The Treponema pallidum subspecies pallidum is the etiologic agent of syphilis. Syphilis is usually transmitted sexually, but can also be passed vertically from mother to child either in utero (congenital syphilis) or perinatally during birth. Regardless of the mode of transmission, untreated cases of syphilis can result in multisystem involvement with significant morbidity. The disease is systemic and is characterized by periods of latency; thus, serologic testing is the preferred method of diagnosis. Two types of serologic tests are used: treponemal and nontreponemal assays. Traditional serologic screening for syphilis begins with a nontreponemal test followed by a treponemal test to confirm reactive results. Reverse screening algorithms have become more popular due to immunoassay automation and begin with treponemal testing followed by nontreponemal testing to confirm reactive results.
Quick Answers for Clinicians
In the United States, two types of serologic tests are most commonly used to provide a presumptive diagnosis of syphilis: treponemal and nontreponemal antibody tests. Diagnosis requires the use of both types of tests because serologic tests can be associated with false-positive results. Either test can be used as the initial screening test, followed by confirmatory testing. Microscopic darkfield examination, a complex direct detection method, is often unavailable in most laboratories. Syphilis can be detected by polymerase chain reaction (PCR), but clinical diagnostic PCR assays cleared by the U.S. Food and Drug Administration (FDA) are not yet available. Additionally, Treponema pallidum cannot be cultured in most laboratories and cannot be viewed on a Gram stain.
Testing for syphilis with serology is a two-step process. The traditional approach involves a nontreponemal screening assay followed by a confirmatory treponemal test. The reverse approach, first described by the CDC in 2008, begins with an automated treponemal test followed by a nontreponemal test. Discordant results should be followed by a different treponemal test. There are advantages and disadvantages to both approaches; refer to the Testing Algorithms section for a discussion.
The initial evaluation for congenital syphilis in infants and children should include a quantitative Venereal Disease Research Laboratory (VDRL) or rapid plasma reagin (RPR) titer; the test should be the same as that which was performed on the mother so that the titers can be compared. A serum VDRL or RPR titer that is fourfold higher than the corresponding maternal titer supports a diagnosis of proven or highly probable congenital syphilis.
Indications for Testing
Persons with signs or symptoms of syphilis infection should be tested. Additionally, asymptomatic persons at high risk for syphilis (or of transmitting the disease to others) should be screened for infection. This includes pregnant women, individuals with a sexual partner who has syphilis, sexually active men who have sex with men, and persons infected with HIV.
Two types of serologic tests are used to diagnose syphilis: treponemal and nontreponemal tests.
Nontreponemal Antibody Tests
Nontreponemal tests detect antibodies formed in response to cellular damage as a result of infection. These tests are semiquantitative, simple, inexpensive, and often used for screening. However, these tests are not specific for syphilis and may produce false-positive results indicative of other infections (eg, HIV, autoimmune conditions); therefore, positive results should be reflexed to a treponemal assay for confirmation.
Nontreponemal tests include rapid plasma reagin (RPR) and Venereal Disease Research Laboratory (VDRL) tests. In addition to screening for syphilis, RPR tests are also useful for monitoring treatment (see Monitoring section). The VDRL may be performed on serum and cerebrospinal fluid (CSF), the necessary specimen for diagnosing neurosyphilis.
Treponemal Antibody Tests
Treponemal tests detect antibodies that specifically target T. pallidum; other conditions are unlikely to cause a positive result. These tests are qualitative and are reported as reactive or nonreactive. Treponemal antibodies often appear earlier than nontreponemal antibodies and usually remain detectable for life, even after treatment. Therefore, a positive treponemal screening result must be followed by a nontreponemal test to discriminate between an active and past infection.
Treponemal tests include fluorescent treponemal antibody absorption (FTA-ABS), T. pallidum particle agglutination assay (TP-PA), and immunoassays (eg, enzyme immunoassays [EIAs], chemiluminescence immunoassays [CIAs]). TP-PA is the CDC’s recommended test to confirm a positive nontreponemal screen; the TP-PA is more specific and produces fewer false-positive results than the FTA-ABS test. Immunoassays are routinely automated now, which makes them the preferred choice for screening purposes.
Testing for syphilis with serology is a two-step process. The traditional or classic approach involves a nontreponemal screening assay followed by a confirmatory treponemal test. This strategy is rapid and inexpensive and able to detect acute syphilis, but false-negative results are possible, especially in early and late syphilis. False-positive results may also occur because nontreponemal tests are not specific for T. pallidum infection and may signal another infection.
The reverse syphilis screening approach begins with an automated treponemal test (eg, EIA or CIA) followed by a nontreponemal test. Discordant results (ie, a reactive treponemal test and a negative nontreponemal test) should be followed by another treponemal test. This approach has gained popularity for multiple reasons: Cost savings are possible due to automation, and early and late syphilis are more likely to be detected than when the traditional algorithm is followed. However, in low-risk/low-prevalence populations, frequent false-positive test results (an initial reactive treponemal test with a negative confirmatory nontreponemal test) require confirmation with a second treponemal test. This reverse approach also cannot differentiate between active and previously treated infections.
Direct detection methods, such as darkfield microscopy, are not often used because they are labor intensive, require the use of specialized and expensive equipment, and may miss the infection if it is not present in the specimen.
Congenital and Perinatal Syphilis
Congenital syphilis is contracted from an infected mother via transplacental transmission of T. pallidum, which can occur at any time during pregnancy; syphilis can also be acquired at birth from contact with maternal lesions. Intrauterine syphilis infection can result in stillbirth, hydrops fetalis, or preterm birth, or infected infants may be asymptomatic at birth. Diagnosing congenital syphilis can be challenging because maternal nontreponemal and treponemal immunoglobulin G (IgG) antibodies may be passed to the fetus through the placenta. All neonates born to mothers who have reactive nontreponemal and treponemal test results should be evaluated with a quantitative nontreponemal serologic test (RPR or VDRL) on neonatal serum. The nontreponemal test that is performed on the infant should be the same as that which was performed on the mother so that the infant’s titers can be compared with the mother’s titers (treponemal testing on neonatal serum is not recommended because of the difficulty it presents for interpretation).
An infant with a serum VDRL or RPR titer that is fourfold higher than the corresponding maternal titer is considered to have proven or highly probable congenital syphilis. Note that the absence of a fourfold or greater titer for the neonate does not exclude congenital syphilis, as an abnormal physical examination that is consistent with congenital syphilis or a positive darkfield test/PCR of lesions or body fluids also supports the criteria for proven or highly probable congenital syphilis.
T. pallidum can infect the central nervous system and result in neurosyphilis. No single laboratory test can diagnose neurosyphilis. The diagnosis depends on a combination of CSF studies in the presence of reactive serologic test results and neurologic signs and symptoms. In a person with neurologic signs or symptoms, a reactive CSF VDRL is considered diagnostic. The FTA-ABS CSF test is less specific for neurosyphilis than the VDRL.
Nonpregnant Adults and Adolescents
The U.S. Preventive Services Task Force (USPSTF) recommends screening for syphilis infection in persons who are at increased risk for infection. Men who have sex with men or persons living with HIV may benefit with more frequent screening (every 3 months) but should be screened at least annually.
Individuals infected with HIV should be screened at an initial visit and then annually, if sexually active. More frequent screening (every 3-6 months) is recommended for those with multiple sex partners and for those who engage in sex and/or illicit drug use.
Most major national organizations, including the CDC, recommend screening all pregnant women for syphilis at the first prenatal visit. Additionally, the CDC, the American Academy of Pediatrics (AAP), and the American College of Obstetricians and Gynecologists (ACOG) endorse repeat screening for women at high risk for syphilis (rescreen early in third trimester and again at delivery).
Titers from nontreponemal tests (RPR or VDRL) generally correlate with the amount of infection present and should be used to monitor treatment success. The frequency of monitoring depends on the stage of disease and presence of HIV coinfection. In patients with early syphilis, monitor 6-12 months after treatment and any time symptoms reoccur. In late syphilis, follow up at 6, 12, and 24 months. In patients with HIV, monitor more frequently.
ARUP Laboratory Tests
CDC-recommended test for the screening and diagnosis of syphilis; follows traditional screening algorithm
Semi-Quantitative Charcoal Agglutination/Semi-Quantitative Particle Agglutination
CDC-recommended screening test for syphilis; begins traditional syphilis screening algorithm
May use as confirmatory test for reverse algorithm
Preferred monitoring test for established syphilis
Semi-Quantitative Charcoal Agglutination
Acceptable initial screen in traditional syphilis screening algorithm (nontreponemal testing)
May be useful as a confirmatory test for reverse algorithm
Acceptable monitoring test for established syphilis
Recommended initial screen in reverse syphilis screening algorithm (treponemal testing)
CDC-recommended confirmatory test for syphilis
Useful to follow up reactive treponemal screening test when reverse algorithm is used
Semi-Quantitative Charcoal Agglutination/Semi-Quantitative Particle Agglutination
Preferred diagnostic assay for CSF specimens in suspected neurosyphilis
May aid in the diagnosis of neurosyphilis
For CSF specimens, VDRL is preferred
Semi-Quantitative Indirect Fluorescent Antibody
Not a preferred confirmatory test
Refer to TP-PA
Semi-Quantitative Indirect Fluorescent Antibody
Not an optimal reflex test
Refer to RPR with reflex to titer and TP-PA confirmation
Soreng K, Levy R, Fakile Y. Serologic Testing for Syphilis: Benefits and Challenges of a Reverse Algorithm. Clin Microbiol Newsl. 2014; 36 (24): 195-202.PubMed
American Academy of Pediatrics. Syphilis. In Kimberlin DW, B. M., Jackson MA, Long SS. Red Book: 2018 Report of the Committee on Infectious Diseases. Syphilis, 31st ed. American Academy of Pediatrics, 2018.Book
U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. 2015 sexually transmitted diseases treatment guidelines: Congenital syphilis. CDC. [Updated: Jun 2015; Accessed: Dec 2019]Online
Miller M, Binnicker MJ, Campbell S, et al. A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2018 Update by the Infectious Diseases Society of America and the American Society for Microbiology. Clin Infect Dis. 2018; 67 (6): 813-816.PubMed
Workowski KA, Bolan GA, Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines, 2015. MMWR Recomm Rep. 2015; 64 (RR-03): 1-137.PubMed
USPSTF - Final recommendation statement: Syphilis infection in nonpregnant adults and adolescents. ScreeningU.S. Preventive Services Task Force. Final recommendation statement: Syphilis infection in nonpregnant adults and adolescents. Screening. [Released: Jun 2016; Accessed: Dec 2019]Online
Dunseth CD, Ford BA, Krasowski MD. Traditional versus reverse syphilis algorithms: A comparison at a large academic medical center. Pract Lab Med. 2017; 8 52-59.PubMed
U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Syphilis - CDC Fact Sheet (Detailed). [Last reviewed: Jan 2017; Accessed: Dec 2019]Online
Binder SR, Theel ES. Syphilis testing algorithms: A review. World J Immunol. [Accessed: Dec 2019]Online
Rhoads DD, Genzen JR, Bashleben CP, et al. Prevalence of Traditional and Reverse-Algorithm Syphilis Screening in Laboratory Practice: A Survey of Participants in the College of American Pathologists Syphilis Serology Proficiency Testing Program. Arch Pathol Lab Med. 2017; 141 (1): 93-97.PubMed
Kaplan JE, Benson C, Holmes KK, et al. Guidelines for prevention and treatment of opportunistic infections in HIV-infected adults and adolescents: recommendations from CDC, the National Institutes of Health, and the HIV Medicine Association of the Infectious Diseases Society of America. MMWR Recomm Rep. 2009; 58 (RR-4): 1-207; quiz CE1-4.PubMed