Evaluation of the Elecsys Syphilis Immunoassay for Detection of Syphilis in Populations at Risk of Disease in the US and Argentina

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1 Evaluation of the Elecsys Syphilis Immunoassay for Detection of Syphilis in Populations at Risk of Disease in the US and Argentina Robert H. Christenson, 1 * Marvin Lessig, 2 Gabrielle Miles, 3 Silke Luebcke, 4 Cheryl Stillions, 3 and Prentiss Jones 5 Background: The Elecsys syphilis immunoassay is an automated, qualitative immunoassay that uses a doubleantigen sandwich format to detect antibodies to Treponema pallidum in human serum and plasma. We aimed to validate performance of the immunoassay in various populations at risk for syphilis infection in the US and Argentina. Methods: Samples were obtained for a number of study cohorts, including participants from routine syphilis testing at high or low risk for syphilis, HIV-positive patients, pregnant women, and patients in various stages of syphilis infection. The primary objective was to validate the Elecsys syphilis immunoassay by comparing it with a composite testing algorithm using US Food and Drug Administration (FDA)-approved tests, including the predicate IMMULITE 2000 syphilis screening assay, the rapid plasma reagin, and the T. pallidum particle agglutination assay. Results: Complete algorithm testing was performed on all 2660 collected samples. Acceptable precision was demonstrated in all samples. Comparison of the Elecsys syphilis immunoassay with the final syphilis status for all samples yielded a diagnostic sensitivity of 99.5% (95% CI, ) and a diagnostic specificity of 99.2% (95% CI, ). Overall, the lower limit of the 95% CIs for sensitivity and specificity met the expected performance of 95%. Conclusion: This is the first study that confirms the high sensitivity and specificity of the Elecsys syphilis immunoassay in US and Argentinian cohorts and highlights the assay's usefulness as an alternative to current tests for the diagnosis of syphilis infection in a broad range of participant cohorts. IMPACT STATEMENT The Elecsys syphilis immunoassay was previously validated in European and Asian samples. We validated the performance of the assay in populations at varying risk for syphilis infection in the US and Argentina, using an FDA-approved composite testing algorithm as a reference standard. Acceptable precision was demonstrated in all 2660 samples. Comparison of the immunoassay with the final syphilis status yielded a sensitivity of 99.5% (95% CI, ) and a specificity of 99.2% (95% CI, ). The Elecsys syphilis immunoassay represents an attractive alternative to current tests for the diagnosis of syphilis in a broad range of participant cohorts. 1 Department of Pathology, University of Maryland School of Medicine, Baltimore, MD; 2 Nationwide Laboratory Services, Plantation, FL; 3 Global Medical and Scientific Affairs CPS, Roche Diagnostics Operations, Indianapolis, IN; 4 R&D, Roche Diagnostics GmbH, Penzberg, Germany; 5 Department of Biomedical Sciences, Western Michigan University Homer Stryker M.D. School of Medicine, Kalamazoo, MI. *Address correspondence to this author at: University of Maryland School of Medicine, 22 South Greene Street, Baltimore, MD Fax ; rchristenson@umm.edu. DOI: /jalm American Association for Clinical Chemistry July : JALM 89

2 Syphilis is a sexually transmitted disease caused by infection with the bacterium Treponema pallidum. It can also be passed from mother to child in utero or at birth (congenital syphilis), often with devastating effects on the fetus if not treated early enough during pregnancy (1, 2). An estimated 5.6 million new cases of syphilis occurred globally in 2012 (3), with an estimated maternal syphilis infections resulting in adverse pregnancy outcomes and infected infants worldwide (4). If diagnosed during the early stages of infection, syphilis can be readily treated with antibiotics (5). However, if left unchecked, the disease can progress through secondary, early and late latent (asymptomatic), and tertiary stages, resulting in multiple clinical conditions that mimic a number of other disease states (6). Approximately 25% of patients will experience at least 1 relapse during early latency, with the majority of these cases occurring within the first year of infection (7). Diagnosis of syphilis can also be delayed due to the lack of obvious symptoms during the earlier disease stages. Commonly used diagnostic tests for syphilis are based on detection of antitreponemal antibodies and include the T. pallidum hemagglutination assay, T. pallidum particle agglutination assay (TPPA), 6 and fluorescent treponemal antibody-absorbed test, as well as a number of immunoassays. The Elecsys syphilis immunoassay is a fully automated, qualitative electrochemiluminescence immunoassay designed for use in conjunction with CDCapproved reverse screening, diagnostic, and treatment algorithms for syphilis (8 11). The assay uses a double-antigen sandwich format to detect antibodies to T. pallidum in human serum and plasma. Biotinylated T. pallidum-specific recombinant antigens (TpN15, TpN17, and TpN47) and T. pallidum-specific recombinant antigens (TpN15, TpN17, and TpN47) labeled with a ruthenium complex react with anti-t. pallidum antibodies to form a sandwich complex. The use of monomeric and polymeric antigens ensures the detection of both IgG and IgM antitreponemal antibodies. Performance evaluation of the Elecsys syphilis immunoassay has been previously reported in samples obtained from European and Asian populations (12, 13). In a multicenter study of more than 8000 samples collected from routine diagnostic requests and blood donations, an overall specificity of 99.9% and sensitivity of 99.6% 100% was reported when compared with the ARCHITECT syphilis TP assay (Abbott Laboratories), LIAISON Treponema screen assay (DiaSorin), Serodia TPPA assay (Fujirebio), Vitros syphilis TPA assay (Ortho Clinical Diagnostics), Enzygnost syphilis assay (Siemens Healthcare Diagnostics), and the IMMULITE 2000 syphilis screen assay (Siemens Healthcare Diagnostics; 12). Comparison of the Elecsys syphilis immunoassay with the ARCHITECT syphilis TP assay, LIAISON Treponema screen assay, Serodia TPPA assay, and the Mediace T. pallidum latex agglutination (TPLA) assay (Sekisui Medical) in 7 independent laboratories, in a broader variety of diagnostic settings and participant populations, yielded an overall diagnostic specificity of 99.9% in 1965 routine clinical samples and 99.9% in 5792 blood donor samples; the sensitivity of the assay was 100% (13). The aim of this analysis was to validate performance of the Elecsys syphilis immunoassay in populations at high or low risk for syphilis infection in the US and Argentina, including samples sent for routine testing, HIV-positive patients, pregnant women, and patients in various stages of syphilis infection. METHODS Study design The Elecsys syphilis immunoassay (Roche Diagnostics) was evaluated at 3 different sites across the US, with samples obtained from the US and 6 Nonstandard abbreviations: TPPA, T. pallidum particle agglutination assay; FDA, US Food and Drug Administration; RPR, rapid plasma reagin; COI, cutoff index; PPA, positive percent agreement; NPA, negative percent agreement.. 90 JALM :01 July 2018

3 ARTICLES Fig. 1. Testing algorithm including Elecsys syphilis assay (left side) and the FDA-accepted composite reference standard (right side). Argentina. Institutional Review Board approval was obtained from each institution before any clinical study work. All investigational sites conducted the study in accordance with the principles of the Declaration of Helsinki and International Conference on Harmonization Good Clinical Practice guidelines. The primary study objective was to validate the Elecsys syphilis assay for regulatory approval by means of comparison with a composite testing algorithm (Fig. 1; 9 11) developed with the US Food and Drug Administration (FDA), which applied FDA-approved tests, including the predicate IMMULITE 2000 syphilis screen assay (Siemens Healthcare), the rapid plasma reagin (RPR) nontreponemal specific assay (Becton, Dickinson and Company), and the TPPA Treponema-specific assay (Fujirebio). A secondary objective was to demonstrate precision performance according to Clinical and Laboratory Standards Institute document EP05-A2 (14). Study samples Samples were collected at 9 investigational sites (Spartanburg, NC; Baltimore, MD; Whittier, July : JALM 91

4 Table 1. Criteria used for categorizing participants. High risk Low risk Healthy population Clinical population Staged value (well-characterized specimens representing the various stages of syphilis) Inclusion criteria Must belong to one of the listed increased-risk groups for syphilis. Occupational risks: Tattoo artists Morticians Commercial sex worker Sexual risks: Individuals with multiple sex partners Individuals sharing sex with partner(s) with a history of syphilis Male-on-male sex partners (male homosexuals) Individuals sharing sex with HIV-infected partner(s) Individuals sharing sex with commercial sex workers (prostitutes) Individuals diagnosed with STDs Behavioral risks: IV drug users (current or past) Individuals sharing straw cocaine Individuals with a history of incarceration No identified occupational, sexual, or behavioral risks as described above under the high-risk cohort. These groups may include specimens from healthy subjects (in which specimens were submitted for well check-ups ), specimens submitted for premarital testing, and/or unselected specimens from hospital patients submitted for routine chemistry. Appropriate answers to a defined health status questionnaire. Must have medical documentation of syphilis stage including treatment or no treatment confirmation. CA; Minneapolis, MN; Miami Beach, FL; 3 sites in Miami, FL; and Buenos Aires, Argentina). All clinical specimens were collected via written informed consent, with an Institutional Review Board-approved Health Insurance Portability and Accountability Act authorization process in the US, in accordance with federal regulations. Samples were collected for a number of different study cohorts: individuals from routine syphilis testing either at high or low risk for syphilis; HIV-positive patients at high risk for syphilis; pregnant women at unknown risk for syphilis; and patients in various stages of syphilis infection represented by primary, secondary, and latent (both treated and untreated) stages (Table 1). Inclusion and exclusion criteria were established for each study cohort. All participants were 18 years or older and could only be enrolled into 1 cohort. Composite testing and classification of patient samples As illustrated by the algorithm in Fig. 1, testing included the Elecsys syphilis assay (left side) and the composite reference standard, which included the IMMULITE 2000 syphilis screen assay and RPR testing on all samples. The TPPA test was used to adjudicate samples with mismatched results for IMMULITE and RPR testing. Elecsys description and interpretation The Elecsys syphilis immunoassay can be run on the same system as other Elecsys tests for infectious diseases (12). The effectiveness of the assay in screening donor blood has not yet been established in the US. Quality control was performed with PreciControl Syphilis reagents (Roche. 92 JALM :01 July 2018

5 ARTICLES Diagnostics). All studies were performed on cobas e 411 analyzers (Roche Diagnostics). The left side of Fig. 1 displays the testing algorithm used for the Elecsys syphilis assay. Samples with a cutoff index (COI; signal from patient sample/cutoff signal) <1.0 were considered nonreactive in the Elecsys syphilis assay, which measures syphilis-specific antibodies; these samples did not undergo further testing, in accordance with the reverse screening algorithm advocated by the CDC. Samples with a COI 1.0 were considered reactive. All initially reactive samples were retested in duplicate with the Elecsys syphilis assay (Fig. 1). If COI values for both replicates were <1.0, then the samples were considered nonreactive for syphilis-specific antibodies (12, 13). Initially reactive samples yielding COI values 1.0 for either or both the retests were considered repeatedly reactive and confirmed according to the recommended composite algorithm (Fig. 1; 8 11). IMMULITE 2000 syphilis screen and RPR testing Fig. 1 also shows the algorithm used for testing of samples with the FDA-cleared IMMULITE syphilis screen assay and the RPR assay. If both these assays yielded a nonreactive result for a sample, then the result was nonreactive. When both the IMMULITE and RPR results were reactive, the sample was determined to be reactive. For discordant samples in which the IMMULITE or RPR results did not match, the sample was tested with the TPPA assay; all TPPA results were categorized as either reactive or nonreactive and considered definitive. Method comparison testing of the Elecsys syphilis assay and the IMMULITE 2000 syphilis screen assay was conducted at 3 testing sites: Plantation, FL; South Bend, IN; and Baltimore, MD, where composite RPR and TPPA reference testing for all samples was also performed. Comparator assay testing was conducted according to the manufacturers' instructions. Analytical performance Intermediate precision (within lab), repeatability (within run), and reproducibility (between labs) were determined for PreciControl materials and human serum pools (near assay cutoff and at a moderate positive level). The 3 testing sites were required to successfully complete a familiarization-testing phase before initiating the imprecision analysis. Imprecision testing was performed on 3 different cobas e 411 analyzers at the 3 external testing sites with a single reagent lot. Five human serum pools and 2 PreciControl pools were tested in replicates of 3, in 2 runs per day for 5 days, according to Clinical and Laboratory Standards Institute EP05-A2 guidance (14). Analysis of variance was conducted on the imprecision data collected over 5 days. Intermediate precision, repeatability, and reproducibility were presented as mean, SD, and percent CV of the COI values. Clinical performance Comparative clinical performance was assessed through the positive percent agreement (PPA; sensitivity) and negative percent agreement (NPA; specificity) values between the Elecsys syphilis assay and the composite testing algorithm using FDA-approved tests (Fig. 1). Statistical analyses All analytical data were captured by the WinCAEv electronic data capture software developed for Roche-sponsored studies. Clinical data were collected with the Medrio database. Statistical analyses using clinical information were performed in the biostatistics department at Roche Diagnostics, Indianapolis, with SAS Software version 9.3 of the SAS System for Windows 7 Enterprise (SAS Institute Inc.). July : JALM 93

6 Table 2. Demographic characteristics for all participant cohorts, except the staged cohort (n = 154). All variables expressed as: n (%) Sex High risk (n = 622) Low risk (n = 902) HIV positive (n = 457) Pregnant (n = 316) Healthy population (n = 209) All (N = 2506) Female 357 (57.4) 551 (61.1) 183 (40.0) 316 (100) 80 (38.3) 1487 (59.3) Male 265 (42.6) 351 (38.9) 274 (60.0) (61.7) 1019 (40.7) Age, years (12.5) 242 (26.8) 35 (7.7) 65 (20.6) 7 (3.4) 427 (17.0) (26.4) 210 (23.3) 37 (8.1) 132 (41.8) 36 ( (23.1) (16.1) 134 (14.9) 46 (10.1) 103 (32.6) 29 (13.9) 412 (16.4) (21.2) 136 (15.1) 100 (21.9) 16 (5.1) 44 (21.1) 428 (17.1) (18.0) 121 (13.4) 196 (42.9) 62 (29.7) 491 (19.6) (5.3) 49 (5.4) 42 (9.2) 28 (13.4) 152 (6.1) (0.5) 10 (1.1) 1 (0.2) 2 (1.0) 16 (0.6) 80 1 (0.5) 1 (0.04) Race Asian 1 (0.2) 29 (3.2) 1 (0.2) 4 (1.3) 0 35 (1.4) Black/African-American 253 (40.7) 296 (32.8) 377 (82.5) 22 (7.0) 120 (57.4) 1068 (42.6) Mixed race 4 (0.6) 5 (0.6) 3 (0.7) 2 (0.6) 6 (2.9) 20 (0.8) Caucasian/White 362 (58.2) 562 (62.3) 64 (14.0) 285 (90.2) 79 (37.8) 1352 (54.0) Other 2 (0.3) 10 (1.1) 12 (2.6) 3 (0.9) 4 (1.9) 31 (1.2) RESULTS Participant samples In total, 2697 study participants were prospectively recruited at 8 sites across the US and 1 site in Buenos Aires, Argentina. Overall, 37 (1.4%) of the participants were excluded, leaving 2660 enrolled individuals as the final study population (see Fig. 1 in the Data Supplement that accompanies the online version of this article at content/vol3/issue1). Complete algorithm testing was performed on all 2660 collected samples, consisting of individuals from routine syphilis testing (n = 1524), either at high risk (n = 622) or low risk for syphilis (n = 902); HIV-positive patients (n = 457); pregnant women at unknown risk for syphilis (n = 316); patients with syphilis infection, represented by primary, secondary, and latent (treated and untreated) stages (n = 154); and apparently healthy individuals at no increased risk for syphilis, referred to as the healthy population cohort (n = 209). Demographic data for all study cohorts, except the staged cohorts, are displayed in Table 2. A somewhat higher percentage of females than males was enrolled in the high-risk cohort (57.4% vs 42.6%, respectively), and higher percentages of males than females were enrolled in the HIVpositive cohort (60.0% vs 40.0%, respectively) and the healthy population (61.7% vs 38.3%, respectively). The age groups and races (Asian, Black/ African American, mixed race, Caucasian/White, and other) that were enrolled in the study are listed in Table 2. Analytical performance Detailed precision and reproducibility results obtained at the 3 test sites are shown in Table 1 in the online Data Supplement and in Table 3, respectively. A total of 21 pools were tested for repeatability (7 sample pools 3 sites 1 reagent lot); 100% of the pools met the expected target values determined on different analyzers (n 2) according to standard operating procedures. The. 94 JALM :01 July 2018

7 ARTICLES Table 3. Reproducibility results for 5 human serum pools and 2 PreciControls from the 3 test sites (n = 90 for each sample). a Mean Within-run repeatability Between run Between day Between site Reproducibility Sample COI SD 95% CI % CV SD % CV SD % CV SD % CV SD 95% CI % CV HSP HSP HSP HSP HSP PreciControl PreciControl a SD of 0 due to variance contributed by particular component was below stated significant figure. HSP 06, low positive; HSP 07, low positive; HSP 08, high negative; HSP 09, negative; HSP 10, high positive; PreciControl 1, negative; PreciControl 2, positive. HSP, human serum pool. SD for negative pools (COI < 1.0) ranged from 0.01 to 0.02, and the SD for positive pools (COI 1.0) ranged from 0.01 to 0.13 (see Table 1 in the online Data Supplement). Overall, 21 pools were also tested for intermediate precision, and all the pools met the expected target. The SD for negative pools ranged from 0.01 to 0.04, and the SD for positive pools ranged from 0.02 to 0.18 (see Table 1 in the online Data Supplement). The reproducibility analyses also met the expected target; the SD for negative pools was consistent at 0.03, and the % CV for positive pools ranged from 2.86% to 3.82% (Table 3). Clinical performance Table 4 shows that the comparison data for the Elecsys syphilis assay with the final syphilis status from the composite testing for all samples yielded a sensitivity (PPA) of 99.5% (95% CI, ) and a specificity (NPA) of 99.2% (95% CI, ). Overall, the lower limit of the 95% CIs for sensitivity (98.2%) and specificity (98.7%) met the expected performance of 95%. Considering different cohorts individually, there was excellent concordance of the Elecsys syphilis assay and the reference standards composite testing in each group. In particular, the PPA between Table 4. Percentage agreement between the Elecsys syphilis assay and the final syphilis status as derived from the composite reference algorithm. Positive percent agreement Negative percent agreement Study cohort % (n/n) 95% CI % (n/n) 95% CI Overall 99.50% (400/402) % (2239/2258) High risk 100 (49/49) (572/573) Low risk 100 (17/17) (883/885) HIV positive 100 (162/162) (282/295) Pregnant 100 (15/15) (301/301) Staged (140/142) (12/12) Healthy population 100 (17/17) (189/192) July : JALM 95

8 Table 5. Discordant results across all cohorts. Final composite syphilis status Reactive Nonreactive Elecsys syphilis immunoassay Reactive 0 19 Nonreactive 2 0 the Elecsys syphilis assay and the final syphilis status in pregnant and HIV-positive samples was 100% (Table 4). The lower end of the 95% CIs for sensitivity ranged from 78.2% in the pregnant cohort to 97.8% in the HIV-positive cohort; the lower end of the 95% CIs for specificity ranged from 73.5% in the staged cohort to 99.2% in the low-risk cohort. It is critical to note that 95% CIs represent a statistical range and not the actual results. For this reason, the 95% CI data for the different cohorts must be interpreted with caution since 95% CIs are extremely dependent on the number of samples in the individual cohort. In total, 21 of the 2660 (0.8%) samples were discordant between the Elecsys syphilis assay and the final composite syphilis status. Of interest, 13 (62%) of these samples had discordant results between the IMMULITE syphilis screen assay and the RPR assay, and underwent further analysis using the TPPA assay in accordance with Fig. 1. The patients showing discordant results with the Elecsys syphilis assay were dispersed among the different cohorts: high risk (n = 1), low risk (n = 2), HIV positive (n = 13), staged (n = 2), and healthy population (n = 3). Of the 21 discordant samples, 19 were reactive for the Elecsys syphilis assay, but were nonreactive for the composite testing algorithm (Table 5). The remaining 2 were nonreactive with the Elecsys syphilis assay, and therefore underwent no further Elecsys testing in accordance with the algorithm (Fig. 1). Our ability to investigate these discordant results was restricted because clinical information regarding the patients from whom the discordant samples were collected was limited to the demographic data shown in Table 2. The samples were from participants between 21 and 68 years of age, with an almost exact 50% split of females and males. All samples went through a single freeze-thaw cycle. Further, performing additional measurements or retrospective postanalytical investigation/assessment of samples was not included in the design of this study. Therefore, we were unable to investigate possible sample-specific issues, as available sample volume collected was used for testing, according to the composite algorithm. DISCUSSION Rates of syphilis infection in the US have been steadily increasing in recent years, with the annual incidence of primary and secondary syphilis cases almost doubling from 8724 in 2005 to in 2013 (15). Men have experienced the largest increase in syphilis infections during this time, particularly in those who have sex with other men (15). To address the need for more accurate diagnostic tests and the lack of a gold standard for syphilis diagnosis, the Elecsys syphilis immunoassay was recently developed and validated in serum and plasma samples obtained from European and Asian populations (12, 13). It is important to note that once an individual has been infected with T. pallidum, they develop circulating antibodies that remain for life. However, if a patient who has been tested with a reliable testing method returns a negative result 90 days after exposure, as was seen in 12 members of the latent syphilis cohort, it is considered a negative diagnosis (16). Thus, the specificity of tests for T. pallidum for recent infection or reinfection may be population specific, which is why it is vital to validate the test in different populations. This is also why the CDC recommends follow-up testing if the initial T. pallidum result is positive in the reverse sequence algorithm.. 96 JALM :01 July 2018

9 ARTICLES In the current analysis, we aimed to validate performance of the Elecsys syphilis assay in cohorts at varying risk for syphilis infection in US and Argentinian populations. Because there is no gold standard for syphilis diagnosis, we assessed performance of the Elecsys assay by comparison with a composite of FDA-approved tests in an algorithm that met with the acceptance of FDA reviewers. Overall, the Elecsys syphilis immunoassay demonstrated acceptable precision results in all samples tested. As expected, the major source of variance originated from the between-run component for both intermediate precision and reproducibility. The Elecsys syphilis immunoassay showed very high concordance with the composite reference standard results, i.e., diagnostic sensitivity of 99.5% (95% CI, ) and diagnostic specificity of 99.2% (95% CI, ). High concordance was also found with each of the individual cohorts tested. However, the number of samples in each individual cohort was relatively limited, so the lower 95% confidence limits of sensitivity and specificity are not particularly informative. The results described here confirm that the previously reported high specificity and sensitivity of the immunoassay also hold in the US and Argentinian populations included in this study (12, 13). A small number of samples (0.8%) showed discordant results between the Elecsys assay and the final composite testing outcome. The demographic information collected from the participants was minimal and, due to the study design, no sample remained for follow-up studies. Most of the discordant samples (90%) were reactive, and the remaining 10% (n = 2) of the discordant results were nonreactive. Although discordant results are never ideal, the pattern observed provides some reassurance that the test has high diagnostic sensitivity, which is critical for minimizing falsely negative results that could leave patients who are reactive for syphilis testing undetected and therefore untreated. We are unable to further investigate the root cause of discordant results, which is a limitation. Our analysis demonstrated the effectiveness of the Elecsys syphilis immunoassay in a variety of participant cohorts, including pregnant women, patients in various stages of syphilis infection, individuals at high or low risk of the disease, and HIV-positive patients. Since syphilis infection is associated with an increased risk of HIV infection (17 19), and HIV adversely affects the serologic response to syphilis (20), there is a particular need for the Elecsys syphilis assay to detect syphilis in HIV-positive patients. The HIV-positive cohort in our study included 457 patients, which is the largest data set yet examined for this patient cohort, and adds important data to the previous evidence base (12, 13). African-American (42.6%), Caucasian/White (54.0%), and Asian (1.4%) populations were well represented in this study and comparable with the proportions of these cohorts in the US as a whole (13.3%, 61.3%, and 5.7%, respectively; 21). However, sites in neither the Southwest nor the West Coast regions of the US were included in the study, and there was no focused effort to recruit patients who described themselves as Latino or Latina. This latter cohort comprises approximately 20% of the US population (21), and the study does not provide specific confirmation of test performance in this Latino/Latina population, which is a limitation. A recent publication reported that the overall sensitivity and specificity of the Elecsys syphilis immunoassay is comparable to the ARCHITECT syphilis TP assay (97.7% and 97.1%, respectively) and the reformulated ARCHITECT syphilis TP assay (99.2% and 99.7%, respectively; 22). The Elecsys syphilis immunoassay's high sensitivity and specificity allows for consistent interpretation of results across a range of participant cohorts and shows that this assay is appropriate for incorporation into the CDC-recommended reverse algorithm. Thus, the Elecsys syphilis immunoassay represents July : JALM 97

10 an attractive automated alternative to current diagnostic tests to aid in the diagnosis of syphilis infection in conjunction with clinical indicators, signs, and symptoms of the disease. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Authors Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Employment or Leadership: S. Luebecke, Roche Diagnostics GmbH; R. H. Christenson, JALM, AACC. Consultant or Advisory Role: R.H. Christenson, Roche Diagnostics, Siemen s Healthcare Diagnostics. Stock Ownership: S. Luebecke, Roche Diagnostics GmbH. Honoraria: None declared. Research Funding: Roche Diagnostics. Expert Testimony: None declared. Patents: None declared. Role of Sponsor: The funding organization, Roche Diagnostics, had a direct role in the design of the study. Acknowledgments: This study was sponsored by Roche Diagnostics. The authors gratefully acknowledge the participation of volunteers enrolled in this study and the assistance from the data and sample collection sites. Third-party medical writing assistance, under the direction of the authors, was provided by Fiona Fernando, PhD, of Gardiner-Caldwell Communications, and was funded by Roche Diagnostics. REFERENCES 1. Newman L, Kamb M, Hawkes S, Gomez G, Say L, Seuc A, Broutet N. Global estimates of syphilis in pregnancy and associated adverse outcomes: analysis of multinational antenatal surveillance data. PLoS Med 2013;10:e Qin J, Yang T, Xiao S, Tan H, Feng T, Fu H. Reported estimates of adverse pregnancy outcomes among women with and without syphilis: a systematic review and meta-analysis. PLoS One 2014;9:e World Health Organization. WHO guidelines for the treatment of Treponema pallidum (syphilis) (Accessed January 2017). 4. Wijesooriya NS, Rochat RW, Kamb ML, Turlapati P, Temmerman M, Broutet N, Newman LM. Global burden of maternal and congenital syphilis in 2008 and 2012: a health systems modelling study. Lancet Glob Health 2016;4:e Clement ME, Okeke NL, Hicks CB. Treatment of syphilis: a systematic review. JAMA 2014;312: Peeling RW, Hook EW 3rd. The pathogenesis of syphilis: the Great Mimicker, revisited. J Pathol 2006;208: Radolf JD, Deka RK, Anand A, Šmajs D, Norgard MV, Yang XF. Treponema pallidum, the syphilis spirochete: making a living as a stealth pathogen. Nat Rev Microbiol 2016;14: Centers for Disease Control and Prevention sexually transmitted diseases treatment guidelines. Syphilis. (Accessed June 2017). 9. Workowski KA, Berman S, Centers for Disease Control and Prevention (CDC). Sexually transmitted diseases treatment guidelines, MMWR Recomm Rep 2010;59: Binnicker MJ. Which algorithm should be used to screen for syphilis? Curr Opin Infect Dis 2012;25: French P, Gomberg M, Janier M, Schmidt B, van Voorst Vader P, Young H; IUST. IUSTI: 2008 European guidelines on the management of syphilis. Int J STD AIDS. 2009;20: Enders M, Hunjet A, Gleich M, Imdahl R, Mühlbacher A, Schennach H, et al. Performance evaluation of the Elecsys syphilis assay for the detection of total antibodies to Treponema pallidum. Clin Vaccine Immunol 2015;22: Kremastinou J, Polymerou V, Lavranos D, Aranda Arrufat A, Harwood J, Martínez Lorenzo MJ, et al. Evaluation of Elecsys syphilis assay for routine and blood screening and detection of early infection. J Clin Microbiol 2016;54: Clinical and Laboratory Standards Institute. Evaluation of precision performance of quantitative measurement methods; approved guideline second edition. Wayne (PA): CLSI; CLSI document EP05-A Patton ME, Su JR, Nelson R, Weinstock H; Centers for Disease Control and Prevention. Primary and secondary syphilis United States, MMWR Morb Mortal Wkly Rep 2014;63: Everything about syphilis and syphilis testing (2015). (Accessed November 2017). 17. Zhang X, Wang C, Hengwei W, Li X, Li D, Ruan Y, et al. Risk factors of HIV infection and prevalence of co-infections among men who have sex with men in Beijing. China. AIDS 2007;21 Suppl 8:S Patterson TL, Semple SJ, Staines H, Lozada R, Orozovich P, Bucardo J, et al. Prevalence and correlates of HIV infection among female sex workers in 2 Mexico-US border cities. J Infect Dis 2008;197: Chen XS, Yin YP, Tucker JD, Gao X, Cheng F, Wang TF, et al. Detection of acute and established HIV infections. 98 JALM :01 July 2018

11 ARTICLES in sexually transmitted disease clinics in Guangxi. China: implications for screening and prevention of HIV infection. J Infect Dis 2007;196: Tobian AAR, Quinn TC. Herpes simplex virus type 2 and syphilis infections with HIV: an evolving synergy in transmission and prevention. Curr Opin HIV AIDS 2009;4: United States Census Bureau (2016) gov/quickfacts/fact/table/us/pst (Accessed November 2017). 22. De Keukeleire S, Desmet S, Lagrou K, Oosterlynck J, Verhulst M, Van Besien J, et al. Analytical and clinical comparison of Elecsys syphilis (Roche ) - ARCHITECT syphilis TP and reformulated ARCHITECT syphilis TP (Abbott ) assay. Diagn Microbiol Infect Dis 2017;87: July : JALM 99