Viability-PCR Shows That NAAT Detects a High Proportion of DNA from Non-Viable Chlamydia trachomatis

Abstract

ObjectivesAccording 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.MethodsTechnical 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.ResultsTechnical 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.ConclusionsV-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.

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

“…Our finding of approximately 20% mRNA negative is similar to those of Janssen and colleagues [17] who developed a viability PCR (V-PCR) assay to assess CT viability and found that 24% of vaginal swabs from women diagnosed CT PCR positive had non-viable DNA only. Unlike our assay which quantified mRNA as a marker of viable CT, their V-PCR assay consisted of propidium monoazide (PMA) treatment prior to DNA extraction followed by quantitative PCR targeting the ompA gene for the detection of CT DNA.…”

Assessment of rectalChlamydia trachomatisviable load in women by viability-PCR

“…The evaluation of CT viability in rectal samples by V-PCR is feasible and valid. Previously, we validated the V-PCR method in vaginal samples from women 12. In the current study, CT culture has been conducted as a validation approximation of CT viability for V-PCR and showed that samples with no evidence for viable CT or samples with an unquantifiable total load were also negative for CT culture.…”

“…Assessment of CT viability was achieved by using the V-PCR method as described previously with minor adaptions12: in the current study, propidium monoazide (PMA) photoactivation was completed by using the PMA-Lite LED Photolysis Device (Biotium, Hayward, California, USA) with an exposure time of 10 min. Furthermore, CT quantification was conducted in a total volume of 50 µL to achieve a higher sensitivity.…”

“…The 2SP V-PCR sample was split into two aliquots of 500 µL; one was used to evaluate the total CT load (untreated) and the other for viable CT load (PMA treated). The resulting cycle of quantification (Cq) values were entered into a master calibration curve and log transformed to calculate the corresponding CT load (log 10 CT/mL), as described previously 12. Samples without a qPCR signal within 42 PCR cycles were defined as unquantifiable (13.2% (n=7) of rectal samples).…”

“…Recently, we validated the viability-PCR (V-PCR) method for the assessment of CT viability in clinical swab samples 12. This method combines the high sensitivity and specificity of PCR with the ability to distinguish between viable and dead bacteria based on membrane integrity 13–15.…”