VDRL Test for Syphilis

The Venereal Disease
Research Laboratory
test (VDRL)
BY
A.MANOJ KUMAR

The Venereal Disease Research
Laboratory test (VDRL)
•It is a screening test for sexually
transmitted infection – syphilis caused
by the spirochete bacterium Treponema
pallidum.
•It is a non-treponemal test, which
detects antibodies IgM and IgG
antibodies to lipoidal material released
from damaged host cells as well as to
lipoprotein-like material, and possibly
cardiolipin released from the
treponemes. These antibodies are
traditionally referred to as ‘reagins’.

•The VDRL test is thus slide micro
flocculation test used to screen for
syphilis in addition to more specific tests
used to diagnose the disease.
•VDRL test becomes positive 1-2 weeks
after appearance of (primary lesion)
chancre. The test becomes reactive (50-
75%) in the late phase of primary
syphilis, becomes highly reactive
(100%) in the secondary syphilis and
reactivity decreases (75%) thereafter.

Principle of VDRL Test
The Venereal Disease Research
Laboratory (VDRL) tests are slide
microflocculation test that detect
antibodies produced against antigens
released by damaged host cells in
patients suffering from syphilis.
It uses antigen (referred to as VDRL
antigen) containing 0.03% cardiolipin,
0.21% lecithin, and 0.9% cholesterol.
The antigen, suspended in a buffered
saline solution, forms flocculates when
combined with lipoidal antibodies in
serum or cerebrospinal fluid from
syphilis patients.

For the test, at first a drop of
antigen is placed on a slide and then a
drop of serum is added to it.
The slide is rotated to mix the
content.
In case of positive test, flocculation
occurs which are read using a
microscope.
Clumping or agglutination indicate
reactive specimen or presence of
autoantibody in patient’s specimen
while non-reactive specimens appear as
homogeneous suspension.

Requirements of VDRL Test
 Patient’s serum,
 water bath,
 freshly prepared cardiolipin antigen,
VDRL slide,
 mechanical rotator,
pipettes, and
hypodermic syringe with unbeveled
needle and microscope.
 Known reactive and non-reactive
serum controls are also required.

Procedure of VDRL Test
The test can be performed both
qualitatively and quantitatively. Those
tests that are reactive by qualitative
test are subjected to quantitative test
to determine the antibody titres.
Qualitative Method
•Patients’ serum is inactivated by
heating at 56oC for 30 minutes in a
water bath to remove non-specific
inhibitors (such as complement).
•VDRL antigen suspension (colloidal
suspension of tissue cardiolipid or
chemically synthesized cardiolipin),
controls and samples are brought to
room temperature.

•One drop (50 µl) of the test specimen,
positive and negative controls is
pipetted onto separate reaction circles
of the disposable slide.
•A drop of diluted antigen suspension is
added to the measured volume of
specimen, positive and negative
controls.
•Using a mixing stick the test specimen
and the VDRL reagent is mixed such
that it thoroughly spreads uniformly
over the entire reaction circle.

•The slide gently rotated and
continuously either manually or on a
mechanical rotor at 180 r.p.m.
•Flocculation is checked microscopically
using 10X objective and eye piece at
about 8 minutes.

Quantitative Method
•Dilute serum samples to an endpoint
titer. Quantitative tests for 3 serum
specimens through the 1:8 dilution may
be performed on one slide.
•Place 50 µl of 0.9% saline in circles
numbered 2 through 4. Do not spread
saline.
•Using a safety pipette device, place 50
µl of serum in circle 1 and 50 µl of
serum in circle 2.
•Mix the saline and the serum in circle
2 by drawing the mixture up and down
in the safety pipette eight times.

•Transfer 50 µl from circle 2 (1:2) to
circle 3, and mix.
•Transfer 50 µl from circle 3 to circle 4,
mix, and then discard the last 50 µl.
•Gently re-suspend the antigen
suspension.
•Add exactly 1 freefalling drop (17 µl)
of antigen suspension to each circle.
•Place the slide on the mechanical
rotator. Rotate the slide for 4 minutes at
180 ±2 rpm.
•Immediately after rotation, read the
test.

•If the highest dilution tested (1:8) is
reactive, continue as follows:
•Prepare a 1:8 dilution of the test
specimen in a test tube. Add 0.1 ml
of serum to 0.7 ml of 0.9% saline.
Mix thoroughly.
•Place 50 µl of 0.9% saline into
paraffin rings 2, 3, and 4. Prepare
additional serial dilutions for strongly
reactive specimens.
•Add 50 µl of the 1:8 dilution of the
test specimen to paraffin rings 1 and
2.
•Prepare serial twofold dilutions
beginning with ring 2.

Result Interpretation of VDRL Test
•Positive test: Presence of antigen-
antibody clumps in the center or the
periphery of the test circle indicate
positive VDRL test.
•Negative test: Absence of antigen-
antibody clumps. Indicated by a
smooth, even light gray appearance
with no aggregates.
•All reactive and weakly reactive serum
requires serial dilution to estimate
antibody titer. The titre is reported as
the reciprocal of the highest dilution,
which shows a positive test result.

Applications of VDRL Test
•It is used mostly as the screening test
for syphilitic infection.
•Screening for syphilis is a routine part
of pregnancy tests.
•VDRL test for syphilis is also
performed if being treated for another
STI such as gonorrhea, infected
with HIV, or if engaged in high-risk
sexual activity.
•If already treated for syphilis, VDRL is
used for follow-up testing on
recommendations of the Center for
Disease Control and Prevention (CDC).

Advantages of VDRL Test
•VDRL test is most widely used simple
and rapid test for syphilis.
•The VDRL tests are fast, easy to
perform, and excellent for screening of
samples.
•VDRL test is also helpful in the
diagnosis of congenital syphilis.
•Since, the organism Treponema
pallidum cannot be cultured in artificial
media, the screening of syphilis via
serological testing such as VDRL
becomes important.

•Patient need not have the symptoms of
syphilis for this test to be accurate. It
can detect syphilis in patients without
symptoms.
• Since it is a non-treponemal test (non-
specific test) it is used to investigate
syphilis along with other treponematose,
Yaws and Pinta.

Limitations of VDRL Test
•Without some other evidence for the
diagnosis of syphilis, a reactive non-
treponemal test such as VDRL does not
confirm pallidum infection. Any reactive
VDRL test must be confirmed with a
specific or treponemal test such as
TPHA, FTA-ABS test.
•The anti lipoidal antibodies detected
are not only produced as a consequence
of syphilis and other treponemal
diseases, but also may be produced in
response to nontreponemal diseases of
an acute and chronic nature in which
tissue damage occurs.

•The VDRL test isn’t always accurate.
For example, false-negative results may
arise if an individual had syphilis for less
than three months, as it could take long
for the body to make antibodies.
•The test is also unreliable in late-stage
syphilis.

•On the other hand, false-positive
results can be seen in conditions like
HIV, Lyme disease, malaria, pneumonia,
systemic lupus erythematosus, IV drug
use and tuberculosis.
•The antibodies produced as a result of
a syphilis infection can stay in the body
even after your syphilis has been
treated.

•A prozone reaction may occur.
• In a prozone reaction, reactivity with
undiluted serum is inhibited.
•The prozone phenomenon may be
suspected when a specimen produces
only a weakly reactive or a rough
nonreactive result in a qualitative test.
•The VDRL may be reactive in persons
from areas where yaws is endemic. As a
rule, residual titers from these
infections will be <1:8.

VDRL Test for Syphilis

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VDRL Test for Syphilis