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Molecular detection of ceftriaxone resistance in Neisseria gonorrhoeae clinical specimens: a tool for public health control
  1. Michaela Joanne Day1,
  2. Dolcibella Boampong2,
  3. Rachel Pitt1,
  4. Aisha Bari3,
  5. Monica Rebec4,
  6. John Saunders5,6,
  7. Helen Fifer6,
  8. Tamyo Mbisa2,
  9. Michelle Jayne Cole1
  1. 1STI Reference Laboratory, UK Health Security Agency, London, UK
  2. 2Antiviral Unit, UK Health Security Agency, London, UK
  3. 3Microbiology Department, Charing Cross Hospital, London, UK
  4. 4Infection and Immunity Laboratory, NHS North West London Pathology, London, UK
  5. 5The National Institute for Health Research Health Protection Research Unit in Blood Borne and Sexually Transmitted Infections at University College London in partnership with the UK Health Security Agency, London, UK
  6. 6Blood Safety, Hepatitis, Sexually Transmitted Infections and HIV Services, UKHSA, London, UK
  1. Correspondence to Dr Michaela Joanne Day, STI Reference Laboratory, UKHSA, London, SW7 4UB, UK;{at}


Objectives This study aimed to validate and implement a rapid screening assay for molecular detection of the penA-60 allele that is associated with ceftriaxone resistance in Neisseria gonorrhoeae for use on both isolate lysates and clinical specimen DNA extracts.

Methods A N. gonorrhoeae penA real-time (RT)-PCR was adapted to include a species-specific pap confirmation target and a commercially available internal control to monitor for PCR inhibition.

The modified assay was validated using N. gonorrhoeae-positive (n=24) and N. gonorrhoeae-negative (n=42) clinical specimens and isolate lysates. The panel included seven samples with resistance conferred by penA alleles targeted by the assay and four samples with different penA alleles. The feasibility of using the penA RT-PCR for molecular surveillance was assessed using clinical specimens from 54 individuals attending a London sexual health clinic who also had a N. gonorrhoeae isolate included in the 2020 Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP).

Results The assay correctly identified N. gonorrhoeae specimens (n=7) with penA-60/64 alleles targeted by the assay. No penA false negatives/positives were detected, giving the penA target 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 other Neisseria species or other urogenital pathogens was detected. The N. gonorrhoeae target (pap) was detected in 73 out of 78 of the N. 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%). No penA-59/60/64 alleles were detected within the clinical specimens from the GRASP 2020 feasibility molecular surveillance study (n=54 individuals).

Conclusion The 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 circulating penA alleles.

  • Diagnostic Techniques and Procedures
  • Drug Resistance, Bacterial
  • Gonorrhea
  • Molecular Diagnostic Techniques

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  • Handling editor Nicola Low

  • Contributors MJD and MJC prepared the draft manuscript and table. All authors reviewed, commented, edited and approved the manuscript. AB and MR provided appropriate specimens for inclusion in the project. DB, RP, MJD and MJC were responsible for the UKHSA laboratory work. The project proposal and funding was conceived by HF, TM, JS and MJC.

  • Funding The research was funded by the National Institute for Health and Care Research Health Protection Research Unit (NIHR HPRU) in Blood Borne and Sexually Transmitted Infections at University College London in partnership with UK Health Security Agency (UKHSA) as well as UKHSA internal funding.

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.