Yetunde Fakile

Increased Susceptibility to Vaginal Simian/Human Immunodeficiency Virus Transmission in Pig-tailed Macaques Coinfected With Chlamydia trachomatis and Trichomonas vaginalis

BACKGROUND Sexually transmitted infections (STIs) are associated with an increased risk of human immunodeficiency virus (HIV) infection, but their biological effect on HIV susceptibility is not fully understood. METHODS Female pig-tailed macaques inoculated with Chlamydia trachomatis and Trichomonas vaginalis (n = 9) or medium (controls; n = 7) were repeatedly challenged intravaginally with SHIVSF162p3. Virus levels were evaluated by real-time polymerase chain reaction, plasma and genital cytokine levels by Luminex assays, and STI clinical signs by colposcopy. RESULTS Simian/HIV (SHIV) susceptibility was enhanced in STI-positive macaques (P = .04, by the log-rank test; relative risk, 2.5 [95% confidence interval, 1.1-5.6]). All STI-positive macaques were SHIV infected, whereas 3 controls (43%) remained uninfected. Moreover, relative to STI-negative animals, SHIV infections occurred earlier in the menstrual cycle in STI-positive macaques (P = .01, by the Wilcoxon test). Levels of inflammatory cytokines (interferon γ, interleukin 6, and granulocyte colony-stimulating factor [G-CSF]) were higher in STI-positive macaques during STI inoculation and SHIV exposure periods (P ≤ .05, by the Wilcoxon test). CONCLUSIONS C. trachomatis and T. vaginalis infection increase the susceptibility to SHIV, likely because of prolonged genital tract inflammation. These novel data demonstrate a biological link between these nonulcerative STIs and the risk of SHIV infection, supporting epidemiological associations of HIV and STIs. This study establishes a macaque model for studies of high-risk HIV transmission and prevention.

Development of a pigtail macaque model of sexually transmitted infection/HIV coinfection using Chlamydia trachomatis, Trichomonas vaginalis, and SHIV(SF162P3).

Background  Sexually transmitted infections (STIs) are associated with an increased risk of HIV infection. To model the interaction between STIs and HIV infection, we evaluated the capacity of the pigtail macaque model to sustain triple infection with Trichomonas vaginalis, Chlamydia trachomatis, and SHIVSF162P3.

A comparison of the analytical level of agreement of nine treponemal assays for syphilis and possible implications for screening algorithms

Objective The serological diagnosis of syphilis requires the detection of two distinct antibodies, the non-treponemal and trepomenal. Center for Disease Control and Prevention (CDC) recommends screening first with a non-treponemal test such as (Rapid Plasma Reagin/Venereal Disease Research Laboratory), and then confirming those results with one of the several treponemal tests (Fluorescent Treponemal Antibody-Absorption (FTA-ABS), Enzyme Immunoassay, chemiluminescence, treponema pallidum particle agglutination (TP-PA) or Point of Care). Owing to the high volume of samples processed by some laboratories using automated systems, the screening with treponemal assays and confirming with non-treponemal tests is becoming the established norm. The purpose of this study was to evaluate eight treponemal assays using TP-PA as the predicate assay. Methods 290 stored serum samples were tested qualitatively according to the manufacturers directions. Results Concordance with specimens tested as reactive or non-reactive using TP-PA was: FTA-ABS 94.5–100%, Trep-Sure 100–98.9%, BioELISA 100–98.9%, INNO-LIA 99.1–99.4%, BIOLINE 100–98.9%, CAPTIA IgG 100–97.2%, Trep-ID 100–100% and LIAISON 100–99.4%. In order to properly evaluate the performance of these assays, the analytical sensitivity was determined by endpoint titration of serial dilutions of the reactive serum samples in normal sera. The median endpoint titre varied from 1:4 for FTA-ABS to 1:512 for Trep-Sure. Conclusions The performance of the treponemal serological assays was comparable while using medium and high-titre sera. However, the varying performance on specimen dilutions suggests that there may be differences in sensitivity with low-titre sera that are more prevalent in primary and late syphilis cases.

Serologic Testing for Syphilis: Benefits and Challenges of a Reverse Algorithm

Syphilis is a human infection of global importance. Its diagnosis can be challenging, requiring construction of a serologic profile based on the results of at least two types of antibody tests: treponemal and nontreponemal. The traditional approach to the serodiagnosis of syphilis has been the use of a nontreponemal screening assay followed by the performance of a treponemal confirmatory test if the initial nontreponemal screening test was reactive. With the increasing availability of automated, easier-to-perform, and rapid treponemal assays, an increasing number of laboratory testing sites are adopting reverse sequence screening for the serodiagnosis of syphilis: screening with a treponemal assay first, then confirmation with a nontreponemal assay and, when necessary, discrepant resolution using another treponemal test. In addition to offering automation and increased throughput, a reverse algorithm can increase disease detection, especially in late latent and early primary stages of infection when the nontreponemal antibody test may be nonreactive. However, a disadvantage to this approach is that there can be an increase in false-positive test results. This article reviews the clinical and workflow benefits and limitations of a reverse testing algorithm and discusses current guidance available from the Centers for Disease Control and Prevention.

Correlation of Treponemal Immunoassay Signal Strength Values With Reactivity of Confirmatory Treponemal Testing

ABSTRACT 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.

P5.091 Head-Head Comparison of Reactivity and Signal Strength Value For Reactivity Among Seven Treponemal Assays: A Preliminary Report

Background Automated immunoassays (AI) for detection of T. pallidum antibodies are increasingly used for syphilis screening in the United States. These assays demonstrate fast performance, reduced labour requirements, and high throughput with walk-away capability. Limited data are available about the relative seroreactivity among commercial treponemal assays, especially in low risk populations. Additionally, it is unknown to what extent the AI signal strength values, used to assess reactivity, are associated with non-AI treponemal reactivity. We compared concordance of seroreactivity among 7 treponemal tests and assessed AI signal strength values associated with reactivity. Methods Previously identified reactive and nonreactive sera (n = 566) were obtained from Kaiser Northern and Southern California regional laboratories. All sera were tested with AIs: BioPlex 2200 Syphilis IgM/IgG (BioRad), treponemal LIAISON (DiaSorin), Advia-Centaur syphilis (Siemens), and non-AIs (INNOLIA syphilis score (INNOGENETICS), TrepSure (Phoenix Biotech), Treponemal Pallidum Particle Agglutination (TP-PA) (Fujirebio), and Fluorescent Treponemal Antibody-Absorption (FTA-ABS) (Zeus Scientific) tests. Reactivity was interpreted according to manufacturers’ instructions. Results Seroreactivity ranged from 40.5 – 43.9% for AIs, and 33.0–42.2% for non-AIs. In all 7 tests, 30% (167/566) were reactive, and positive agreement among assays was 82.3%. The overall seroreactivity among AIs was 38.9% (220/566) and positive agreement was 92.6%. Minimum signal strength values of 11.72 (Centaur, range: 1.1–45), 4.4 (BioPlex, range: 1.1–8) and 9.4 (Liaison, range: 1.1–70) correlated 100% with TPPA reactivity. The proportion of AI-seroreactive specimens that were also TP-PA reactive were: 86.5% (198/229) for BioPlex, 85.2% (202/237) for ADVIA-Centaur, and 81.6% (200/245) for LIAISON. Conclusion Although there is some variation in seroreactivity among the 7 tests, there is good correlation. A large proportion of AI tests with a minimal signal-to-cutoff ratio were associated with a positive TP-PA, suggesting that a second treponemal test may not be necessary to confirm AI-reactive, RPR-nonreactive sera.

O3-S1.04 Performance characteristics of bioplex 2200 syphilis IgG and Liaison Treponema automated assays for detection of antibodies to Treponema pallidum

Background Serological testing continues to be a crucial tool for syphilis diagnosis and control. The commonly accepted syphilis screening algorithm is screening with non-treponemal tests such as RPR or VDRL, and confirming with treponemal tests such as TP-PA. Recently, automation has been introduced whereby serological screening using treponemal tests has resulted in reduced labour time and removal of the subjectivity associated with the traditional testing algorithm. The objective of this study was to compare the performance characteristics of two FDA approved automated tests, the BioRad BioPlex 2200 Syphilis IgG and the DiaSorin LIAISON treponemal assays, with known predicate tests. The BioPlex 2200 syphilis IgG is a multiplex test that utilises three analytes (15-, 17-, & 47-kDa) to detect specific IgG antibodies, whereas the LIASION treponemal assay uses only one analyte (17 kDa) in a single step sandwich method to detect both syphilis IgG and IgM antibodies. Methods A total of 1086 commercially obtained sera tested in this study consisted of: 430 from pregnant women, 409 from HIV positive individuals, and 111 from known syphilis patients of various disease stages. Characterised syphilis samples (n=140) were also obtained from the CDC serum repository. All samples were screened by the Bioplex IgG, Liaison, RPR and TP-PA tests. Any indeterminate results were repeated at least once. Results Of the 1086 samples tested, the syphilis reactivities were the following: 551 (50.7%) by BioPlex IgG, 528 (48.6%) by LIAISON, and 509 (46.9%) by TP-PA. The sensitivity and specificity when compared to TP-PA for LIASION was 98.8% and 90.5% respectively. The BioPlex IgG sensitivity and specificity when compared to TP-PA was 85.1% and 80% respectively. Overall, 443 (40.8%) samples were found to be reactive and 450 (41.4%) non reactive to both LIAISON and BioPlex IgG. All three tests agreed on 877 (81%) samples. On the 209 discordant samples TP-PA agreed with LIAISON 85.2% (n=178), BioPlex 7.2% (n=15), but disagreed with both tests 7.7% (n=16). Conclusion Both tests have high throughput, walk-away capability, and would be useful in low prevalence settings. There was good agreement between the LIAISON and the BioPlex IgG in 893 (82%) samples (Cohens κ=0.64). The LIAISON had higher sensitivity most likely due to its detection of both IgG and IgM, while the BioPlex detected only IgG antibodies. Both tests show significant promise in the future of syphilis serology.

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.

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.

P1.42 A new magnetic particle-based agglutination assay for anti-cardiolipin antibody detection in syphilis

Introduction A magnetic particle based assay was developed for the detection of nontreponemal anti-cardiolipin antibodies in sera of suspected syphilis cases. The presence of this group of antibodies in combination with a reactive treponemal test indicates active syphilis. In this study, we aimed to overcome technical difficulties with attaching cardiolipin to solid support. The newly developed assay potentially offers advantages of better result interpretation, accuracy, and minimum equipment need compared to traditional nontreponemal tests in diagnosing syphilis. Methods To develop the nontreponemal magnetic agglutination assay (NT-MAA), cardiolipin antigen was modified first through a chemical oxidation process. The oxidised antigen was later covalently linked to magnetic particles. To test the beads, serum samples were mixed with cardiolipin-magnetic particle complex, and incubated in round bottom well microplates. The test was interpreted as reactive when agglutination was observed. Non-reactive sample demonstrated a “button” in the centre of a microwell. The NT-MAA was evaluated using a panel of previously characterised human sera (n=80) and results were compared to rapid plasma reagin (RPR, ASI) and Treponema pallidum particle agglutination tests (TP-PA, Fujirebio). A true positive sample was defined as being reactive for both RPR and TP-PA, while a true negative as both RPR and TP-PA non-reactive. Results Out of 80 sera tested, 48 were found true positive and 32 true negative with the reference tests. In comparison, the NT-MAA, demonstrated a sensitivity and specificity of 100% and 96.8%, respectively. Conclusion Magnetic particle-based assays offer high flexibility because they work with different assay formats. This exploratory study, describes technical advances for development of nontreponemal test (NT-MAA), and also demonstrated an encouraging performance with the studied samples. Additional evaluation with syphilis samples from defined clinical stages of syphilis will help further validate test performance.