Viability-PCR Shows That NAAT Detects a High Proportion of DNA from Non-Viable Chlamydia trachomatis.
OBJECTIVE
According to the current guidelines for laboratory diagnosis of sexually transmitted infections (STIs), nucleic acid amplification tests (NAATs) are the preferred diagnostic method for Chlamydia trachomatis (CT) infections. However, NAATs amplify the available target DNA without discriminating between DNA originating from viable or non-viable CT. Assessing CT viability will provide more insights in the clinical and public health relevance of a CT positive test result. The aim of this study was to technically validate and implement viability-PCR (V-PCR) to asses CT viability.


METHODS
Technical validation of V-PCR was performed by the assessment of predefined viability ratios of CT. Samples were subjected to V-PCR which consisted of propidium monoazide (PMA) treatment prior to DNA extraction followed by quantitative PCR (qPCR) targeting the ompA gene for the detection of CT DNA. Finally, V-PCR was applied to vaginal swabs of 50 CT positive patients, as indicated by routine NAAT, collected at our outpatient STD clinics before antimicrobial treatment.


RESULTS
Technical validation of V-PCR showed that PMA treatment of heat-inactivated CT culture resulted in an almost complete loss of qPCR signal. PMA treated samples of the fresh viable CT culture showed no marked reduction of PCR signal, indicating that all DNA from viable CT could be detected. Applying V-PCR to clinical samples showed that in 36% of samples (18/50) less than 1% of CT DNA originated from viable bacteria.


CONCLUSIONS
V-PCR showed to be a fast and easy method to assess CT viability in clinical samples, without the need of traditional challenging cell culture methods. Furthermore, V-PCR results of clinical samples have indicated that a substantial amount of the amplified CT DNA originated from non-viable cells. Although results might be influenced by cell death during transport, this study suggests that there is a potential overestimation of quantitative CT positivity by currently used NAATs.
Department of Medical Microbiology, School of Public Health and Primary Care (CAPHRI), Maastricht University Medical Center (MUMC+), Maastricht, The Netherlands.
Kevin J H
KJ
Janssen
Department of Medical Microbiology, School of Public Health and Primary Care (CAPHRI), Maastricht University Medical Center (MUMC+), Maastricht, The Netherlands.|Department of Sexual Health, Infectious Diseases and Environmental Health, South Limburg Public Health Service (GGD South Limburg), Geleen, The Netherlands.
Christian J P A
CJ
Hoebe
Department of Medical Microbiology, School of Public Health and Primary Care (CAPHRI), Maastricht University Medical Center (MUMC+), Maastricht, The Netherlands.|Department of Sexual Health, Infectious Diseases and Environmental Health, South Limburg Public Health Service (GGD South Limburg), Geleen, The Netherlands.
Nicole H T M
NH
Dukers-Muijrers
Department of Medical Microbiology, School of Public Health and Primary Care (CAPHRI), Maastricht University Medical Center (MUMC+), Maastricht, The Netherlands.|Department of Sexual Health, Infectious Diseases and Environmental Health, South Limburg Public Health Service (GGD South Limburg), Geleen, The Netherlands.
Lisanne
L
Eppings
Department of Medical Microbiology, School of Public Health and Primary Care (CAPHRI), Maastricht University Medical Center (MUMC+), Maastricht, The Netherlands.
Mayk
M
Lucchesi
Department of Medical Microbiology, School of Public Health and Primary Care (CAPHRI), Maastricht University Medical Center (MUMC+), Maastricht, The Netherlands.
Petra F G
PF
Wolffs
D002692:Chlamydia trachomatis / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology*; D004269:DNA, Bacterial / Q000235:genetics*; D005260:Female; D006367:HeLa Cells; D006801:Humans; D050296:Microbial Viability / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D055815:Young Adult
D004269:DNA, Bacterial