the analysis of A. fumigatus isolates based on their microsatellite profile, which we have incorporated into a freely available, user-friendly R Shiny application (AfumID) that provides clinicians and researchers with a method for the fast, automated characterization of A. fumigatus genetic relatedness. Our study highlights the effect that azole drug resistance is having on the genetic diversity of A. fumigatus and emphasizes its global importance upon this medically important pathogenic fungus. IMPORTANCE Azole drug resistance in the human-pathogenic fungus Aspergillus fumigatus continues to emerge, potentially leading to untreatable aspergillosis in immunosuppressed hosts. Two dominant, environmentally associated resistance mechanisms, which are thought to have evolved through selection by the agricultural application of azole fungicides, are now distributed globally. Understanding the effect that azole resistance is having on the genetic diversity and global population of A. fumigatus will help mitigate drug-resistant aspergillosis and maintain the azole class of fungicides for future use in both medicine and crop protection.

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One-Step Triplex High-Resolution Melting Analysis for Rapid Identification and Simultaneous Subtyping of Frequently Isolated Salmonella Serovars

Applied and Environmental Microbiology ◽

10.1128/aem.07668-11 ◽

2012 ◽

Vol 78 (9) ◽

pp. 3352-3360 ◽

Cited By ~ 15

Author(s):

Josef Zeinzinger ◽

Ariane T. Pietzka ◽

Anna Stöger ◽

Christian Kornschober ◽

Renate Kunert ◽

...

Keyword(s):

High Resolution ◽

Salmonella Enterica ◽

High Resolution Melting ◽

Melting Curve ◽

Cost Effective ◽

Melting Curve Analysis ◽

Nucleotide Polymorphisms ◽

Content Type ◽

High Resolution Melting Curve ◽

Gene Scanning

ABSTRACTSalmonellosis is one of the most important food-borne diseases worldwide. For outbreak investigation and infection control, accurate and fast subtyping methods are essential. A triplex gene-scanning assay was developed and evaluated for serotype-specific subtyping ofSalmonella entericaisolates based on specific single-nucleotide polymorphisms in fragments offljB,gyrB, andycfQ. Simultaneous gene scanning offljB,gyrB, andycfQby high-resolution melting-curve analysis of 417Salmonellaisolates comprising 46 different serotypes allowed the unequivocal, simple, and fast identification of 37 serotypes. Identical melting-curve profiles were obtained in some cases fromSalmonella entericaserotype Enteritidis andSalmonella entericaserotype Dublin, in all cases fromSalmonella entericaserotype Ohio andSalmonella entericaserotype Rissen, fromSalmonella entericaserotype Mbandaka andSalmonella entericaserotype Kentucky, and fromSalmonella entericaserotype Bredeney,Salmonella entericaserotype Give, andSalmonella entericaserotype Schwarzengrund. To differentiate the most frequentSalmonellaserotype, Enteritidis, from someS. Dublin isolates, an additional single PCR assay was developed for specific identification ofS. Enteritidis. The closed-tube triplex high-resolution melting-curve assay developed, in combination with anS. Enteritidis-specific PCR, represents an improved protocol for accurate, cost-effective, simple, and fast subtyping of 39Salmonellaserotypes. These 39 serotypes represent more than 94% of all human and more than 85% of all nonhumanSalmonellaisolates (including isolates from veterinary, food, and environmental samples) obtained in the years 2008 and 2009 in Austria.

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A Novel Combination of CYP51A Mutations Confers Pan-Azole Resistance in Aspergillus fumigatus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02501-19 ◽

2020 ◽

Vol 64 (8) ◽

Cited By ~ 5

Author(s):

Daiana Macedo ◽

Tomás Brito Devoto ◽

Santiago Pola ◽

Jorge L. Finquelievich ◽

María L. Cuestas ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Gene Replacement ◽

Azole Resistance ◽

New Mutation ◽

Content Type ◽

The Third ◽

Resistant Phenotype ◽

Intensive Use ◽

Selection Of

ABSTRACT The treatment of invasive and chronic aspergillosis involves triazole drugs. Its intensive use has resulted in the selection of resistant isolates, and at present, azole resistance in Aspergillus fumigatus is considered an emerging threat to public health worldwide. The aim of this work is to uncover the molecular mechanism implicated in the azole resistance phenotype of three Aspergillus fumigatus clinical strains isolated from an Argentinian cystic fibrosis patient under long-term triazole treatment. Strain susceptibilities were assessed, and CYP51A gene sequences were analyzed. Two of the studied Aspergillus fumigatus strains harbored the TR34-L98H allele. These strains showed high MIC values for all tested triazoles (>16.00 μg/ml, 1.00 μg/ml, 1.00 μg/ml, and 2.00 μg/ml for itraconazole, isavuconazole, posaconazole, and voriconazole, respectively). The third strain had a novel amino acid change (R65K) combined with the TR34-L98H mutations. This new mutation combination induces a pan-azole MIC augment compared with TR34-L98H mutants (>16 μg/ml, 4.00 μg/ml, 4.00 μg/ml, and 8.00 μg/ml for itraconazole, isavuconazole, posaconazole, and voriconazole, respectively). The strain harboring the TR34-R65K-L98H allele showed no inhibition halo when voriconazole susceptibility was evaluated by disk diffusion. The effect of these mutations in the azole-resistant phenotype was confirmed by gene replacement experiments. Transformants harboring the TR34-L98H and TR34-R65K-L98H alleles mimicked the azole-resistant phenotype of the clinical isolates, while the incorporation of the TR34-R65K and R65K alleles did not significantly increase azole MIC values. This is the first report of the TR34-L98H allele in Argentina. Moreover, a novel CYP51A allele (TR34-R65K-L98H) that induces a pan-azole MIC augment is described.

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First Description of Azole-Resistant Aspergillus fumigatus Due to TR46/Y121F/T289A Mutation in France

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00127-15 ◽

2015 ◽

Vol 59 (7) ◽

pp. 4331-4335 ◽

Cited By ~ 42

Author(s):

Rose-Anne Lavergne ◽

Florent Morio ◽

Loïc Favennec ◽

Stéphane Dominique ◽

Jacques F. Meis ◽

...

Keyword(s):

Public Health ◽

Cystic Fibrosis ◽

Aspergillus Fumigatus ◽

Environmental Samples ◽

Health Concern ◽

Azole Resistance ◽

Public Health Concern ◽

Content Type ◽

High Level ◽

Level Resistance

ABSTRACTAzole resistance inAspergillus fumigatusis an emerging public health concern. Recently, a novel fungicide-driven mutation in thecyp51Agene and its promoter, TR46/Y121F/T289A, leading to high-level resistance to voriconazole has been identified in The Netherlands, Belgium, Germany, Denmark, Tanzania, and India in both clinical and environmental samples. Here we report the first description ofA. fumigatuscarrying this mutation in France, in a cystic fibrosis patient, underlining the need for extensive monitoring ofAspergillusresistance.

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