Laboratory diagnosis of Syphilis

Last updated on June 4th, 2021

Syphilis is a sexually transmitted disease caused by the bacterium Treponema pallidum. Syphilis has diverse clinical manifestations and shares many clinical features with other treponemal and nontreponemal diseases (because of this, syphilis has been called “The Great Pretender”). The etiological agent, Treponema pallidum, cannot be cultured, and there is no single optimal alternative test.

Commonly used laboratory diagnosis testing available for syphilis are:

Direct diagnosis

Darkfield micrograph of Treponema pallidum (source: CDC)
Darkfield micrograph of Treponema pallidum
(source: CDC)

Direct laboratory methods used for the laboratory diagnosis of syphilis include the detection of Treponema pallidum by microscopic examination of fluid or smears from lesions, histological examination of tissues or nucleic acid amplification methods such as polymerase chain reaction (PCR).
Tests used for the direct detection of Treponema pallidum are;

Dark-field microscopy

Dark-field microscopy is used to demonstrate the presence of motile Treponema pallidum in lesions or aspirates in early-stage (primary or secondary) syphilis prior to healing
of lesions.

Direct fluorescent antibody test for Treponema pallidum

Nucleic acid amplification (PCR based) methods

Indirect diagnosis/serological methods

It is based on serological tests for the detection of antibodies. Serological testing is the mainstay in the laboratory diagnosis and follow-up of syphilis. Serological tests fall into two categories: nontreponemal tests for screening, and treponemal tests for confirmation.

Nontreponemal tests

They measure both immunoglobulin (IgG and IgM) antiphospholipid antibodies 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 itself.  Commonly used nontreponemal tests are

Rapid plasma reagin (RPR) test

Toluidine red unheated serum test (TRUST)

Venereal Disease Research Laboratory (VDRL) test

Treponemal Tests

All treponemal tests use Treponema pallidum or its components as the antigen.

Treponemal tests are used as confirmatory tests to verify reactivity in non-treponemal tests. Once positive, treponemal tests remain positive throughout life with or without treatment, so these tests can not be used to know the response to treatment.

Commonly used treponemal tests are:

Fluorescent treponemal antibody absorption test (FTA-ABS) test

Treponema pallidum particle agglutination (TP-PA) test

Treponema pallidum Hemagglutination Assay (TPHA)

Flow-chart for laboratory diagnosis of syphilis

If lesion exudate or tissue is available, the direct examination is performed, followed by a nontreponemal serology test. A reactive nontreponemal test is then confirmed by a treponemal test.Testing algorithm for Primary Syphilis

A confirmed serological test result is indicative of the presence of treponemal antibodies but does not indicate the stage of disease and, depending on the test, may not differentiate between past and current infection.

About Acharya Tankeshwar 476 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.


  1. Thanks for your sharing knowledge about microbiology, i wonder if the patient has VDRL neg (-) but TPHA positive(+) 1/80 and clinical symptom is abscent is the patient suffering syphillis or we have to do confirmation test FTA-Abs first before treatment or we can direct give the patient treatment?from Sandi widjaja.

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