Multiplex High-Resolution Melting Assay for Simultaneous Identification of Molecular Markers Associated with Extended-spectrum Cephalosporins and Azithromycin Resistance in Neisseria gonorrhoeae

Abstract Antimicrobial resistance (AMR) in Neisseria gonorrhoeae persists as a major public health concern globally. The objective of current study was to develop and evaluate a multiplex assay relied on high-resolution melting (HRM) technology as a rapid, simple and cost-effective method for simultaneously detecting and identifying different molecular markers associated with extended-spectrum cephalosporins (ESCs) and azithromycin (AZM) resistance in N. gonorrhoeae. Forty-eight well-characterized N. gonorrhoeae clinical isolates were selected for initial assay establishment. The multiplex HRM assays were able to accurately identify different nucleotide variations of the AMR determinants related to ESCs and AZM resistance. Specificity and cross-reactivity were assessed by testing 15 non-gonococcal strains. Then the assay was validated on 218 archived DNA specimens that had been sequenced using whole genome sequencing (WGS) technology. Compared with WGS, these assays had a sensitivity of 98.6%, with specificity 99.2%. For further validation of the assay’s performance, a total of 338 samples (156 clinical isolates and 182 clinical specimens) were screened using the multiplex HRM assay. The results showed good concordance with the results of PCR-sequencing. Given its rapidity (within 90 min), ease of performing, and low cost (less than $1.00 per sample), our method potentially could be applied to large-scale epidemiological programs for increasing surveillance of ESCs and AZM resistance in N. gonorrhoeae.