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Multiplex real-time PCR assay with high-resolution melting analysis for characterization of antimicrobial resistance in neisseria gonorrhoeae
Institute for Infectious Diseases, University of Bern, Bern, Switzerland.
Institute for Infectious Diseases, University of Bern, Bern, Switzerland.
Institute for Infectious Diseases, University of Bern, Bern, Switzerland.
Institute for Infectious Diseases, University of Bern, Bern, Switzerland.
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2016 (English)In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 54, no 8, 2074-2081 p.Article in journal (Refereed) Published
Abstract [en]

Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detecting AMR determinants could provide valuable tools for surveillance, epidemiological studies and to inform individual case management. We developed a fast (<1.5 hrs) SYBR-green based real-time PCR method with high resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully-characterized N. gonorrhoeae strains, 19 commensal Neisseria spp., and an additional panel of 193 gonococcal isolates. Results were compared with culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with non-gonococcal Neisseria species and the detection limit was 10(3)-10(4) 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 and spectinomycin (both sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations generating 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 with current culture-based AMR testing.

Place, publisher, year, edition, pages
Washington, USA: American Society for Microbiology , 2016. Vol. 54, no 8, 2074-2081 p.
National Category
Microbiology
Research subject
Microbiology
Identifiers
URN: urn:nbn:se:oru:diva-50490DOI: 10.1128/JCM.03354-15ISI: 000381278200021PubMedID: 27225407Scopus ID: 2-s2.0-84979622689OAI: oai:DiVA.org:oru-50490DiVA: diva2:931843
Note

Funding Agency:

SwissTransMed (RaDAR-Go)

Available from: 2016-05-30 Created: 2016-05-30 Last updated: 2017-10-17Bibliographically approved

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