Screening and algorithmic diagnosis

Screening for syphilis involves serologic tests that can be combined with reflex testing according to guideline-recommended diagnostic algorithms, as described below.

Serologic tests

Serologic tests are used most often to indirectly screen for and diagnose syphilis in lieu of directly detecting T pallidum9,10 and are an important tool when no lesions are present. Serologic testing for syphilis includes treponemal and nontreponemal serology. Treponemal serologic testing detects antibodies specific to T pallidum, whereas nontreponemal serologic testing detects antibodies that are broadly reactive to phospholipid antigens shared by both the host and T pallidum. Diagnosis of syphilis requires both types of serologic tests (Figures 1 and 2).8,10,11

Figure 1. Traditional Algorithm for Serologic Diagnosis of Syphilis
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Figure 2. Reverse Algorithm for Serologic Diagnosis of Syphilis
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Treponemal tests

Treponemal tests detect antibodies that target T pallidum. These antibodies are detectable within 2 to 6 weeks of infection and typically remain reactive for life regardless of treatment.12 However, 15% to 25% of patients treated for primary syphilis serorevert and become nonreactive to treponemal tests after 2 to 3 years.8,10

The T pallidum particle agglutination assay (TP-PA) (test code 653) is a traditional treponemal test that detects agglutination, or aggregation, of particles carrying antigens from lysed T pallidum. Aggregation only occurs when reactive antibodies are present in a specimen.12,13 Test results are qualitative and depend on subjective interpretation.

More recently, automated treponemal immunoassays (IAs) (test code 13646) detect antibodies in the specimen that react to recombinant T pallidum antigens. These automated IAs have higher throughput than traditional treponemal tests and objectively quantify reactivity.12 Sensitivity of treponemal IAs for all stages of syphilis ranges from 95% to 100%.14

A nonreactive treponemal test result indicates a lack of detectable T pallidum antibodies and is consistent with a negative syphilis result. However, treponemal tests may be nonreactive early during infection, before treponemal antibodies are present or detectable. Treponemal antibody titers do not predict treatment response and therefore should not be used for this purpose.8,10

A reactive treponemal test result is consistent with any stage of syphilis as well as syphilis that has been treated in the past. False-positive results can be caused by cross-reactive antibodies that target antigens from other Treponema species (eg, Treponema carateum), nonsyphilis-causing T pallidum subspecies that cause bejel and yaws, or spirochetes (eg, Borrelia).9,12

Nontreponemal tests

Nontreponemal tests detect antibodies that target a combination of lipoidal antigens (cardiolipin, phosphatidylcholine, and cholesterol) found in both T pallidum and host cell membranes.10 Due to these antigen characteristics, CDC updated their terminology to refer to nontreponemal tests as nontreponemal (lipoidal antigen) tests.10 However, this Clinical Focus will continue to use nontreponemal tests until the new terminology is extensively adopted.

Nontreponemal antibodies are made in response to T pallidum infection and are typically made after treponemal antibodies, appearing within 6 to 8 weeks of infection.12 Unlike treponemal antibodies, nontreponemal antibody titers reflect disease activity. Patients typically have decreased titers or complete seroreversion upon syphilis treatment. Even if syphilis is not treated, titers can decrease over time as the disease progresses into the late stage.2,12

The Venereal Disease Research Laboratory (VDRL) and rapid plasma reagin (RPR) tests detect nontreponemal antibodies by aggregation of antigen particles when these antibodies are present in a specimen. The VDRL and RPR tests have comparable performance that varies by disease stage (eg, 62%-78% sensitivity for primary syphilis, 97%-100% sensitivity for secondary syphilis).15

A nonreactive nontreponemal test result is consistent with no active syphilis. However, patients with early primary syphilis or late syphilis may be nonreactive to nontreponemal tests owing to the natural history of nontreponemal antibodies. High antibody titers can cause false-negative results because of the prozone reaction, which occurs when antibodies saturate antigen particles in the assay and prevent aggregation.12

A reactive nontreponemal test result is consistent with active syphilis, although it is not sufficient for diagnosis. Up to 2% of people have false-positive nontreponemal test results caused by a wide range of biological conditions (eg, autoimmune disease, cancer, infectious diseases, old age, and pregnancy).9,12

Diagnostic algorithms

The CDC recommends using both a treponemal test and a nontreponemal test to screen for and diagnose syphilis, using either the traditional or reverse algorithm.8,10 Treponemal tests are specific for T pallidum but can indicate present or past infections, both treated and untreated. On the other hand, nontreponemal tests indicate active disease but are not specific to T pallidum. Quest offers diagnostic panels that align with the traditional algorithm (test code 36126) and the reverse algorithm (test code 90349). Clinical evaluation and patient history can help determine the age of infection, stage of syphilis disease, and treatment history, which are important for interpreting the results of either algorithm.

Traditional algorithm

The traditional algorithm for syphilis diagnosis (Figure 1) begins with a nontreponemal test. A reactive result reflexes to a treponemal test to confirm the reactive nontreponemal result.

Using the traditional algorithm, a nonreactive nontreponemal test result is consistent with no active syphilis. Reactive results from both the nontreponemal and treponemal tests are consistent with current or past syphilis.

Discordant results (eg, reactive RPR with nonreactive IA) indicate that syphilis is unlikely and possibly represent a false-positive nontreponemal test. For individuals with nonreactive or discordant results who have suspected recent exposure to syphilis, repeat testing on a new specimen obtained after 2 to 4 weeks is recommended.11

Reverse algorithm

The reverse algorithm for syphilis diagnosis (Figure 2) is becoming more widely adopted as it allows for the utilization of automated treponemal IAs.9 This algorithm begins with a treponemal test, often an automated IA. A reactive result will reflex to a nontreponemal test (RPR). Specimens with discordant results are subjected to a second treponemal test (TP-PA).

In the reverse algorithm, a nonreactive treponemal test result is consistent with no current or past syphilis. Reactive results from both the treponemal and nontreponemal tests are consistent with current or past syphilis, and a clinical evaluation should be performed to identify past infection or current signs and symptoms of disease.

Discordant results from the treponemal and nontreponemal tests with a reactive second treponemal test result (eg, reactive IA, nonreactive RPR, reactive TP-PA) are consistent with current (potential early) syphilis or past syphilis. Discordant results with a nonreactive second treponemal test result (eg, reactive IA, nonreactive RPR, nonreactive TP-PA) are inconclusive for syphilis and may be caused by an early infection or a false-positive result from the initial treponemal test.11 Consistent with the traditional algorithm, repeat testing after 2 to 4 weeks is recommended for individuals with nonreactive or discordant results who have suspected recent exposure.11

Comparison of traditional and reverse algorithms

The CDC lists both algorithms in guidelines, and both are acceptable for the screening and diagnosis of syphilis.8,10,16 Each algorithm has important advantages and limitations. The preferred algorithm should be based on resources in the laboratory to perform the testing, the volume of testing, and the patient population.10,17

The reverse algorithm detects more T pallidum infections than the traditional algorithm because some stages of syphilis (eg, primary and late) can present with reactive treponemal but nonreactive nontreponemal serology.17,18 Nontreponemal antibody tests may be nonreactive in patients with primary syphilis who are tested shortly after infection, before nontreponemal antibodies are made. Nontreponemal antibody titers can also decrease to nonreactive levels in patients with untreated or unsuccessfully treated late syphilis. The traditional algorithm stops after a nonreactive nontreponemal test, so it misses specimens that would be reactive to a subsequent treponemal test.

One caveat of the reverse algorithm is that it also detects past treated syphilis. Thus, patient history is important for interpreting nonreactive nontreponemal results. In some high-prevalence settings, the reverse algorithm may be less useful owing to higher proportions of patients with past treated syphilis, among other factors.19-21

The reverse algorithm may produce more false-positive results at the initial step than the traditional algorithm.9,22 Therefore, the second treponemal test is important to confirm reactive treponemal results in patients with nonreactive nontreponemal serology. The false-positive rate of the initial treponemal test may be related to the syphilis prevalence within a population. One CDC study on the performance of the reverse algorithm reported nearly 3 times as many initial false positives among low-prevalence populations as among high-prevalence populations.23

Guidelines for syphilis screening

Both the CDC and the United States Preventive Services Task Force (USPSTF) recommend routine syphilis screening for men who have sex with men (MSM), individuals with HIV, and other individuals at increased risk for syphilis (Table 3).8,24 Syphilis screening is also recommended for individuals being considered for or receiving HIV PrEP because syphilis is associated with higher risk of HIV acquisition.25 MSM are highly impacted by syphilis, accounting for 45% of male primary and secondary syphilis cases in 2022.1 The prevalence of syphilis is also increased in certain geographic regions and among certain races and ethnicities.8,24 For most at-risk populations, screening is recommended at least annually, with more frequent screening (eg, every 3-6 months) considered based on individual risk and local prevalence.8,24

Table 3. Individuals Recommended for Screening Due to Increased Risk for Syphilis [return to contents]

The CDC, USPSTF, and American College of Obstetricians and Gynecologists (ACOG) recommend screening pregnant individuals for syphilis at the first prenatal visit because of the serious potential complications of syphilis during pregnancy and the risk of congenital syphilis.8,26,27 Recent guidance from ACOG further recommends universal screening of pregnant individuals again during the third trimester and at delivery.27 This guidance differs from previous recommendations that indicated rescreening only for those at high risk for syphilis.8,26 Screening enables timely treatment, which reduces the rate of adverse pregnancy outcomes from about 77% to 24% among pregnant individuals with syphilis.28

Either the traditional (test code 36126) or reverse algorithm (test code 90349) can be used to screen for syphilis.24,26 Quest offers test panels for obstetric care (test codes 20210, 93802, and 12075) that include the traditional algorithm for syphilis screening (Appendix). Quest also offers HIV PrEP panels for baseline testing and monitoring that include syphilis screening. Details can be found in HIV Pre-Exposure Prophylaxis (PrEP): Laboratory Testing | Test Guide | Quest Diagnostics.

Other diagnostic testing

In addition to the reflexive algorithmic approach, diagnosis of syphilis can involve serology-based diagnostics for neurosyphilis and congenital syphilis or direct detection of T pallidum DNA in lesions or cerebrospinal fluid (CSF), as described below.

Neurosyphilis

Neurosyphilis occurs when T pallidum invades the CNS. Diagnosis of neurosyphilis is based on reactive nontreponemal and treponemal serologic tests, clinical signs or symptoms that are consistent with neurosyphilis, and a combination of CSF tests (including cell count, protein, and/or reactive CSF-VDRL test).8 The CDC recommends testing for neurosyphilis in syphilis patients with neurologic signs and symptoms (eg, meningitis, stroke, change in mental status, loss of vibration sense), those with tertiary syphilis, and those with suspected ocular syphilis who have cranial nerve dysfunction.8

CSF VDRL (test code 4128) is about 99% specific, but only 77% sensitive for neurosyphilis.29 Thus, a reactive result is consistent with neurosyphilis, but a nonreactive result does not rule out neurosyphilis.8 In contrast, CSF FTA-ABS (test code 17088) is sensitive but not specific for neurosyphilis (about 95% and 62%, respectively).8,29 Thus, a nonreactive result is useful for ruling out the condition, especially for individuals with nonspecific neurologic signs and symptoms.8,10 T pallidum can also be directly detected from CSF by PCR (test code 16595), but CDC guidelines indicate that insufficient evidence is available to recommend this testing approach.10

Congenital syphilis

Among newborns (<30 days old) and infants, the interpretation of serologic tests can be difficult because parental IgG antibodies cross the placenta and can be detected in the infant’s serum. Therefore, diagnosis of congenital syphilis in newborns relies on a combination of a syphilis diagnosis in the pregnant individual, the pregnant individual’s treatment history, evidence of syphilis in the newborn, and the comparison of nontreponemal antibody titers from the newborn and the pregnant individual (at delivery).8

Newborns born from individuals who had syphilis during pregnancy (ie, reactive nontreponemal and treponemal tests) should be tested for congenital syphilis using a nontreponemal test (RPR [test code 799] or VDRL [test code 30509]).8,10 Those born from individuals with reactive nontreponemal tests at delivery should be examined for additional evidence of congenital syphilis, including a physical examination and PCR analysis of specimens from lesions or body fluids.8

A newborn’s reactive nontreponemal test indicates confirmed proven/highly probable congenital syphilis if the newborn’s titer is at least 2 dilutions higher than the pregnant individual’s titer at delivery.8 Below this threshold, other factors such as the physical examination results and the pregnant individual’s treatment history must be considered to determine the likelihood of congenital syphilis (classified as confirmed proven/highly probable, possible, less likely, or unlikely).8 For newborns with confirmed proven/highly probable or possible congenital syphilis, a CSF examination, including a VDRL test (test code 4128), is recommended for neurosyphilis.8

Diagnosis of congenital syphilis in infants (≥1 month old) and children can be done using the traditional or reverse algorithm in conjunction with physical examination and review of the birthing parent’s health records to distinguish between congenital and acquired syphilis.8 Testing for treponemal IgM antibodies (test code 34323) can also help diagnose congenital syphilis in infants, but is not recommended for newborns <30 days old.8 Infants or children at risk for congenital syphilis should also receive a CSF evaluation, including a VDRL test (test code 4128).8

Direct molecular detection

According to CDC guidelines, laboratory-developed nucleic acid amplification tests (NAATs) can be used on specimens collected from suspected primary or possible secondary syphilis lesions in seronegative patients, if performed in CLIA-compliant laboratories.10 Direct molecular detection using a NAAT (test code 16595) might provide a more timely diagnosis of primary syphilis than serologic testing.10 This testing can also be used for the differential diagnosis of T pallidum and herpes simplex virus infections as a component of the genital lesion panel (test code 38286).

Additional testing after diagnosis

Additional testing is indicated for syphilis patients and their sex partners following diagnosis. Patients with syphilis are often co-infected with HIV at the time of diagnosis, and syphilis increases risk of HIV acquisition.2,30 Thus, syphilis patients should be tested for HIV and other sexually transmitted infections, as indicated.8

Sex partners of syphilis patients are at risk for T pallidum infection. Because lesions typically occur during the first year of infection, the CDC recommends clinical and serological evaluation of sex partners for patients diagnosed with primary, secondary, or early nonprimary nonsecondary syphilis.8 Testing criteria are based on the time since sexual contact and the disease stage of the patient (Table 4). Serologic testing can guide the treatment of sex partners with sexual contact >90 days before the diagnosis, whereas presumptive treatment is recommended for those with sexual contact <90 days before the diagnosis.8

Table 4. Criteria for Testing Sex Partners of Syphilis Patients [return to contents]

Monitoring

Nontreponemal antibody titers typically decrease upon syphilis treatment, so nontreponemal tests can be used to monitor a patient’s response to treatment (Table 5). Each monitoring test should use the same assay (either RPR or VDRL) and be performed by the same laboratory so that results are comparable over time.8,10 Quest offers both RPR (test code 799) and VDRL (test code 30509) nontreponemal testing that can be used to monitor treatment response.

Table 5. Frequency of Follow-Up Nontreponemal Tests to Monitor Response to Syphilis Treatment [return to contents]

Treatment response is generally defined as a reduction in nontreponemal titer by at least 2 dilutions within the monitoring period.8,10 However, titers may decrease by fewer than 2 dilutions (ie, inadequate serologic response), including in about 10% to 20% of primary and secondary syphilis patients.8 Inadequate serologic response is more likely to occur in patients who are older, have low baseline titers, or have later stages of disease.8 These patients are recommended to receive additional clinical and serologic evaluations, including reevaluation for HIV infection.8

Treatment failure can also cause nontreponemal titers to decrease by fewer than 2 dilutions, or even to increase.8 One cause of treatment failure is neurosyphilis; CNS infection requires a different treatment regimen and can act as a reservoir for T pallidum. When treatment failure is suspected, the CDC recommends a CSF examination to test for neurosyphilis.8 Increasing nontreponemal titers after treatment can also be caused by reinfection, which serologic tests cannot distinguish from treatment failure.

Appendix [return to contents]

Screening Panels Including Syphilis Serologic Testing [return to contents]