Syphilis Serology

Treponema pallidum are spirochete bacteria that  is transmitted by sexual contact and causes syphilis. Vertical transmission can cause congenital syphilis, which might result in spontaneous abortions, miscarriages, or stillbirths. Infants with congenital syphilis can manifest clinical signs of infection at birth or months to years after birth. 

Syphilis infections progress through three stages.Primary syphilis appears about 3 weeks after exposure and is characterized by single or multiple ulcers (chancres) in the mouth, vagina, or rectum. They are often painless and might be unnoticed. Chancres can persist for 2–6 weeks before healing spontaneously. 

Secondary syphilis typically begins 2–24 weeks after most primary lesions heal. This stage is commonly characterized by a mucocutaneous rash on the trunk, palms, and soles. One-fourth of patients develop mucous patches in the mouth or condylomata lata on the genitals or rectum. The secondary stage can also include lymphadenopathy, alopecia, and occasionally neurologic and ocular manifestations. Signs and symptoms of secondary syphilis typically resolve in approximately 3 months (range of 1–12 months). Patients with an HIV infection may experience the symptoms of primary and secondary syphilis symptoms concurrently. 

Moist primary and secondary syphilis lesions contain infectious T. pallidum that can be transmitted through sexual contact to susceptible persons. 

The interval between primary to secondary and secondary to tertiary syphilis is known as latency because no symptoms or signs of syphilis are present. The latent period between secondary and tertiary syphilis can last for years or decades before symptoms appear. In up to two thirds of patients, the disease can remain latent for life and never progress to tertiary syphilis. 

During tertiary syphilis, treponemal spirochetes can multiply in the thoracic aorta and coronary arteries, resulting in stenosis or aneurysms. Syphilitic gummas, which are soft granulomatous infections, can cause tissue destruction in any organ system including bones and cartilage.  Neurosyphilis includes tabes dorsalis and general paresis.

Treponema pallidum, cannot be cultured in the clinical microbiology lab. Therefore, serologic tests are necessary for the diagnosis of the disease in any stage. Serologic tests for syphilis are classified as either nontreponemal or treponemal.

Nontreponemal tests are used as a screening test for syphilis, as a diagnostic test in patients with signs or symptoms suggestive of syphilis or a known sexual contact, when assessing possible reinfections, and when monitoring treatment outcome. 

Nontreponemal tests include RPR and VDRL. These tests detect IgM and IgG antibodies to a combination of cardiolipin, cholesterol, and phosphatidylcholine. They are flocculation tests that detect antibody-antigen complexes that precipitate out of solution. VDRL tests are read microscopically at 100x magnification. The RPR test uses charcoal to aid in detection of the flocculant, and the results can be read macroscopically because the antigen-antibody lattice traps the charcoal particles. 

Nontreponemal tests are usually performed manually, but some RPR tests have been automated. The automated systems digitally analyze the density and size of antigen-antibody complexes. Automated RPR tests may have a limited serum dilution ranged. Serum samples that are outside the dilution range of an automated test should be reflex tested using a manual RPR.

Results of nontreponemal tests should be reported as an endpoint titer and not with greater or less than values. Endpoint titers are the highest dilution yielding a reactive result. Endpoint titers from RPR and VDRL should not be used interchangeably to manage patients because they are different test methods and the subjective titer results can vary by laboratory. Therefore, patient specimens should be tested using the same nontreponemal test method and specimen type.

Nontreponemal tests are negative or have low titers in the early stages of primary syphilis. RPR sensitivity is 62 to 76% and VDRL sensitivity is 62 to 78%. They also have low sensitivity in latent syphilis; RPR sensitivity is 61% and VDRL sensitivity is 64%.

Titers increase as the disease progresses. Antibody titers can correlate with infection status and can be used to monitor response to therapy. A fourfold change in titer between two results with the same nontreponemal tests is considered clinically significant. Nontreponemal antibody titers usually decrease at least fourfold during the 12 months after syphilis treatment and might become nonreactive over time, especially among patients treated before the secondary stage of syphilis. 

Approximately 30% of patients remain weakly seropositive. This serofast state occurs most commonly in persons treated more than 1 year after acquiring syphilis or in persons with multiple episodes of syphilis. Titers are typically equal to or less than 1:8, but higher titers have been observed. 

Excess antibodies in serum can produce a false negative result that is known as the prozone or hook effect. A prozone can be avoided by diluting serum prior to testing. Prozone rarely occurs during screening for syphilis. Therefore, routine dilution of all nonreactive samples is not recommended. Clinicians should request a prozone rule out if a patient with signs or symptoms suggestive of syphilis has a nonreactive, undiluted nontreponemal test result. 

Nontreponemal tests are not highly specific for syphilis. Biological false positive reactions (BFP) occur in about 1 in 4,000 persons in the general population and 1 in 2,000 pregnant women.  BFP are associated with medical conditions other than syphilis including malaria, leprosy, HIV, recent vaccinations, injection drug use, and autoimmune disorders. Titers are usually low and remain low or decrease with time. 

Treponemal tests are clinically used to confirm the results of reactive nontreponemal tests and to evaluate patients with signs suggestive of syphilis in early primary infection when nontreponemal tests might not yet be reactive. 

Manual treponemal tests include FTA-ABS, TPPA, Captia Syphilis IgG EIA, Trep-Sure EIA, and Zeus Scientific EIA. FTA-ABS is based on florescence microscopy and uses a fluorescein isothiocyanate-labeled antihuman immunoglobulin to detect antibody binding to whole T. pallidum that has been fixed on a glass slide. TPPA is a particle agglutination assay with T. pallidum antigens bound to gelatin particles. TPPA has replaced the MHA-TP assay. 

At least 12 automated treponemal tests have been approved by FDA. They include enzyme immunoassays, chemiluminescent immunoassays, and multiplex flow immunoassays. The reading output is typically an index value calculated as a signal to cutoff ratio (S/CO) or fluorescence ratio using values between the specimen and calibrator controls. Their main advantage is higher throughput and reduced labor costs. Automated tests are often run as the initial test in a reverse sequence screening algorithm. 

TPPA sensitivity is 86 to 100% in primary syphilis and 91 to 98% in latent syphilis. Treponemal tests become reactive earlier in primary syphilis than the RPR and remain reactive in greater than 90% of patients with latent syphilis, irrespective of treatment.  Therefore, treponemal tests cannot be used to monitor response to therapy. 

Treponemal tests can be falsely positive due to Epstein Barr virus infections, Lyme disease, and autoimmune disease. However, a reactive treponemal test in addition to a reactive nontreponemal test is highly specific for Treponema pallidum infection. 

Serologic tests have variable sensitivity at each stage of disease. The % sensitivity of each serologic test by stage of infection is:

Traditionally, laboratories have screened serum samples for syphilis using a non-treponemal test and only used a treponemal assay to confirm screen-positive samples. This algorithm was orginally recommended by CDC due in part to the fact that nontreponemal tests are relatively inexpensive, fairly easy to perform, show good correlation with disease status, and may be used to follow a patient’s response to therapy. Despite these advantages, non-treponemal tests are subjective and non-specific, and may result in false-positive results, especially in areas with a low prevalence for the disease. Furthermore, tests such as RPR require manual processing which is labor intensive.

Following the advent of automated treponemal immunoassays, many laboratories adopted a reverse sequence alogrithm. The reverse algorithm begins with an automated treponemal test. Any sample with a positive result must be followed by a quantitative nontreponemal test. If RPR is positive, a diagnosis of syphilis is likely. Samples that are positive by the screening test but negative by a nontreponemal test should then be tested by a second treponemal assay that has a different format and includes different antigens.

A positive treponemal test and negative RPR could mean that the treponemal screen was a false positive or that the patient had a past infection that was treated, or the patient could have primary or latent syphilis. Most often, the second treponemal assay is TP-PA. If it is negative, the first treponemal test can be considered a false positive. 

Each algorithm has advantages and disadvantages and both are acceptable. From a clinical perspective, the reverse algorithm is preferable because of its increased sensitivity in primary and latent disease compared to the traditional algorithm. However, up to 17% of the treponemal positive results do not confirm with the RPR and require a second treponemal assay. 

Serum, plasma, and CSF are specimen types that have been used in syphilis assays that detect antibodies against T. pallidum. Serum should be stored at 2°C–8°C (35.6°F–46.4°F) and tested within 5 days or frozen. Plasma is acceptable for certain qualitative and quantitative syphilis serologic assays. CSF should be tested within 4 hours of testing or stored refrigerated for up to 5 days. 

VDRL testing on CSF for the diagnosis of neurosyphilis is highly specific (99.8%), but has low sensitivity (50%), therefore a reactive CSF VDRL is diagnostic, but a non-reactive result does not rule out neurosyphilis. FTA-ABS can also be performed on CSF, and has 100% sensitivity, but lower specificity than VDRL (94%).

References

Papp JR, Park IU, Fakile Y, Pereira L, Pillay A, Bolan GA. CDC Laboratory Recommendations for Syphilis Testing, United States, 2024. MMWR Recomm Rep 2024;73(No. RR-1):1–32. DOI: http://dx.doi.org/10.15585/mmwr.rr7301a1

Lopes Almeida Gomes L, Stone CJ, Shaw KS. Two-Stage Syphilis Testing. JAMA. Published online June 14, 2024. doi:10.1001/jama.2024.10505

Mangione  CM, Barry  MJ, Nicholson  WK,  et al; US Preventive Services Task Force.  Screening for syphilis infection in nonpregnant adolescents and adults: US Preventive Services Task Force reaffirmation recommendation statement.   JAMA. 2022;328(12):1243-1249. doi:10.1001/jama.2022.15322

Satyaputra  F, Hendry  S, Braddick  M, Sivabalan  P, Norton  R.  The laboratory diagnosis of syphilis.   J Clin Microbiol. 2021;59(10):e0010021. doi:10.1128/JCM.00100-21