Venereal Disease Research Laboratory (VDRL) Test

Venereal Disease Research Laboratory (VDRL) test is a rapid and simple test used for screening of syphilis. It is caused by Treponema pallidum. This test is non-treponemal test because it does not detect the organism directly. It detects reagin antibodies which are produced in the body against lipoidal material. These antibodies are mainly IgG and IgM type.

In VDRL test, the patient serum is heated and then it is taken on a glass slide. Sometimes unheated CSF is also used in suspected nervous system infection. Then VDRL antigen is added with the sample. The antigen is made of cardiolipin, cholesterol and lecithin. When the antibody is present in the sample, it reacts with this antigen and forms small clumps. This clumping reaction is called flocculation. It is seen under microscope.

The test is useful for early screening and also for knowing the response of treatment. But this test is not fully specific for syphilis. False positive result may occur in pregnancy, autoimmune disease and some other infection. So, positive VDRL test is not taken as final diagnosis. It should be confirmed by specific treponemal test.

Principle of Venereal Disease Research Laboratory (VDRL) Test

Principle of Venereal Disease Research Laboratory (VDRL) Test is based on slide microflocculation reaction between reagin antibodies and VDRL antigen. The reagin antibodies are non-specific antibodies, mainly IgG and IgM, which are produced in the body due to cellular damage during syphilis infection. These antibodies do not act directly against Treponema pallidum, but they react with lipoidal antigen.

In this test, heated serum or unheated cerebrospinal fluid (CSF) of patient is mixed with antigen suspension on a glass slide. The antigen is made up of cardiolipin, lecithin and cholesterol in buffered saline. Cardiolipin reacts with the reagin antibody. Cholesterol helps in forming larger visible particles and lecithin helps to make the reaction proper and standard.

When antibody is present in the sample, antigen and antibody combine with each other and small clumps are formed. This clumping is called flocculation. It is observed under microscope. If flocculation is present, the test is called reactive. If the mixture remains smooth and grey without any clumping, the test is called non-reactive.

Requirements of Venereal Disease Research Laboratory (VDRL) Test

The following are the requirements of VDRL test–

1. Patient serum.
2. Water bath, used for inactivation of serum.
3. Freshly prepared cardiolipin antigen.
4. VDRL slide, which is a glass slide with concave depressions.
5. Mechanical rotator.
6. Pipettes.
7. Hypodermic syringe with unbeveled needle.
8. Microscope.
9. Known reactive serum control.
10. Known non-reactive serum control.

Procedure of Venereal Disease Research Laboratory (VDRL) Test

A. Preparation of sample and reagent

1. Patient blood sample is collected and serum is separated.
2. The serum is heated in water bath at 56°C for 30 minutes. This is done for inactivation of complement and other non-specific inhibitors.
3. Freshly prepared VDRL cardiolipin antigen suspension, test serum and control serum are brought to room temperature.
4. Known reactive control serum and non-reactive control serum are also kept ready before doing the test.

B. Qualitative method

1. One drop or 0.05 ml of inactivated patient serum is placed into one reaction circle of VDRL slide.
2. One drop of reactive control serum and non-reactive control serum are placed in separate circles.
3. One drop or 1/60 ml of VDRL antigen suspension is added to each circle by using hypodermic syringe with unbeveled needle.
4. The serum and antigen are mixed uniformly and spread inside the whole reaction circle.
5. The slide is rotated on mechanical rotator at 180 rpm for 4 minutes.
6. After rotation, the slide is observed immediately under microscope by using low power objective.
7. First the control serum reactions are checked. Then the patient sample is observed for flocculation.
8. If visible clumping is present, it is reported as reactive. If slight clumping is present, it is reported as weakly reactive. If no clumping is present and smooth suspension is seen, it is reported as non-reactive.

C. Quantitative method

1. Quantitative method is done only when the sample is reactive in qualitative test.
2. Serial two-fold dilutions of reactive serum are prepared by using 0.9% saline.
3. The dilutions are made as 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 and further if needed.
4. One drop or 0.05 ml of each diluted serum is placed in separate reaction circles of VDRL slide.
5. One drop or 1/60 ml of freshly re-suspended VDRL antigen is added to each diluted serum.
6. The slide is rotated again on mechanical rotator at 180 rpm for 4 minutes.
7. After rotation, each circle is observed immediately under microscope for flocculation.
8. The highest dilution of serum showing clear flocculation is taken as the VDRL titer.
9. The result is reported as reciprocal of the highest reactive dilution. For example, if 1:32 dilution shows flocculation, then the titer is reported as 32.

Result Interpretation of VDRL Test

The following are the result interpretation of VDRL test–

A. Qualitative interpretation

• Reactive result – In this result, visible antigen-antibody clumps are seen in the test circle. This clumping is called flocculation. It may be present in the center or margin of the circle.
• Weakly reactive result – In this result, small or partial clumps are seen. It is also taken as reactive type result. Serial dilution is needed to know the antibody titer.
• Non-reactive result – In this result, no clumping is seen. The mixture remains smooth and even grey suspension. It indicates no detectable reagin antibody in the sample.

B. Clinical interpretation

• True positive result – A reactive VDRL test only indicates possible syphilis infection. Since it is a non-specific test, it must be confirmed by specific treponemal test like TP-PA or FTA-ABS.
• Quantitative titer – In reactive sample, serum dilution is done to find the highest dilution which still gives flocculation. The result may be reported as 1:4, 1:8, 1:16 etc. Four fold rise of titer, such as 1:4 to 1:16, indicates new infection, reinfection or treatment failure. Four fold decrease of titer indicates good response to treatment.
• Biological false positive – Sometimes VDRL test becomes reactive but confirmatory treponemal test becomes negative. This is called biological false positive. It may occur in pregnancy, old age, autoimmune diseases like lupus and rheumatoid arthritis, immunization, IV drug use and infections like malaria, HIV, hepatitis C, tuberculosis and yaws.
• False negative result – Sometimes VDRL test may be non-reactive even in syphilis. It may occur in very early infection when antibody is not formed properly. It may also occur in late syphilis and in prozone phenomenon. In prozone phenomenon, antibody level is very high and proper lattice formation does not occur, so visible flocculation is not formed.
• Serofast state – Some treated patients may show low level reactive titer for long time. It may remain as 1:4 or less for many years. This does not always mean treatment failure.

Applications of VDRL Test

The following are the applications of VDRL test–

• It is used as a primary screening test for detection of syphilis infection. It is rapid, simple and commonly used in routine laboratory.
• It is used in prenatal screening during pregnancy. This helps to detect syphilis in mother and prevent infection to the baby.
• It is used for screening of high risk persons. It includes HIV infected persons, patients treated for other sexually transmitted disease like gonorrhea, and persons with high risk sexual activity.
• It is used for diagnosis of neurosyphilis by testing cerebrospinal fluid (CSF). In this condition, the infection enters into central nervous system.
• It is useful in evaluation of congenital syphilis. The newborn serum titer is compared with mother serum titer for helping in diagnosis.
• It is used for monitoring treatment response and prognosis of syphilis. In successful treatment, antibody titer decreases, so quantitative VDRL test helps to follow the disease condition.
• It is also used for investigation of non-venereal treponemal diseases like yaws and pinta, because it is a non-specific test.

Advantages of VDRL Test

The following are the advantages of VDRL test–

• VDRL test is a rapid and simple test. It can be performed easily in routine laboratory and it is widely used for screening of samples.
• It is an inexpensive test, so it is useful for routine screening and for testing of large number of patients.
• It is useful for monitoring treatment response and reinfection. After successful treatment, the antibody titer usually decreases, so the progress of disease can be followed.
• It can detect syphilis in patient without clear symptoms. So, it is useful in latent or asymptomatic infection also.
• It is an important serological test because Treponema pallidum cannot be cultured in artificial media. So, serological diagnosis is needed.
• It is useful in evaluation and diagnosis of congenital syphilis in infants.
• It can also be used for investigation of other non-venereal treponemal diseases like yaws and pinta, because it is a non-specific test.

Limitations of VDRL Test

The following are the limitations of VDRL test–

• VDRL test is not specific for syphilis, because it detects reagin antibodies released due to damaged host cells and not the specific antibodies of Treponema pallidum. So, false positive result may occur in HIV, Lyme disease, malaria, tuberculosis, systemic lupus erythematosus, pregnancy and old age.
• A reactive VDRL test does not confirm syphilis infection by itself. It should be confirmed by specific treponemal test like TPHA or FTA-ABS for proper diagnosis.
• VDRL test cannot differentiate sexually transmitted syphilis from non-venereal treponemal infections. So, it may become reactive in diseases like yaws and pinta also.
• False negative result may occur in early stage of syphilis, because enough antibodies are not formed in the body. It is also less reliable in late stage of syphilis.
• Prozone phenomenon may give false negative or weakly reactive result. It occurs when antibody concentration is very high, commonly in secondary syphilis or HIV co-infection, and proper clumping is not formed.
• The antibodies detected by VDRL test may remain in the patient body even after successful treatment of syphilis. So, reactive result does not always indicate active infection.
• Diagnosis of congenital syphilis is difficult by VDRL test, because maternal antibodies can cross the placenta and produce reactive result in infant. Testing of umbilical cord blood may also give false positive due to maternal blood contamination or false negative due to Wharton’s jelly.
• CSF VDRL test is highly specific for neurosyphilis, but it has low sensitivity. So, negative CSF VDRL does not completely rule out neurosyphilis.
• VDRL test has some technical limitations also. The VDRL antigen suspension is unstable and should be prepared freshly, serum must be heated at 56°C for 30 minutes, and plasma cannot be used because heating causes clotting which interferes with microscopic reading.

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