TPHA / TPPA

Principle

• Treponemal tests: detect antibodies specific to Treponema pallidum antigens.
• TPHA: preserved avian or sheep red cells sensitised with T. pallidum (Nichols strain) antigens agglutinate in the presence of specific antibody.
• TPPA: uses coloured gelatin particles instead of red cells, avoiding reactions against red cell components.
• Serum is diluted in an absorbing diluent that removes non-specific antibodies (e.g. against non-pathogenic treponemes and red cell components).

Specimen

• Serum or plasma; CSF in specialised protocols.

Procedure

• Dilute serum in absorbing diluent in U-well microtitre plate.
• Add sensitised (test) cells or particles and unsensitised (control) cells or particles to separate wells.
• Final screening dilution is typically 1:80 for the test well; the control well is used at the stated dilution.
• Incubate at room temperature (about 45–60 minutes for TPHA; about 2 hours for TPPA), free from vibration.
• Positive samples may be titrated.

Results

• Positive: smooth mat of cells or particles covering the well bottom.
• Negative: compact button with smooth edges (or ring for TPPA).
• Agglutination in the unsensitised control well invalidates the test (non-specific reaction); absorb or use another method.

Quality control and pitfalls

• Include positive and negative controls each run.
• Usually stays positive for life after infection, even after treatment, so it cannot distinguish active from past infection or monitor therapy.
• Cannot distinguish syphilis from yaws, pinta or bejel.
• Less sensitive than FTA-ABS in early primary syphilis.
• Occasional false positives in leprosy, infectious mononucleosis, autoimmune disease and injecting drug users.

Clinical use

• Confirmation of reactive VDRL/RPR, or first-line screen in the reverse algorithm, followed by RPR to assess activity.