Advancing COVID-19 diagnostics: rapid detection of intact SARS-CoV-2 using viability RT-PCR assay.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Commonly used methods for both clinical diagnosis of SARS-CoV-2 infection and management of infected patients involve the detection of viral RNA, but the presence of infectious virus particles is unknown. Viability PCR (v-PCR) uses a photoreactive dye to bind non-infectious RNA, ideally resulting in the detection of RNA only from intact virions. This study aimed to develop and validate a rapid v-PCR assay for distinguishing intact and compromised SARS-CoV-2. Propidium monoazide (PMAxx) was used as a photoreactive dye. Mixtures with decreasing percentages of intact SARS-CoV-2 (from 100% to 0%) were prepared from SARS-CoV-2 virus stock and a clinical sample. Each sample was divided into a PMAxx-treated part and a non-PMAxx-treated part. Reverse transcription-PCR (RT-PCR) using an in-house developed SARS-CoV-2 viability assay was then applied to both sample sets. The difference in intact SARS-CoV-2 was determined by subtracting the cycle threshold (Ct) value of the PMAxx-treated sample from the non-PMAxx-treated sample. Mixtures with decreasing concentrations of intact SARS-CoV-2 showed increasingly lower delta Ct values as the percentage of intact SARS-CoV-2 decreased, as expected. This relationship was observed in both high and low viral load samples prepared from cultured SARS-CoV-2 virus stock, as well as for a clinical sample prepared directly from a SARS-CoV-2 positive nasopharyngeal swab. In this study, a rapid v-PCR assay has been validated that can distinguish intact from compromised SARS-CoV-2. The presence of intact virus particles, as determined by v-PCR, may indicate SARS-CoV-2 infectiousness. IMPORTANCE: This study developed a novel method that can help determine whether someone who has been diagnosed with coronavirus disease 2019 (COVID-19) is still capable of spreading the virus to others. Current tests only detect the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, but cannot tell whether the particles are still intact and can thus infect cells. The researchers used a dye that selectively blocks the detection of damaged virions and free RNA. They showed that this viability PCR reliably distinguishes intact SARS-CoV-2 capable of infecting from damaged SARS-CoV-2 or free RNA in both cultured virus samples and a clinical sample. Being able to quickly assess contagiousness has important implications for contact tracing and safely ending isolation precautions. This viability PCR technique provides a simple way to obtain valuable information, beyond just positive or negative test results, about the actual risk someone poses of transmitting SARS-CoV-2 through the air or surfaces they come into contact with.

Incident urogenital and anorectal Chlamydia trachomatis in women: the role of sexual exposure and autoinoculation: a multicentre observational study (FemCure).

BACKGROUND: Anorectal infections with Chlamydia trachomatis (CT) are common in women visiting STI outpatient clinics. We here evaluated the risk posed by sexual exposure and by alternate anatomical site infection for incident anorectal and urogenital CT. METHODS: Prospective multicentre cohort study, FemCure. Participants were treated for CT, and after 4, 6, 8, 10 and 12 weeks, they self-collected anorectal and urogenital samples (swabs) for CT-DNA testing. We calculated the proportion with incident CT, that is, CT incidence (at weeks 6-12) by 2-week time-periods. Compared with no exposure (A), we estimated the risk of incident CT for (B) sexual exposure, (C) alternate site anatomic site infection and (D) both, adjusted for confounders and expressed as adjusted ORs with 95% CIs. RESULTS: We analysed data of 385 participants contributing 1540 2-week periods. The anorectal CT incidence was 2.9% (39/1343) (95 CI 1.8 to 3.6); 1.3% (A), 1.3% (B), 27.8% (C) and 36.7% (D). The ORs were: 0.91 (95% CI 0.32 to 2.60) (B), 26.0 (95% CI 7.16 to 94.34) (C), 44.26 (95% CI 14.38 to 136.21) (D).The urogenital CT incidence was 3.3% (47/1428) (95% CI 2.4 to 4.4); 0.7% (A), 1.9% (B), 13.9% (C) and 25.4% (D). The ORs were: 2.73 (95% CI 0.87 to 8.61) (B), 21.77 (95% CI 6.70 to 70 71) (C) and 49.66 (95% CI 15.37 to 160.41) (D). CONCLUSIONS: After initial treatment, an alternate anatomical site CT infection increased the risk for an incident CT in women, especially when also sex was reported. This may suggest a key role for autoinoculation in the re-establishment or persistence of urogenital and anorectal chlamydia infections.

Oropharyngeal Chlamydia trachomatis in women; spontaneous clearance and cure after treatment (FemCure).

OBJECTIVES: Women attending STI clinics are not routinely tested for oropharyngeal Chlamydia trachomatis (CT) infections. We aimed to assess spontaneous clearance of oropharyngeal CT and cure after antibiotic treatment in women. METHODS: Women with vaginal or rectal CT (n=560) were recruited at STI clinics in 2016-2017, as part of the FemCure study (prospective cohort study). We included participants' data from week -1, that is, the diagnosis at initial visit, when clinics applied selective oropharyngeal testing. At week -1, a total of 241 women were oropharyngeally tested (30 positive) and 319 were untested. All FemCure participants provided nurse-collected oropharyngeal samples at study enrolment, that is, week 0, just prior to treatment (n=560), and after treatment at weeks 4 (n=449), 8 (n=433) and 12 (n=427). Samples were tested by nucleic acid amplification test, and at week 0 also by viability testing by viability PCR. Proportions of oropharyngeal CT test results were presented to represent spontaneous clearance and cure. RESULTS: Of 30 women diagnosed with oropharyngeal CT at week -1, fifteen (50%) were negative at week 0 after a median of 9 days, that is, 'spontaneous clearance'. At week 0, a total of 560 participants were tested, and 46 (8.8%) were oropharyngeal CT positive; 12 of them (26.1%) had viable CT. Of the 46 positive, 36 women had an oropharyngeal test after treatment; 97.2% (35/36) were negative at week 4, that is, 'cure'. Of all women with follow-up visits, the proportion of oropharyngeal CT positive was between 0.5% and 1.6% between weeks 4 and 12. Of those not tested at week -1 (n=319), 8.5% (n=27) were oropharyngeal positive at week 0. CONCLUSIONS: The clinical importance of oropharyngeal CT in women is debated. We demonstrated that spontaneous clearance of oropharyngeal CT among women is common; of those who did not clear for CT, three-quarters had non-viable CT. After regular treatment with azithromycin or doxycycline, cure rate (97%) of oropharyngeal CT is excellent. TRIAL REGISTRATION NUMBER: NCT02694497.

How to Count Our Microbes? The Effect of Different Quantitative Microbiome Profiling Approaches.

Next-generation sequencing (NGS) has instigated the research on the role of the microbiome in health and disease. The compositional nature of such microbiome datasets makes it however challenging to identify those microbial taxa that are truly associated with an intervention or health outcome. Quantitative microbiome profiling overcomes the compositional structure of microbiome sequencing data by integrating absolute quantification of microbial abundances into the NGS data. Both cell-based methods (e.g., flow cytometry) and molecular methods (qPCR) have been used to determine the absolute microbial abundances, but to what extent different quantification methods generate similar quantitative microbiome profiles has so far not been explored. Here we compared relative microbiome profiling (without incorporation of microbial quantification) to three variations of quantitative microbiome profiling: (1) microbial cell counting using flow cytometry (QMP), (2) counting of microbial cells using flow cytometry combined with Propidium Monoazide pre-treatment of fecal samples before metagenomics DNA isolation in order to only profile the microbial composition of intact cells (QMP-PMA), and (3) molecular based quantification of the microbial load using qPCR targeting the 16S rRNA gene. Although qPCR and flow cytometry both resulted in accurate and strongly correlated results when quantifying the bacterial abundance of a mock community of bacterial cells, the two methods resulted in highly divergent quantitative microbial profiles when analyzing the microbial composition of fecal samples from 16 healthy volunteers. These differences could not be attributed to the presence of free extracellular prokaryotic DNA in the fecal samples as sample pre-treatment with Propidium Monoazide did not improve the concordance between qPCR-based and flow cytometry-based QMP. Also lack of precision of qPCR was ruled out as a major cause of the disconcordant findings, since quantification of the fecal microbial load by the highly sensitive digital droplet PCR correlated strongly with qPCR. In conclusion, quantitative microbiome profiling is an elegant approach to bypass the compositional nature of microbiome NGS data, however it is important to realize that technical sources of variability may introduce substantial additional bias depending on the quantification method being used.

Assessment of rectal Chlamydia trachomatis viable load in women by viability-PCR.

OBJECTIVES: In recent years, studies have demonstrated frequent rectal Chlamydia trachomatis (CT) detection in women, irrespective of reported anal sex or rectal symptoms. However, the clinical relevance and public health implication of rectal CT detection in women remain under debate. Therefore, evaluating CT viability may provide more insight into the relevance of standard routine nucleic acid amplification test (NAAT)-positive results. METHODS: In this cross-sectional explorative study, a convenience sample of female patients at our STI clinic aged 18 years or older, diagnosed with vaginal and/or rectal CT, were invited to participate. On return for treatment, rectal CT-diagnosed women were instructed to self-collect rectal swab samples before being treated. Standard COBAS 4800 CT/NG routine NAAT testing was applied for CT diagnosis. Rectal viable CT load was evaluated by using viability-PCR (V-PCR). RESULTS: 53 women with rectal CT were included in this study; 86.8% (46/53) had a quantifiable rectal total CT load. Of women with quantifiable samples, 52.2% (24/46) had viable CT detected from rectal swabs by V-PCR, with a mean rectal viable CT load of 3.31 log10 CT/mL (range 1.16-6.22). No statistically significant difference (p=0.73) was observed in the mean rectal viable CT load of women with an indication for rectal testing (n=9) and without (n=15), 3.20 log10 CT/mL (range 2.06-4.36) and 3.38 log10 CT/mL (range 1.16-6.22), respectively. CT culture yielded positive test results from rectal swabs in 22.6% (12/53) of rectal CT NAAT-diagnosed women. Of women with viable rectal CT by V-PCR (n=24), 50% (12/24) were positive by CT culture. CONCLUSIONS: Overall, the detection of high rectal viable CT loads in this study indicates that rectal CT in some women might represent a currently ongoing infection rather than just the presence of remnant DNA from dead bacteria or only contamination from an active vaginal CT infection.

Treatment Effectiveness of Azithromycin and Doxycycline in Uncomplicated Rectal and Vaginal Chlamydia trachomatis Infections in Women: A Multicenter Observational Study (FemCure).

BACKGROUND: Rectal infections with Chlamydia trachomatis (CT) are prevalent in women visiting a sexually transmitted infection outpatient clinic, but it remains unclear what the most effective treatment is. We assessed the effectiveness of doxycycline and azithromycin for the treatment of rectal and vaginal chlamydia in women. METHODS: This study is part of a prospective multicenter cohort study (FemCure). Treatment consisted of doxycycline (100 mg twice daily for 7 days) in rectal CT-positive women, and of azithromycin (1 g single dose) in vaginally positive women who were rectally untested or rectally negative. Participants self-collected rectal and vaginal samples at enrollment (treatment time-point) and during 4 weeks of follow-up. The endpoint was microbiological cure by a negative nucleic acid amplification test at 4 weeks. Differences between cure proportions and 95% confidence intervals (CIs) were calculated. RESULTS: We analyzed 416 patients, of whom 319 had both rectal and vaginal chlamydia at enrollment, 22 had rectal chlamydia only, and 75 had vaginal chlamydia only. In 341 rectal infections, microbiological cure in azithromycin-treated women was 78.5% (95% CI, 72.6%-83.7%; n = 164/209) and 95.5% (95% CI, 91.0%-98.2%; n = 126/132) in doxycycline-treated women (difference, 17.0% [95% CI, 9.6%-24.7%]; P < .001). In 394 vaginal infections, cure was 93.5% (95% CI, 90.1%-96.1%; n = 246/263) in azithromycin-treated women and 95.4% (95% CI, 90.9%-98.2%; n = 125/131) in doxycycline-treated women (difference, 1.9% [95% CI, -3.6% to 6.7%]; P = .504). CONCLUSIONS: The effectiveness of doxycycline is high and exceeds that of azithromycin for the treatment of rectal CT infections in women. CLINICAL TRIALS REGISTRATION: NCT02694497.

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

OBJECTIVES: 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.

Design of the FemCure study: prospective multicentre study on the transmission of genital and extra-genital Chlamydia trachomatis infections in women receiving routine care.

BACKGROUND: In women, anorectal infections with Chlamydia trachomatis (CT) are about as common as genital CT, yet the anorectal site remains largely untested in routine care. Anorectal CT frequently co-occurs with genital CT and may thus often be treated co-incidentally. Nevertheless, post-treatment detection of CT at both anatomic sites has been demonstrated. It is unknown whether anorectal CT may play a role in post-treatment transmission. This study, called FemCure, in women who receive routine treatment (either azithromycin or doxycycline) aims to understand the post-treatment transmission of anorectal CT infections, i.e., from their male sexual partner(s) and from and to the genital region of the same woman. The secondary objective is to evaluate other reasons for CT detection by nucleic acid amplification techniques (NAAT) such as treatment failure, in order to inform guidelines to optimize CT control. METHODS: A multicentre prospective cohort study (FemCure) is set up in which genital and/or anorectal CT positive women (n = 400) will be recruited at three large Dutch STI clinics located in South Limburg, Amsterdam and Rotterdam. The women self-collect anorectal and vaginal swabs before treatment, and at the end of weeks 1, 2, 4, 6, 8, 10, and 12. Samples are tested for presence of CT-DNA (by NAAT), load (by quantitative polymerase chain reaction -PCR), viability (by culture and viability PCR) and CT type (by multilocus sequence typing). Sexual exposure is assessed by online self-administered questionnaires and by testing samples for Y chromosomal DNA. Using logistic regression models, the impact of two key factors (i.e., sexual exposure and alternate anatomic site of infection) on detection of anorectal and genital CT will be assessed. DISCUSSION: The FemCure study will provide insight in the role of anorectal chlamydia infection in maintaining the CT burden in the context of treatment, and it will provide practical recommendations to reduce avoidable transmission. Implications will improve care strategies that take account of anorectal CT. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02694497 .