TPHA Test: Introduction, Principle, Procedure, Result-Interpretation, Clinical Significance, and Keynotes

Introduction of TPHA Test

TPHA test  stands for Treponema pallidum hemagglutination. This is a confirmatory test for syphilis that is caused by a bacterium, T. pallidum. TPHA test is a sensitive indirect hemagglutination test, that detects specific Treponema pallidum antibodies in serum within one hour. It uses in combination, with the VDRL(Venereal Disease Research Laboratory test) or RPR (rapid plasma reagin) and TPHA tests to provide accurate and reliable confirmation of active syphilis infection. No specialized equipment is required and the results are clearly visible and easily interpreted.

Principle of TPHA Test

TPHA is an indirect hemagglutination assay carried out on microplates for the qualitative and semi-qualitative detection of anti-Treponema pallidum-specific antibodies in human serum. Avian blood cells stabilized and sensitized with a solution of T. pallidum antigen (Nichols strain) agglutinate in the presence of anti-T. Pallidum antibodies, exhibit a typical agglutination pattern (i.e. hemagglutination).

Test Requirements

• Patient blood (plasma/serum)
• Test Controls (negative and positive)
• Sample diluent
• Microtiter well
• Timer

TPHA Test Procedure

Antigen suspension contains avian red cells sensitized with Nichols strain of Treponema pallidum mixes 1:20 dilution of serum in a microtiter well to make a final dilution of serum 1:80. Now incubate the plate at room temperature for an hour. Finally, observe for hemagglutination.

Result Interpretations of TPHA (Treponema pallidum hemagglutination) Test

The upper, left-hand well contains a positive control test. The red cells have had treponemal antigens attached and antibodies in the serum have caused these cells to agglutinate and form a mat across the bottom of the well. These antibodies can be presumed to be specific for Treponemes since otherwise identical red cells that have not had the treponemal antigens attached do not cause hemagglutination, as seen in the bottom, left-hand well. A negative serum test is shown in the center and a patient’s test is on the right as shown above image. This result supports a diagnosis of syphilis. In brief, positive control (PC): is positive, negative control (NC) is negative and the test is also negative as shown in the above footage.

Clinical Significance of THPA test

• TPHA is a confirmatory test of syphilis.
• Sensitivity of Serological Tests in Untreated Syphilis- Stage of disease (% positive-Range)

Test	Primary	Secondary	Latent	Tertiary
VDRL	78(74-87)	100	95(88-100)	71(37-94)
RPR	86(77-99)	100	98(95-100)	73
FTA-ABS	84(70-100)	100	97(97-100)	94
EIA	93	100	100	–

Interpretations of the TPHA Test

RPR	TPHA	IgM EIA
–	–	–	No syphilis or incubating syphilis
–	–	+	Early primary syphilis
+	+	+	Primary or secondary
+	–	+	Early infection
+	+	–	Late secondary or latent syphilis
+	–	–	Biological false positive, late syphilis
–	+	–	Late infection, treated syphilis, or false positive treponemal test
Increasing	+	Increasing	Re-infection, relapse

Keynotes on TPHA

1. This is a qualitative method of the TPHA test.
2. The positive test indicates a 1:80 titer.
3. A quantitative method of the TPHA test is also available. The procedure is nearly the same but there is a need for serial dilution.
4. TPPA stands for Treponema pallidum particle agglutination
5. FTA-ABS stands for fluorescent treponemal antibody absorption test.
6. EIA stands for Enzyme immunoassay.

Bibliography

1. http://atlasmedical.com/upload/productFiles/202003/TPHA%20Package%20Insert.pdf
2. https://apps.who.int/iris/bitstream/handle/10665/262186/PMC2396117.pdf?sequence=1&isAllowed=y
3. http://www.biolabo.fr/biolabo/pdfs/noticesE/syphilisE/AT-45-00-100-TPHA.pdf
4. http://www.biolifeit.com/public/cartellina-allegati-schede-certificazioni/schede-tecniche-inglese/TS-UC80515.pdf
5. https://www.e-ijd.org/article.asp?issn=0019 5154;year=2018;volume=63;issue=6;spage=479;epage=483;aulast=Kashyap