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No widespread dissemination of Chlamydia trachomatis diagnostic-escape variants and the impact of Neisseria gonorrhoeae positivity on the Aptima Combo 2 assay
  1. Michelle Jayne Cole1,
  2. Grahame S Davis1,
  3. Helen Fifer1,
  4. John Michael Saunders1,2,
  5. Magnus Unemo3,
  6. Ronza Hadad3,
  7. David J Roberts1,
  8. Mohammed Fazal1,
  9. Michaela Joanne Day1,
  10. Jack Minshull1,
  11. Peter Muir4,
  12. Paddy J Horner5,6,
  13. Noel O Gill1,
  14. Kate Folkard1
  1. 1 National Infection Service, Public Health England, London, UK
  2. 2 Research Department of Infection and Population Health, University College London, London, UK
  3. 3 WHO Collaborating Centre for Gonorrhoea and Other STIs, National Reference Laboratory for Pathogenic Neisseria, Department of Laboratory Medicine, Microbiology, Faculty of Medicine and Health, Örebro University, Orebro, Sweden
  4. 4 Bristol Public Health Laboratory, Public Health England, Bristol, UK
  5. 5 School of Social and Community Medicine, University of Bristol, Bristol, UK
  6. 6 Bristol Sexual Health Centre, University Hospitals Bristol NHS Foundation Trust, Bristol, UK
  1. Correspondence to Dr Michelle Jayne Cole, Public Health England, London NW9 5EQ, UK; Michelle.Cole{at}


Objectives A Finnish Chlamydia trachomatis (CT) new variant was detected in 2019 that escaped detection in the Hologic Aptima Combo 2 (AC2) assay due to a C1515T mutation in the CT 23S rRNA target region. Reflex testing of CT-negative/CT-equivocal specimens as well as those positive for Neisseria gonorrhoeae (NG) with the Hologic Aptima CT (ACT) assay was recommended to identify any CT variants.

Methods From June to October 2019, specimens with discrepant AC2/ACT CT results were submitted to Public Health England and screened for detectable CT DNA using an inhouse real-time (RT)-PCR. When enough DNA was present, partial CT 23S rRNA gene sequencing was performed. Analysis of available relative light units and interpretative data was performed.

Results A total of 317 discordant AC2/ACT specimens were collected from 315 patients. Three hundred were tested on the RT-PCR; 53.3% (n=160) were negative and 46.7% (n=140) were positive. Due to low DNA load in most specimens, sequencing was successful for only 36 specimens. The CT 23S rRNA wild-type sequence was present in 32 specimens, and two variants with C1514T or G1523A mutation were detected in four specimens from three patients. Of the discordant specimens with NG interpretation, 36.6% of NG-negative/CT-negative AC2 specimens had detectable CT DNA on the inhouse RT-PCR vs 53.3% of NG-positive/CT-negative specimens.

Conclusions No widespread dissemination of AC2 diagnostic-escape CT variants has occurred in England. We however identified the impact of NG positivity on the discordant AC2/ACT specimens; a proportion appeared due to NG positivity and the associated NG signal, rather than any diagnostic-escape variants or low DNA load. Several patients with gonorrhoea may therefore receive false-negative AC2 CT results. Single diagnostic targets and multiplex diagnostic assays have their limitations such as providing selection pressure for escape mutants and potentially reduced sensitivity, respectively. These limitations must be considered when establishing diagnostic pathways.

  • Chlamydia trachomatis
  • Neisseria gonorrhoeae
  • molecular diagnostic techniques
  • chlamydia infections
  • nucleic acid amplification techniques

Data availability statement

Data are available upon reasonable request. Data are available upon reasonable request by contacting the corresponding author of the manuscript.

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Data availability statement

Data are available upon reasonable request. Data are available upon reasonable request by contacting the corresponding author of the manuscript.

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  • Handling editor Deborah Williamson

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  • Contributors MJC, GSD, HF, JMS, DJR, PM, PJH, ONG and KF were involved in the design of the study. MJC, MU, RH, MF, MJD and JM were responsible for the laboratory investigations. MJC and GSD analysed the data. MJC, GSD, HF, JMS, MU and KF drafted the paper, which was commented on and approved by all the authors. MJC is responsible for the overall content of the manuscript.

  • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

  • Competing interests None declared.

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