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 extend 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.
Chlamydia trachomatis (chlamydia) is the most commonly diagnosed bacterial sexually transmitted infection (STI) worldwide. The advancement of molecular techniques has made chlamydia diagnostics infinitely easier. However, molecular techniques lack the information on chlamydia viability. Where in routine diagnostics the detection of chlamydia DNA or RNA might suffice, in other patient scenarios, information on the viability of chlamydia might be essential. Areas covered: In this review, the authors discuss the specific strengths and limitations of currently available methods to evaluate chlamydia viability: conventional cell culture, messenger RNA (mRNA) detection and viability-PCR (V-PCR). PubMed and Google Scholar were searched with the following terms: Chlamydia trachomatis, Treatment failure, Anal chlamydia, Microbial viability, Culture, Viability-PCR, Messenger RNA, and Molecular diagnostics Expert commentary: Several techniques are currently available to determine chlamydia viability and thus the clinical relevance of a positive test result in clinical samples. Depending on the underlying research question, all three discussed techniques have their merits when testing for viability. However, mRNA methods show the most promise in determining the presence of a true infection, in case the chlamydia reticulate body can be specifically detected. Further research is needed to understand how to best apply viability testing in current chlamydia diagnostics.
Spontaneous resolution (clearance) of Chlamydia trachomatis (CT) infections can occur between diagnosis by nucleic acid amplification assays (NAAT) and treatment. Moreover, viability polymerase chain reaction (V-PCR) techniques showed that part of non-resolved NAAT positives represent non-viable CT. This may impact clinic policies aiming to restrict antibiotic treatment (i.e. to viable CT only). We followed 560 CT diagnosed women to assess the proportion without viable CT at follow-up, and associated risk factors.
Methods
Vaginal (vCT) or rectal (rCT) NAAT positive adult women, negative for HIV, syphilis and Neisseria gonorrhoeae, who not recently used antibiotics, were included at three STI outpatient-clinics (Netherlands, 2016–2017; FemCure). At clinic-diagnosis women were (a) vCT positive, rCT untested (n=351), (b) vCT, rCT positive (n=155), (c) vCT positive, rCT negative (n=25), (d) vCT negative, rCT positive (n=29). After a median of 8 [IQR:7–12] days, before treatment, samples were tested using NAAT and V-PCR. We present percentages of women without viable CT at follow-up, and tested which factors (group [a-d], age, education, non-western-background, symptoms, anal/vaginal sex, sexpartners) were associated, using logistic regression.
Results
At follow-up, percentages of women NAAT negative at both anatomic sites were 5.4% (a), 0.6% (b), 32.0% (c), and 27.6% (d). Percentages of women without viable CT (i.e. NAAT negative or NAAT positive and V-PCR undetectable) at both anatomic sites were 9.4% (33/351, a), 3.9% (6/155, b), 52.0% (13/25, c), and 41.4% (12/29, d). Alongside group (p<0.001), older age was independently associated (odds ratio: 1.07 per year (95%CI: 1.01–1.13; p=0.029) with lack of viable CT.
Conclusion
Less than ten percent of STI-clinic women diagnosed with vaginal and rectal CT (or were rectally untested) did not have viable CT one week after diagnosis (when they return for treatment). Yet, this percentage was higher in women with single vaginal or rectal infection and in older women; this may affect treatment-choices.
Spontaneous clearance of Chlamydia trachomatis (CT) infections can occur between diagnosis and treatment. We followed CT patients to assess clearance using a conventional definition (no total CT-DNA, assessed by routine quantitative PCR methods) and a definition accounting for viability, assessed by viability PCR testing.Three outpatient STI clinics included CT-diagnosed women (The Netherlands, 2016-2017, FemCure study); participants had vaginal CT (vCT) and rectal CT (rCT) (group A: n=155), vCT and were rectally untested (group B: n=351), single vCT (group C: n=25) or single rCT (group D: n=29). Follow-up (median interval 9 days) vaginal and rectal samples underwent quantitative PCR testing (detecting total CT-DNA). When PCR positive, samples underwent V-PCR testing to detect 'viable CT' (CT-DNA from intact CT organisms; V-PCR positive). 'Clearance' was the proportion PCR-negative patients and 'clearance of viable CT' was the proportion of patients testing PCR negative or PCR positive but V-PCR negative. We used multivariable logistic regression analyses to assess diagnosis group (A-D), age, days since initial CT test (diagnosis) and study site (STI clinic) in relation to clearance and clearance of viable CT.Clearance and clearance of viable CT at both anatomic sites were for (A) 0.6% and 3.9%; (B) 5.4% and 9.4%; (C) 32.0% and 52.0% and (D) 27.6% and 41.4%, respectively. In multivariate analyses, women with single infections (groups C and D) had higher likelihood of clearance than women concurrently infected with vCT and rCT (p<0.001).Of rectally untested women (group B), 76.9% had total CT-DNA and 46.7% had viable CT (V-PCR positive) at the rectal site.Of untreated female vCT patients who had CT also at the rectal site, or who were rectally untested, only a small proportion cleared CT (in fact many had viable CT) at their follow-up visit (median 9 days). Among single site infected women clearance was much higher.NCT02694497.
Chlamydia trachomatis (CT) is routinely diagnosed by nucleic acid amplification tests (NAATs), which are unable to distinguish between nucleic acids from viable and non-viable CT organisms.We applied our recently developed sensitive PCR (viability PCR) technique to measure viable bacterial CT load and explore associated determinants in 524 women attending Dutch sexual health centres (STI clinics), and who had genital or rectal CT.We included women participating in the FemCure study (Netherlands, 2016-2017). At the enrolment visit (pre-treatment), 524 were NAAT positive (n=411 had genital and rectal CT, n=88 had genital CT only and n=25 had rectal CT only). We assessed viable rectal and viable genital load using V-PCR. We presented mean load (range 0 (non-viable) to 6.5 log10 CT/mL) and explored potential associations with urogenital symptoms (coital lower abdominal pain, coital blood loss, intermenstrual bleeding, altered vaginal discharge, painful or frequent micturition), rectal symptoms (discharge, pain, blood loss), other anatomical site infection and sociodemographics using multivariable regression analyses.In genital (n=499) CT NAAT-positive women, the mean viable load was 3.5 log10 CT/mL (SD 1.6). Genital viable load was independently associated with urogenital symptoms-especially altered vaginal discharge (Beta=0.35, p=0.012) and with concurrent rectal CT (aBeta=1.79; p<0.001). Urogenital symptoms were reported by 50.3% of women; their mean genital viable load was 3.6 log10 CT/mL (vs 3.3 in women without symptoms). Of 436 rectal CT NAAT-positive women, the mean rectal viable load was 2.2 log10 CT/mL (SD 2.0); rectal symptoms were reported by 2.5% (n=11) and not associated with rectal viable load.Among women diagnosed with CT in an outpatient clinical setting, viable genital CT load was higher in those reporting urogenital symptoms, but the difference was small. Viable genital load was substantially higher when women also had a concurrent rectal CT.ClinicalTrials.gov NCT02694497.
Current routine diagnostic methods for the detection of Chlamydia trachomatis (CT) do not provide information on CT viability. Previously, detection of messenger-RNA (mRNA) has been utilized as a marker for bacterial viability, as mRNA molecules are generally short-lived (half-life of minutes). However, only one study evaluated CT mRNA half-life times [t1/2] of two clinical isolates (serovar L2b and E) which ranged from 1 to ≥5000 minutes. Here we assess and confirm mRNA half-life times of serovar D to further facilitate evaluation of CT viability.
Methods
CT serovar D was propagated in HeLa cells until 30 h post infection and treated with rifampicin to arrest gene transcription. Total RNA was isolated at 0 min (before treatment), and 10 min, 30 min and 60 min after treatment. RNA was converted to cDNA using random hexamers. RT-qPCR was used to amplify fragments of the unprocessed 16S (intermediate molecules in 16S rRNA synthesis), 16S (early), rpoD (early), omcB (mid), and hctA (late) gene transcripts. Half-life time was based on the fit of an exponential decay between values obtained at before and after transcriptional arrest.
Results
In this study showed that the obtained t1/2 values for CT serovar D mRNA (median t1/218 min) were similar as previously reported for the CT serovars L2b and E (median t1/2 15 and 17 min, respectively). The observed half-lifes of rpoD (9 min), hctA (12 min), omcB (18 min), and unprocessed 16S (18 min) transcripts were relatively short, while 16S gene transcripts were more stable over time (t1/254 min).
Conclusion
The detection of rpoD, hctA, omcB, and unprocessed 16S gene transcripts showed the most promising results as a potential marker for an active CT infection. Currently we are evaluating the time to clearance of mRNA molecules in patients after being treated.
Nucleic acid amplification tests (NAATs) have revolutionized our ability to diagnose Chlamydia trachomatis (CT). Sometimes, in addition, assessment of CT viability would help to gain more insight in the clinical impact of a positive NAAT. Methods to assess the CT viability have become available in research settings (e.g. viability-PCR; V-PCR). Here we assess viability in six different anatomic sites in women.
Methods
Immediately prior to treatment (STI clinic South Limburg), 28 vaginal NAAT-CT-positive (COBAS4800 CT/NG) adult women, were included in the 'CHLAMOUR' study. We used V-PCR to assess CT viable load (log10 CT/ml) in same clinician taken standardized samples from the cervix, vagina, perineum, anus, optional rectum, and pharynx. Mean loads were compared using t-tests.
Results
Twenty-eight women were included of whom 68% (19/28) consented to proctoscopic examination (rectal). NAAT-CT-positive rate was 75% for cervix, 79% vagina, 64% perineum, 64% anus, 74% rectum, and 21% for pharynx. Viable load was detected in 90% (19/21) CT positive cervix, 77% (17/22) vagina, 11% (2/18) perineum, 61% (11/18) anal, 93% (13/14) rectal, and 0% (0/6) pharynx samples. The mean viable load was marginally higher in cervical compared to vaginal samples (4.37 [SD:1.35] vs. 3.45 [SD:1.05], p=0.055). Mean viable load was higher in rectal compared to anal samples (3.51 [SD:0.51] vs. 2.70 [SD:0.42], p=0.01). Viable load was 2.72 [SD:1.69] CT positive perineum samples.
Conclusions
The amount of viable CT varied by anatomic site, and were highest 'upward in the body', which is thus likely to represent actual site of infection. Still, the vaginal and anal sites (that are usually self-sampled) had high concordance with the cervical and rectal sites. CT at the perineum may indicate autoinoculation. Notably, the presence of viable CT in anorectal samples indicated the presence of an active anorectal infection, which should be accounted for in comprehensive CT management.
Abstract Chlamydia trachomatis (CT) increases its plasmid numbers when stressed, as occurs in clinical trachoma samples. Most CT tests target the plasmid to increase the test sensitivity, but some only target the chromosome. We investigated clinical urogenital samples for total plasmid copy numbers to assess its diagnostic value and intra-bacterial plasmid copy numbers to assess its natural variation. Both plasmid and chromosome copies were quantified using qPCR, and the plasmid:chromosome ratio (PCr) calculated in two cohorts: (1) 383 urogenital samples for the total PCR (tPCr), and (2) 42 vaginal swabs, with one half treated with propium-monoazide (PMA) to prevent the quantification of extracellular DNA and the other half untreated to allow for both tPCr and intra-bacterial PCr (iPCr) quantification. Mann–Whitney U tests compared PCr between samples, in relation to age and gender. Cohort 1: tPCr varied greatly (1–677, median 16). Median tPCr was significantly higher in urines than vaginal swabs (32 vs. 11, p < 0.001). Cohort 2: iPCr was more stable than tPCr (range 0.1–3 vs. 1–11). To conclude, tPCr in urogenital samples was much more variable than previously described. Transport time and temperature influences DNA degradation, impacting chromosomal DNA more than plasmids and urine more than vaginal samples. Data supports a plasmid target in CT screening assays to increase clinical sensitivity.
