Syphilis: Evolving screening algorithms and the role of automated immunoassays

Syphilis, once trending toward elimination in the United States, has resurged in the last decade; incidence rose 36% between 2006 and 2010.¹ Infections occur predominantly in men (7.9:1.1, males:females), especially in populations engaging in high-risk sexual behavior (e.g., men who have sex with men [MSM]).^1,2 Untreated syphilis increases the chance of acquiring other sexually transmitted diseases (STDs), such as HIV.³

Caused by the bacterium Treponema pallidum (Figure 1), syphilis is commonly acquired as an STD, but it also can be transmitted vertically by expectant mothers.^3,4 Routine maternal screening and treatment for syphilis is critical to avoid serous perinatal consequences. Without screening, ~69% of vertical transmissions result in adverse outcomes (~40% infant mortality; serious birth defects in survivors).^3,4 Screening may reduce mortality attributable to congenital syphilis by up to 50%.⁴

Figure 1.Treponema pallidum (Courtesy of Siemens Healthcare Diagnostics)

Syphilis is a multistage disease. In primary syphilis, an ulcer (chancre) may be present, but is generally painless, and often goes unnoted. If untreated, infection progresses to secondary syphilis, where signs may include a rash (typically nonpruritic), often presenting on the palms of the hand or soles of the feet. In both stages, signs and symptoms resolve without therapy. Untreated syphilis progresses to the latent stage, which is asymptomatic and can last for years or even decades. About 15% of cases progress to the late (tertiary) stage, an often severe disease state where outcomes can include cardiovascular or neurologic damage or death.^3,5

Syphilis responds well to antibiotic treatment. A single penicillin injection often resolves infections of less than a year; longer term or resistant infections may require additional doses or second line antibiotics. Treatment does not protect from reinfection.³

Diagnosing syphilis with treponemal and nontreponemal tests

T. pallidum cannot be cultured by routine methods.⁵ DNA-based tests using ulcerative swabs or whole-blood samples have limitations (e.g., costs and often low positivity rate in secondary and latent syphilis) and are not broadly used for screening.⁶

Two types of serologic assays are required to diagnose syphilis. Results must always be interpreted in context with patient history and exam.^5,7 Nontreponemal tests detect antibodies against lipoidal material from T. pallidum and syphilis-damaged human cells. Since these antibodies also arise in other conditions (e.g., autoimmune disease, acute viral infection, advancing age), a treponemal test helps determine if the damage is most likely a result of syphilis. Treponemal assays detect antibodies that recognize specific treponemal antigens. If both test results are positive, active syphilis infection is likely, and treatment is necessary. While many partially or fully automated options are now available for treponemal assays, nontreponemal tests remain primarily manual. The order in which the tests are run can vary.^7-9

Traditional vs. reverse-screening algorithms

Traditional algorithms recommend a nontreponemal assay for screening followed by a treponemal antibody test for confirmation of any initially reactive samples (Figure 2a).^7-13

Many automated tests detect both IgM and IgG antibodies to treponemal-specific antigen, allowing for sensitive detection of primary infection (Table 1).⁹ Seeing advantages to automation, many labs are adopting a reverse algorithm, employing a treponemal test to detect infection, followed by a nontreponemal test to assess active disease (Figure 2b).^8,9 As the majority of patients screened are negative, only a small percentage of samples require the manual nontreponemal test in this approach.

Table 1. Examples of syphilis serologic tests (adapted from the CDC)⁹

Besides workflow advantages, evidence suggests an increased detection rate of late-stage syphilis with a reverse-screening algorithm. Treponemal antibody is found in the majority of syphilis-infected individuals (~85% remain positive for life).^7,13 In contrast, up to 30%^11,12 of untreated, late-stage infected individuals become undetectable for nontreponemal antibody, but remain positive for treponemal antibody. Increased late-stage identification allows for treatment intervention and minimizes potential for disease progression.

Qualitative positivity on both the treponemal and nontreponemal tests is consistent with a diagnosis of syphilis. Nontreponemal assays can also be used quantitatively, allowing assessment of treatment efficacy (decreasing titer indicates therapeutic response).

Discordant results

Regardless of which algorithm is used, a small percent of total screens may produce discordant results. Differential test sensitivity may account for a subset of cases. For example, early primary syphilis may be nontreponemal assay-reactive, but treponemal assay-non-reactive using an IgG-only test.⁷ However, some treponemal assays detecting both IgM and IgG may have greater sensitivity than nontreponemal assays.

Disease unrelated to syphilis may cause false positives (FP) in either testing modality. As mentioned, nontreponemal FP can result from autoimmune disease or acute viral infection. Reversion in some untreated infections to a seronegative status for nontreponemal antibody can lead to discordant results using the reverse algorithm.^7,8 Antibody to other treponemal bacteria (such as the agents of yaws or pinta) cannot be differentiated using treponemal assays. Given the complexities of diagnosis, algorithm annotations can be a useful guide to proper interpretation.^7,8

Recently, the Centers for Disease Control and Prevention (CDC) reported on five labs employing a reverse screening approach. Data (from both low- and high-risk populations) showed that, overall, 96.6% of samples could be screened out using a reverse algorithm, and increased detection of possible syphilis was observed (consistent with previous observations).¹⁴ Reactive samples (3.4% overall) were tested with RPR and alternate treponemal testing (TP-PA or FTA-ABS). While initial reactivity was confirmed for 68.4% of these samples, 31.6% of discordant samples failed to show reactivity and were designated FP.⁸

To address potential reverse screen FPs, the CDC suggests confirmation of discordant samples using the TP-PA assay: TP-PA reactivity is necessary to rule out an FP. Other treponemal assays, e.g., FTA-ABS, were not recommended&emdash;TP-PA was cited as likely providing greater specificity.⁸ In a follow-up CDC webinar, Gail Bolan, MD, discussed results of the study and reinforced this guidance. While the CDC has not formally abandoned the traditional approach, it has provided a clear path for labs wishing to adopt a reverse screen.⁹

It is important to understand that FP rates vary with testing populations, and though driven predominantly by prevalence, other factors can contribute.⁷ The 2011 CDC data showed a higher rate of presumed FP compared to the 2008 study (31.6% compared to 17.2%).^8,14 This likely reflects population differences, as well as the mix of treponemal and nontreponemal assays used.⁸ Significant differences in treponemal assay design and detection methodology could account, in part, for some of the discrepancies seen. The CDC plans to pursue studies to better characterize head-to-head performance of various treponemal assays.⁸

Current treponemal and nontreponemal assays enable laboratories to produce results rapidly in both low- and high-throughput environments. Moving from a traditional to a reverse screening algorithm may result in increased detection of syphilis cases, mitigate subjective interpretation, and improve workflow. Clear guidance is now available that enables labs to discriminate presumed positives when using a reverse screening approach.

References

1. STD trends in the United States: 2010 national data for gonorrhea, chlamydia, and syphilis. Snapshot: Sexually transmitted diseases in 2010. www.cdc.gov/std/stats10/tables/trends-snapshot.htm. Accessed May 5, 2012.
2. STD trends in the United States: 2010 national data for gonorrhea, chlamydia, and syphilis. www.cdc.gov/std/stats10/syphilis.htm. Accessed May 5, 2012.
3. Syphilis: CDC fact sheet. www.cdc.gov/std/syphilis/STDFact-Syphilis.htm. Accessed April 2012.
4. Hawkes S, Matin N, Broutet N, Low N. Effectiveness of interventions to improve screening for syphilis in pregnancy: a systematic review and meta-analysis. Lancet Infect Dis. 2011;11(9):684-691.
5. LaFond RE, Lukehart SA. Biological basis for syphilis. Clin Micro Rev. 2006;19(1):29-49.
6. Martin IE, Tsang RSW, Sutherland K, Tilley P, Read R, Anderson B, et al. Molecular characterization of syphilis in patients in Canada: azithromycin resistance and detection of Treponema pallidum DNA in whole-blood samples versus ulcerative swabs. JCM. 2009;47(6):1668-1673.
7. Ratnam S. The laboratory diagnosis of syphilis. Can J Infec Dis Med Microbiol. 2005;16(1):45-51.
8. Discordant results from reverse sequence syphilis screening&emdash;five laboratories, United States, 2006-2010. MMWR Morb Mortal Wkly Rep. 2011;60(5):133-137.
9. Hoover K, Park I. CDC: National Center for HIV/AIDS, viral hepatitis, STD, and TB prevention. Webinar: February 2012.
10. Mayo Clinic Laboratories. Serologic testing for syphilis. www.mayomedicallaboratories.com/articles/hottopics/transcripts/2011/04-syphilis/07.html. Accessed May 5, 2012.
11. Diggory P. Role of the venereal disease research laboratory test in the detection of syphilis. Br J Vener Dis. 1983;59:8-10.
12. Talwar S, Jha PK, Tiwari VD. VDRL titres in early syphilis before and aftertreatment Genitourin Med. 1992;68:120-122.
13. Larsen SA, Steiner BM, Rudolph AH. Laboratory diagnosis and interpretation of tests for syphilis. Clin Microbiol Rev. 1995;8(1):1-21.
14. Syphilis testing algorithms using treponemal tests for initial screening&emdash;four laboratories, New York City, 2005-2006. MMWR Morb Mortal Wkly Rep 2008 Aug 15; 57:872.

Dr. Katherine Soreng received a PhD in Immunology and Molecular Pathogenesis from Emory University and completed a two-year post-doctoral fellowship at the Centers for Disease Control. She currently manages the Clinical and Scientific Marketing team at Siemens Healthcare Diagnostics. Connie Mardis, MEd, is a prolific writer on many clinical topics who frequently speaks to various professional groups in her role as Director, Global Marketing Education at Siemens.