title: Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.
abstract: 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 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 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 species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of 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 nongonococcal Neisseria species, and the detection limit was 10(3) to 10(4) genomic 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.
authors.affiliation: Institute for Infectious Diseases, University of Bern, Bern, Switzerland.
authors.forename: Valentina
authors.initials: V
authors.lastname: Donà
authors.affiliation: Institute for Infectious Diseases, University of Bern, Bern, Switzerland.
authors.forename: Sara
authors.initials: S
authors.lastname: Kasraian
authors.affiliation: Institute for Infectious Diseases, University of Bern, Bern, Switzerland.
authors.forename: Agnese
authors.initials: A
authors.lastname: Lupo
authors.affiliation: Institute for Infectious Diseases, University of Bern, Bern, Switzerland.
authors.forename: Yuvia N
authors.initials: YN
authors.lastname: Guilarte
authors.affiliation: Department of Infectious Diseases, Bern University Hospital and University of Bern, Bern, Switzerland.
authors.forename: Christoph
authors.initials: C
authors.lastname: Hauser
authors.affiliation: Department of Infectious Diseases, Bern University Hospital and University of Bern, Bern, Switzerland.
authors.forename: Hansjakob
authors.initials: H
authors.lastname: Furrer
authors.affiliation: Örebro University, Örebro, Sweden.
authors.forename: Magnus
authors.initials: M
authors.lastname: Unemo
authors.affiliation: Institute of Social and Preventive Medicine, University of Bern, Bern, Switzerland.
authors.forename: Nicola
authors.initials: N
authors.lastname: Low
authors.affiliation: Institute for Infectious Diseases, University of Bern, Bern, Switzerland aendimiani@gmail.com.
authors.forename: Andrea
authors.initials: A
authors.lastname: Endimiani
mesh_terms: D024881:Drug Resistance, Bacterial; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009344:Neisseria gonorrhoeae / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044366:Transition Temperature