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.

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.

Ocular Chlamydia trachomatis Infection Under the Surgery, Antibiotics, Facial Cleanliness, and Environmental Improvement Strategy in Amhara, Ethiopia, 2011-2015.

Background: World Health Organization (WHO) recommendations for starting and stopping mass antibiotic distributions are based on a clinical sign of trachoma, which is indirectly related to actual infection with the causative agent, Chlamydia trachomatis. Methods: This study aimed to understand the effect of SAFE (surgery, antibiotics, facial cleanliness, and environmental improvement) interventions on ocular chlamydia in Amhara, Ethiopia, by describing the infection prevalence in a population-based sample of children aged 1-5 years. Trachoma surveys were conducted in all districts of Amhara, from 2011 to 2015 following approximately 5 years of SAFE. Ocular swabs were collected from randomly selected children to estimate the zonal prevalence of chlamydial infection. The Abbott RealTime polymerase chain reaction assay was used to detect C. trachomatis DNA. Results: A total of 15632 samples were collected across 10 zones of Amhara. The prevalence of chlamydial infection in children aged 1-5 years was 5.7% (95% confidence interval, 4.2%-7.3%; zonal range, 1.0%-18.5%). Chlamydial infection and trachomatous inflammation-intense (TI) among children aged 1-9 years were highly correlated at the zonal level (Spearman correlation [r] = 0.93; P < .001), while chlamydial infection and trachomatous inflammation-follicular were moderately correlated (r = 0.57; P = .084). Conclusions: After 5 years of SAFE, there is appreciable chlamydial infection in children aged 1-5 years, indicating that transmission has not been interrupted and that interventions should continue. The sign TI was highly correlated with chlamydial infection and can be used as a proxy indicator of infection.

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.

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.

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.

Ribosomal RNA evidence of ocular Chlamydia trachomatis infection following 3 annual mass azithromycin distributions in communities with highly prevalent trachoma.

Twelve trachoma-hyperendemic communities were treated with 3 annual mass azithromycin distributions. Children aged 0-9 years were monitored 1 year following the third treatment. An RNA-based test detected ocular chlamydial infection in more children than did a DNA-based test (6.9% vs 4.2%), and in a larger number of communities (8 vs 7).

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.

Lack of macrolide resistance in Chlamydia trachomatis after mass azithromycin distributions for trachoma.

We investigated antimicrobial drug resistance in ocular Chlamydia trachomatis 18 months after 4 biannual communitywide distributions of antimicrobial drugs in a region of Ethiopia where ocular strains of C. trachomatis are highly endemic. We found no significant differences in susceptibilities to azithromycin and doxycycline in 6 posttreatment and 4 pretreatment samples.

Evaluation of self-collected glans and rectal swabs from men who have sex with men for detection of Chlamydia trachomatis and Neisseria gonorrhoeae by use of nucleic acid amplification tests.

Self-collected glans and rectal swab specimens from men who have sex with men (MSM) may be appropriate, convenient specimens for testing. We evaluated the use of self-collected swabs for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae by a transcription-mediated amplification test (AC2; Aptima Combo 2; Gen-Probe Inc.) and a strand displacement amplification test (SDA; ProbeTec; Becton Dickinson Co.) in MSM seen at the city sexually transmitted disease clinic in San Francisco, CA. For the glans swab specimen, subjects enrolled early in the study rolled a Dacron swab across the meatus three times (method 1). A slightly more invasive procedure was performed later in the study: the subjects inserted the swab 1/4 in. into the urethra, rotated the swab, and then withdrew the swab (method 2). MSM self-collected a rectal swab specimen and also provided first-catch urine (FCU). Additional rectal swab samples were then obtained by the clinician. For the detection of C. trachomatis and N. gonorrhoeae, all swabs were evaluated by AC2 and SDA, FCU was tested by AC2, and the clinician-collected rectal swabs were cultured. A rectal true-positive (TP) result was defined as a culture-positive result for C. trachomatis or N. gonorrhoeae, two or more positive nucleic acid amplification test (NAAT) results, or a single NAAT-positive result confirmed by an alternate amplification method (the Aptima C. trachomatis or N. gonorrhoeae test). A glans TP result was defined as a positive result for FCU, positive results for both glans specimens (one tested by AC2 and one tested by SDA), or a positive result for a single glans specimen confirmed by an alternate amplification method. The prevalence rates of C. trachomatis and N. gonorrhoeae by testing of FCU were 6.8% (60/882 specimens) and 12.2% (108/882 specimens), respectively. Mixed results were obtained with the glans swab: N. gonorrhoeae detection by AC2 and SDA (method 1) had the best performance (sensitivities, >92%) with samples from a population with a higher prevalence of infection, but their performance for the detection of C. trachomatis was poor and varied by collection method (sensitivities, 56 to 68%). The prevalence rates of C. trachomatis and N. gonorrhoeae in the rectum were 7.3% (66/907 specimens) and 9.4% (83/882 specimens), respectively. The sensitivities of the tests with self-collected and clinician-collected rectal swab specimens were comparable (for C. trachomatis, 41% and 44%, respectively, by SDA and 82% and 71%, respectively, by AC2; for N. gonorrhoeae, 77% and 68%, respectively, by SDA and 84% and 78%, respectively, by AC2). AC2 and SDA were far superior to culture for the detection of C. trachomatis and N. gonorrhoeae in the rectum, with both tests detecting at least twice as many infections. While we found self-collected rectal swabs from MSM to be valid specimens for testing, the sensitivities of the tests with glans swab specimens were disappointing except for those from patients with symptomatic N. gonorrhoeae infections. Self-collected glans swab specimens may not be appropriate for the detection of C. trachomatis or for the detection of N. gonorrhoeae in low-risk or asymptomatic patients by AC2 and SDA, and we would not recommend their use on the basis of our results. Further studies are needed.

Complete local elimination of infectious trachoma from severely affected communities after six biannual mass azithromycin distributions.

OBJECTIVE: To determine whether infectious trachoma can be completely eliminated from severely affected villages. DESIGN: Cross-sectional survey of 2 villages previously enrolled and monitored over 42 months as part of a larger, group-randomized clinical trial. PARTICIPANTS: A total of 758 individuals residing in 2 villages with high baseline trachoma prevalence, of a total population of 768 (98.7%). METHODS: All members of the 2 villages were offered 6 biannual mass treatments with oral azithromycin. At 42 months, each current village member was examined. The right upper tarsal conjunctiva was everted and swabbed. Samples were processed for evidence of Chlamydia trachomatis RNA. MAIN OUTCOME MEASURES: Clinical activity by World Health Organization simplified grading scale for trachoma and laboratory evidence of chlamydial RNA. RESULTS: Average antibiotic coverage over the study period was 90% and 94% in the 2 villages. Clinical trachoma activity in children aged 1 to 5 years decreased from 78% and 83% in the 2 villages before treatment to 17% and 24% at 42 months. Polymerase chain reaction (PCR) evidence of infection in the same age group decreased from 48% to 0% in both villages at 42 months. When all age groups were examined, there were zero cases with evidence of chlamydial RNA among 758 total villagers tested. CONCLUSIONS: Biannual mass distribution of azithromycin can locally eliminate ocular chlamydial infection from severely affected communities.

Evaluation of CDC-recommended approaches for confirmatory testing of positive Neisseria gonorrhoeae nucleic acid amplification test results.

We evaluated three of the CDC approaches for confirming Neisseria gonorrhoeae (gonococcus [GC])-positive nucleic acid amplification test (NAAT) results: (i) repeating the original test on the original specimen, (ii) testing the original specimen with a different test, and (iii) performing a different test on a duplicate specimen collected at the same visit. For the first approach, clinical specimens were initially tested by Aptima Combo 2 (AC2) (Gen-Probe Inc., San Diego, CA), ProbeTec (strand displacement amplification [SDA]) (Becton Dickinson Co., Sparks, MD), and Amplicor (PCR) (Roche Molecular Systems, Branchburg, NJ). The original GC-positive specimens were then retested by the same NAAT for confirmation. For the second approach, specimens initially positive by AC2, SDA, or PCR were retested by different NAATs (SDA, PCR, AC2, and Aptima Neisseria gonorrhoeae assay [AGC]; Gen-Probe Inc.). For the third approach, duplicate urethral swabs and first-catch urine (FCU) samples from men and duplicate cervical swabs and FCU samples from women were each tested by SDA, AC2, and AGC in parallel. We found that 89 to 96% of samples positive by SDA, PCR, and AC2 were confirmed by repeat testing and that 85 to 98% of SDA, PCR, and AC2 results were confirmed by using different NAATs on the original specimen. For FCU samples from men, any NAAT can be used for confirmation. However, for all other specimen types, some NAATs cannot be used to confirm positive results from other NAATs. Thus, a single repeat test appears to be a reliable method for confirmation, but by doing more extensive testing, an additional 5% were confirmed. With >90% of all GC-positive NAATs being confirmed, our results show that confirmatory testing is not warranted for these genital specimens.

Comparing ceftriaxone plus azithromycin or doxycycline for pelvic inflammatory disease: a randomized controlled trial.

OBJECTIVE: To evaluate the equivalence of ceftriaxone plus doxycycline or azithromycin for cases of mild pelvic inflammatory disease (PID). METHODS: Patients with PID received an intramuscular injection of 250 mg of ceftriaxone, and were randomly assigned to receive 200 mg/d of doxycycline for 2 weeks, or 1 g of azithromycin per week, for 2 weeks. The degree of pain was assessed on days 2, 7, and 14 and clinical cure was assessed on day 14. RESULTS: From 133 patients eligible for the study, 13 were excluded for having conditions other than PID, 11 were lost on follow-up, and three had oral intolerance to the antibiotics, yielding 106 for protocol analysis. No significant difference was observed regarding the degree of pain between the doxycycline and azithromycin groups. Clinical cure per protocol was 98.2% (56 of 57; 95% confidence interval [CI], 0.9-0.99) with azithromycin, and 85.7% (42 of 49; 95% CI, 0.72-0.93) with doxycycline (P=0.02). In a modified intention to treat analysis, clinical cure was 90.3% (56 of 62; 95% CI, 0.80-0.96) with azithromycin, and 72.4% (42 of 58; 95% CI, 0.58-0.82) with doxycycline (P=.01); a relative risk of 0.35, and a number needed to treat of six for benefit with azithromycin. CONCLUSION: When combined with ceftriaxone, 1g of azithromycin weekly for 2 weeks is equivalent to ceftriaxone plus a 14-day course of doxycycline for treating mild PID.

Comparison of an rRNA-based and DNA-based nucleic acid amplification test for the detection of Chlamydia trachomatis in trachoma.

BACKGROUND/AIM: The World Health Organisation (WHO) hopes to achieve global elimination of trachoma, still the leading cause of preventable blindness worldwide, in part through mass antibiotic treatment. DNA-based nucleic acid amplification tests (NAATs) are currently used to evaluate the success of treatment programmes by measuring the prevalence of C trachomatis infection. Some believe that newer ribosomal RNA (rRNA)-based tests may be much more sensitive since bacterial rRNA is present in amounts up to 10 000 times that of genomic DNA. Others believe that rRNA-based tests are instead less sensitive but more specific, due to the presence of dead or subviable organisms that the test may not detect. This study compares an rRNA-based test to a DNA-based test for the detection of ocular C trachomatis infection in children living in trachoma-endemic villages. METHODS: An rRNA-based amplification test and DNA-based polymerase chain reaction (PCR) were performed on swab specimens taken from the right upper tarsal conjunctiva of 56 children aged 0-10 years living in two villages in Amhara, Ethiopia. RESULTS: The rRNA-based test detected ocular C trachomatis infection in 35 (63%) subjects compared with 22 (39%) detected by PCR (McNemar's test, p = 0.0002). The rRNA-based test gave positive results for all subjects that were positive by PCR, and also detected infection in 13 (23%) additional subjects. CONCLUSION: The rRNA-based test appears to have significantly greater sensitivity than PCR for the detection of ocular chlamydial infection in children in trachoma-endemic villages. Using the rRNA-based test, we may be able to detect infection that was previously missed with PCR. Past studies using DNA-based tests to assess prevalence of infectious trachoma following antibiotic treatment may have underestimated the true prevalence of infection.

Sociodemographic markers and behavioral correlates of sexually transmitted infections in a nonclinical sample of adolescent and young adult women.

BACKGROUND: We examined sociodemographic markers and 3-month behavioral correlates of sexually transmitted infections (STIs) in a nonclinical cross-section of adolescent and young adult women. METHODS: All women (N=2288) enrolled in recruit training for the US Marine Corps during a 1-year period were asked to voluntarily participate in either a cognitive-behavioral, skills-building intervention to prevent STIs and unintended pregnancies or a nutrition and fitness program. Participants (94.2%) completed a self-administered questionnaire and were screened for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis infection. The present study presents only the baseline data from the larger study. RESULTS: One or more STIs was diagnosed in 14.1% of participants. Results of a logistic-regression model indicated that the presence of an STI at screening was significantly (Por=2 years), frequency of hormonal contraceptive use (never and sometimes), perception that their sex partners had other concurrent sex partners, and the race or ethnicity of their last sex partner (African American and Native American). CONCLUSIONS: The high prevalence of STIs in this nonclinical sample of young women suggests the need for ongoing screening and prevention interventions that target young, healthy, sexually active women.

What sexually transmitted disease screening method does the adolescent prefer? Adolescents' attitudes toward first-void urine, self-collected vaginal swab, and pelvic examination.

OBJECTIVE: To assess sexually active adolescents' attitudes toward 3 screening collection techniques for detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis using first-void urine (FVU), self-collected vaginal swab specimens, and pelvic examination with clinician-collected endocervical swab specimens. DESIGN: Participants completed a preexamination health survey, provided FVU and self-collected vaginal swab samples, and had a pelvic examination with endocervical swab specimen collection. In a confidential postexamination interview, patients ranked the 3 screening techniques according to preference and responded to qualitative positive and negative descriptors to evaluate each technique. SETTING: San Francisco area health maintenance organization and university clinics. PARTICIPANTS: A convenience sample of 155 ethnically diverse females aged 12 to 21 years, who were sexually active and were to have a pelvic examination. MAIN OUTCOME MEASURES: Adolescents' preferences for and evaluations of 3 sexually transmitted disease screening techniques. RESULTS: Participants preferred the FVU test for sexually transmitted disease screening over the pelvic examination and the self-administered vaginal swab test (P<.001). These results were consistent when controlling for potentially mitigating experiences, including previous pelvic examination, tampon or condom use, and prior pregnancy. In evaluating what they liked and disliked about each of the 3 screening methods, participants described the FVU most positively, the pelvic examination most negatively, and the vaginal swab technique slightly less positively than the FVU. CONCLUSION: Most sexually active adolescents attending clinics for pelvic examination prefer to be screened for sexually transmitted diseases first by the FVU, second by the self-collected vaginal swab test, and last by the pelvic examination.