Abstract Serological diagnosis of syphilis depends on assays that detect treponemal and non-treponemal

Transcription

1 JCM Accepted Manuscript Posted Online 18 July 2018 J. Clin. Microbiol. doi: /jcm This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply Laboratory Evaluation of a Commercially Available Rapid Syphilis Test Lara E. Pereira 1*, Joshua McCormick 2, Tandin Dorji 1, Joseph Kang 1, Yongcheng Sun 1, Mayur Shukla 3, Andre Hopkins 1, John Deutsch 2, Ellen N. Kersh 1, Kyle Bernstein 1, and Yetunde F. Fakile 1 1 Division of STD Prevention, Centers for Disease Control and Prevention, Atlanta, GA; 2 Oak Ridge Institute for Science and Education, Oak Ridge, TN; 3 Atlanta Research and Education Foundation Inc, Decatur, GA *Corresponding author: Lara E. Pereira, vvg3@cdc.gov Abstract Serological diagnosis of syphilis depends on assays that detect treponemal and non-treponemal 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 TM (SHC) assay is the only Food and Drug Administration (FDA)-cleared and Clinical Laboratory Improvement Amendments (CLIA)-waived RST in the US. In this study, 1,406 archived human sera were tested using SHC and traditional treponemal and non-treponemal 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 non-treponemal assays for a given specimen, or nonreactive by both types of assays. Sensitivity and specificity of SHC when compared with treponemal tests alone were 88.7% ( %) and 93.1% ( %), respectively, while comparison with the laboratory test panel consensus showed 95.7% ( %) sensitivity and 93.2% ( %) specificity. The data were further stratified based on age, sex, pregnancy and HIV status. Sensitivity and specificity of SHC ranged from 66.7% ( %) to 91.7% ( %) and 88% ( %) to 100% ( %), respectively, across groups when compared to traditional treponemal assays, generally increasing for all groups except the HIV+ population when factoring the laboratory test panel consensus. These data contribute to current knowledge of SHC performance for distinct populations and may guide use in various settings. 1

2 29 30 Introduction Syphilis cases and rates are increasing across all regions of the United States (1, 2). Recent estimates indicate rising rates among men and women in every age group 15 years, particularly among men who have sex with men (MSM) (1, 2). Early and accurate diagnosis of syphilis are key for treatment and prevention. Direct detection of the causative agent of syphilis, T. pallidum, is limited to darkfield microscopy examination of lesion exudate or tissues, and no commercial molecular detection tests are currently Food and Drug Administration (FDA)-cleared for diagnosis. While some laboratories provide locally developed and validated polymerase chain reaction (PCR) tests, serology-based methods that are FDA-cleared are recommended for antibody detection and screening purposes, and are currently used to diagnose and manage syphilis (1, 3). Serodiagnosis of syphilis involves two categories of tests nontreponemal and treponemal (1, 3-8). Nontreponemal tests detect IgG and IgM antibodies that are not specific to T. pallidum, instead targeting lipoidal antigens released from damaged host cells caused by T. pallidum, or from damaged T. pallidum cell wall, and host immune responses (9, 10). Antibodies specific to T. pallidum antigen(s) are also produced and these are detected by treponemal tests (10). Nontreponemal and treponemal serological tests can yield false positive or false negative results in part due to the complex pathology of syphilis and individual assay limitations (1, 4-21). Thus to identify syphilis both types of tests must be performed, and for many years testing has followed a traditional algorithm that involves nontreponemal testing first, then a treponemal test(s). Due to cost and workflow factors, a reverse algorithm has been adopted by some laboratories, which first uses automated treponemal tests to facilitate high throughput and quick turnaround, followed by a nontreponemal test and reflex testing to confirm discordant results (1, 3). In addition to a patient s clinical and immune status, specimen type (blood versus serum versus plasma) and quality can also affect downstream laboratory test results (6, 7, 10). Regardless of the algorithm used, clinicians must also consider epidemiologic risk, 2

3 patient history and clinical signs to determine whether treatment or follow-up testing is the next appropriate step (1, 3, 10, 11). Current FDA-cleared non-treponemal tests include laboratory-based manual assays such as the Rapid Plasma Reagin (RPR) and Venereal Disease Research Laboratory (VDRL) tests. FDA-cleared laboratory treponemal assays also include manual procedures such as the fluorescent treponemal antibody absorbed (FTA-ABS) test and T. pallidum passive particle agglutination (TP-PA) test, as well as automated or automatable enzyme immunoassays (EIA), chemiluminescence immunoassays (CIA) and immunoblots (3, 5, 7, 10, 11). In recent years, rapid syphilis tests (RST) have been gaining interest globally, with one FDA-cleared and CLIA-waived qualitative lateral flow immunochromatographic assay available for use in the US (Syphilis Health Check TM [SHC], Trinity Biotech USA Inc, NY US) (22, 23). Unlike traditional laboratory-based syphilis treponemal tests, RST are portable for use in the field, relatively inexpensive, do not involve complex training, and yield near instantaneous results that allow for immediate point-of-care (POC) treatment and/or linkage to care (15, 24-30). The manufacturer s insert for the SHC test, for example, indicates that results can be read within minutes (22). Traditional serological testing can still be performed at well-equipped off-site laboratories but risks of delayed treatment and/or loss of patient follow-up could be minimized with RST in the interim. Other RST have been evaluated in international settings (31-39), while only three reports have described SHC performance in the US and in Uganda since its release in 2011 (40-42). The goal of the current study is to therefore gather additional data for this RST within the scope of existing serological tests and recommendations. The study design involves comparative performance evaluation of SHC (treponemal test) and traditional treponemal (TP-PA, EIA, CIA) and non-treponemal (RPR) assays using a collection of archived clinical and commercially-procured human serum samples. Further analyses of serological test results among distinct populations were also done to gain insight on SHC performance among specific group(s). 3

4 80 Materials and Methods Serum specimens. A total of 1,406 archived, unlinked (de-identified) frozen (-20 0 C to C) serum specimens spanning years were provided by Bio-Rad Laboratories (Hercules, CA), healthcare organizations, and public health laboratories to the Centers for Disease Control and Prevention (CDC, Atlanta, GA). Specifically, 455 and 200 samples were provided by the Association of Public Health Laboratories (Silver Spring, MD) and Georgia Public Health Laboratory (Decatur, GA), respectively, through joint collaborative efforts with the CDC. Bio-Rad Laboratories donated 506 specimens that were initially procured commercially by Bio-Rad based on FDA recommendations for their internal FDA 510(k) study. Their remnant specimens were gifted to the CDC. Kaiser Permanente Northern California (KPNC), Kaiser Permanente Southern California (KPSC) and the San Francisco Department of Public Health (SFDPH) provided 245 samples to the CDC. A combination of screening and diagnostic specimens were provided by KPNC and KPSC, whose laboratories utilize reverse sequence screening. Specimens from SFDPH were from the city s municipal STD clinic, which performs point-of-care nontreponemal RPR testing at the clinic followed by treponemal testing in the laboratory; specimens sent to the CDC included those with reactive serology and clinical diagnosis of syphilis. There was no preselection sample criteria used by the CDC, with all specimens being accepted as is from these suppliers, and later tested at CDC using treponemal and nontreponemal tests in parallel. Serology results from the suppliers were also blinded until after testing was completed at CDC. If available, information pertaining to age, sex, pregnancy status and HIV status were provided by each supplier. For a given population category, not all information was available across the remaining categories. Negative/non-reactive specimens were obtained from normal healthy subjects though information related to previous history and/or treatment of diseases other than syphilis and HIV was not available. Approvals in accordance with federal regulations, state laws, ethics guidelines, and CDC policies were obtained prior to CDC laboratory testing. Samples received at the CDC were stored at C until ready for testing, at which time they were maintained at 4 C until all testing was complete to ensure that analyses were performed in the same 4

5 freeze-thaw cycle. Specimens had undergone up to four freeze-thaw cycles from the time of collection by suppliers to time of testing at CDC Syphilis serological assays. Of the 1,406 serum samples, 754 were previously tested in a different CDC study as treponemal reactive while 652 were treponemal non-reactive based on TP-PA (Fujirebio Diagnostics Inc., Malvern PA), EIA, and CIA (McCormick et al, unpublished data). Non-treponemal results, obtained from testing by RPR (Arlington Scientific Inc, Springville UT), were provided for 752 of the 1,406 samples by the serum suppliers. Repeat RPR testing for these specimens could not be performed at CDC due to insufficient sample volume but RPR tests were conducted for the remaining 654 samples at CDC. All 1,406 samples were tested using the rapid lateral flow immunochromatographic assay, Syphilis Health Check TM (SHC) during the same freeze-thaw cycle as the other treponemal tests, according to manufacturer s instructions (Trinity Biotech USA Inc., Jamestown NY). Samples and SHC test cassettes were allowed to equilibrate to room temperature before testing. Briefly, for each specimen, 25 µl of serum was dispensed into the sample well of the test cassette followed by 200 µl of the kit s diluent containing saline buffer, detergent and 0.1% sodium azide. Following a 10 minute incubation at room temperature (15 minutes maximum), results were read by evaluating the development and intensity of indicator lines in the cassette s control and test windows. Onsite training for CDC laboratorians on the use of SHC was provided by Trinity Biotech USA Inc. CDC laboratorians performing the RPR and SHC tests were blinded to results from each test, and to previous results obtained at CDC and provided by the suppliers. The nontreponemal and treponemal tests described above were performed in parallel at the CDC and an algorithm was not followed since the goal was to test all specimens by all assays regardless of outcome, in order to fully evaluate SHC test performance. Data and statistical analyses. Data obtained from TP-PA, EIA and CIA tests were considered as treponemal variables (TR) while data from SHC testing at CDC were considered as SHC variables. SHC and TR variables were converted to 0 if recorded as non-reactive (-) and 1 if it was recorded as reactive 5

6 (+). A laboratory test panel consensus (LT) variable was also created in this study to consider specimens reactive by both treponemal and non-treponemal tests, or non-reactive by both tests. Based on this laboratory-derived definition, 194 samples with discordant treponemal and non-treponemal results (i.e. TR+/NTR- and vice versa) were excluded from LT variable analyses. The non-treponemal (NTR) variables were converted to 0 if recorded as non-reactive and 1 if recorded as reactive or minimally reactive (Rm). If both TR and NTR variables were recorded as 1 then LT is 1 and if both TR and NTR variables were recorded as 0 then LT is 0. Statistical analysis was performed using the R statistical programming software (The R Foundation, Vienna, Austria) (43, 44). The R package popepi was used to convert the factor variables into numerical values. Sensitivity and specificity were determined by comparing SHC results to either treponemal results alone, or to the LT variable described above. The 95% confidence intervals for both the sensitivity and specificity were obtained using the R package epir. Population stratification. The age variable in the dataset was grouped into three different categories; (1) subjects aged 11-20, (2) 21-30, and (3) 31. No restrictions (sex, HIV status, or pregnancy status) were considered for the three age stratifications. Information regarding pregnancy status is as reported by the serum supplier(s). Results Overall analysis. A total of 1,406 serum specimens were tested by the rapid SHC assay and results were first compared with standard laboratory treponemal assay results (i.e. reactive or nonreactive to TP-PA, EIA and CIA) previously determined at the CDC. As shown in Table 1 (left panel), the sensitivity and specificity of SHC was 88.7% (95% CI: %) and 93.1% (95% CI: %), respectively, when compared with the treponemal only test as a reference standard. When performing analysis based on the laboratory test panel consensus defined in Materials and Methods, 1,212 samples that met this criteria 6

7 showed that sensitivity increased to 95.7% (95% CI: %) (Table 1, right panel). However, specificity remained relatively unchanged at 93.2% (95% CI: ) Population-specific analysis. With the availability of information pertaining to age, sex, pregnancy status, and HIV status for some of the samples, serological test results were collated for these archived specimens and the dataset was stratified into four distinct groups (Table 2). Analyses similar to those described above were then performed for each population. As shown in Table 2, SHC sensitivity and specificity ranged from % and %, respectively, among the three age groups when compared to the reference treponemal assays, with highest values being noted for the group aged 31. Consistent with findings obtained for the overall analysis above, all parameters generally improved when defining positives and negatives based on the laboratory test panel consensus that includes both treponemal and non-treponemal assay results. Similar trends were observed for the populations stratified based on sex, pregnancy and HIV infection (Table 2). Of note, SHC testing of the HIV positive sample set showed the lowest sensitivity when compared to treponemal results (66.7%). Although results for both male and female populations were similar for the rapid SHC test and the traditional assays, most of the SHC performance parameters were lower for the female population when compared to males; indeed, 100% specificity was observed for male samples testing when comparing SHC results to laboratory test panel consensus. However, high specificity values of 100% were observed for females when this population was further stratified based on pregnancy status, while sensitivity was higher for the pregnant group, regardless of whether SHC results were compared to the reference treponemal assays only or both treponemal and non-treponemal test results. In summary, the majority of the data set showed sensitivity and specificity above 90% across all groups, and confidence intervals often overlapped among the groups. With the exception of the HIV positive population, the performance parameters evaluated here generally improved further when considering agreement of results among all three tests the rapid SHC test, and the reference treponemal and non-treponemal assays. 7

8 Discussion In our study, testing and analysis of 1,406 archived human sera showed that overall, SHC had >85% sensitivity and >90% specificity when compared to traditional treponemal assays (TP-PA, EIA and CIA), with sensitivity increasing further to >95% when the laboratory test panel consensus was considered (Table 1), while specificity remained relatively unchanged. These findings are similar to two previous reports (40, 41) that utilized smaller sample sizes (<250), albeit one of the studies (40) included a comparison with a single treponemal test and differences in sample matrix (whole blood versus serum) that limits direct comparisons with the current study. Nonetheless, Nakku-Joloba et al (41) demonstrated that both specificity and sensitivity of SHC improved considerably when compared to both treponemal (TP-HA) and non-treponemal (RPR) standards, which is consistent with our findings. A caveat of the laboratory test panel consensus used in this study is that discordant results (TR+/NTR- and vice versa) were excluded from analysis. The CDC was not privy to information regarding patient history or confirmed clinical syphilis diagnosis for these sample sets, thus it is possible that true positives or true negatives were missed. However, since TR+/NTR+ individuals are typically the most likely to require treatment, high sensitivity among this subset, as shown in this study, would be beneficial for identifying those who need treatment. Our analysis included a large sample of sera, allowing for stratified analyses. These stratified analyses identified variability in performance characteristics based on four categories age, sex, pregnancy, and HIV status - with sensitivity and specificity ranging from 60.0%-97.2% and %, respectively, depending on population and reference tests. Confidence intervals were also noted to overlap among groups, and should be taken into consideration when interpreting these findings. Parameters with the exception of specificity improved for most of the examined populations when the laboratory test panel consensus was considered, which is consistent with patterns obtained for the overall analysis and with previous reports (41). A limitation of the stratified analyses is that differences in sample source or screening criteria among the suppliers and its effect, if any, on downstream test performance 8

9 comparisons cannot be ruled out. In addition, for a given category (age, sex, pregnancy or HIV status), information was available for some or none of the remaining categories meaning that there is overlap across groups in some cases, with no overlap in others. Thus, there could be other characteristics to consider for a given population, which might influence sensitivity and specificity outcomes. Analysis of samples from the HIV positive population showed the lowest sensitivity (<70%), however it is important to note that the sample size is relatively small for the HIV positive datasets that include SHC test comparison to treponemal results only (N=79) and both treponemal and non-treponemal results (N=56). The confidence intervals for sensitivity were also noted to be wide ( %), and so there is a need for a study with larger test groups to determine if this trend holds true. The reason(s) for the lower sensitivity of SHC observed for the HIV positive population in this study are unclear, however missed or underestimated syphilis diagnoses in HIV positive individuals have been reported for other serological assays, and also depends on the algorithm used (7, 12, 45, 46). Several confounding factors that include HIV viremia, CD4 T-cell count and subsequent impact on inflammatory and/or humoral immune responses may influence treponemal antibody levels, as well as affinity and/or avidity of antibodies. Reactivity of these antibodies in treponemal assays may vary further depending on the test platform and T.pallidum antigen(s) used in the assays, while also recognizing that reference assays themselves may yield a certain degree of false negative and false positive results. The disease stage and immune status of the HIV positive population considered in this study are unknown. The scope of our analysis is also limited in that other high risk groups that include immunocompromised or immunosuppressed individuals, and men who have sex with men (MSM) were not evaluated. Given the increased rate of reported primary and secondary syphilis cases among MSM in the US in recent years (2), additional SHC test evaluation for these populations is needed. Specimens from populations with a previous history of other treponemal/spirochete infections, or diseases other than syphilis and HIV, would also help further delineate test sensitivity and specificity in a future study. Due to variations in sensitivity and specificity among the overall and stratified data sets, multiple measures of association that include concordance, phi coefficient and equivalence were also calculated to 9

10 gain additional perspective on test similarities (data not shown). Results suggested that while the SHC test is not equivalent to the reference treponemal and non-treponemal assays evaluated in this study, test performance did not differ by a significant degree and indeed showed strong positive relationships. However, agreement among assays based on these parameters is not necessarily accurate or conclusive due to the impact of epidemiologic factors such as prevalence (47). There is also an additional study limitation to consider, as the archived sera had undergone up to four freeze-thaw cycles. Of note, a previous CDC study has shown that both IgG and IgM treponemal antibodies show no change in reactivity for up to ten freeze-thaw cycles (48). Despite study limitations, the current findings show that the rapid SHC test performance aligns with traditional treponemal and non-treponemal tests though performance varies among specific populations and can also change depending on the serological assay(s) used as reference standards as shown in previous reports. Furthermore, the influence of differences in sample type or quality, potential assay modifications and subjective interpretations across laboratories cannot be ruled out (28). As noted above, the SHC tests in this study were performed by laboratorians that received specific training by the test manufacturer and serum instead of whole blood was used as the test medium. Additional studies would be needed to address test performance in point of care settings. It is also important to note that patients with a previous history of treated syphilis could still test as seroreactive, as observed with other treponemal tests. Thus, appropriate diagnostic and follow-up steps should be considered. Regardless, RST such as the SHC assay address an important need in the syphilis diagnostics sphere, particularly in the U.S. where the SHC test is currently the only FDA-cleared RST. Rapid tests like the SHC have the potential to improve access to immediate diagnosis and treatment particularly during outreach programs, in resource-limited settings, or among highly vulnerable populations such as pregnant women, where immediate treatment is an urgent concern so as to prevent loss to follow up and potential fetal complications. Indeed, outreach screening has been reported for at least four states in the US at the community level, STD clinics, and other facilities that include high risk populations, with treatment being administered in those showing reactive RST results while confirmatory laboratory tests are performed 10

11 (28). More research is needed to determine if this approach effectively addresses loss to follow-up and reduces syphilis transmission in high risk groups. Importantly, data from this study are consistent with previous reports and contribute to our understanding of SHC efficacy and utility as an alternative serodiagnostic method, while reaffirming existing recommendations that laboratory-based reference serological assays should not be replaced (1-3, 28). Continued independent study of SHC performance in various laboratory and field settings will add to a body of data intended to provide perspectives on new and existing syphilis screening practices, and guide future RST development. Acknowledgements We would like to thank our partners at Bio-Rad Laboratories, the Association of Public Health Laboratories, Georgia Public Health Laboratory, Kaiser Permanente Northern California, Kaiser Permanente Southern California, and the San Francisco Department of Public Health for providing serum samples. We would also like to thank Trinity BioTech USA Inc. for supplying SHC kits and providing onsite training for CDC laboratorians. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Use of trade names is for identification purposes only and does not constitute endorsement by the CDC or the US Department of Health and Human Services. 11

12 Table 1. Comparative analysis of Syphilis Health Check (SHC) testing of 1,406 archived human sera with treponemal (TR) testing only, and with the laboratory test panel consensus that includes both treponemal and nontreponemal (NTR) results. Treponemal only* TR+ TR SHC Sensitivity (95% CI): (0.862, 0.909) Specificity (95% CI): (0.909, 0.949) Test panel consensus** TR+/NTR+ TR-/NTR SHC Sensitivity (95% CI): (0.936, 0.972) Specificity (95% CI): (0.910, 0.951) * Treponemal assays include TP-PA, EIA and CIA platforms. **Test panel consensus refers to results that were either reactive by both treponemal and nontreponemal (RPR) tests or nonreactive by both treponemal and nontreponemal tests; discordant treponemal and non-treponemal results were excluded from analysis. Downloaded from on September 21, 2018 by guest 12

13 Table 2. Population-specific determination of sensitivity and specificity of Syphilis Health Check (SHC) test performance compared to treponemal (TR) assays only or with a laboratory test panel consensus that includes both treponemal and non-treponemal (NTR) assays. Age Sex Pregnancy status HIV positive Population* Assay results Sensitivity (95% CI) SHC SHC 31 Female Male Nonpregnant Pregnant SHC SHC SHC SHC SHC SHC Treponemal only** Test panel consensus*** TR+ TR- TR+/NTR+ TR-/NTR Treponemal only (0.845, 0.934) (0.777, 0.896) (0.877, 0.947) (0.775, 0.878) (0.870, 0.936) (0.653, 0.877) (0.731, 0.879) (0.460, 0.835) Test panel consensus*** (0.931, 0.992) (0.907, 0.990) (0.910, 0.971) (0.880, 0.972) (0.939, 0.985) (0.749, 0.953) (0.835, 0.994) (0.147, 0.947) Treponemal only (0.824, 0.980) (0.688, 0.975) (0.908, 0.953) (0.868, 0.964) (0.555, 0.997) (0.478, 1.000) (0.863, 0.962) (0.815, 0.973) Specificity (95% CI) Test panel consensus*** (0.811, 0.978) (0.676, 0.973) (0.909, 0.954) (0.865, 0.963) (0.478, 1.000) (0.292, 1.000) (0.862, 0.962) (0.811, 0.978) *1,406 sample results were stratified based on age, sex, pregnancy and HIV status. Populations may overlap across categories. For a given category, not all information was available for the remaining categories. **TR assays include TP-PA, EIA and CIA platforms. ***Test panel consensus refers to results that were either reactive by both treponemal and nontreponemal (RPR) tests or nonreactive by both treponemal and nontreponemal tests; discordant treponemal and nontreponemal results were excluded from analysis.

14 References 1. Centers for Disease Control and Prevention Syphilis testing algorithms using treponemal tests for initial screening- Four laboratories, New York City, , vol 57, p Centers for Disease Control and Prevention Sexually transmitted diseases surveillance: Syphilis US Department of Health and Human Services, CDC 3. Workowski KA, Bolan GA Centers for Disease Control and Prevention: 2015 Sexually Transmitted Diseases Treatment Guidelines: Syphilis.. MMWR Recomm Rep 64: Centers for Disease C, Prevention Discordant results from reverse sequence syphilis screening--five laboratories, United States, MMWR Morb Mortal Wkly Rep 60: Henao-Martinez AF, Johnson SC Diagnostic tests for syphilis: New tests and new algorithms. Neurol Clin Pract 4: Morshed MG Current trend on syphilis diagnosis: issues and challenges. Adv Exp Med Biol 808: Morshed MG, Singh AE Recent trends in the serologic diagnosis of syphilis. Clin Vaccine Immunol 22: Soreng K, Levy R, Fakile Y Serologic Testing for Syphilis: Benefits and Challenges of a Reverse Algorithm. Clin Microbiol Newsl 36: Kahn RL Syphilis serology with lipoidal antigen: the meaning of positive reactions. J Natl Med Assoc 64: passim. 10. Ratnam S The laboratory diagnosis of syphilis. Can J Infect Dis Med Microbiol 16: Larsen SA, Steiner BM, Rudolph AH Laboratory diagnosis and interpretation of tests for syphilis. Clin Microbiol Rev 8: Knaute DF, Graf N, Lautenschlager S, Weber R, Bosshard PP Serological response to treatment of syphilis according to disease stage and HIV status. Clin Infect Dis 55: Goh BT Syphilis in adults. Sex Transm Infect 81: McMillan A, Young H Reactivity in the Venereal Diseases Research Laboratory test and the Mercia IgM enzyme immunoassay after treatment of early syphilis. Int J STD AIDS 19: Sena AC, White BL, Sparling PF Novel Treponema pallidum serologic tests: a paradigm shift in syphilis screening for the 21st century. Clin Infect Dis 51: el-zaatari MM, Martens MG, Anderson GD Incidence of the prozone phenomenon in syphilis serology. Obstet Gynecol 84: Post JJ, Khor C, Furner V, Smith DE, Whybin LR, Robertson PW Case report and evaluation of the frequency of the prozone phenomenon in syphilis serology - an infrequent but important laboratory phenomenon. Sex Health 9: Sidana R, Mangala HC, Murugesh SB, Ravindra K Prozone phenomenon in secondary syphilis. Indian J Sex Transm Dis 32: Berkowitz K, Baxi L, Fox HE False-negative syphilis screening: the prozone phenomenon, nonimmune hydrops, and diagnosis of syphilis during pregnancy. Am J Obstet Gynecol 163: Shields M, Guy RJ, Jeoffreys NJ, Finlayson RJ, Donovan B A longitudinal evaluation of Treponema pallidum PCR testing in early syphilis. BMC Infect Dis 12: Geusau A, Kittler H, Hein U, Dangl-Erlach E, Stingl G, Tschachler E Biological false-positive tests comprise a high proportion of Venereal Disease Research Laboratory reactions in an analysis of 300,000 sera. Int J STD AIDS 16: Syphilis Health Check Syphilis Health Check Rapid Immunochromatographic Test [Package Insert]. Stone Harbor, NJ: Diagnostics Direct. 23. Food and Drug Administration FDA grants CLIA waiver expanding the availability of rapid screening test for syphilis. Accessed 14

15 Jafari Y, Peeling RW, Shivkumar S, Claessens C, Joseph L, Pai NP Are Treponema pallidum specific rapid and point-of-care tests for syphilis accurate enough for screening in resource limited settings? Evidence from a meta-analysis. PLoS One 8:e Kay NS, Peeling RW, Mabey DC State of the art syphilis diagnostics: rapid point-of-care tests. Expert Rev Anti Infect Ther 12: Klepser DG, Klepser ME Point-of-care testing in the pharmacy: how is the field evolving? Expert Rev Mol Diagn 18: Peeling RW, Holmes KK, Mabey D, Ronald A Rapid tests for sexually transmitted infections (STIs): the way forward. Sex Transm Infect 82 Suppl 5:v Peterman TA, Fakile YF What Is the Use of Rapid Syphilis Tests in the United States? Sex Transm Dis 43: Rogozinska E, Kara-Newton L, Zamora JR, Khan KS On-site test to detect syphilis in pregnancy: a systematic review of test accuracy studies. BJOG 124: Tucker JD, Bu J, Brown LB, Yin YP, Chen XS, Cohen MS Accelerating worldwide syphilis screening through rapid testing: a systematic review. Lancet Infect Dis 10: Benzaken AS, Sabido M, Galban E, Pedroza V, Araujo AJ, Peeling RW, Mabey D Field performance of a rapid point-of-care diagnostic test for antenatal syphilis screening in the Amazon region, Brazil. Int J STD AIDS 22: Benzaken AS, Sabido M, Galban EG, Pedroza V, Vasquez F, Araujo A, Peeling RW, Mayaud P Field evaluation of the performance and testing costs of a rapid point-of-care test for syphilis in a red-light district of Manaus, Brazil. Sex Transm Infect 84: Bocoum FY, Ouedraogo H, Tarnagda G, Kiba A, Tiendrebeogo S, Bationo F, Liestman B, Diagbouga S, Zarowsky C, Traore RO, Kouanda S Evaluation of the diagnostic performance and operational characteristics of four rapid immunochromatographic syphilis tests in Burkina Faso. Afr Health Sci 15: Causer LM, Kaldor JM, Fairley CK, Donovan B, Karapanagiotidis T, Leslie DE, Robertson PW, McNulty AM, Anderson D, Wand H, Conway DP, Denham I, Ryan C, Guy RJ A laboratory-based evaluation of four rapid point-of-care tests for syphilis. PLoS One 9:e Dlamini NR, Phili R, Connolly C Evaluation of rapid syphilis tests in KwaZulu-Natal. J Clin Lab Anal 28: Lee JH, Lim CS, Lee MG, Kim HS Evaluation of a Rapid Immunochromatographic Treponemal Antibody Test Comparing the Treponema Pallidum Particle Agglutination Assay. J Clin Lab Anal 29: Mabey D, Peeling RW, Ballard R, Benzaken AS, Galban E, Changalucha J, Everett D, Balira R, Fitzgerald D, Joseph P, Nerette S, Li J, Zheng H Prospective, multi-centre clinic-based evaluation of four rapid diagnostic tests for syphilis. Sex Transm Infect 82 Suppl 5:v West B, Walraven G, Morison L, Brouwers J, Bailey R Performance of the rapid plasma reagin and the rapid syphilis screening tests in the diagnosis of syphilis in field conditions in rural Africa. Sex Transm Infect 78: Zorzi A, Cordioli M, Gios L, Del Bravo P, Toskin I, Peeling RW, Blondeel K, Cornaglia G, Kiarie J, Ballard R, Mirandola M Field evaluation of two point-of-care tests for syphilis among men who have sex with men, Verona, Italy. Sex Transm Infect 93:S51-S Matthias J, Dwiggins P, Totten Y, Blackmore C, Wilson C, Peterman TA Notes from the Field: Evaluation of the Sensitivity and Specificity of a Commercially Available Rapid Syphilis Test - Escambia County, Florida, MMWR Morb Mortal Wkly Rep 65: Nakku-Joloba E, Kiragga A, Mbazira JK, Kambugu F, Jett-Goheen M, Ratanshi RP, Gaydos C, Manabe YC Clinical Evaluation of 2 Point-of-Care Lateral Flow Tests for the Diagnosis of Syphilis. Sex Transm Dis 43: Goza M, Kulwicki B, Akers J, Klepser ME Syphilis Screening: A Review of the Syphilis Health Check Rapid Immunochromatographic Test. J Pharm Technol 33: Team RC R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria Team RS RStudio: Integrated development for R. RStudio Inc, Boston, MA 15

16 Chen B, Peng X, Xie T, Jin C, Liu F, Wu N The tradition algorithm approach underestimates the prevalence of serodiagnosis of syphilis in HIV-infected individuals. PLoS Negl Trop Dis 11:e Johnson PD, Graves SR, Stewart L, Warren R, Dwyer B, Lucas CR Specific syphilis serological tests may become negative in HIV infection. AIDS 5: Food and Drug Administration Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests. US Department of Health and Human Services. 48. Castro AR, Jost HA Effect of multiple freeze and thaw cycles on the sensitivity of IgG and IgM immunoglobulins in the sera of patients with syphilis. Sex Transm Dis 40: Downloaded from on September 21, 2018 by guest 16