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. G onorrhea is the second most common bacterial sexually transmitted infection worldwide, with an estimated 78 million new cases in 2012 (1). Moreover, Neisseria gonorrhoeae has developed resistance to most current and past treatment options. Antimicrobial-resistant (AMR) gonorrhea is a major public health concern about which the World Health Organization (WHO) emphasizes the importance of global surveillance to identify emerging resistance, monitor trends, and inform revisions of treatment guidelines (2, 3).At a molecular level, the mechanisms that confer resistance to the most common treatment options have been well characterized. For instance, the acquisition of mosaic penA alleles, with or without substitutions at amino acid position 501 of the encoded penicillin-binding protein 2 (PBP2), has been linked to decreased susceptibility or resistance to the extended-spectrum cephalosporins (ESCs) cefixime (CFX) and ceftriaxone (CRO) (4, 5). In particular, strains harboring a mosaic pattern XXXIV penA gene, including the internationally spreading N. gonorrhoeae multiantigen sequence typing (NG-MAST) genogroup 1407, have been responsible for ESC treatment failures in severa...

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