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Year : 2019  |  Volume : 40  |  Issue : 2  |  Page : 186-191

Serological tests for syphilis

1 Department of Skin and VD, Medical College Baroda, SSG Hospital, Vadodara, Gujarat, India
2 SBKS Medical Institute and Research Centre, Vadodara, Gujarat, India

Date of Web Publication26-Nov-2019

Correspondence Address:
Dr. Yogesh S Marfatia
2, Nandvan Society, Yogidhara Flats, Alkapuri, Vadodara, Gujarat
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijstd.IJSTD_86_19

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How to cite this article:
Shah D, Marfatia YS. Serological tests for syphilis. Indian J Sex Transm Dis 2019;40:186-91

How to cite this URL:
Shah D, Marfatia YS. Serological tests for syphilis. Indian J Sex Transm Dis [serial online] 2019 [cited 2022 Nov 29];40:186-91. Available from:

   Introduction Top

  • T. pallidum subsp. pallidum is the causative organism for syphilis. Three morphologically identical subspecies of T. pallidum have been classified as follows:

    • T. pallidum subsp. pallidum (syphilis)
    • T. pallidum subsp. pertenue (yaws), and
    • T. pallidum subsp. endemicum (bejel).

  • The immune response pattern in syphilis and the nonvenereal treponematoses is similar. This complicates the interpretation of reactive serologic tests for syphilis (both nontreponemal, e.g., Venereal Disease Research Laboratory [VDRL] and treponemal tests, e.g., T. pallidum hemagglutination assay [TPHA]) in patients who have lived in areas where endemic treponematoses are still prevalent
  • Syphilis has diverse clinical manifestations and shares many clinical features with other treponemal and nontreponemal diseases. Therefore, it is mandatory that the clinical diagnosis is always supported by appropriate laboratory tests and that the test results are interpreted with reference to the patient's history and physical examination findings.

   History Top

Wassermann test or Wassermann reaction

  • It was the First blood test for syphilis and the first test in the nontreponemal tests (NTT) category developed by Wassermann, Julius Citron, and Albert Neisser in 1906
  • It is based on Complement fixation principle in which a sample of blood or CSF was taken and introduced to the antigen which was cardiolipin extracted from bovine muscle or heart. Treponemal nonspecific antibodies react with the lipid – the Wassermann reaction (WR) of antiphospholipid antibodies (APAs)
  • The intensity of the reaction (classed 1, 2, 3, or 4) indicates the severity of the condition
  • Newer NTTs, such as the rapid plasma reagin (RPR) and VDRL tests, have mostly replaced it.

   Investigations for Syphilis Top

Although Treponema pallidum cannot be grown in culture, there are many tests for the direct and indirect diagnosis of syphilis [Table 1].[1]
Table 1: Diagnostic tests for syphilis

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   Nontreponemal Tests Top

Venereal Disease Research Laboratory test

  • Mechanism: This detects both immunoglobulin (Ig) G and IgM APA (Ab against cardiolipin) formed by the host in response to lipoidal material released by damaged host cells early in infection and lipid from the cell surfaces of the treponeme itself
  • Seroconversion occurs from 21 days of exposure till about up to 6 weeks after infection.

Variants of Venereal Disease Research Laboratory test

  • RPR test: Results are available within 5 min

  • [INLINE:1]

    Conventional RPR card test[2] Automated RPR test

    Uses cardiolipin Ag with carbon Immunoassay technique using latex

    Particle to detect regain Ab Particles coated with lecithin and cardiolipin

  • Toludine red unheated serum test
  • Unheated serum reagin test
  • Reagin screen test
  • Automated reagin test.


  • Qualitative test for screening syphilis
  • Results are reported as nonreactive, reactive, and weakly reactive
  • In reactive NTT:

    1. A fourfold increase in titer indicates infection, reinfection, or treatment failure
    2. A fourfold decrease in titer indicates effective therapy.

    • Quantitative VDRL: The VDRL test can be quantitated by examining serial dilutions of serum and can be used to follow the course of illness, including the response to therapy
    • Patients with early syphilis who have been treated with appropriate doses and preparations of benzathine penicillin should be evaluated clinically and serologically, using a NTT, after 3 months to assess the results of therapy
    • A second evaluation should be performed after 6 months and, if indicated by the results at this point, again after 12 months to reassess the condition of the patient and detect possible reinfection.[3]

  • When a nontreponemal serologic test shows persistent reactivity with no signs of decline of titer at 6 months after adequate therapy or if it fails to show a fourfold decrease of initial high titer within 1 year, it is considered a seroresistance or serofast state.


  1. Inexpensive and simple
  2. Suitable for mass screening
  3. The baseline titer can be used to follow-up the treatment response.


  1. Reduced sensitivity in primary syphilis and late latent syphilis
  2. False-positive results due to technical error and variations in normal population, due to antiphospholipid autoantibodies (biologic false positivity) [Table 2]
  3. False-negative results: In VDRL test, the optimal ratio of the antigen antibody yields an insoluble precipitate that is visible, thus rendering the test positive. The zone of equivalence defines this optimal ratio
Table 2: Causes of biologic false-positive serological test

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  • However, undiluted serum specimens have a high quantity of reagin antibody which will give false-negative reaction. This is known as PROZONE phenomenon.[4] Further, on serial dilutions, the test becomes positive.

  • It is clinically significant in certain populations at risk like:

    • Those who are on continuous immunosuppressive drugs
    • Those who are HIV seropositive
    • Antigen excess can also result in false-negative result which is known as POSTZONE phenomenon [Figure 1].
Figure 1: Pro and post zone phenomenon

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Thus, confirmation by a treponemal test is required.

   Treponemal Tests Top

Treponema pallidum immobilization test

  • This test uses the virulent T. pallidum (Nichol's strain) obtained from rabbits
  • It detects antibody which inhibits the normal movements of T. pallidum in the presence of complement (specific treponemal immobilizing antibody). The reaction of treponemes in the presence of patient's serum is observed by dark field microscopy
  • The test is positive if 50% or more of the treponemes are immobilized
  • It becomes positive few days to a week later than the VDRL test
  • Specificity is 100%, but as the test is time consuming, expensive, and hazardous, it is not performed nowadays.

Currently used treponemal tests are as follows:

Treponema pallidum hemagglutination assay

  • Microhemagglutination assay for IgM and IgG antibodies in which sensitized sheep erythrocytes are coated with T. pallidum (Nichol's strain)
  • Results are reported as reactive if agglutination occurs in a dilution of 1:80 or more
  • Reactivity can be expected around the 4th to 5th week of infection
  • It is more sensitive and specific than VDRL and fluorescent treponemal antibody absorption (FTA-Abs) test except in primary syphilis.

Fluorescent treponemal antibody absorption test

  • Indirect Immunofluorescence antibody test
  • Presence of antibody in patient's serum is indicated by fluorescence. Intensity of fluorescence is reported as nonreactive, borderline, or reactive
  • Reactivity begins in the 3rd week of infection
  • It is the most sensitive serological test in the early stage of syphilis.

Fluorescent treponemal antibody absorption double-staining test

  • A fluorochrome-labeled counterstain for T. pallidum and antihuman IgG conjugate labeled with tetramethylrhodamine isothiocyanate to detect antibody in patient's serum are employed in this test
  • False positivity can occur in 1% of sera.[5]

Treponemal enzyme immunoassay

  • In this test, antigen is fixed to wells in microtiter plates, and then serum is added and rinsed off after 30–60 min
  • Antibody to T. pallidum binds to antigen and reacts in the 2nd incubation with an enzyme labeled antihuman globulin
  • Result is available in 3–4 h.


  1. Automated (or semi-automated) processing[6]
  2. Objective reading of results
  3. Interfacing with the laboratory computer system to allow electronic report generation

    • New algorithm suggests screening with treponemal enzyme immunoassay alone followed by a NTT.

Western blot technique

  • Based on the whole T. pallidum lysate antigen
  • The presence of antibodies to the immunodeterminants with molecular weights 15.5, 17, 44.5, and 47 kDa appears to be diagnostic for acquired syphilis
  • When an IgM-specific conjugate is used, the test has value in the diagnosis of congenital syphilis.


  1. Treponemal tests may remain reactive for years with or without treatment, and treponemal test antibody titers correlate poorly with disease activity

  2. Therefore, treponemal tests should not be used to evaluate response to therapy, relapse, or reinfection in previously treated patients
  3. Treponemal tests do not differentiate venereal syphilis from endemic syphilis (yaws and pinta).

Sensitivity and specificity of commonly used serologic tests

  • Sensitivity: It is defined as the proportion of people who test positive for the disease among those who have the disease[7]
  • Specificity: It is defined as the proportion of healthy patients known not to have the disease who will test negative for it [Table 3], [Table 4] and [Figure 2].
Figure 2: Sensitivity and specificity of various serological tests

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Table 3: Sensitivity and specificity of various serological tests

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Table 4: Window period of various serological tests

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Other tests

Oral fluid test for syphilis

  • Time-resolved fluorescence immunoassay to detect antibodies to T. pallidum recombinant antigens in the oral fluid specimens collected using “Oracol” swab
  • In early syphilis – Sensitivity: 100%

  • Specificity: 97.9%.

  • Patients with positive syphilis serology – Sensitivity: 76.5%

  • Specificity: 96.9%.

  • Potentially useful when collection of blood is not practicable.

   Cerebrospinal Fluid Examination in Syphilis Top


  1. Neurological, ophthalmic, or auditory symptoms and signs
  2. Other clinical evidence of active infection – aortitis, gumma, or iritis
  3. Treatment failure
  4. HIV infection
  5. A nontreponemal serum titer of >32 if the duration of syphilis is over 1 year
  6. A nonpenicillin-based treatment regimen is planned
  7. All infants suspected of prenatal syphilis.

    • The typical CSF findings of neurosyphilis are:

      1. Moderate mononuclear pleiocytosis (10–400 cells/mL)
      2. Elevated total protein (0.46–2.0 g/L)
      3. Positive CSF VDRL criteria.

    • The CSF VDRL test is highly specific, and false-positive results are rare in the absence of blood contamination
    • Most venerologists consider an examination of CSF unnecessary in early syphilis and prefer to examine CSF after 1–2 years of posttreatment follow-up
    • In untreated asymptomatic late syphilis, a CSF examination should always be done
    • A normal CSF is by definition an essential prerequisite for a diagnosis of latent syphilis.

   Testing Policy Top

  • NTTs such as RPR test detect almost all cases of early syphilis, but false positives are possible
  • Treponemal tests, such as TPHA and FTA-Ab, are used to confirm NTT results[8]
  • Quantitative RPR titers can help evaluate the response to treatment [Table 5]
  • When additional tests are not available, all clients with reactive RPR should be treated
  • The Centers for Disease Control and Prevention (CDC) has proposed an algorithm for evaluating syphilis. Patients who have a reactive treponemal test on two different tests with a negative NTT are candidates for treatment if they have not been treated in the past [Figure 3].
Figure 3: Algorithm for evaluation of syphilis

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   Guidelines for Screening of Syphilis in Pregnancy and Newborns Top

  • The WHO STI guideline recommends screening all pregnant women for syphilis during the first antenatal care (ANC) visit and to be repeated early in the 3rd trimester[9]
  • In areas with a high prevalence of syphilis, the CDC has recommended re-screening in the 3rd trimester and again at the time of delivery to detect new infections during pregnancy
  • For all syphilis-positive women detected during ANC, their newborns should be tested by RPR using infant serum and not cord blood as it can yield false-positive result
  • All newborns showing fourfold rise in titer compared to that of their mothers' titer need to be hospitalized to initiate penicillin treatment for 10 days.

Serological tests in HIV

  • Syphilis is common among patients with HIV infection and vice versa. The course of clinical disease and serologic markers may be altered or more aggressive in co-infected patients
  • Serologic tests for syphilis are still the cornerstone of diagnosing untreated syphilis infection, independent of the HIV status
  • Unusual serologic responses in co-infected patients can be seen such as:

    • Increased biological false positivity
    • Higher-than-expected serologic VDRL titers
    • Seronegative cases of syphilis.

  • Delayed titer responses after treatment
  • Return of titers to nonreactivity as immunosuppression advances
  • Currently, routine periodic screening (at least annually and 2–4 times yearly among high-risk groups, such as Men who have sex with Men (MSM) is strongly recommended
  • Irrespective of signs and symptoms, all HIV-positive patients should have baseline VDRL screening and follow-up at 3 months to rule out the possibility of false-negative results, as seroconversion generally takes about 4–6 weeks after infection
  • In the course of reactive VDRL, the diagnosis of syphilis should be confirmed by the specific treponemal test
  • According to the current CDC guidelines, indications of lumbar puncture in HIV and syphilis co-infection are as follows:[10]

    1. Patients with neurologic symptoms should undergo immediate lumbar puncture to rule out neurosyphilis
    2. HIV-infected patients who present with late, latent syphilis or syphilis of unknown duration
    3. Patients with evidence of serologic failure after receiving syphilis therapy

  • Risk of neurosyphilis is increased substantially – three- to fivefold – in HIV-infected patients with RPR titers 1:32 or in whom CD4 counts are <350
  • HIV testing is critical for all patients with a new diagnosis of syphilis.

Syphilis screening using rapid point-of-care tests

  • Operational requirements for RPR/VDRL testing are not available at most primary care. Delay in obtaining test results through referrals to offsite laboratories can delay or result in missed opportunities for treatment. Very often, patients may not return for follow-up, particularly if they are asymptomatic
  • Aim: To integrate rapid, simple, and technologically appropriate syphilis testing at venues with limited resources
  • Six test kits validated by the WHO.

   Conclusion Top

Every patient suspected of syphilis should be subjected to a NTT (i.e., qualitative VDRL) followed by a treponemal test (i.e., TPHA). VDRL test indicates recent infection and is also useful in follow-up to monitor response of therapy. While treponemal tests are more specific tests, due to the limitation that they remain positive lifelong, they are not indicative of recent infection.

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Conflicts of interest

There are no conflicts of interest.

   References Top

Ratnam S. The laboratory diagnosis of syphilis. Can J Infect Dis Med Microbiol 2005;16:45-51.  Back to cited text no. 1
Lee JH, Lim CS, Lee MG, Kim HS. Comparison of an automated rapid plasma reagin (RPR) test with the conventional RPR card test in syphilis testing. BMJ Open 2014;4:e005664.  Back to cited text no. 2
Nayak S, Acharjya B. VDRL test and its interpretation. Indian J Dermatol 2012;57:3-8.  Back to cited text no. 3
[PUBMED]  [Full text]  
Sidana R, Mangala HC, Murugesh SB, Ravindra K. Prozone phenomenon in secondary syphilis. Indian J Sex Transm Dis AIDS 2011;32:47-9.  Back to cited text no. 4
Young H. Guidelines for serological testing for syphilis. Sex Transm Infect 2000;76:403-5.  Back to cited text no. 5
Young H, Aktas G, Moyes A. Enzywell recombinant enzyme immunoassay for the serological diagnosis of syphilis. Int J STD AIDS 2000;11:288-91.  Back to cited text no. 6
Holmes KK, Sparling PF. Clinical Manifestation of syphilis; Sexually Transmitted Diseases; 4th Edition; The Mcgrow Hill Companies, New York; 2008. p. 672-81.  Back to cited text no. 7
Department of AIDS Control, Ministry of Health & Family Welfare, Government of India. National Guidelines on Prevention, Management and Control of Reproductive Tract Infections and Sexually Transmitted Infections. Department of AIDS Control, Ministry of Health & Family Welfare, Government of India; 2014.  Back to cited text no. 8
Shahrook S, Mori R, Ochirbat T, Gomi H. Strategies of testing for syphilis during pregnancy. Cochrane Database Syst Rev 2014;10:CD010385.  Back to cited text no. 9
Ghanem KG, Moore RD, Rompalo AM, Erbelding EJ, Zenilman JM, Gebo KA, et al. Lumbar puncture in HIV-infected patients with syphilis and no neurologic symptoms. Clin Infect Dis 2009;48:816-21.  Back to cited text no. 10


  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

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