Real-time PCR for pharmacodynamic studies of Chlamydia trachomatis

Storm, Martin; Gustafsson, Ingegerd; Herrmann, Björn; Engstrand, Lars

doi:10.1016/j.mimet.2004.12.015

Cited by 8 publications

(16 citation statements)

References 14 publications

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“…Real-time PCR is performed in a closed system, amplification and detection of target are done in a single step, and there is the potential for automation. Real-time PCR has been used for pharmacodynamic studies for C. trachomatis (19,23). Two real-time PCR assays for the detection of C. trachomatis have also been described (8,13).…”

mentioning

confidence: 99%

Development and Validation of a Rotor-Gene Real-Time PCR Assay for Detection, Identification, and Quantification of Chlamydia trachomatis in a Single Reaction

et al. 2006

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confidence: 99%

Development and Validation of a Rotor-Gene Real-Time PCR Assay for Detection, Identification, and Quantification of Chlamydia trachomatis in a Single Reaction

et al. 2006

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“…When antibiotic treatment kills chlamydia, it can take 3 weeks to clear the nucleic acid from the genital tract [59], so any NAAT conducted during this time may detect non-viable organism only and be a false positive. Chlamydial mRNA assays use a quantitative real-time chlamydia molecular test (see p7) and will quantitatively measure mRNA transcription of omp2 (omcB) outer membrane protein in chlamydia as an expression of viable organisms (active infection) [37]. In addition, we will collect comprehensive sexual practice data to identify men at risk of re-infection using SMS.…”

Section: Discussionmentioning

confidence: 99%

“…Using the algorithm (Fig. 2), our secondary outcomes will be classified as: i) microbial cure based on a negative chlamydia NAAT test result at week 4; ii) false positive diagnosis if chlamydia NAAT positive at week 4, but no evidence of mRNA detected [37]; iii) new infection if infected with a different organism (based on sequencing results) OR if the same organism and the participant reports unprotected anal receptive sex between tests; iv) chlamydia treatment failure if infected with exactly the same organism and no reports of unprotected sex between recruitment and week 4 follow up. [NOTE: This algorithm assumes that if a man has a repeat infection with exactly the same organism confirmed by sequencing and reports unprotected sex, he will be classified as having a new infection rather than treatment failure.…”

Section: Methodsmentioning

confidence: 99%

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Treatment efficacy of azithromycin 1 g single dose versus doxycycline 100 mg twice daily for 7 days for the treatment of rectal chlamydia among men who have sex with men – a double-blind randomised controlled trial protocol

et al. 2017

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BackgroundRectal infection with Chlamydia trachomatis is one of the most common bacterial sexually transmissible infections among men who have sex with men (MSM) with diagnosis rates continuing to rise. Current treatment guidelines recommend either azithromycin 1 g single dose or doxycycline 100 mg twice daily for 7 days. However, there are increasing concerns about treatment failure with azithromycin. We are conducting the first randomised controlled trial (RCT) to compare treatment efficacy of azithromycin versus doxycycline for the treatment of rectal chlamydia in MSM.Methods/DesignThe Rectal Treatment Study will recruit 700 MSM attending Australian sexual health clinics for the treatment of rectal chlamydia. Participants will be asked to provide rectal swabs and will be randomised to either azithromycin 1 g single dose or doxycycline 100 mg twice daily for 7 days. Participants will be asked to complete questionnaires about adverse drug reactions, sexual behaviour and drug adherence via short message service and online survey. The primary outcome is the treatment efficacy as determined by a negative chlamydia nucleic acid amplification test at 4 weeks post treatment. Secondary outcomes will utilise whole genome sequencing and mRNA assay to differentiate between treatment failure, reinfection or false positive results.DiscussionRectal chlamydia is an increasing public health concern as use of pre-exposure prophylaxis against HIV becomes commonplace. Optimal, evidence-based treatment is critical to halting ongoing transmission. This study will provide the first RCT evidence comparing azithromycin and doxycycline for the treatment of rectal chlamydia. The results of this trial will establish which treatment is more efficacious and inform international management guidelines.Trial registrationAustralian New Zealand Clinical Trials Registry ACTRN12614001125617.

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“…To overcome potential miss-classification bias, the detection of mRNA in addition to DNA could allow the differentiation between viable infection and non-viable DNA. In this study, we modified a previously published CT mRNA detection (omp2) method from reverse transcriptase qPCR (RTqPCR) to digital PCR (dPCR), (allowing for direct quantification of the target molecule using microfluidic generated droplets and capillary flow fluorescence detection) [11] and aimed to evaluate it using a sample of CT DNA PCR positive women to differentiate between viable and non-viable DNA. Previous studies have shown that dPCR has better sensitivity with as much as a 50-fold improvement on current real time PCR assays and provides quantitative bacterial load data on every sample [12].…”

Section: Introductionmentioning

confidence: 99%

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

Phillips

Vodstrcil

Huston

et al. 2018

Eur J Clin Microbiol Infect Dis

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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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Real-time PCR for pharmacodynamic studies of Chlamydia trachomatis

Cited by 8 publications

References 14 publications

Development and Validation of a Rotor-Gene Real-Time PCR Assay for Detection, Identification, and Quantification of Chlamydia trachomatis in a Single Reaction

Development and Validation of a Rotor-Gene Real-Time PCR Assay for Detection, Identification, and Quantification of Chlamydia trachomatis in a Single Reaction

Treatment efficacy of azithromycin 1 g single dose versus doxycycline 100 mg twice daily for 7 days for the treatment of rectal chlamydia among men who have sex with men – a double-blind randomised controlled trial protocol

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

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