Molecular detection of ceftriaxone resistance inNeisseria gonorrhoeaeclinical specimens: a tool for public health control

Abstract

ObjectivesThis study aimed to validate and implement a rapid screening assay for molecular detection of thepenA-60 allele that is associated with ceftriaxone resistance inNeisseria gonorrhoeaefor use on both isolate lysates and clinical specimen DNA extracts.MethodsAN. gonorrhoeae penAreal-time (RT)-PCR was adapted to include a species-specificpapconfirmation target and a commercially available internal control to monitor for PCR inhibition.The modified assay was validated usingN. gonorrhoeae-positive (n=24) andN. gonorrhoeae-negative (n=42) clinical specimens and isolate lysates. The panel included seven samples with resistance conferred bypenAalleles targeted by the assay and four samples with differentpenAalleles. The feasibility of using thepenART-PCR for molecular surveillance was assessed using clinical specimens from 54 individuals attending a London sexual health clinic who also had aN. gonorrhoeaeisolate included in the 2020 Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP).ResultsThe assay correctly identifiedN. gonorrhoeaespecimens (n=7) withpenA-60/64 alleles targeted by the assay. NopenAfalse negatives/positives were detected, giving thepenAtarget of the assay a sensitivity, specificity, positive and negative predicted values (PPV, NPV) of 100% (95% CIs; sensitivity; 56.1–100%, specificity; 93.6–100%, PPV; 56.1–100%, NPV; 93.6–100%).No cross-reactivity with otherNeisseriaspecies or other urogenital pathogens was detected. TheN. gonorrhoeaetarget (pap) was detected in 73 out of 78 of theN. gonorrhoeae-positive specimens, resulting in 92.6% sensitivity (95% CI 83.0% to 97.3%), 100% specificity (95% CI 75.9% to 100%) and PPV, and a NPV of 89.4% (95% CI 52.5% to 90.9%). NopenA-59/60/64 alleles were detected within the clinical specimens from the GRASP 2020 feasibility molecular surveillance study (n=54 individuals).ConclusionThe implementation of this PCR assay for patient management, public health and surveillance purposes enables the rapid detection of gonococcal ceftriaxone resistance conferred by the most widely circulatingpenAalleles.