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.

Workflow and Throughput of Commercial Assays to Detect Mycoplasma genitalium and Macrolide Resistance-Mediating Mutations.

ABSTRACT: Aptima Mycoplasma genitalium (MG) required the shortest and STD6 the longest time to detect MG in clinical samples. ResistancePlus MG detected MG and macrolide resistance-mediating mutations simultaneously. Times were influenced by specimen numbers. M. genitalium positives from the other 2 assays required increased time for macrolide resistance-mediating mutation sequencing.

Comparison of Assays for the Diagnosis of Mycoplasma genitalium and Macrolide Resistance Mutations in Self-Collected Vaginal Swabs and Urine.

BACKGROUND: The objective was to compare commercial assays on clinical specimens for Mycoplasma genitalium (MG) detection and macrolide resistance mutation (MRM) frequency. METHODS: Three self-collected vaginal swabs (VS) and a first-void urine (FVU) from 300 consented women were tested by Aptima MG (AMG), ResistancePlus MG (RPMG) and Seeplex STD6 ACE (STD6) for detection of MG. Aptima MG and STD6 MG positives were tested for MRM using MG 23S rRNA polymerase chain reaction with Sanger sequencing (23SMGSS) compared with MRM determination in the RPMG assay. Unique AMG positives were tested with confirmatory Aptima assays. RESULTS: M. genitalium prevalence ranged from 7.1% to 19.7%, influenced by the assay used and the specimen tested. Overall agreements for MG detection were 96.3% (κ = 0.91) for VS and 93.3% (κ = 0.72) for FVU between AMG and RPMG with lower agreements with STD6. Using a rotating reference standard, sensitivities on VS and FVU were 100% and 100% for AMG, 100% and 83.3% for RPMG, and 54.2% and 48.4% for STD6. Specificities were high for RPMG and STD6 and AMG detected extra positives, most of which were confirmed. Macrolide resistance mutation frequency rates testing VS and FVU were 50% (24/48) and 58.1% (18/31) by RPMG compared with 52.5% (31/59) and 23.5% (12/51) by 23SMGSS. MRM overall agreements between RPMG and 23SMGSS were 73.2% (κ = 0.41) for VS and 76.0% (κ = 0.52) for FVU. CONCLUSIONS: Aptima MG detected more cases of MG infections. ResistancePlus MG detection was more effective on VS than on FVU. Seeplex STD6 ACE performance was inferior. The MRM detection component of RPMG agreed with results from 23SMGSS most of the time.

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.

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).

Urinary Meatal Swabbing Detects More Men Infected With Mycoplasma genitalium and Four Other Sexually Transmitted Infections Than First Catch Urine.

Urinary meatal swabs compared with urine showed higher infection rates for Mycoplasma genitalium (15.3% vs 12.6%, P = 0.035), Chlamydia trachomatis (11.3% vs 9.3%, P = 0.039), Neisseria gonorrhoeae (1.4% vs 1.1%, P = 1.00), Trichomonas vaginalis (8.0% vs 1.7%, P < 0.001), and high-risk human papillomavirus (5.9% vs 3.4%, P = 0.078) respectively.

Comparison of cobas 4800, m2000, Viper XTR, and Infinity 80 Automated Instruments When Processing Urine Specimens for the Diagnosis of Chlamydia trachomatis and Neisseria gonorrhoeae.

OBJECTIVES: North American and European advisory groups recommend testing for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) with nucleic acid amplification tests. Testing is often performed on automated instruments. The objectives of this study were to process urines for the diagnosis of CT and NG and to examine workflow procedures and outcomes. METHODS: While processing 1, 24, 48, 96, and 192 urine specimens on 3 batch-mode systems which use 96-well plates: cobas 4800, m2000, and Viper XTR and the random access cartridge testing GeneXpert Infinity 80, we measured assay performance, hands-on time for processing and maintenance, reagents and plastics consumption, time required to obtain results, and testing accuracy. RESULTS: The Infinity 80 required the least hands-on time for single specimens and smaller batches, whereas the Viper XTR and m2000 required the most hands-on time for all batch sizes. Cumulative daily, weekly, and monthly maintenance was highest for the Viper XTR and lowest for Infinity 80. All batch-mode instruments consumed large amounts of disposables. Time to results was shortest for the Infinity 80, and the Viper XTR provided the shortest time for the batch-mode instruments. All systems showed similar diagnostic accuracy. CONCLUSIONS: Because detection performances were similar, issues of hands-on time, maintenance, time to results, and consumables are important operational factors for the diagnosis and treatment of CT/NG infections.

Trichomonas vaginalis Prevalence and Correlates in Women and Men Attending STI Clinics in Western Canada.

Trichomonas vaginalis prevalence (2.8%) in female sexually transmitted infection clinic attendees was within the prevalence of chlamydia (5.8%) and gonorrhea (1.8%), while being very low for male attendees (0.2%). Correlates among women were indigenous ethnicity, other ethnicity, and being symptomatic.

Mycoplasma genitalium Antibiotic Resistance-Mediating Mutations in Canadian Women With or Without Chlamydia Trachomatis Infection.

Testing remnant Aptima specimens from women infected with Chlamydia trachomatis detected 13.4% (53/396) with Mycoplasma genitalium compared with 5.4% (22/406) in matched C. trachomatis-negative women. Overall, 9.4% (provincial ranges of 3-20%) were infected with M. genitalium and resistance mediating mutations were found in 47.3% (26/55) to macrolides and 1.9% (1/53) to fluoroquinolones by sequencing.

Ease, Comfort, and Performance of the HerSwab Vaginal Self-Sampling Device for the Detection of Chlamydia trachomatis and Neisseria gonorrhoeae.

BACKGROUND: Many sexually transmitted diseases are asymptomatic in the lower genital tract and can cause upper tract complications if left untreated. Self-collected vaginal (SCV) swabs enable the accurate detection of many sexually transmitted infections and give women the option of collecting their own samples while providing them with privacy and convenience. METHODS: We compared SCV samples collected and transported dry using the HerSwab device to physician-collected vaginal (PCV) Aptima swabs for the detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG), and measured patients' ease and comfort with self-collection. A total of 189 women aged 16 to 41 years were consented into the study and answered a standardized anonymized questionnaire regarding self-collection with the HerSwab device. RESULTS: Women reported self-collection with HerSwab to be easy (97.1%) and comfortable (88.3%). They preferred self-collection over physician collection (80.9%) and would consider using HerSwab for self-collection at home (79.7%). Samples of SCV and PCV showed an overall agreement of 94.7% (κ = 0.64) for CT and of 98.4% (κ = 0.56) for NG, and HerSwab collection detected 7 more positive patients than PCV collection. The overall prevalence of infection was 10.6% for CT and 2.6% for NG. CONCLUSION: HerSwab SCV samples are suitable for the diagnosis of CT and NG.

Comparison of Workflow, Maintenance, and Consumables in the GeneXpert Infinity 80 and Panther Instruments While Testing for Chlamydia trachomatis and Neisseria gonorrhoeae.

BACKGROUND: The 2015 Sexually Transmitted Diseases Treatment Guidelines from the Centers for Disease Control and Prevention recommend testing for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) using nucleic acid amplification tests, and prompt treatment of infected persons on site under direct observation. Faster time to results may enable treatment and management outcomes. METHODS: Workflow parameters for processing 1, 10, 48, 96, and 192 tests were determined in the GeneXpert Infinity 80 (Cepheid) and Panther (Hologic) instruments. RESULTS: In an Xpert CT/NG cartridge, the time to first results on the Infinity 80 was 1 hour 30 minutes for single or multiple tests and final results for 10, 48, 96, and 192 tests were available at 1 hour 37 minutes, 1 hour 54 minutes, 3 hour 17 minutes, and 5 hour 7 minutes, respectively. With the Aptima CT/GC assay on the Panther, the respective times were 3 hr 45 min for the first test result, and 3 hour 51 minutes, 4 hour 38 minutes, 5 hour 26 minutes, and 7 hour 4 minutes to final results. The Panther required more time for maintenance and consumed a greater variety of plastics and reagents but required less hands-on time when testing larger numbers of specimens. CONCLUSIONS: The Infinity 80 is a versatile instrument for continuous random access testing of small or large numbers of clinical specimens and may provide diagnostic results, in some settings, in time for treatment of CT and NG infections.

Rapid Diagnosis of Trichomonas vaginalis by Testing Vaginal Swabs in an Isothermal Helicase-Dependent AmpliVue Assay.

BACKGROUND: The AmpliVue Trichomonas Assay (Quidel) is a new Federal Drug Administration-cleared rapid test for qualitative detection of Trichomonas vaginalis (TV) DNA in female vaginal specimens. The assay is based on BioHelix's helicase-dependent amplification isothermal technology in conjunction with a disposable lateral-flow detection device, with a total turnaround time of approximately 45 minutes. OBJECTIVE: The objective of this study was to compare the performance of this new assay to wet preparation and culture as well as to another Federal Drug Administration-cleared nucleic acid amplification assay. METHODS: Four clinician collected vaginal swabs were obtained from women attending sexually transmitted disease, family planning, and OB/GYN clinics and tested by AmpliVue Trichomonas Assay and comparator tests: saline microscopy, TV culture (InPouch), and Aptima TV. AmpliVue Trichomonas Assay results were compared with a composite positive comparator (CPC) as determined by the results from culture and/or wet mount microscopic examination. At least one of either the wet preparation or culture reference test results was required to be positive to establish CPC. RESULTS: A total of 992 patients, 342 symptomatic and 650 asymptomatic patients, were included in the study. Results for AmpliVue for all women combined compared with saline microscopy and culture as a CPC yielded a sensitivity of 100%. Specificity for all women was 98.2%. Overall percent agreement versus Aptima TV was 97.8%. Sensitivity for AmpliVue compared with Aptima was 90.7% %, whereas specificity was 98.9%. CONCLUSIONS: The rapid AmpliVue Trichomonas Assay performed as well as microscopy and culture, and had comparable sensitivity and specificity to another nucleic acid amplification test for the detection of TV. This study provided evidence of new diagnostic options and indicated very good performance of amplified testing for detection of TV in symptomatic and asymptomatic women.

Canadian Public Health Laboratory Network laboratory guidelines for the use of point-of-care tests for the diagnosis of syphilis in Canada.

Syphilis point-of-care tests (POCT) are widely available in developing countries enabling early diagnosis, treatment and support. The majority of commercially available tests use treponemal antigens and the presence of antibodies does not distinguish between current and past infection, which may lead to unnecessary antibiotic use and stigmatization of having a current STI. In hard-to-reach populations, the benefits may outweigh the risks. Available studies show reasonable performance of POCT with median sensitivity of 86%, specificity of 99% and positive predictive values >80% when prevalence was >0.3%. Although no syphilis POCT are approved in Canada at this time, a single study in an outreach setting in Alberta showed limited benefit due to a high prevalence of previous infection but more studies are needed. Newer dual tests employing treponemal and nontreponemal antigens look promising.

Les tests au point de service (TPdS) de la syphilis sont largement répandus dans les pays en voie développement, ce qui favorise un diagnostic, un traitement et un soutien rapides. La majorité des tests offerts sur le marché font appel aux antigènes tréponémiques. Toutefois, la présence d'anticorps ne permet pas de distinguer une infection en cours d'une infection antérieure, ce qui peut entraîner l'utilisation inutile d'antibiotiques et une stigmatisation liée à l'ITS. Dans des populations difficiles à joindre, les avantages dépassent peut-être les risques. Selon les études existantes, les TPdS donnent des résultats raisonnables, à la sensibilité médiane de 86 %, à la spécificité de 99 % et aux valeurs prédictives positives de plus de 80 % lorsque la prévalence est supérieure à 0,3 %. Même si aucun TDdS de la syphilis n'est approuvé au Canada, une seule étude, réalisée dans un milieu communautaire en Alberta, en a démontré les avantages limités en raison de la forte prévalence d'infection antérieure, mais d'autres études s'imposent. De nouveaux doubles tests, faisant appel à des antigènes tréponémiques et non tréponémiques, semblent prometteurs.

Canadian Public Health Laboratory Network laboratory guidelines for the use of direct tests to detect syphilis in Canada.

Treponema pallidum subsp. pallidum and/or its nucleic acid can be detected by various methods such as microscopy, rabbit infectivity test or polymerase chain reaction (PCR) tests. The rabbit infectivity test for T. pallidum, although very sensitive, has been discontinued from most laboratories due to ethical issues related to the need for animal inoculation with live T. pallidum, the technically demanding procedure and long turnaround time for results, thus making it impractical for routine diagnostic use. Dark-field and phase-contrast microscopy are still useful at clinic- or hospital-based laboratories for near-bedside detection of T. pallidum in genital, skin or mucous lesions although their availability is decreasing. The lack of reliable and specific anti-T. pallidum antibodies and its inferior sensitivity to PCR may explain why the direct fluorescent antibody test for T. pallidum is not widely available for clinical use. Immunohistochemical staining for T. pallidum also depends on the availability of specific antibodies, and the method is only applicable for histopathological examination of biopsy and autopsy specimens necessitating an invasive specimen collection approach. With recent advances in molecular diagnostics, PCR is considered to be the most reliable, versatile and practical for laboratories to implement. In addition to being an objective and sensitive test for direct detection of Treponema pallidum subsp. pallidum DNA in skin and mucous membrane lesions, the resulting PCR amplicons from selected gene targets can be further characterized for antimicrobial (macrolide) susceptibility testing, strain typing and identification of T. pallidum subspecies.

Diverses méthodes, telles que la microscopie, le test d'infectivité du lapin et la réaction en chaîne de la polymérase (PCR), permettent de déceler le Treponema pallidum sous-espèce pallidum et/ou son acide nucléique. Même s'il est très sensible, le test d'infectivité du lapin n'est plus utilisé dans la plupart des laboratoires pour déceler le T. pallidum. En effet, des raisons éthiques liées à la nécessité d'inoculer le T. pallidum vivant à l'animal, l'intervention exigeante sur le plan technique et la longue attente avant d'obtenir les résultats le rendent peu pratique pour un usage diagnostique régulier. Dans les laboratoires des cliniques ou des hôpitaux, la microscopie à fond noir et la microscopie à contraste de phase contribuent toujours à déceler le T. pallidum dans les lésions génitales, cutanées ou muqueuses près du chevet du patient, mais elles sont de moins en moins offertes. Le test d'immunofluorescence directe est peu utilisé pour diagnostiquer le T. pallidum en milieu clinique, peut-être en raison de l'absence d'anticorps anti-T. pallidum fiables et spécifiques et de sa faible sensibilité par rapport au PCR. La coloration immunohistochimique du T. pallidum dépend également de la présence d'anticorps spécifiques, et la méthode est applicable seulement à l'examen histopathologique des prélèvements invasifs de biopsies et d'autopsies. Étant donné les progrès récents des diagnostics moléculaires, la PCR est considérée comme le test le plus fiable, le plus polyvalent et le plus pratique à utiliser en laboratoire. Le PCR est objectif et spécifique pour la détection directe de l'ADN du Treponema pallidum sous-espèce pallidum dans les lésions de la peau et des muqueuses ; ses amplicons provenant de cibles géniques précises peuvent être caractérisés en vue de tests de susceptibilité antimicrobienne (aux macrolides), du typage des souches et du dépistage des sousespèces de T. pallidum.

Canadian Public Health Laboratory Network laboratory guidelines for the diagnosis of neurosyphilis in Canada.

Neurosyphilis refers to infection of the central nervous system by Treponema pallidum, which may occur at any stage. Neurosyphilis has been categorized in many ways including early and late, asymptomatic versus symptomatic and infectious versus non-infectious. Late neurosyphilis primarily affects the central nervous system parenchyma, and occurs beyond early latent syphilis, years to decades after the initial infection. Associated clinical syndromes include general paresis, tabes dorsalis, vision loss, hearing loss and psychiatric manifestations. Unique algorithms are recommended for HIV-infected and HIV-uninfected patients, as immunocompromised patients may present with serologic and cerebrospinal fluid findings that are different from immunocompetent hosts. Antibody assays include a VDRL assay and the FTA-Abs, while polymerase chain reaction for T. pallidum can be used as direct detection assays for some specimens. This chapter reviews guidelines for specimen types and sample collection, and identifies two possible algorithms for use with immunocompromised and immunocompetent hosts using currently available tests in Canada, along with a review of treatment response and laboratory testing follow-up.

La neurosyphilis désigne l'infection du système nerveux central par le Treponema pallidum à tout stade de la maladie. Elle est classée de diverses façons, y compris précoce ou tardive, asymptomatique ou symptomatique, infectieuse ou non infectieuse. La neurosyphilis tardive touche principalement le parenchyme du système nerveux central et se manifeste après une syphilis latente précoce, des années ou même des décennies après l'infection initiale. Des syndromes cliniques s'y associent, y compris la parésie générale, le tabes dorsalis, la perte d'acuité visuelle et auditive et les manifestations psychiatriques. Des algorithmes différents sont recommandés pour les patients infectés ou non infectés par le VIH, car les manifestations sérologiques et céphalorachidiennes des patients immunodéprimés peuvent différer de celles des patients immunocompétents. Les tests de détection des anticorps incluent le VDRL et le FTA-Abs, tandis que pour certains prélèvements, la réaction en chaîne de la polymérase peut servir de test de détection du T. pallidum. Ce chapitre traite des directives sur les types de prélèvement et leur collecte et présente deux algorithmes qui peuvent être utilisés auprès des hôtes immunodéprimés et immunocompétents à l'aide des tests offerts au Canada. Il contient également une analyse de la réponse thérapeutique et du suivi des tests de laboratoire.

Comparison of flocked and aptima swabs and two specimen transport media in the aptima combo 2 assay.

Self-collected vaginal Aptima swabs and flocked swabs in Aptima specimen transport medium and ESwabs in ESwab medium detected all 37 Chlamydia trachomatis-infected patients from 287 women tested by the Aptima Combo assay. Prevalence rates of C. trachomatis, Neisseria gonorrhoeae, and dual infection were 12.8%, 3.1%, and 2.4%, respectively.

Workflow and maintenance characteristics of five automated laboratory instruments for the diagnosis of sexually transmitted infections.

The choice of a suitable automated system for a diagnostic laboratory depends on various factors. Comparative workflow studies provide quantifiable and objective metrics to determine hands-on time during specimen handling and processing, reagent preparation, return visits and maintenance, and test turnaround time and throughput. Using objective time study techniques, workflow characteristics for processing 96 and 192 tests were determined on m2000 RealTime (Abbott Molecular), Viper XTR (Becton Dickinson), cobas 4800 (Roche Molecular Diagnostics), Tigris (Hologic Gen-Probe), and Panther (Hologic Gen-Probe) platforms using second-generation assays for Chlamydia trachomatis and Neisseria gonorrhoeae. A combination of operational and maintenance steps requiring manual labor showed that Panther had the shortest overall hands-on times and Viper XTR the longest. Both Panther and Tigris showed greater efficiency whether 96 or 192 tests were processed. Viper XTR and Panther had the shortest times to results and m2000 RealTime the longest. Sample preparation and loading time was the shortest for Panther and longest for cobas 4800. Mandatory return visits were required only for m2000 RealTime and cobas 4800 when 96 tests were processed, and both required substantially more hands-on time than the other systems due to increased numbers of return visits when 192 tests were processed. These results show that there are substantial differences in the amount of labor required to operate each system. Assay performance, instrumentation, testing capacity, workflow, maintenance, and reagent costs should be considered in choosing a system.

Head-to-head comparison of second-generation nucleic acid amplification tests for detection of Chlamydia trachomatis and Neisseria gonorrhoeae on urine samples from female subjects and self-collected vaginal swabs.

In a comparison of 4 second-generation nucleic acid amplification tests performed with self-collected vaginal swab (SCVS) and first-void urine (FVU) specimens from 575 women, SCVS specimens indicated more infections than did FVU specimens in all assays. The prevalence rates were 9% (53/575 patients) for Chlamydia trachomatis and 2% (11/575 patients) for Neisseria gonorrhoeae. The clinical sensitivities for testing SCVS specimens for C. trachomatis were 98.1% on a Tigris system and 96.2% on a Panther system for the Aptima Combo 2 assay (Hologic Gen-Probe), 98.0% for the RealTime CT/NG assay on an m2000 instrument (Abbott), 90.6% for the ProbeTec CT/GC Q(x) assay on the Viper system (Becton Dickinson), and 84.6% for the cobas CT/NG assay on the cobas 4800 platform (Roche). Clinical sensitivities for C. trachomatis in FVU specimens were 88.7% (Tigris) and 88.0% (Panther) for the Aptima Combo 2 assay, 76.9% for the RealTime CT/NG assay, 75.5% for the ProbeTec CT/GC Q(x) assay, and 81.1% for the cobas CT/NG assay. Clinical sensitivities of the assays for N. gonorrhoeae, with limited positive results, ranged from 63.6% to 100%. Specificities for both infections ranged from 98.4 to 100%. Differences in analytical sensitivities and levels of molecular targets in clinical samples but not inhibitors of amplification may explain the differences in clinical sensitivities.