External quality assessment to support the WHO ProSPeRo study for the evaluation of two dual HIV/syphilis point-of-care tests in seven countries.

BACKGROUND: Sexually transmitted infections (STIs) such as syphilis and HIV remain to be a significant public health issue worldwide. Dual rapid point-of-care tests (POCTs) have shown promise for detecting antibodies to HIV and syphilis but have not been fully evaluated in the field. Our study supported the WHO ProSPeRo study on Sexually Transmitted Infection Point-of-Care Testing (STI POCT) by providing external quality assessment (EQA) for HIV and syphilis testing in reference laboratories and their associated clinical sites in seven countries. METHODS: HIV/syphilis serum liquid and dried tube specimen (DTS) panels were prepared by CDC. Liquid panels were distributed to the reference laboratories for three rounds of testing using commercially and locally available laboratory-based serological tests. DTS panels were sent to the clinical testing sites for 8 rounds of POC testing using the Abbott SD BIOLINE HIV/Syphilis Duo test (hereafter referred to as SD BIOLINE) and the Chembio Dual Path Platform (DPP) HIV-Syphilis assay. EQA panels were tested at CDC using the Rapid Plasma Reagin (RPR) test and the Treponema pallidum Particle Agglutination assay (TP-PA) for syphilis antibodies. Genetic Systems HIV-1/HIV-2 Plus O EIA, Geenius HIV Supplemental Assay and the Oraquick Advance HIV test were used to detect HIV antibodies in the EQA panels. Results from the reference laboratories and POCT sites were compared to those obtained at the CDC and a percentage agreement was calculated. RESULTS: Qualitative RPR and TP-PA performed at the reference laboratories demonstrated 95.4-100% agreement with CDC results while quantitative RPR and TP-PA tests demonstrated 87.7% and 89.2% agreement, respectively. A 93.8% concordance rate was observed for qualitative HIV testing in laboratories. EQA testing at clinical sites using dual tests showed 98.7% and 99.1% agreement for detection of HIV antibodies and eight out of 10 sites had > 95.8% agreement for syphilis testing. However, two clinical sites showed only 65.0-66.7% agreement for SD BIOLINE and 84.0-86.7% for DPP, respectively, for syphilis testing. CONCLUSIONS: Overall, laboratories demonstrated high EQA performance in this study. Both HIV/syphilis POCTs gave expected results in the clinic-based evaluations using DTS. However, testing errors were identified in a few testing sites suggesting the necessity for continuous training and monitoring the quality of POC testing.

Factors Associated With Syphilis Transmission and Acquisition Among Men Who Have Sex With Men: Protocol for a Multisite Egocentric Network Study.

BACKGROUND: In the United States, the rates of primary and secondary syphilis have increased more rapidly among men who have sex with men (MSM) than among any other subpopulation. Rising syphilis rates among MSM reflect changes in both individual behaviors and the role of sexual networks (eg, persons linked directly or indirectly by sexual contact) in the spread of the infection. Decades of research examined how sexual networks influence sexually transmitted infections (STIs) among MSM; however, few longitudinal data sources focusing on syphilis have collected network characteristics. The Centers for Disease Control and Prevention, in collaboration with 3 sites, enrolled a prospective cohort of MSM in 3 US cities to longitudinally study sexual behaviors and STIs, including HIV, for up to 24 months. OBJECTIVE: The Network Epidemiology of Syphilis Transmission (NEST) study aimed to collect data on the factors related to syphilis transmission and acquisition among MSM. METHODS: The NEST study was a prospective cohort study that enrolled 748 MSM in Baltimore, Maryland; Chicago, Illinois; and Columbus, Ohio. NEST recruitment used a combination of convenience sampling, venue-based recruitment, and respondent-driven sampling approaches. At quarterly visits, participants completed a behavioral questionnaire and were tested for syphilis, HIV, gonorrhea, and chlamydia. The participants also provided a list of their sexual partners and described their 3 most recent partners in greater detail. RESULTS: The NEST participants were enrolled in the study from July 2018 to December 2021. At baseline, the mean age of the participants was 31.5 (SD 9.1) years. More than half (396/727. 54.5%) of the participants were non-Hispanic Black, 29.8% (217/727) were non-Hispanic White, and 8.8% (64/727) were Hispanic or Latino. Multiple recruitment strategies across the 3 study locations, including respondent-driven sampling, clinic referrals, flyers, and social media advertisements, strengthened NEST participation. Upon the completion of follow-up visits in March 2022, the mean number of visits per participant was 5.1 (SD 3.2; range 1-9) in Baltimore, 2.2 (SD 1.6; range 1-8) in Chicago, and 7.2 (SD 2.9; range 1-9) in Columbus. Using a community-based participatory research approach, site-specific staff were able to draw upon collaborations with local communities to address stigma concerning STIs, particularly syphilis, among potential NEST participants. Community-led efforts also provided a forum for staff to describe the NEST study objectives and plans for research dissemination to the target audience. Strategies to bolster data collection during the COVID-19 pandemic included telehealth visits (all sites) and adaptation to self-collection of STI specimens (Baltimore only). CONCLUSIONS: Data from NEST will be used to address important questions regarding individual and partnership-based sexual risk behaviors among MSM, with the goal of informing interventions to prevent syphilis in high-burden areas. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR1-10.2196/40095.

Development of a Bead-Based Multiplex Assay for Use in Multianalyte Screening and Surveillance of HIV, Viral Hepatitis, Syphilis, and Herpes.

Diagnostic assays that can simultaneously determine the presence of infection with multiple pathogens are key for diagnosis and surveillance. Current multiplex diagnostic assays are complex and often have limited availability. We developed a simple, multianalyte, pathogen detection assay for screening and serosurveillance using the Luminex Magpix platform that is high throughput and can be helpful in monitoring multiple diseases. The Luminex bead-based 10-plex immunoassay for the detection of HIV-1, HIV-2, Treponema pallidum, hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus 1 (HSV-1), and HSV-2 infections was accomplished by coupling beads with specific antigens to detect IgG antibodies in plasma or serum samples. Each coupled antigen was systematically optimized, and the performance was evaluated using a panel of well-characterized specimens (n = 417) that contained antibodies to HIV-1, HIV-2, T. pallidum, HBV, HCV, HSV-1, and HSV-2. The multiplex assay had a sensitivity of 92.2% (95% Clopper-Pearson confidence interval [CI], 90.2 to 94.0%) and a specificity of 98.1% (95% CI, 97.6 to 98.7%). The sensitivities and specificities for disease-specific biomarker detection ranged from 68.7 to 100% and 95.6 to 100%, respectively. The results showed that the 10-plex immunoassay had an overall agreement of 96.7% (95% CI, 96.7 to 97.3%) with reference tests and a corresponding kappa value of 0.91 (95% CI, 0.90 to 0.93). Kappa values for the individual pathogens ranged from 0.69 to 1.00. The assay is robust and allows the simultaneous detection of antibodies to multiple antigens using a small sample volume in a high-throughput format. This assay has the potential to simplify disease surveillance by providing an alternative to expensive and highly specialized individual tests.

Development of a novel magnetic particle-based agglutination immunoassay for anticardiolipin antibody detection in syphilis.

OBJECTIVES: Serological tests of non-treponemal and treponemal types are the most frequently used for syphilis diagnosis. Treponemal tests are available in wide variety of assay formats; however, limited advances have been made for the improvement of conventional non-treponemal tests. The objective of this work was to develop a novel non-treponemal magnetic particle-based agglutination assay (NT-MAA) and evaluate its feasibility for syphilis testing. METHODS: Cardiolipin was modified and coupled to magnetic microbeads. Serum diluted in phosphate-buffered saline was mixed with cardiolipin-coupled beads and incubated in a round bottom microplate for 90-120 min followed by visual inspection. A panel of reported syphilis (n=127) and non-reactive (n=244) specimens was prepared to evaluate the NT-MAA performance in comparison to conventional rapid plasma reagin (RPR). Treponema pallidum particle agglutination (TP-PA) assay and enzyme immunoassay (EIA) were included. Analytical sensitivity and reproducibility of NT-MAA were also determined. RESULTS: The non-treponemal NT-MAA and RPR showed sensitivity of 90.6% and 88.2% and specificity of 96.7% and 100%, respectively. The treponemal TP-PA and EIA yielded sensitivity of 100% and 99.2%, respectively, and 100% specificity by both assays. The per cent agreement between NT-MAA and RPR was 97% (kappa=0.931, 95% CI 0.891 to 0.971). Analytical sensitivity determined with IgM anticardiolipin antibody (ACA) was 2.6 µg/mL for both NT-MAA and RPR, while IgG ACA yielded 0.9 µg/mL and 1.7 µg/mL for NT-MAA and RPR, respectively. Qualitative results of intra-assay and interassay reproducibility revealed 100% consistency for NT-MAA. CONCLUSION: Preliminary evaluation of the novel NT-MAA validated proof of concept using laboratory-characterised syphilis sera and demonstrated performance comparable to RPR. Further validation of NT-MAA using additional specimens with better clinical staging may broaden the scope of developed test for syphilis diagnosis.

Evaluation of the WHO/CDC Syphilis Serology Proficiency Programme to support the global elimination of mother-to-child transmission of syphilis: an observational cross-sectional study, 2008-2015.

OBJECTIVES: Syphilis morbidity is high among pregnant women in lower income countries with limited laboratory capacity. We evaluated a long-standing global Syphilis Serology Proficiency Programme (SSPP) that supports testing quality in national reference laboratories to determine if participation affects congenital syphilis elimination strategies. DESIGN: In this observational cross-sectional study, we calculated coverage on type, frequency and quality of syphilis testing reported by laboratories enrolled in the SSPP from 2008 to 2015. We used country-reported data to WHO on four congenital syphilis (CS) indicators and World Bank country economic data to compare coverage and completeness of reporting of indicators in lower income countries with and without an SSPP-enrolled laboratory. PARTICIPANTS: From 2008-2015, 78 laboratories from 51 countries participated in >1 SSPP evaluation; 56% were national reference laboratories, of which most (93%) participated for >3 years and 11 (22%) in all 24 cycles. RESULTS: Median proficiency performance score was >95% regardless of test conducted. Of the 51 countries with an SSPP-enrolled laboratory, 22 (43%) were lower-income countries, of which 21 reported CS data during 2008-2015. Comparing CS data from 87 (90% of total) lower income countries with and without an SSPP-enrolled laboratory, countries with an SSPP-laboratory had stronger reporting on antenatal syphilis testing (p=0.04). For 2015, an estimated 74% of prenatal syphilis tests and 63% of positive tests reported to WHO from countries with an SSPP-enrolled laboratory. CONCLUSION: The SSPP has focused well on national reference laboratories, but has been only partially successful in recruiting laboratories from lower income countries. The finding that over half of syphilis infections in pregnant women living in countries with SSPP-enrolled laboratories suggests wide reach of the current quality assurance programme. However, reach could expand with focussed recruitment of laboratories from lower income countries.

Evaluation of a Rapid Syphilis Test in an Emergency Department Setting in Detroit, Michigan.

BACKGROUND: Syphilis transmission can be prevented by prompt diagnosis and treatment of primary and secondary infection. We evaluated the performance of a point-of-care rapid syphilis treponemal (RST) test in an emergency department (ED) setting. METHODS: Between June 2015 and April 2016, men aged 18 to 34 years seeking services in a Detroit ED, and with no history of syphilis, were screened for syphilis with the RST test, rapid plasma reagin (RPR) test, and Treponema pallidum particle agglutination assay (TP-PA). A positive reference standard was both a reactive RPR and a reactive TP-PA. We compared test results in self-reported men who have sex with men (MSM) to non-MSM. RESULTS: Among 965 participants, 10.9% of RST tests were reactive in MSM and only 1.5% in non-MSM (P < 0.001). Sensitivity of the RST test was 76.9% and specificity was 99.0% (positive predictive value, 50.0%) compared with the positive reference standard. Three discordant specimens found negative with the RST test but positive with the reference standard had an RPR titer of 1:1, compared with 10 specimens with concordant positive results that had a median RPR titer of 1:16. The RST sensitivity was 50.0% (positive predictive value, 68.4%) compared to the TP-PA test alone. Among men seeking care in an ED, the RST detected 76.9% of participants with a reactive RPR and TP-PA. CONCLUSIONS: The RST test detected all of the participants with an RPR titer ≥1:2 but less than 20% of participants with a positive TP-PA and negative RPR. The RST test was useful to detect a high proportion of participants with an active syphilis in an urban ED.

Performance of the Syphilis Health Check in Clinic and Laboratory-Based Settings.

BACKGROUND: In this study, we evaluate the performance of the Syphilis Health Check (SHC) in clinical and laboratory settings using fingerstick whole blood and serum. METHODS: Fingerstick whole blood and serum specimens from adult patients (n = 562) without prior syphilis history presenting at 2 county health department STD clinics in North Carolina were tested. Fingerstick specimens were tested with the SHC in clinic, and serum specimens were tested at the North Carolina State Laboratory of Public Health with: (1) qualitative rapid plasma reagin, (2) treponemal EIA, and (3) SHC. Sensitivity and specificity were calculated with 95% confidence intervals. RESULTS: The fingerstick whole blood had a sensitivity of 100% (7 of 7) and specificity of 95.7% (531 of 555), compared with consensus reference testing (CRT) (rapid plasma reagin and EIA reactive), but a sensitivity of 50% (8 of 16), and specificity of 95.9% (523 of 546), when compared with the treponemal EIA. Both laboratory-based SHC on serum and whole-blood SHC performed similarly, compared with CRT, and the treponemal EIA alone. Twenty-four specimens SHC reactive on whole blood were nonreactive by CRT. In 8 of these 24 cases, STD clinic staff reported difficulty reading the test line for the SHC. Of the fingerstick whole-blood SHC reactive specimens, only 14 of 31 were also serum SHC reactive. CONCLUSIONS: The SHC on whole blood appears to be sensitive at detecting patients likely to have syphilis and could be an option for testing among high-risk populations. However, given challenges in interpreting SHC test results, adequate training of persons performing testing and ongoing quality assurance measures are key.

Performance of Treponemal Tests for the Diagnosis of Syphilis.

BACKGROUND: Treponemal immunoassays are increasingly used for syphilis screening with the reverse sequence algorithm. There are few data describing performance of treponemal immunoassays compared to traditional treponemal tests in patients with and without syphilis. METHODS: We calculated sensitivity and specificity of 7 treponemal assays: (1) ADVIA Centaur (chemiluminescence immunoassay [CIA]); (2) Bioplex 2200 (microbead immunoassay); (3) fluorescent treponemal antibody absorption test (FTA-ABS); (4) INNO-LIA (line immunoassay); (5) LIAISON CIA; (6) Treponema pallidum particle agglutination assay (TPPA); and (7) Trep-Sure (enzyme immunoassay [EIA]), using a reference standard combining clinical diagnosis and serology results. Sera were collected between May 2012-January 2013. Cases were characterized as: (1) current clinical diagnosis of syphilis: primary, secondary, early latent, late latent; (2) prior treated syphilis only; (3) no evidence of current syphilis, no prior history of syphilis, and at least 4 of 7 treponemal tests negative. RESULTS: Among 959 participants, 262 had current syphilis, 294 had prior syphilis, and 403 did not have syphilis. FTA-ABS was less sensitive for primary syphilis (78.2%) than the immunoassays or TPPA (94.5%-96.4%) (all P ≤ .01). All immunoassays were 100% sensitive for secondary syphilis, 95.2%-100% sensitive for early latent disease, and 86.8%-98.5% sensitive in late latent disease. TPPA had 100% specificity. CONCLUSIONS: Treponemal immunoassays demonstrated excellent sensitivity for secondary, early latent, and seropositive primary syphilis. Sensitivity of FTA-ABS in primary syphilis was poor. Given its high specificity and superior sensitivity, TPPA is preferred to adjudicate discordant results with the reverse sequence algorithm over the FTA-ABS.

Laboratory Evaluation of a Commercially Available Rapid Syphilis Test.

Serological diagnosis of syphilis depends on assays that detect treponemal and nontreponemal antibodies. Laboratory certification and trained personnel are needed to perform most of these tests, while high costs and long turnaround time can hinder treatment initiation or linkage to care. A rapid treponemal syphilis test (RST) that is simple to perform, accessible, and inexpensive would be ideal. The Syphilis Health Check (SHC) assay is the only Food and Drug Administration (FDA)-cleared and Clinical Laboratory Improvement Amendments (CLIA)-waived RST in the United States. In this study, 1,406 archived human serum samples were tested using SHC and traditional treponemal and nontreponemal assays. Rapid test results were compared with treponemal data alone and with a laboratory test panel consensus defined as being reactive by both treponemal and nontreponemal assays for a given specimen, or nonreactive by both types of assays. The sensitivity and specificity of the SHC assay compared with treponemal tests alone were 88.7% (95% confidence interval [CI], 86.2 to 90.0%) and 93.1% (95% CI, 90.0 to 94.9%), respectively, while comparison with the laboratory test panel consensus showed 95.7% (95% CI, 93.6 to 97.2%) sensitivity and 93.2% (95% CI, 91.0 to 95.1%) specificity. The data were further stratified based on age, sex, pregnancy, and HIV status. The sensitivity and specificity of the SHC assay ranged from 66.7% (95% CI, 46.0 to 83.5%) to 91.7% (95% CI, 87.7 to 94.7%) and 88% (95% CI, 68.8 to 97.5%) to 100% (95% CI, 47.8 to 100%), respectively, across groups compared to traditional treponemal assays, generally increasing for all groups except the HIV-positive (HIV+) population when factoring in the laboratory test panel consensus. These data contribute to current knowledge of the SHC assay performance for distinct populations and may guide use in various settings.

A Nonhuman Primate Model for Rectally Transmitted Syphilis.

Among men who have sex with men (MSM), those with a diagnosis of syphilis or other rectal sexually transmitted infections (STIs) are at a higher risk for human immunodeficiency virus acquisition, which is concerning given the large increase in recently reported syphilis cases in the United States. We have developed the first nonhuman primate model for rectally transmitted syphilis by exposing simian/human immunodeficiency virus-infected and naive rhesus macaques to Treponema pallidum in the rectum. All animals showed mucosal lesions, systemic dissemination, and seroconversion (treponemal antibodies). This model would be valuable for studying the manifestations of and interventions for T. pallidum infection, with and without human immunodeficiency virus coinfection.

Correlation of Treponemal Immunoassay Signal Strength Values with Reactivity of Confirmatory Treponemal Testing.

Automated treponemal immunoassays are used for syphilis screening with the reverse-sequence algorithm; discordant results (e.g., enzyme immunoassay [EIA] reactive and reactive plasma reagin [RPR] nonreactive) are resolved with a second treponemal test. We conducted a study to determine automated immunoassay signal strength values consistently correlating with reactive confirmatory treponemal testing. We conducted a cross-sectional analysis of four automated immunoassays (BioPlex 2200 microbead immunoassay [MBIA], Liaison chemiluminescence immunoassay [CIA], Advia-Centaur CIA, and Trep-Sure EIA) and three manual assays (Treponema pallidum particle agglutination [TP-PA], fluorescent treponemal antibody absorption [FTA-ABS] test, and Inno-LIA line immunoassay). We compared signal strength values of automated immunoassays and positive and negative agreement. Among 1,995 specimens, 908 (45.5%) were true positives (≥4/7 tests reactive) and 1,087 (54.5%) were true negatives (≥4/7 tests nonreactive). Positive agreement ranged from 86.1% (83.7 to 88.2%) for FTA-ABS to 99.7% (99.0 to 99.9%) for Advia-Centaur CIA; negative agreement ranged from 86.3% (84.1 to 88.2%) for Trep-Sure EIA to 100% for TP-PA (99.6 to 100%). Increasing signal strength values correlated with increasing reactivity of confirmatory testing (P < 0.0001 for all automated immunoassays by Cochran-Armitage test for trend). All automated immunoassays had signal strength cutoffs corresponding to ≥4/7 reactive treponemal tests. BioPlex MBIA and Liaison CIA had signal strength cutoffs correlating with ≥99% and 100% TP-PA reactivity, respectively. The Advia-Centaur CIA and Trep-Sure EIA had signal strength cutoffs correlating with at least 95% TP-PA reactivity. All automated immunoassays had signal strength cutoffs correlating with at least 95% FTA-ABS reactivity. Assuming that a 95% level of confirmation is adequate, these signal strength values can be used in lieu of confirmatory testing with TP-PA and FTA-ABS.