Indian Journal of Sexually Transmitted Diseases and AIDS
: 2019  |  Volume : 40  |  Issue : 2  |  Page : 186--191

Serological tests for syphilis

Dhiral Shah1, Yogesh S Marfatia2,  
1 Department of Skin and VD, Medical College Baroda, SSG Hospital, Vadodara, Gujarat, India
2 SBKS Medical Institute and Research Centre, Vadodara, Gujarat, India

Correspondence Address:
Dr. Yogesh S Marfatia
2, Nandvan Society, Yogidhara Flats, Alkapuri, Vadodara, Gujarat

How to cite this article:
Shah D, Marfatia YS. Serological tests for syphilis.Indian J Sex Transm Dis 2019;40:186-191

How to cite this URL:
Shah D, Marfatia YS. Serological tests for syphilis. Indian J Sex Transm Dis [serial online] 2019 [cited 2020 Sep 22 ];40:186-191
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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), andT. 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 prevalentSyphilis 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.


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 1906It 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 conditionNewer NTTs, such as the rapid plasma reagin (RPR) and VDRL tests, have mostly replaced it.

 Investigations for Syphilis

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}

 Nontreponemal Tests

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 itselfSeroconversion 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


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 testUnheated serum reagin testReagin screen testAutomated reagin test.


Qualitative test for screening syphilisResults are reported as nonreactive, reactive, and weakly reactiveIn reactive NTT:

A fourfold increase in titer indicates infection, reinfection, or treatment failureA 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 therapyPatients 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 therapyA 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.


Inexpensive and simpleSuitable for mass screeningThe baseline titer can be used to follow-up the treatment response.


Reduced sensitivity in primary syphilis and late latent syphilisFalse-positive results due to technical error and variations in normal population, due to antiphospholipid autoantibodies (biologic false positivity) [Table 2]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}

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 drugsThose who are HIV seropositiveAntigen excess can also result in false-negative result which is known as POSTZONE phenomenon [Figure 1].{Figure 1}

Thus, confirmation by a treponemal test is required.

 Treponemal Tests

Treponema pallidum immobilization test

This test uses the virulent T. pallidum (Nichol's strain) obtained from rabbitsIt 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 microscopyThe test is positive if 50% or more of the treponemes are immobilizedIt becomes positive few days to a week later than the VDRL testSpecificity 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 moreReactivity can be expected around the 4th to 5th week of infectionIt 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 testPresence of antibody in patient's serum is indicated by fluorescence. Intensity of fluorescence is reported as nonreactive, borderline, or reactiveReactivity begins in the 3rd week of infectionIt 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 testFalse 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 minAntibody to T. pallidum binds to antigen and reacts in the 2nd incubation with an enzyme labeled antihuman globulinResult is available in 3–4 h.


Automated (or semi-automated) processing[6]Objective reading of resultsInterfacing 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 antigenThe presence of antibodies to the immunodeterminants with molecular weights 15.5, 17, 44.5, and 47 kDa appears to be diagnostic for acquired syphilisWhen an IgM-specific conjugate is used, the test has value in the diagnosis of congenital syphilis.


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

Therefore, treponemal tests should not be used to evaluate response to therapy, relapse, or reinfection in previously treated patientsTreponemal 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} {Table 3}{Table 4}

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” swabIn 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


Neurological, ophthalmic, or auditory symptoms and signsOther clinical evidence of active infection – aortitis, gumma, or iritisTreatment failureHIV infectionA nontreponemal serum titer of >32 if the duration of syphilis is over 1 yearA nonpenicillin-based treatment regimen is plannedAll infants suspected of prenatal syphilis.

The typical CSF findings of neurosyphilis are:

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

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

 Testing Policy

NTTs such as RPR test detect almost all cases of early syphilis, but false positives are possibleTreponemal 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 treatedThe 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].{Table5}{Figure 3}

 Guidelines for Screening of Syphilis in Pregnancy and Newborns

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 pregnancyFor 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 resultAll 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 patientsSerologic tests for syphilis are still the cornerstone of diagnosing untreated syphilis infection, independent of the HIV statusUnusual serologic responses in co-infected patients can be seen such as:

Increased biological false positivityHigher-than-expected serologic VDRL titersSeronegative cases of syphilis.

Delayed titer responses after treatmentReturn of titers to nonreactivity as immunosuppression advancesCurrently, 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 recommendedIrrespective 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 infectionIn the course of reactive VDRL, the diagnosis of syphilis should be confirmed by the specific treponemal testAccording to the current CDC guidelines, indications of lumbar puncture in HIV and syphilis co-infection are as follows:[10]

Patients with neurologic symptoms should undergo immediate lumbar puncture to rule out neurosyphilisHIV-infected patients who present with late, latent syphilis or syphilis of unknown durationPatients 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 <350HIV 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 asymptomaticAim: To integrate rapid, simple, and technologically appropriate syphilis testing at venues with limited resourcesSix test kits validated by the WHO.


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.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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