Treponemal Tests - labsstudies

Treponemal Tests

Treponemal Tests

Treponemal Tests

Treponemal tests rely on the detection of antibodies formed specifically against the treponemes’ antigenic determinants.

Anti-treponemal antibodies can be detected using a variety of methods, including agglutination, EIA, immunofluorescence, immunoblotting, and immunochromatography. Treponema pallidum or one of its components is used as the antigen in all treponemal tests.

Treponema pallidum is the one of gram negative Bacteria that cause Syphilis disease.

The treponemal tests should be reserved for confirmatory testing when clinical signs and/or history contradict the reactive Nontreponemal test results.. Example of nontreponemal tests are, VDRL and Rapid Plasma Reagin. 

Treponemal tests are almost always positive in secondary and latent syphilis. In most cases, once the treponemal tests are reactive, they remain reactive for the rest of the patient’s life. In fact, in some late-stage syphilis patients, a reactive treponemal test may be the only way to confirm the suspected diagnosis. None of the treponemal tests are currently recommended for use with CSF.

Laboratory Techniques for Detection of Treponema pallidum

There are several Laboratory Techniques for diagnosis Treponema pallidum, These include: 

  1. Fluorescent Treponemal Antibody absorption (FTA-ABS) test,
  2. Treponema pallidum Hemagglutination Assay (TPHA)
  3. Treponema pallidum particle agglutination test (TP-PA)
  4. Enzyme Immunoassay (EIA)
  5. Immunoblotting
  6. Immunochromatographic card test
  7. Treponemal tests for specific IgM
  8. Polymerase Chain Reaction Testing for Treponema pallidum

1. Fluorescent Treponemal Antibody Absorption (FTA – ABS)

It is the most sensitive of all syphilis tests, but it is also the most difficult to perform. Both the test performance and the reading of results must be thoroughly checked.

The antigen in the FTA-ABS is a killed suspension of T. pallidum spirochetes. Overlaying whole treponemes fixed to a slide with serum from patients suspected of having syphilis due to a previously positive VDRL or RPR test is used in this procedure.

To reduce nonspecific cross-reactivity, the patient’s serum is first absorbed with non-T. pallidum treponemal antigen.

The Treponema Pallidum Hemagglutination (TPHA) Test Is also extremely sensitive and has the advantage of being simple to perform.

 2. The Treponema Pallidum Hemagglutination (TPHA) Test

Is extremely sensitive and has the advantage of being simple to perform.


  • In the TPHA test, diluted serum samples from the patient are mixed in the wells of a microtiration plate with sheep or avian red cells coated (sensitized) with T. pallidum antigen.
  • Unsensitized cells added to a second well serve as a control. If an antibody is present, the sensitized cells agglutinate and settle in a distinctive mat pattern at the bottom of the well.
  • Unagglutinated cells in a negative test and control well form a button or smooth ring at the bottom of the well.

Reading result

The TPHA results are reported down as reactive (1+, 2+, 3+, 4+,) or non-reactive (, -).

Completely negative results range in pattern from a solid compact button of cell to a circle of cells with a small central hole.

3. Treponema Pallidum Particle Agglutination (TPPA) Test

The most recent modification to TPHA is the use of gelatin or polymer particles as carriers for the T. pallidum antigen rather than erythrocytes. The Treponema pallidum particle agglutination (TPPA) test, for example, uses colored gelatin particles as antigen carriers and can be performed on serum or plasma.

The use of gelatin particles has nearly eliminated non-specific agglutination reactions, and the sensitivity of the test in primary syphilis is said to have increased due to the improved IgM binding capacity of the sensitized gel particles. As a result, many laboratories have switched from TPHA to TPPA.

The Reporting result and quality control are similar to TPHA.

4. Enzyme Immunoassay (EIA)

The enzyme immunoassay technique for detecting treponemal antibodies was described for the first time in 1975. Indirect, competitive, and capture EIAs have been developed for the detection of anti-treponemal IgG, IgM, or IgG and IgM, and numerous commercial kits are available.

The benefits of EIA include its suitability for automation, objective reading of results, and the ability to connect EIA plate readers to laboratory computer systems, reducing the possibility of transcription errors. According to recent research, certain new recombinant antigen-based EIAs are now among the most sensitive and specific treponemal tests.

5. Immunoblotting

Immunoblotting detects antibodies to specific proteins. Solubilized T. pallidum proteins are separated by gel electrophoresis according to their molecular size in a Treponemal Western blot. The separated proteins are then transferred to a nitrocellulose membrane, which is dried and cut into strips. After incubating the strips with the patient’s serum, antigen-antibody complexes are visualized by adding enzyme-conjugated anti-human globulin followed by substrate, which causes a color reaction.


It is widely accepted that antibodies to immune determinants with molecular masses of 15, 17, 44.5, and 47kd are diagnostic for acquired syphilis. According to studies, the assay (using IgG conjugate) is more sensitive and specific than the FTA-ABS test.


The use of recombinant antigens rather than fractionated proteins is a recent advancement. For example, the INNO-LIA Syphilis test (Innogenetics) is a line immunoassay that employs three recombinant antigens and one synthetic peptide derived from T. pallidum proteins.

6. Immunochromatographic card test

A drop of whole blood, serum, or plasma is placed on the card, and as the sample migrates through the card, it reconstitutes and mixes with selenium-conjugated T. pallidum. The mixture then migrates to the “patient window,” which contains immobilized T. pallidum antigen. If T. pallidum antibodies are present in the sample, they bind to the conjugated antigen as well as the immobilized antigen, forming a red line at the patient window site. The test can be read after a minimum of 15 minutes and up to 24 hours.

In general, all of the treponemal tests described thus far detect anti-treponemal IgM, IgG, or IgG and IgM, and once reactive, are usually reactive for life unless treated early in infection. As a result, they indicate that the patient has had a treponemal infection at some point in the past but are unable to differentiate between past and present infection. Furthermore, they cannot be used to track treatment response.

 7. Treponemal tests for specific IgM

Anti-treponemal IgM detection is helpful in the diagnosis of neonatal congenital syphilis because IgM does not cross the placenta and thus cannot be maternally derived. Its presence also suggests active disease in adults with no recent treatment history.

Most laboratories test for treponema-specific IgM now use an IgM EIA, specifically an IgM capture EIA.

Anti-human IgM is attached to the wells of a microtitration plate in this technique. These fixed antibodies “capture” any IgM present in the serum. Any Treponema-specific IgM that is bound is then detected by an indicator system that includes a treponemal antigen labeled with an enzyme.

The IgM capture EIA has a sensitivity of 88-92% and a specificity of 95% in symptomatic congenital syphilis.

 8. Polymerase Chain Reaction Testing for Treponema pallidum

Polymerase Chain Reaction testing on a swab taken from a genital ulcer or mucous membrane lesion (and sent to the laboratory in a dry tube) is becoming more common, and it has high sensitivity and specificity.

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