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

Donà, Valentina; Kasraian Fard, Sara; Lupo, Agnese; Guilarte, Yuvia N; Hauser, Christoph; Furrer, Hansjakob; Unemo, Magnus; Low, Nicola; Endimiani, Andrea (2016). A Multiplex Real-Time PCR with High Resolution Melting Analysis for the Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae. Journal of clinical microbiology, 54(8), JCM.03354-15. American Society for Microbiology 10.1128/JCM.03354-15

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Service Sector > Institute for Infectious Diseases
04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Social and Preventive Medicine
04 Faculty of Medicine > Department of Haematology, Oncology, Infectious Diseases, Laboratory Medicine and Hospital Pharmacy (DOLS) > Clinic of Infectiology

UniBE Contributor:

Donà, Valentina; Kasraian Fard, Sara; Lupo, Agnese; Hauser, Christoph; Furrer, Hansjakob; Low, Nicola and Endimiani, Andrea

Subjects:

500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health
300 Social sciences, sociology & anthropology > 360 Social problems & social services

ISSN:

0095-1137

Publisher:

American Society for Microbiology

Language:

English

Submitter:

Annelies Luginbühl

Date Deposited:

05 Jul 2016 10:48

Last Modified:

01 Dec 2016 02:30

Publisher DOI:

10.1128/JCM.03354-15

PubMed ID:

27225407

BORIS DOI:

10.7892/boris.83454

URI:

https://boris.unibe.ch/id/eprint/83454

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