APPLIED MICROBIOLOGY

LABORATORY DIAGNOSIS OF SYPHILIS

Syphilis is caused by a treponeme, Treponema pallidum. The disease runs in stages. The diagnosis
may be required in its primary stage, secondary stage, tertiary stage or late syphilis as well as treated
and congenital syphilis.

Laboratory diagnosis is indicated for:

a. Diagnosing a suspected case of syphilis

b. Assessment of efficacy of treatment
c. Screening of blood and preventing spread

The diagnosis of syphilis can be established by:

a. Demonstration of treponema by microscopy in congenital syphilis and primary and secondary

stages
b. Demonstration of reaginic and treponemal antibodies.

Direct Examination

Detection of treponemes from the lesion is the key aid to diagnosis. Dark-field microscopy is recom-
mended. Samples should be collected under aseptic conditions before antibiotics are started.
Organisms of T. pallidum are actively motile filamentous helicals with evenly spaced coils.

Immunofluorescence

Tissue fluid or exudate is spread on a glass slide, air dried and sent to the laboratory. It is fixed and
stained with fluorescent labelled anti-treponeme serum and examined by means of
immunofluorescent microscopy. This gives a higher positivity rate than the direct microscopy.

Serological Tests

These tests form the mainstay of laboratory diagnosis. It is useful to carry out at least two different
tests on each specimen. Two types of antigens are available are:

a. Cardiolipin: not derived from spirochaetes and used in VDRL (Venereal Diseases Research
Laboratory) tests which are non-treponemal test.

b. Specific antigens derived from T. pallidum which are used in TPHA (Treponema pallidum
haemagglutination test) and fluorescent treponema antibody absorbed (FTA-Abs) tests.

In routine three serological tests (VDRL, TPHA, FTA-Abs) are used.

VDRL Test

Venereal Diseases Research Laboratory (VDRL) test is based on the fact that the particles of lipid
antigens remain dispersed with normal serum but form visible clumps when combine with reagin.
VDRL is a type of flocculation test which can be performed on a slide as well as in a tube. In slide test,
0.5 ml of serum of the patient is taken in special slides with depressions (cavity slides) or slides
prepared with paraffin rings.
One drop of freshly prepared antigen is added with a syringe delivering 60 drops from one ml. The
slide is shaken in a VDRL shaker for 4 minutes at 180 rotations. It is then examined under the micros-
cope with low power objective. Formation of clumps indicates positive reaction designated as
reactive while uniform distribution of crystals in the drop indicates that the serum is non-reactive. A
weakly positive reaction may also occur (weakly reactive).

The reactive sera can be diluted and results quantified. VDRL test can also be done in tubes as tube
flocculation test.

Advantages of VDRL test:

a. Simple and rapid test

b. Reasonably sensitive and specific
c. Requires small quantity of serum
d. Can be easily quantified
e. Results are reproducible
f. Reagents are cheap, easily available and have long shelf life
g. Quality control of tests can be performed.

Biological False Positive Results

In spite of simplicity of test and numerous advantages there are various biological false positive
(BFP) results. These can be classified as acute (when they disappear in six months time) or chronic
when they are positive for more than six months), include the following:

Systemic lupus erythematosus, Malaria, Lepromatous leprosy, Infectious mononucleosis, Tropical

eosinophilia, Relapsing fever, Hepatitis, Tissue regeneration, Collagen disorders, Severe trauma,
Coronary artery disease, Repeated blood loss, Menstruation, Immunization, Pregnancy, Haemolytic
anaemia and Heroin addiction.

TPHA Test

Treponema pallidum haemagglutination (TPHA) test is performed by using extract of T. pallidum.

Red blood cells are treated to adsorb treponemes on their surface. When mixed with serum
containing antitreponemal anti bodies, the cells become clumped. This test is similar to FTA-ABS test
in specificity and sensitivity, but become positive somewhat later in the infection.

Micro haemagglutination -T. pallidum (MHA-TP) is automated version of this test and so also is
haemagglutination treponemal test for syphilis (HATTS). However, these two tests lack sensitivity in
primary syphilis.

FTA-ABS Test

Fluorescent antibody test uses an indirect immunofluorescence test in which Nichol's strain of T.
pallidum is employed as antigen. The patient's serum is diluted 1:5 and reacted with the antigen
which has been smeared on the slide. The combination is covered with antihuman immunoglobulin
fluorescent conjugate. After proper washing, the smear is examined under fluorescent microscope.

In a positive test fluorescent treponemes are observed. When patient serum is diluted to 1:200 instead
of 1:5 (as is done now a days), the test is called as FTA200. Patient's serum is adsorbed with an extract
of Reiter's treponemes to avoid false positive results and then the test is performed (FTA-ABS test).
This is extremely sensitive and specific test.
 Interpretation of Serological Tests for Syphilis

Stage of disease VDRL TPHA FT A-ABS

Primary syphilis +/- +/- +
Secondary syphilis + + +
Tertiary syphilis + + +
Late syphilis + + +
Congenital syphilis + + +
Treated syphilis - + +

The nontreponemal tests are mainly used for:

a. screening procedures
b. to detect reinfection
c. to evaluate response to therapy
d. to diagnose neurosyphilis.

The treponemal tests are mainly used for:

a. confirmation of diagnosis of syphilis

b. detection of latent syphilis and
c. diagnosis in patients with negative non treponemal tests.