Enhanced Detection of Syphilis Infection: Implementation of a Reverse Testing Algorithm

Scott Bainbridge, CLS, Special Chemistry/Toxicology Supervisor
Jennifer Brown, MD, Division of Infectious Diseases
Nam K. Tran, PhD, MS, FACB, Clinical Chemistry and POCT

Background: Syphilis is caused by the spirochete bacteria Treponema pallidum. This organism cannot be cultured, therefore diagnosis relies primarily on serologic techniques.1 These serologic techniques are include the rapid plasma reagin (RPR), venereal disease research laboratory (VRDL), fluorescent treponemal antibody –absorbed (FTA-ABS), Treponema pallidum particle agglutination assay (TPPA), enzyme immunoassay (EIA), and multiplex flow immunoassay (MFI) tests. Each of these methods has advantages and disadvantages. Current best practice dictates that serologic screening techniques should not be used independently, but in defined sequences or algorithms. The “Traditional Algorithm” involves serologic screening for Syphilis using a nontreponemal test (e.g., VDRL and RPR) first and followed by a treponemal specific test such as EIA, TPPA, or FTA-ABS. This traditional method has a high positive predictive value, but often misses early primary and treated infections. From a clinical laboratory perspective, the traditional algorithm is also labor intensive and involves subjective interpretation by laboratory scientists.

More recently, it has been recommended to employ a “Reverse Algorithm” where the serologic screening method for Syphilis involves the use of a treponemal specific test (e.g., EIA) first to detect IgG antibodies to Treponema pallidum.2-4 This is followed by a nontreponemal test such as RPR. As noted, the reverse algorithm has the advantage of detecting primary and treated infections as well as reducing the false positive results associated with the traditional algorithm. However, a disadvantage to the reverse algorithm is some patients may have discordant results where the initial IgG screen is reactive and the RPR is non-reactive.5,6 This situation can arise from past, successfully treated syphilis, latent, untreated syphilis or early syphilis. The United States Center for Disease Control and Prevention (CDC) recommends performing a second, confirmatory treponemal specific antibody test such as TPPA to differentiate between a false positive and past syphilis.

Laboratory Best Practice: UC Davis Health currently employs the reverse algorithm. The initial screen is performed on serum and tests for IgG antibodies specific for Treponema pallidum. This test is performed on the Bioplex 2200 using multiplex flow Immunoanalyzer and has the advantage of greater clinical sensitivity and specificity compared to other older EIA tests. Table 1 summarizes the results interpretation for the reverse algorithm. In brief, IgG antibodies to Treponema pallidum are present for life, even after Syphilis has been successfully treated. To assist in the diagnosis of untreated infection, sera screening reactive for IgG antibodies to Treponema pallidum and reflex to the RPR test performed. If the RPR test is reactive, this suggests active, untreated syphilis. If the RPR is non-reactive, this suggests past, successfully treated syphilis. To rule out potential false positive results, we also reflexively perform the TPPA on Treponema pallidum IgG reactive but RPR non-reactive samples. In this situation, if the TPPA test is reactive this confirms the initial IgG screen and suggests past, successfully treated syphilis. If the TPPA test is non-reactive, this suggests that the initial IgG screen was falsely positive, although it does not rule out early Syphilis infections. If Syphilis is clinically suspected, a second specimen should be submitted at a later date. For additional information, please refer to the CDC education materials (https://www.cdc.gov/std/syphilis/syphilis-webinar-slides.pdf) and the UC Davis Health Testing Directory (https://www.testmenu.com/ucdavis).

Table 1 Syphilis Reverse algorithm serology interpretation table

EIA/MFI RPR TPPA Interpretation
Non-Reactive N/A N/A Syphilis not detected. If clinically suspected, retest at a later date
Reactive Reactive N/A Untreated or newly acquired Syphilis
Reactive Non-reactive Non-reactive Possible false reactive initial screen. If clinically suspected, retest at a later date
Reactive Non-reactive Reactive Successfully treated or past Syphilis infection. Historical and clinical evaluation required.

Abbreviations: EIA – Enzyme Immunoassay, MFI – multiplex flow immunoassay, RPR – rapid plasma reagin, TPPA – Treponema pallidum particle agglutination assay


  1. Romanowski B, Sutherland R, Fick GH, Mooney D, Love EJ. Serologic response to treatment of infectious syphilis. Ann Intern Med 1991;114:1005-9.
  2. Sena AC, White BL, Sparling PF. Novel Treponema pallidum serologic tests: a paradigm shift in syphilis screening for the 21st century. Clin Infect Dis 2010;51:700-8
  3. Centers for Disease Control and Prevention Reverse Algorithm Education: https://www.cdc.gov/std/syphilis/syphilis-webinar-slides.pdf, Accessed on February 7, 2017.
  4. Pope V. Use of treponemal tests to screen for syphilis. Infect Med 2004;8:399-404.
  5. Binnicker MJ, Jespersen DJ, Rollins LO: Direct comparison of the traditional and reverse syphilis screening algorithms in a population with a low prevalence of syphilis. J Clin Microbiol 2012 Jan;50(1):148-150
  6. Mortality and Morbidity Weekly Report (MMWR), Centers for Disease Control and Prevention website: https://www.cdc.gov/mmwr/preview/mmwrhtml/mm6005a1.htm, Accessed on February 7, 2017.


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