Multicenter Comparison of Nucleic Acid Amplification Tests for the Diagnosis of Rectal and Oropharyngeal Chlamydia trachomatis and Neisseria gonorrhoeae Infections.

Research using nucleic acid amplification tests (NAATs) have repeatedly found rectal and oropharyngeal infections with Chlamydia trachomatis and Neisseria gonorrhoeae to be common and potentially more difficult to treat than genital infections. Unfortunately, public health and patient care efforts have been hampered by the lack of FDA-cleared NAATs with claims for anorectal or oropharyngeal samples. At the time of the initiation of this study, no commercially available assays had these claims. We formed a novel partnership among academic institutions and diagnostic manufacturers to address this public health need. From May 2018 through August 2019, we recruited 1108 women, 1256 men, and 26 transgender persons each of whom provided 3 anal and 3 oropharyngeal swab specimens. The 3 anal swabs were pooled into a single transport tube as were the 3 oropharyngeal swabs. The performance of each of three study assays was estimated by comparison to the composite result and relative to one another. Percent positivity for chlamydia was 5.9 and 1.2% from anal and oropharyngeal specimens, respectively, compared to 4.2 and 4.1% for gonorrhea. Sensitivity for chlamydia detection ranged from 81.0 to 95.1% and 82.8 to 100% for anal and oropharyngeal specimens, respectively. Gonorrhea sensitivity ranged from 85.9 to 99.0% and 74.0 to 100% for anal and oropharyngeal samples, respectively. Specificity estimates were ≥ 98.9% for all assays, organisms, and sample types. Although there was heterogeneity between sensitivity estimates, these assays offer better ability to detect extragenital infections than culture and potential solutions for providing appropriate sexual health care for populations in which these infections are of concern.

Precision of the Abbott RealTime Assay in the Detection of Ocular Chlamydia trachomatis in a Trachoma-Endemic Area of Ethiopia.

Nucleic acid amplification tests are increasingly used to detect ocular chlamydia infection in trachoma research and programs. To evaluate the reliability of Chlamydia trachomatis detection by the Abbott RealTime CT/NG assay (Abbott Molecular, Inc., Des Plaines, IL) on the m2000 platform, three conjunctival samples were collected from each of 200 children aged 0-9 years in Ethiopia: two from the right eye and one from the left eye. Four aliquots were processed for each child: two from the first right eye sample, one from the second right eye sample, and one from the left eye sample. Sixty-nine swabs were processed in a U.S. laboratory and 131 in an Ethiopian laboratory. Intra-class correlation coefficients (ICCs) were high when comparing two aliquots from the same swab (ICC ranged from 0.96 to 0.99), two separate swabs from the right eye (0.89-0.91), and one right and one left eye swab (0.87-0.89), indicating reliable chlamydial load assessment across different samples and laboratory settings.

Ocular Chlamydia trachomatis infection and infectious load among pre-school aged children within trachoma hyperendemic districts receiving the SAFE strategy, Amhara region, Ethiopia.

BACKGROUND: After approximately 5 years of SAFE (surgery, antibiotics, facial cleanliness, environmental improvement) interventions for trachoma, hyperendemic (trachomatous inflammation-follicular (TF) ≥30%) districts remained in Amhara, Ethiopia. This study's aim was to characterize the epidemiology of Chlamydia trachomatis (Ct) infection and load among pre-school aged children living under the SAFE strategy. METHODS: Conjunctival swabs from a population-based sample of children aged 1-5 years collected between 2011 and 2015 were assayed to provide Ct infection data from 4 endemic zones (comprised of 58 districts). Ct load was determined using a calibration curve. Children were graded for TF and trachomatous inflammation-intense (TI). RESULTS: 7,441 children were swabbed in 4 zones. TF and TI prevalence were 39.9% (95% confidence Interval [CI]: 37.5%, 42.4%), and 9.2% (95% CI: 8.1%, 10.3%) respectively. Ct infection prevalence was 6.0% (95% CI: 5.0%, 7.2%). Infection was highest among children aged 2 to 4 years (6.6%-7.0%). Approximately 10% of infection occurred among children aged 1 year. Ct load decreased with age (P = 0.002), with the highest loads observed in children aged 1 year (P = 0.01) vs. aged 5 years. Participants with TF (P = 0.20) and TI (P<0.01) had loads greater than individuals without active trachoma. CONCLUSIONS: In this hyperendemic setting, it appears that the youngest children may contribute in meaningful ways towards persistent active trachoma.

Mycoplasma genitalium, Chlamydia trachomatis, and Neisseria gonorrhoeae Detected With Aptima Assays Performed on Self-Obtained Vaginal Swabs and Urine Collected at Home and in a Clinic.

Self-obtained vaginal swabs, first-void urine and pooled specimens were collected at home and in a clinic. Percent prevalence and collection site concordance was 30.3 and 100 for Mycoplasma genitalium (74.4% azithromycin resistant) 15.1 and 96.7 for Chlamydia trachomatis and 6.6 and 100 for Neisseria gonorrhoeae (27% ciprofloxacin-resistant).

Alternate Nucleic Acid Targets Can Be Used To Create a Composite Standard To Evaluate Clinical Performance of Nucleic Acid Amplification Tests.

Evaluating the clinical performance of a new nucleic acid amplification test (NAAT) for Mycoplasma genitalium, B. Kirkconnell, B. Weinbaum, K. Santos, T. Le Nguyen, et al. (J Clin Microbiol 57:e00264-19, 2019, https://doi.org/10.1128/JCM.00264-19) created 3 alternate NAATs that detected other unique M. genitalium gene targets. Lacking a reference standard, they used the consensus of results with those 3 NAATs as the comparator. This approach could be a new paradigm to evaluate new NAATs when there is no previously defined reference standard.

Comprehensive global genome dynamics of Chlamydia trachomatis show ancient diversification followed by contemporary mixing and recent lineage expansion.

Chlamydia trachomatis is the world's most prevalent bacterial sexually transmitted infection and leading infectious cause of blindness, yet it is one of the least understood human pathogens, in part due to the difficulties of in vitro culturing and the lack of available tools for genetic manipulation. Genome sequencing has reinvigorated this field, shedding light on the contemporary history of this pathogen. Here, we analyze 563 full genomes, 455 of which are novel, to show that the history of the species comprises two phases, and conclude that the currently circulating lineages are the result of evolution in different genomic ecotypes. Temporal analysis indicates these lineages have recently expanded in the space of thousands of years, rather than the millions of years as previously thought, a finding that dramatically changes our understanding of this pathogen's history. Finally, at a time when almost every pathogen is becoming increasingly resistant to antimicrobials, we show that there is no evidence of circulating genomic resistance in C. trachomatis.

Stability Studies on Dry Swabs and Wet Mailed Swabs for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Aptima Assays.

The Aptima Combo 2 (AC2) and Aptima CT (ACT) (Hologic Inc., San Diego, CA) are nucleic acid amplification tests (NAATs) that detect Chlamydia trachomatis AC2 also detects Neisseria gonorrhoeae Storage and temperature conditions may impact the utility of NAATs in some settings and screening programs. We evaluated specimen stability for use beyond the Aptima package insert specifications for temperature and duration of storage (between 2°C and 30°C and 60 days, respectively) in two studies: (i) dry C. trachomatis-seeded swabs were used with ACT after storage at 4°C, 23°C, or 36°C for up to 84 days and (ii) swabs seeded with C. trachomatis and N. gonorrhoeae and then placed in transport medium were tested with AC2, after being mailed via the U.S. Postal Service to three different sites. Prolonged storage of samples had no effect, and samples stored at 4°C, 23°C, and 36°C for up to 84 days yielded comparable ACT positivities, although there was a drop in signal intensity for virtually all specimens under all storage/shipping conditions after day 21. In the mailing study, 80%, 52% and 29% of seeded swabs were exposed to temperatures of >30°C during three rounds in transit, and 2% reached temperatures of >40°C. No evidence of signal degradation in the AC2 assay for detection of C. trachomatis or N. gonorrhoeae was observed, although some mailed swabs took more than 5 weeks to reach the laboratory site. These two studies support the potential use of swabs at temperatures above 36°C and storage beyond 60 days and provide confidence regarding this commercially available NAAT for testing of specimens after mailing.

What is needed to guide testing for anorectal and pharyngeal Chlamydia trachomatis and Neisseria gonorrhoeae in women and men? Evidence and opinion.

BACKGROUND: Anorectal and pharyngeal infections with Chlamydia trachomatis (CT) and Neisseria gonorrheae (NG) are commonly observed in men who have sex with men (MSM). There is increasing evidence that such infections at extra-genital sites are also common in women. In both sexes, these infections are largely overlooked as they are not routinely tested for in regular care. Testing based on sexual behavior or symptoms would only detect half of these extra-genital infections. This paper elucidates the differences and similarities between women and MSM, regarding the epidemiology of extra-genital CT and NG. It discusses the clinical and public health impact of untested extra-genital infections, how this may impact management strategies, and thereby identifies key research areas. DISCUSSION: Extra-genital CT is as common in women as it is in MSM; NG in women is as common at their extra-genital sites as it is at their genital sites. The substantial numbers of extra-genital CT and NG being missed in women and MSM indicate a need to test and treat more patients and perhaps different choices in treatment and partner management strategies. Doing so will likely contribute to reduced morbidity and transmission in both sexes. However, in our opinion, it is clear that there are several knowledge gaps in understanding the clinical and public health impact of extra-genital CT and NG. Key research areas that need to be addressed concern associated morbidity (anorectal and reproductive morbidity due to extra-genital infections), 'the best' management strategies, including testing and treatment for extra-genital CT, extra-genital treatment resistance, transmission probabilities between partners and between anatomic sites in a woman, and impact on transmission of other infections. Data are also lacking on cost-effectiveness of pharyngeal testing, and of NG testing and anorectal CT testing in women. Gaps in the management of extra-genital CT and NG may also apply for other STIs, such Mycoplasma genitalium. Current management strategies, including testing, to address extra-genital CT and NG in both sexes are suboptimal. Comparative data on several identified key themes in women and MSM are lacking and urgently needed to guide better management of extra-genital infections.

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Rectal and Oropharyngeal Swabs and Urine Specimens from Men Who Have Sex With Men with Abbott's M2000 RealTime.

We evaluated Abbott's RealTime assay for the detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) in the urethra, oropharynx, and rectum of 260 men who have sex with men. Compared with Hologic's AC2, RealTime had good agreement for detecting CT and GC. Overall, there were 25 CT and 44 GC AC2 positives, and 26 CT and 38 GC RealTime positives. For total negatives, there were 742 CT and 725 GC for AC2, 744 CT and 724 GC for RealTime.

Is there evidence for resistance of ocular Chlamydia trachomatis to azithromycin after mass treatment for trachoma control?

BACKGROUND: Trachoma, caused by repeated infections with ocular Chlamydia trachomatis, is targeted for elimination using multiple annual rounds of mass drug administration (MDA) in endemic communities. Infection rates do not decline as expected in some communities, leading to concerns about azithromycin resistance. METHODS: After 3 yearly MDAs in 32 communities in Tanzania, 107 children were identified 1 year later with infection. All were provided MDA again, and 90 were seen again at 2 months, of whom 30 had infection. Chlamydia trachomatis isolates were obtained before and after MDA in 15 paired samples and were tested for antimicrobial susceptibility. The infectious load of C. trachomatis before MDA was determined in 30 children who had infection at both times and 60 whose infection cleared. RESULTS: The median load was 8.6 genome copies per polymerase chain reaction in the consistently infected, and 8.4 in those whose infection cleared (P = .86). For the consistently infected, the average minimum inhibitory concentration was 0.26 µg/mL for azithromycin before and 0.20 µg/mL after MDA. All isolates had minimum inhibitory concentration ≤0.50 µg/mL. CONCLUSIONS: There is no evidence that continued infection after MDA was due either to resistance to azithromycin or to a heavier load of organism before treatment. Other potential causes of persistent infection need to be evaluated.

Will droplet digital PCR become the test of choice for detecting and quantifying ocular Chlamydia trachomatis infection? Maybe not.

Evaluation of: Roberts CH, Last A, Molina-Gonzalez S et al. Development and evaluation of a next-generation digital PCR diagnostic assay for ocular chlamydia trachomatis infections. J. Clin. Microbiol. 51(7), 2195-2203 (2013). Trachoma is the leading infectious cause of blindness in developing countries. Currently, there is no program to eliminate blinding trachoma as a public health problem. We need better diagnostic tests for research and to assess progress in control programs. Roberts et al. adapted droplet digital PCR (ddPCR), an emulsion PCR process that performs absolute quantitation of nucleic acids, to detect and quantify Chlamydia trachomatis infections. They compared the results with ddPCR on conjunctival swab specimens collected in trachoma-endemic area to results using Roche's Amplicor® C. trachomatis/Neisseria gonorrhoeae (CT/NG) PCR and found that ddPCR sensitivity was 73.3%. The authors concluded that 'ddPCR is an effective diagnostic technology suitable for both research and clinical use in diagnosing ocular C. trachomatis infections'. This reviewer disagrees, feeling that if the stated sensitivity is accurate, it is too low, and suggests there may be good reasons to adapt commercially available tests for this purpose.

Is there evidence of the new variant Chlamydia trachomatis in the United States?

A specific real-time polymerase chain reaction followed by melt curve analysis was developed for the detection of the Swedish variant (nvCT) strain of Chlamydia trachomatis (CT). Surveillance was performed on 476 CT-positive clinical specimens obtained from 15 laboratories around the United States using nucleic acid amplification test assays, which would not miss the nvCT. All were negative for nvCT; thus, there is no evidence of the nvCT in the United States.

Incentivising safe sex: a randomised trial of conditional cash transfers for HIV and sexually transmitted infection prevention in rural Tanzania.

OBJECTIVE: The authors evaluated the use of conditional cash transfers as an HIV and sexually transmitted infection prevention strategy to incentivise safe sex. DESIGN: An unblinded, individually randomised and controlled trial. SETTING: 10 villages within the Kilombero/Ulanga districts of the Ifakara Health and Demographic Surveillance System in rural south-west Tanzania. PARTICIPANTS: The authors enrolled 2399 participants, aged 18-30 years, including adult spouses. INTERVENTIONS: Participants were randomly assigned to either a control arm (n=1124) or one of two intervention arms: low-value conditional cash transfer (eligible for $10 per testing round, n=660) and high-value conditional cash transfer (eligible for $20 per testing round, n=615). The authors tested participants every 4 months over a 12-month period for the presence of common sexually transmitted infections. In the intervention arms, conditional cash transfer payments were tied to negative sexually transmitted infection test results. Anyone testing positive for a sexually transmitted infection was offered free treatment, and all received counselling. MAIN OUTCOME MEASURES: The primary study end point was combined prevalence of the four sexually transmitted infections, which were tested and reported to subjects every 4 months: Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and Mycoplasma genitalium. The authors also tested for HIV, herpes simplex virus 2 and syphilis at baseline and month 12. RESULTS: At the end of the 12-month period, for the combined prevalence of any of the four sexually transmitted infections, which were tested and reported every 4 months (C trachomatis, N gonorrhoeae, T vaginalis and M genitalium), unadjusted RR for the high-value conditional cash transfer arm compared to controls was 0.80 (95% CI 0.54 to 1.06) and the adjusted RR was 0.73 (95% CI 0.47 to 0.99). Unadjusted RR for the high-value conditional cash transfer arm compared to the low-value conditional cash transfer arm was 0.76 (95% CI 0.49 to 1.03) and the adjusted RR was 0.69 (95% CI 0.45 to 0.92). No harm was reported. CONCLUSIONS: Conditional cash transfers used to incentivise safer sexual practices are a potentially promising new tool in HIV and sexually transmitted infections prevention. Additional larger study would be useful to clarify the effect size, to calibrate the size of the incentive and to determine whether the intervention can be delivered cost effectively. TRIAL REGISTRATION NUMBER: NCT00922038 ClinicalTrials.gov.

Diagnostic characteristics of tests for ocular Chlamydia after mass azithromycin distributions.

PURPOSE: Although trachoma control programs frequently use the World Health Organization (WHO) simplified grading system for trachoma to monitor the clinical response after repeated mass azithromycin treatments, the programmatic relevance of this evaluation after multiple rounds of antibiotic treatments is unclear. METHODS: Three rounds of annual mass azithromycin were distributed to 12 villages in Ethiopia. Twelve months after the third treatment, children were assessed for follicular trachomatous inflammation (TF) and intense trachomatous inflammation (TI) using the WHO simplified grading system and for ocular chlamydial infection using DNA-based and RNA-based tests. Test characteristics for predicting chlamydial infection were computed assuming a chlamydial RNA-based gold standard. As a secondary analysis, test characteristics were also assessed using a latent class analysis. RESULTS: The prevalence of RNA evidence of ocular chlamydia was 7.1% (95% confidence interval [CI], 2.7-17.4). A DNA-based test and TF had sensitivities of 61.0% (95% CI, 47.1-73.3) and 65.9% (95% CI, 41.6-83.9), specificities of 100% (95% CI, 99.3-100) and 67.5% (95% CI, 61.0-73.5), and positive predictive values of 100% (95% CI, 86.3-100) and 13.4% (95% CI, 5.5-29.3) compared with an RNA-based gold standard. The latent class analysis confirmed that the RNA-based test was a reasonable choice for a gold standard, with a sensitivity of 100% (95% CI, 67.1-100) and specificity of 99.6% (95% CI, 98.1-100). CONCLUSIONS: Basing treatment decisions after mass azithromycin distributions on the WHO simplified grading system will maximize the treatment of infected persons compared with a DNA-based test but will also result in more uninfected persons being treated. The RNA-based test was considerably more sensitive, and almost equivalently specific, compared with a DNA-based test. (ClinicalTrials.gov number, NCT00322972.).