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Detection of Chlamydia trachomatis mRNA using digital PCR as a more accurate marker of viable organism

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European Journal of Clinical Microbiology & Infectious Diseases Aims and scope Submit manuscript

Abstract

Spontaneous resolution of urogenital Chlamydia trachomatis (CT) without treatment has previously been described, but a limitation of these reports is that DNA or RNA-based amplification tests used do not differentiate between viable infection and non-viable DNA. We modified a previously published CT mRNA detection (omp2) method to differentiate between viable infection and non-viable DNA in a sample of CT DNA PCR positive women. We modified a CT mRNA detection (omp2) method from reverse transcriptase qPCR (RTqPCR) to digital PCR (dPCR) and evaluated it in samples from CT DNA positive women. Firstly, CT infected McCoy B cells treated with azithromycin in vitro identified detectable mRNA levels disappeared <2 days, while DNA persisted up to 6 days. We used 55 self-collected vaginal swabs from a cohort of women diagnosed as DNA positive for chlamydia obtained pre- and 7 days of post-azithromycin treatment. Concordance with DNA results was higher for dPCR than RTqPCR (74.5% versus 65.5%). At visit 1, there was a strong linear relationship between DNA and mRNA (r = 0.9, p < 0.000); 24 samples had both mRNA and DNA detected (82.8%) and 5 had only DNA detected with a potential false positive proportion of 17.2% (95%CI: 5.8, 35.8). At visit 2, there was poor correlation between DNA and mRNA (r = 0.14, p = 0.55); eight specimens had only DNA detected (42.1%; 95%CI: 20.25, 66.50) and one had mRNA detected. DNA detection methods alone may detect non-viable DNA. Consideration should be given to further develop mRNA assays as ancillary tests to improve detection of viable chlamydia.

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Acknowledgements

This study was conducted on behalf of the Australian Chlamydia Treatment Study (ACTS) investigator team, which includes Prof Basil Donovan, Prof Christopher Fairley, A/Prof Rebecca Guy, Prof John Kaldor, Prof Malcolm McConville, Dr. Anna McNulty, Dr. David Regan, and A/David Wilson.

Funding

This study has been externally funded by the Australian Government funding body, the National Health and Medical Research Council (NHMRC—project grant number APP1023239).

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Authors and Affiliations

  1. Murdoch Children’s Research Institute, Parkville, Victoria, 3052, Australia

    Samuel Phillips, Lenka A. Vodstrcil & Marcus Y. Chen

  2. Department of Microbiology and Infectious Diseases, The Royal Women’s Hospital, Parkville, Victoria, 3052, Australia

    Samuel Phillips, Suzanne M. Garland & Sepehr N. Tabrizi

  3. University of Sunshine Coast, 90 Sippy downs drive, Sippy Downs, Queensland, 4556, Australia

    Samuel Phillips & Peter Timms

  4. Melbourne School of Population and Global Health, University of Melbourne, Level 3, 207 Bouverie St, Carlton, Victoria, 3053, Australia

    Lenka A. Vodstrcil, Suzanne M. Garland, Sepehr N. Tabrizi & Jane S. Hocking

  5. Melbourne Sexual Health Centre, 580 Swanston Street, Carlton, Victoria, 3053, Australia

    Lenka A. Vodstrcil, Marcus Y. Chen, Karen Worthington & Catriona S. Bradshaw

  6. School of Life Sciences, University of Technology Sydney, City Campus, Broadway, Ultimo, New South Wales, 2007, Australia

    Wilhelmina M. Huston

  7. Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Ave, Kelvin Grove, Brisbane, Queensland, 4057, Australia

    Amba Lawerence

  8. Sydney Sexual Health Centre, Sydney Hospital, Macquarie Street, Sydney, New South Wales, 2001, Australia

    Ruthy McIver

  9. Department of Obstetrics and Gynaecology, University of Melbourne, Carlton, Victoria, 3053, Australia

    Suzanne M. Garland & Sepehr N. Tabrizi

Authors
  1. Samuel Phillips
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  2. Lenka A. Vodstrcil
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  3. Wilhelmina M. Huston
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  4. Amba Lawerence
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  5. Peter Timms
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  6. Marcus Y. Chen
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  7. Karen Worthington
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  8. Ruthy McIver
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  9. Catriona S. Bradshaw
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  10. Suzanne M. Garland
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  11. Sepehr N. Tabrizi
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  12. Jane S. Hocking
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Corresponding author

Correspondence to Samuel Phillips.

Ethics declarations

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Ethical approval

Ethical approval for this study was granted by the Alfred Hospital Ethics Committee (HREC No. 223/12) and the Southern Eastern Sydney Local Health District Human Research Ethics Committee (Southern Sector) (HREC No. 12/143).

Informed consent

Informed consent was obtained from all individual participants included in the study.

Conflict of interest

All authors declare that they have no conflicts of interest.

Additional information

Sepehr N. Tabrizi and Jane S. Hocking joint last author

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Phillips, S., Vodstrcil, L.A., Huston, W.M. et al. Detection of Chlamydia trachomatis mRNA using digital PCR as a more accurate marker of viable organism. Eur J Clin Microbiol Infect Dis 37, 2117–2122 (2018). https://doi.org/10.1007/s10096-018-3347-y

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  • DOI: https://doi.org/10.1007/s10096-018-3347-y

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