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Immunology

Treponemal Syphilis

The fundamental principle of a treponemal test is that it detects antibodies directed specifically against antigens of the Treponema pallidum bacterium itself. Unlike nontreponemal tests that detect a non-specific surrogate marker (reagin), these assays are designed to be highly specific. Their primary role in the traditional testing algorithm is to confirm that a reactive RPR result is a true positive due to a syphilis infection and not a biological false positive

The most critical concept to understand about treponemal tests is this: once a person is infected with syphilis, the antibodies detected by these tests will typically remain present for the rest of their life, regardless of successful treatment. They create a permanent “serological scar.” This means they cannot be used to monitor response to therapy or to distinguish between an active infection and a past, cured infection

Classic Confirmatory Methods

These are the historical gold standards. While largely replaced by automated methods in high-volume labs, the principles are essential to know, and they are still used in some reference labs

1. Fluorescent Treponemal Antibody Absorption (FTA-ABS)

This was the first widely used confirmatory test and is a classic example of an Indirect Immunofluorescence Assay (IFA)

  • The Principle: This test visually detects if a patient’s antibody can bind to whole, fixed T. pallidum organisms on a microscope slide
  • The Reagents
    • Antigen Slide: A microscope slide with fixed spirochetes of the Nichols strain of T. pallidum
    • Sorbent: A diluent containing an extract of non-pathogenic treponemes. This is the critical “absorption” step. It is used to pre-treat the patient’s serum to remove any non-specific, cross-reacting antibodies that might bind to common antigens shared by other spirochetes. This dramatically increases the test’s specificity
    • Conjugate: A fluorescein-labeled anti-human immunoglobulin
  • The Procedure
    1. Patient serum is pre-treated with the sorbent
    2. The absorbed serum is incubated on the antigen slide. If anti-T. pallidum antibodies are present, they will bind to the spirochetes
    3. The slide is washed, and the fluorescent conjugate is added. It binds to the patient’s antibodies
    4. The slide is washed again and viewed under a fluorescent microscope
  • Reading the Result
    • Reactive: The spirochetes will glow a distinct apple-green
    • Nonreactive: The spirochetes will be invisible or appear as faint, red outlines (due to the counterstain)
  • Limitations: Highly subjective, technically demanding, and labor-intensive. Reading requires significant expertise

2. T. pallidum Particle Agglutination (TP-PA) & MHATP

This category of tests, including the Microhemagglutination Assay for T. pallidum (MHATP), replaced the FTA-ABS as the confirmatory test of choice for many years because they are much easier to perform and read

  • The Principle: This is a passive agglutination assay. We are using microscopic particles as carriers for the treponemal antigens
  • The Reagents
    • Antigen Particles: Gelatin particles (in TP-PA) or red blood cells (in MHATP) that have been coated with antigens extracted from T. pallidum
    • Control Particles: Particles coated with no treponemal antigens, used to check for non-specific agglutination
  • The Procedure
    1. The patient’s diluted serum is added to the wells of a microtiter plate containing the antigen-coated particles
    2. If the patient’s serum contains specific anti-treponemal antibodies, they will bind to the antigens on multiple particles, cross-linking them and forming a lattice structure
    3. The plate is incubated, allowing the particles to settle
  • Reading the Result: The interpretation is based on the settling pattern in the U-bottom well
    • Reactive: The cross-linked particles form a smooth, flat “mat” or “lawn” covering the bottom of the well
    • Nonreactive: The particles are not cross-linked, so they roll down the sides of the well and form a tight, compact “button” or ring at the bottom
  • Advantages: Objective, easy-to-read endpoint. More specific than early FTA-ABS tests

Modern Automated Treponemal Tests: EIA and CLIA

These are the methods that dominate modern syphilis testing, especially in the context of the “reverse algorithm.”

  • The Principle: These are Enzyme Immunoassays (EIA) or Chemiluminescence Immunoassays (CLIA) performed on high-throughput automated analyzers. They use recombinant T. pallidum antigens (specific proteins like TpN17 and TpN47) rather than whole bacterial extracts, which further improves specificity
  • The Procedure: The format is typically a sandwich or capture ELISA. For example, wells of a plate or microbeads are coated with treponemal antigens. Patient serum is added, and if specific antibodies are present, they bind. An enzyme-labeled secondary antibody is then added, followed by a substrate that produces a measurable color (EIA) or light (CLIA) signal
  • Advantages
    • Automation: Can be fully automated for high-volume screening
    • Objectivity: Results are read by a spectrophotometer or luminometer, eliminating subjective interpretation
    • High Sensitivity: Can detect very low levels of antibody, making them excellent screening tools
  • Role in the Reverse Algorithm: Because these tests are so sensitive and automatable, many labs now use a treponemal EIA/CLIA as their initial screening test. If it is reactive, they then perform a quantitative RPR to determine the disease’s activity and titer. This approach is better at detecting very early or old, latent syphilis but can create confusing results (e.g., Trep +, RPR -) that require further testing

Putting It All Together: The Clinical Interpretation

The power of syphilis serology comes from using nontreponemal and treponemal tests together

  • RPR Reactive, TP-PA Reactive: Consistent with an active or recent, untreated syphilis infection
  • RPR Nonreactive, TP-PA Reactive: This is a classic pattern. It usually means one of two things:
    1. A successfully treated past infection. The RPR became nonreactive after treatment, but the treponemal antibody “scar” remains
    2. Very early or very late-stage syphilis, where the reagin response is weak or absent
  • RPR Reactive, TP-PA Nonreactive: This indicates a biological false positive RPR. The patient does not have syphilis; their reactive RPR is due to another condition (like lupus or pregnancy). This perfectly illustrates the confirmatory role of the treponemal test