CDC Laboratory Recommendations for Syphilis …

Recommendations for Syphilis Testing in the United States

Nontreponemal (lipoidal antigen) tests typically have been used as a screening test for syphilis, as a diagnostic test when patients have signs or symptoms suggestive of syphilis or have a known sexual contact, when assessing possible reinfections, and when monitoring treatment outcome (Figure 1). RPR and VDRL tests are still the primary screening methods used in public health laboratories in the United States (56); other FDA-cleared nontreponemal (lipoidal antigen) tests (e.g., the toluidine red unheated serum test [TRUST] and unheated serum reagin test [USR]) are available but are less commonly used in the United States. Regardless of which test method is applied, serum antibody titers from RPR, VDRL, and other nontreponemal (lipoidal antigen) tests 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 (lipoidal antigen) test method and specimen type.

The manual nontreponemal (lipoidal antigen) tests are flocculation tests that detect antibody-antigen complexes that fall out of solution as a precipitate. Microscopic or macroscopic procedures have been developed to detect the precipitate that forms after specific binding of antibodies to a combination of cardiolipin, cholesterol, and phosphatidylcholine that are used as antigens in nontreponemal (lipoidal antigen) tests. VDRL tests are read microscopically at 100x magnification (51). 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. The TRUST test uses toluidine red dye in place of charcoal.

Nontreponemal (lipoidal antigen) tests are usually performed manually; however, certain RPR tests have been automated for higher throughput. The automated systems digitally analyze the density and size of antibody-antigen flocculation and store results for future retrieval (57–59). Results from any nontreponemal (lipoidal antigen) test should be reported as an endpoint titer, and not with greater or less than values, to allow for optimal clinical interpretation. Certain automated RPR tests have a constrained serum dilution range (e.g., 1:40–1:64) that might be incapable of generating an endpoint titer beyond this range. In these situations, the titer range of the automated test must be considered, and specimens should require reflex testing using a manual RPR procedure to establish an endpoint titer at either the lower or upper bounds before reporting.

Whether automated or manual, performance depends on multiple factors, including specimen type and quality, stage of syphilis, presence of autoimmune or other diseases, and presence of infections or coinfections with organisms other than T. pallidum. Nontreponemal (lipoidal antigen) tests might be less sensitive than treponemal tests in early primary syphilis and tend to wane with time regardless of treatment. Before testing, test and specimen type should be carefully considered because serum and plasma cannot always be used interchangeably, and certain nontreponemal (lipoidal antigen) tests require heat treatment of specimens.

The subjective nature of results interpretation for manual tests as well as variability among laboratories and technicians pose challenges for clinicians who compare titers with stage of syphilis for treatment purposes, especially when assessing possible reinfection or monitoring treatment outcomes. One caveat of nontreponemal (lipoidal antigen) tests is that a reactive result could be a false positive because of recent conditions (e.g., infections, vaccinations or injection drug use, or underlying autoimmune or other chronic conditions). Nonetheless, when performed by an experienced laboratory technician and used in conjunction with treponemal tests, clinical history, physical examination, and contact history, the nontreponemal (lipoidal antigen) tests are a highly reliable testing method for screening and determining the endpoint titer for subsequent serologic monitoring posttreatment.

Serologic Response After Treatment

Nontreponemal antibody titers usually decrease at least fourfold during the 12 months after syphilis treatment (Figure 1), particularly among persons treated during the early stages of infection, and might become nonreactive over time, especially among patients treated before the secondary stage of syphilis (60–62). However, in certain persons, the decrease in nontreponemal antibody titers is less than fourfold despite recommended treatment. A prospective randomized, double-blind, multisite study of therapy for early syphilis (n = 541) found that 14% of patients had a less than fourfold serologic titer decline 12 months posttreatment; patients living with HIV infection who had primary or secondary syphilis were more likely to have an inadequate response than those without HIV infection (60). In addition, titers might not serorevert to a nonreactive result after treatment and remain persistently reactive, often referred to as the serofast state. This state is most common in persons treated ≥1 year after acquiring syphilis or in persons with multiple episodes of syphilis. Titers are typically ≤1:8, but higher titers also have been observed (63,64). Additional recommendations regarding clinical interpretation of nontreponemal titers are available in CDC’s Sexually Transmitted Infections Treatment Guidelines, 2021 (55). Clinicians can consult with the STD Clinical Consultation Network for assistance with complex cases of titer interpretation (https://stdccn.org/render/Public).

Recommendation for endpoint titers. Endpoint titers (the highest dilution yielding a reactive result) should be determined and clearly reported when testing serum with nontreponemal (lipoidal antigen) assays that detect antibodies to lipoidal antigens (i.e., RPR and VDRL). Reports should not contain mathematical symbols such as or < signs (Box).

Comment and evidence summary. Antibody titers measured by nontreponemal (lipoidal antigen) tests can correlate with infection status and are the only tests available to monitor treatment outcome (60,62). A fourfold change in titer between two results with the same nontreponemal (lipoidal antigen) tests is considered clinically significant (55). Titers need to be reported for appropriate clinical management. Serum samples tested with certain automated RPR tests that are outside the dilution range of the test should be reflex tested using a manual RPR.

Prozone

The detection of antigen-antibody interactions in agglutination or flocculation assays is dependent on the formation of antigen-antibody complexes that clump cells in agglutination tests or aggregates of small particles known as floccules. Many epitopes on an antigen can be bound by an antibody specific to the antigen. Immunoglobulin G (IgG) antibodies have two binding sites and immunogloubulin M (IgM) antibodies have 10 binding sites that can bind up to 10 identical antigens, respectively. As these interactions continue, a lattice structure can develop and become sufficiently large to cause agglutination or flocculation. The level of agglutination or flocculation varies depending on the relative concentrations of antigen and specific antibodies. Agglutination and flocculation assays standardize the antigen concentrations to maximize the formation of a lattice in a reactive test. Excess antibodies in serum or antigens in the assay can interfere with the development of a lattice if each antibody molecule binds to a single (instead of two) antigen epitope (Figure 2). In this case, cross-linking fails to occur and a lattice will not form, which can occur especially in an undiluted serum specimen. This false-negative phenomenon is referred to as a prozone or hook effect because it occurs before the zone of equivalence where the concentration of antibodies and antigens are sufficient for agglutination or flocculation. A prozone can be avoided if the serum sample is diluted before testing. False-negative results attributable to a prozone have been reported for nontreponemal (lipoidal antigen) but not for agglutination-based treponemal tests (51,65).

In two studies of 4,328 and 46,856 patients who had specimens referred for syphilis testing, false-negative RPR tests caused by a prozone were rare (<0.85%) (65,66). In one study, prozone in an RPR test occurred at all stages of syphilis but was more common during primary and secondary syphilis (4.7% and 1.8%, respectively) (65). Diluting serum can remove the prozone; however, no specific dilution values can ensure all effects of a prozone are removed. In the same study, among 36 serum samples with a prozone, 11 required serial dilutions from 1:8 to 1:16 to remove the prozone; 22 of these 36 samples required dilutions ranging from 1:32 to 1:128 for the optimal concentration of antibodies and antigens for agglutination (65). Two samples continued to have a prozone until they were diluted to 1:256 and one to 1:512. Because the prozone phenomenon is considered rare in a general population screened for syphilis, routinely diluting all nonreactive, undiluted nontreponemal (lipoidal antigen) tests is not recommended. However, laboratories should rule out a prozone using a dilution series for a nontreponemal (lipoidal antigen) test when requested by a clinician. A clinician should request a prozone rule out if a patient with signs or symptoms suggestive of syphilis has a nonreactive, undiluted nontreponemal (lipoidal antigen) test result or when unusual graininess is observed in the test of undiluted serum.

Biologic False Positive

A nontreponemal (lipoidal antigen) test that is reactive for conditions other than syphilis is referred to as a biologic false positive (BFP). Persons with antibodies that are reactive in the nontreponemal (lipoidal antigen) tests, but are nonreactive in a confirmatory treponemal test, are defined as BFP reactors. Health departments frequently retain records of persons with known BFP reactions; these data can assist clinicians in a future evaluation of possible syphilis infection in such persons. Reactive nontreponemal (lipoidal antigen) tests attributable to BFP have been estimated to occur in 0.2%–0.8% of the population and are associated with medical conditions other than syphilis (67–71). BFP reactions are attributable to other infections including malaria, leprosy, and HIV; recent vaccinations; autoimmune disorders; and injection drug use (51).

Treponemal Tests

Treponemal tests are clinically used to confirm results of reactive nontreponemal (lipoidal antigen) tests and evaluate patients with signs suggestive of syphilis in early primary infection when nontreponemal (lipoidal antigen) tests might not yet be reactive. Treponemal tests can also be automated for high throughput screening in blood banks and in large laboratories for routine screening using the reverse sequence algorithm. Antibodies detected in treponemal tests typically persist for life despite treatment unless treatment occurs early in the course of infection; approximately 15%–25% of patients treated for primary syphilis can revert to a nonreactive treponemal test (FTA-ABS and MHA-TP) result within 2–3 years after treatment (61,62). In these two studies, no patients treated for secondary syphilis or stages of longer duration of infection seroreverted the reactive treponemal test. Seroreversion of treponemal tests can also occur in patients with advanced HIV disease and AIDS (72,73).

No published data are available that examined whether reversion to a nonreactive treponemal test occurs with an enzyme immunoassay (EIA) or a chemiluminescence immunoassays (CIA) after treatment for syphilis. Treponemal tests, unlike nontreponemal (lipoidal antigen) tests, cannot be used to monitor response to therapy because they remain reactive indefinitely. In patients with a history of treated syphilis and reactive treponemal test results, additional treponemal testing is not helpful for detecting reinfection and is not recommended. In this case, nontreponemal (lipoidal antigen) testing titers along with clinical history of syphilis, physical examination, and sexual risk assessment, including contact history, must be used to determine infection status.

Manual treponemal tests include FTA-ABS, TPPA, Captia Syphilis IgG EIA, Trep-Sure EIA, and Zeus Scientific EIA. Manual assays are typically used as reflex tests to confirm reactive nontreponemal (lipoidal antigen) specimens in the traditional testing algorithm. The FTA-ABS test 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 an indirect agglutination assay with T. pallidum antigens bound to gelatin particles.

The manual TPHA and MHA-TP tests are no longer available for in vitro diagnostics in the United States but are still used in certain international settings. TPHA and MHA-TP are indirect agglutination with T. pallidum antigens bound to avian or ovine erythrocytes. MHA-TP is a microplate version of TPHA.

As of December 31, 2021, a total of 12 FDA-cleared automated treponemal immunoassays were available for clinical use, including EIA, CIA, and multiplex flow (microbead) immunoassays (MFIA). In contrast to the manual assays, the treponemal immunoassays are often run as the initial test in a reverse sequence screening algorithm. All FDA-cleared treponemal tests can be performed on serum; certain tests also can be performed on plasma, including heparin, EDTA, and citrate plasma. Certain laboratories also have also validated use of treponemal tests with dried blood spots (DBS); however, no available tests have been cleared by FDA for this specimen type, nor have data been published on DBS specimens collected in the United States to aid in the diagnosis of syphilis.

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. Equivocal results should be retested according to algorithms in the package insert. The raw reading outputs and index values can be stored for future retrieval. The strength of the S/CO from immunoassays is an estimate of relative binding between molecules in the assay and has been researched as a predictor for positivity in hepatitis C and HIV confirmatory tests (74–78). When applied to treponemal immunoassays, multiple studies reported strong correlation between increasing index value strength and reactive results from an independent treponemal test or a combination of nontreponemal (lipoidal antigen) and treponemal tests, with most studies demonstrating 91%–100% correlation between S/CO cutoffs and TPPA positivity (79–84). Additional research is needed to establish test-specific cutoff values that are likely to be true positives for each of the FDA-cleared immunoassays. S/CO cutoff values could eliminate the need to adjudicate discrepant results between treponemal immunoassays and nontreponemal (lipoidal antigen) tests with a second TPPA.

For discordant nontreponemal (lipoidal antigen) and treponemal test results, an additional treponemal test is recommended using a different type of treponemal test assay and target (e.g., TPPA). Until further data are available regarding the role of S/CO cutoffs in a screening algorithm, the cutoff value could be an additional data point to assess likelihood of infection in complex situations (e.g., among pregnant persons with low risk for syphilis). Clinicians with these types of cases should contact the STD Clinical Consultation Network for assistance (https://stdccn.org/render/Public).

Blood Bank Screening

Blood donations are required to be tested for antibodies to T. pallidum as outlined in 21 CFR 610.40(a)(2). Persons that donate blood found to be serologically reactive are deferred (21 CFR 610.41[a]) and notified (21 CFR 630.40). Updated FDA recommendations for screening blood donors for syphilis are available at https://www.fda.gov/media/85283/download. The list of tests to screen blood donations for infectious agents is available at https://www.fda.gov/vaccines-blood-biologics/complete-list-donor-screening-assays-infectious-agents-and-hiv-diagnostic-assays.

Traditional and Reverse Algorithms for Syphilis Screening

The traditional algorithm for syphilis serologic screening begins with a nontreponemal (lipoidal antigen) test, and any reactive specimens are tested for confirmation by a treponemal test (Figure 3). This sequence has been widely used for decades because nontreponemal (lipoidal antigen) tests were relatively inexpensive and treponemal tests were manual, labor intensive, more costly, and limited in number. However, automated treponemal immunoassays, which were originally cleared by FDA for blood bank screening, are now cleared by FDA for clinical screening, leading to the reverse sequence algorithm. Initial screening with an automated treponemal test of a sample with a positive result must be followed by a quantitative nontreponemal (lipoidal antigen) test. When the reverse sequence algorithm is used, any discordant results should be adjudicated by a second treponemal assay (e.g., TPPA) that has a different format and includes different antigens (85).

The number of clinical laboratories performing traditional, reverse, or both algorithms was assessed among 2,360 laboratories participating in the 2015 College of American Pathologists (CAP) syphilis serology proficiency testing program in the United States (86). Of the 1,911 laboratories that responded, 81.1% (n = 1,550) offered only one algorithm, 9.5% (n = 181) offered different algorithms depending on patient demographics or clinician preference, and 9.4% (n = 180) reported being uncertain whether a single algorithm was offered. Approximately two thirds of laboratories (63.1%; n = 1,205) reported using the traditional algorithm, 15.9% (n = 304) reported using the reverse sequence algorithm, 2.5% (n = 47) reported using both algorithms, 5.9% reported that they did not know, and 3.9% reported “other.” Of responding laboratories, 8.8% (n = 169) stated that they did not reflexively perform a confirmation test. A 2017 survey by APHL reported that 58 of 73 (79.5%) public health laboratories used the traditional algorithm, and 20.5% used the reverse algorithm (https://www.aphl.org/aboutAPHL/publications/Documents/ID-2020Jan-2017-STD-Testing-Survey-Report.pdf). The CAP and APHL surveys should be updated to track changes in clinical laboratory practices over time.

A prospective comparison of 1,000 patient samples from a population with a low prevalence of syphilis tested with both algorithms found 15 (1.5%) that were reactive by the reverse sequence algorithm starting with the BioPlex IgG and four (0.4%) that were reactive by the traditional algorithm with RPR as the first test (87). The four samples that were reactive by RPR were confirmed to be positive by TPPA. The false-positive EIA rate (e.g., EIA reactive, RPR nonreactive, and TPPA nonreactive) was higher in the reverse sequence algorithm than the traditional algorithm (0.6% versus 0%). CDC reported a similar false-positive rate for treponemal immunoassay (0.6%; 866 of 140,176) when using the reverse sequence algorithm during 2006–2010 (85).

Data are conflicting regarding the cost-effectiveness of the traditional versus the reverse sequence algorithm. The traditional algorithm might be more cost-effective (lower cost per adverse event prevented) in settings with a low prevalence of syphilis (approximately 0.5%) and cost saving in higher-prevalence settings (approximately 10%) (88,89). These data are not consistent with a…