Natural Course of Chlamydia trachomatis Bacterial Load in the Time Interval between Screening and Treatment in Anogenital Samples

Dirks, Jeanne A. M. C.; Liere, Geneviève A F S van; Bogers, Saskia J; Dukers-Muijrers, Nicole H. T. M.; Wolffs, Petra; Hoebe, Christian J. P. A.

doi:10.1371/journal.pone.0145693

Cited by 10 publications

(27 citation statements)

References 34 publications

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“…In line with our results, natural clearance of CT DNA and/or RNA during the period between CT detection and treatment has previously been assessed and varied between 9–44% in urogenital samples [25, 26]. Furthermore, in a recent study by our research group we have reported that CT load during the period between CT detection and treatment decreased more than 90% in one out of five of vaginal swab samples [27]. …”

Section: Discussionsupporting

confidence: 89%

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

et al. 2016

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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.

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Section: Discussionsupporting

confidence: 89%

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

et al. 2016

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“…A general limitation of CT load-based studies is that the variability of low and high CT loads, and therefore high and low Cq values, in populations and individuals seems dependent of different factors, including time of diagnosis since infection; this hampers interpretation. For example, our study group assessed the natural course of the CT load during infection between screening and treatment and observed a decrease in CT load in 17–41% of the STI clinic patients dependent on sample type [26]. A limitation of the current study was that we were unable to validate Cq values for (1) other populations than the STI clinic population and (2) for urine samples of men.…”

Section: Discussionmentioning

confidence: 96%

“…We tested the use of the Cq value as a proxy for CT load by comparing derived Cq values with quantified CT load values from our previous studies [11, 25, 26]. CT load values were available from a subset of the STI clinic population, i.e.…”

Section: Methodsmentioning

confidence: 99%

Chlamydia trachomatis bacterial load, estimated by Cq values, in urogenital samples from men and women visiting the general practice, hospital or STI clinic

et al. 2019

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Background The bacterial load of Chlamydia trachomatis (CT) is assumed to play a role in transmission and sequelae. We assessed urogenital CT cycle quantification (Cq) values, as an indicator for CT load, of men and women diagnosed by general practitioners (GPs), hospital physicians and the STI clinic. Methods Urogenital CT-positive samples (n = 2,055 vaginal swabs, n = 77 cervical swabs, n = 1,519 urine samples and n = 19 urethral swabs) diagnosed by GPs, hospital physicians and the STI clinic from the Maastricht Medical Microbiology Laboratory were included (2012–2016). The outcome measure ‘urogenital Cq values’ was used as an inversely proportional measure for CT load. Among all patients, multivariate linear regression analyses were used to assess primary determinants for mean urogenital Cq values, stratified by sex. Additional clinical determinants were assessed among STI clinic patients. Results In men, mean urogenital Cq values were similar between GPs, hospital physicians and the STI clinic (32.7 and 33.5 vs. 32.7; p>0.05). Women visiting the GP had lower urogenital Cq values than women visiting the STI clinic (30.2 vs. 30.9; p = <0.001). Women visiting the hospital had higher urogenital Cq values than women visiting the STI clinic (32.4 vs. 30.9; p = <0.001). Among STI clinic women, urogenital Cq values were lower in women with concurrent anorectal CT and in rectally untested women compared to anorectal CT-negative women (30.7 and 30.6 vs. 33.9; p = <0.001). Conclusion Men visiting different STI care providers had similar urogenital Cq values, which could be an indicator for similar CT loads. The lower Cq values of women visiting the GP compared to women visiting the STI clinic could be an indicator for higher CT loads and likely higher transmission potential. Notably, urogenital Cq values of STI clinic women were much lower (>3 Cq) when STI clinic women also had anorectal CT. This finding could indicate higher urogenital CT loads and likely higher chances of transmission and sequelae.

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“…Whilst diagnosis for many infectious diseases requires the detection of pathogenic DNA/RNA concentrations as low as 10 copies/mL, there are some diseases where infectious pathogens are present at much higher concentrations and are within the scope of what the Subzyme PMB method can detect [20,21]. For example, one study reports that 85.8% (254/296) of positive C. trachomatis specimens had a bacterial load above the quantification limit of the qPCR (1 × 10 6 copies/mL) [22]. Furthermore, pathogens that are known to cause urinary tract infections (UTIs) such as Escherichia coli, Klebsiella spp., Pseudomonas aeuroginosa and Staphylococcus saprophyticus are typically present in urine samples at concentrations ranging between 1 pM-1 nM, which are also within the current detectable concentration range of the Subzyme feedback method [21,23].…”

Section: Subzyme Feedback Cascade Using Pmbsmentioning

confidence: 99%

Sensitive Detection of Nucleic Acids Using Subzyme Feedback Cascades

Hasick

Lawrence²,

Ramadas³

et al. 2020

Molecules

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The development of Subzymes demonstrates how the catalytic activity of DNAzymes can be controlled for detecting nucleic acids; however, Subzymes alone lack the sensitivity required to detect low target concentrations. To improve sensitivity, we developed a feedback system using a pair of cross-catalytic Subzymes. These were individually tethered to microparticles (MP) and separated by a porous membrane rendering them unable to interact. In the presence of a target, active PlexZymes® cleave a first Subzyme, which separates a first DNAzyme from its MP, allowing the DNAzyme to migrate through the membrane, where it can cleave a second Subzyme. This releases a second DNAzyme which can now migrate through the membrane and cleave more of the first Subzyme, thus initiating a cross-catalytic cascade. Activated DNAzymes can additionally cleave fluorescent substrates, generating a signal, and thereby, indicating the presence of the target. The method detected 1 fM of DNA homologous to the ompA gene of Chlamydia trachomatis within 30 min, demonstrating a 10,000-fold increase in sensitivity over PlexZyme detection alone. The Subzyme cascade is universal and can be triggered by any target by modifying the target sensing arms of the PlexZymes. Further, it is isothermal, protein-enzyme-free and shows great potential for rapid and affordable biomarker detection.

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Natural Course of Chlamydia trachomatis Bacterial Load in the Time Interval between Screening and Treatment in Anogenital Samples

Cited by 10 publications

References 34 publications

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

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

Chlamydia trachomatis bacterial load, estimated by Cq values, in urogenital samples from men and women visiting the general practice, hospital or STI clinic

Sensitive Detection of Nucleic Acids Using Subzyme Feedback Cascades

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