Venereal Disease Research Laboratory (VDRL) Test

Syphilis is a sexually transmitted infection (STI) caused by the bacterium Treponema pallidum. It can cause serious complications if left untreated, such as damage to the brain, heart, and other organs. Therefore, it is important to diagnose and treat syphilis as early as possible.

The Venereal Disease Research Laboratory test (VDRL) is one of the screening tests for syphilis. It is a blood test that detects antibodies that your body produces in response to antigens released by cells damaged by the syphilis bacteria. Antigens are substances that trigger an immune response in your body. Antibodies are proteins that bind to and neutralize antigens.

The VDRL test does not directly look for the syphilis bacteria, but rather for the presence of antibodies that indicate a past or current infection. The test can be performed on blood or cerebrospinal fluid (CSF), which is the fluid that surrounds your brain and spinal cord. The test can be done on CSF if there is a suspicion of neurosyphilis, which is a severe form of syphilis that affects the nervous system.

The VDRL test is a simple and rapid test that can be done in a laboratory or at a point-of-care setting. It is also inexpensive and widely available. However, the VDRL test is not specific for syphilis and can give false-positive or false-negative results in some cases. Therefore, a positive VDRL test must be confirmed by another test that is more specific for syphilis, such as the treponemal antibody test (TPA).

The VDRL test is useful for screening people who are at risk of syphilis, such as those who have symptoms of syphilis, have had sexual contact with someone who has syphilis, are pregnant, have HIV, or engage in high-risk sexual behaviors. The test can also be used to monitor the response to treatment and to detect possible reinfection or relapse.

The Venereal Disease Research Laboratory (VDRL) test is a type of non-treponemal test that detects antibodies produced against antigens released by damaged host cells in patients suffering from syphilis. Syphilis is a sexually transmitted infection (STI) caused by the bacterium Treponema pallidum. The VDRL test does not directly identify the bacterium, but rather the immune response to it.

The VDRL test uses an antigen (referred to as VDRL antigen) containing cardiolipin, lecithin, and cholesterol. These are lipids that are similar to those found in the cell membranes of humans and bacteria. The antigen, suspended in a buffered saline solution, forms flocculates (clumps) when combined with lipoidal antibodies (also called reagin) in serum or cerebrospinal fluid (CSF) from syphilis patients. Lipoidal antibodies are non-specific antibodies that react with lipids from both the host and the pathogen.

The VDRL test can be performed on either blood or CSF samples. Blood samples need to be heat-inactivated at 56°C for 30 minutes to remove non-specific inhibitors (such as complement) that may interfere with the test. CSF samples do not require heat-inactivation. A drop of antigen is added to a drop of sample on a slide and mixed by rotating the slide. The slide is then examined under a microscope for the presence or absence of flocculation. Flocculation indicates a positive test result, meaning that the sample contains lipoidal antibodies. No flocculation indicates a negative test result, meaning that the sample does not contain lipoidal antibodies.

The VDRL test can be performed qualitatively or quantitatively. A qualitative test gives a simple positive or negative result, while a quantitative test measures the antibody titre (the highest dilution of the sample that still gives a positive result). A quantitative test can help monitor the course of infection and treatment response.

The VDRL test is one of the screening tests for syphilis, but it is not specific for syphilis. It can also give positive results in other conditions that cause tissue damage or inflammation, such as lupus, leprosy, malaria, HIV, and pregnancy. Therefore, a positive VDRL test needs to be confirmed by a treponemal test, which is specific for T. pallidum. Examples of treponemal tests are fluorescent treponemal antibody absorption (FTA-ABS) test and Treponema pallidum particle agglutination (TPPA) test.

To perform a VDRL test, you will need the following materials and equipment:

• Patient`s serum or cerebrospinal fluid (CSF) sample. Serum is the liquid part of the blood that remains after clotting. CSF is the fluid that surrounds the brain and spinal cord. You can collect serum by drawing blood from a vein using a needle and a syringe. You can collect CSF by performing a lumbar puncture or spinal tap, which involves inserting a needle into the lower back and withdrawing some fluid from the spinal canal.
• Water bath. This is a device that maintains a constant temperature of water for heating or cooling purposes. You will need a water bath to inactivate the serum by heating it at 56°C for 30 minutes. This step removes non-specific inhibitors that may interfere with the test results.
• Freshly prepared cardiolipin antigen. This is the reagent that reacts with the antibodies in the serum or CSF sample. Cardiolipin antigen is a colorless alcoholic solution containing 0.03% cardiolipin, 0.21% lecithin, and 0.9% cholesterol. Cardiolipin is a lipid molecule that is found in damaged host cells and mimics the antigens released by T. pallidum, the bacterium that causes syphilis.
• VDRL slide. This is a disposable glass slide that has several circular wells or reaction circles where you can place the sample and the antigen. Each well can hold about 50 microliters (µl) of liquid.
• Mechanical rotator. This is a device that rotates the slide at a fixed speed and angle to mix the sample and the antigen. The rotation also enhances the formation of flocculation or clumping, which indicates a positive reaction.
• Pipettes and hypodermic syringe with unbeveled needle. These are tools that allow you to measure and transfer small volumes of liquid accurately and precisely. You will need pipettes to transfer the sample and the antigen to the slide wells, and a syringe with an unbeveled needle to prepare serial dilutions of the sample for quantitative testing.
• Microscope. This is an instrument that magnifies small objects using lenses and light. You will need a microscope to examine the slide for flocculation after rotating it for four minutes.
• Known reactive and non-reactive serum controls. These are samples that have known results for the VDRL test. You will need them to check the validity and accuracy of your test procedure and reagents. A reactive control should show flocculation, while a non-reactive control should show no flocculation.

These are the basic requirements for performing a VDRL test. However, you should also follow proper safety precautions, such as wearing gloves, goggles, and lab coats, when handling biological samples and chemicals. You should also dispose of used materials properly and sanitize your work area after completing the test.

The VDRL test can be performed on blood or cerebrospinal fluid (CSF) samples, depending on the stage and symptoms of syphilis. The test can be done qualitatively or quantitatively. Qualitative tests are used to screen for syphilis infection, while quantitative tests are used to measure the antibody levels and monitor the treatment response.

Qualitative Method

1. Patients’ serum is inactivated by heating at 56°C for 30 minutes in a water bath to remove non-specific inhibitors (such as complement).
2. VDRL antigen suspension (colloidal suspension of tissue cardiolipin or chemically synthesized cardiolipin), controls and samples are brought to room temperature.
3. One drop (50 µl) of the test specimen, positive and negative controls is pipetted onto separate reaction circles of the disposable slide.
4. A drop of diluted antigen suspension is added to the measured volume of specimen, positive and negative controls.
5. Using a mixing stick the test specimen and the VDRL reagent is mixed such that it thoroughly spreads uniformly over the entire reaction circle.
6. The slide gently rotated and continuously either manually or on a mechanical rotor at 180 r.p.m.
7. Flocculation is checked microscopically using 10X objective and eye piece at about 8 minutes.

Quantitative Method

1. Dilute serum samples to an endpoint titer. Quantitative tests for 3 serum specimens through the 1:8 dilution may be performed on one slide.
2. Place 50 µl of 0.9% saline in circles numbered 2 through 4. Do not spread saline.
3. Using a safety pipette device, place 50 µl of serum in circle 1 and 50 µl of serum in circle 2.
4. Mix the saline and the serum in circle 2 by drawing the mixture up and down in the safety pipette eight times.
5. Transfer 50 µl from circle 2 (1:2) to circle 3, and mix.
6. Transfer 50 µl from circle 3 to circle 4, mix, and then discard the last 50 µl.
7. Gently re-suspend the antigen suspension.
8. Add exactly 1 freefalling drop (17 µl) of antigen suspension to each circle.
9. Place the slide on the mechanical rotator. Rotate the slide for 4 minutes at 180 ±2 rpm.
10. Immediately after rotation, read the test.
11. 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. Complete the test as described above.

The VDRL test results are either reactive or nonreactive. A reactive result means that the test detected antibodies to syphilis in the blood or CSF sample. A nonreactive result means that the test did not detect any antibodies to syphilis.

However, a reactive result does not necessarily confirm a syphilis infection, and a nonreactive result does not necessarily rule out a syphilis infection. This is because the VDRL test is a nontreponemal test, which means that it is not specific for syphilis and can yield false-positive or false-negative results.

Some possible causes of false-positive results are:

• Other infections, such as HIV, Lyme disease, malaria, tuberculosis, etc.
• Autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, etc.
• Pregnancy, especially in the third trimester
• IV drug use
• Vaccination
• Old age

Some possible causes of false-negative results are:

• Early or late stage of syphilis infection
• Prozone phenomenon, which occurs when the antibody concentration is too high and interferes with the antigen-antibody reaction
• Antibiotic treatment
• Immunosuppression

Therefore, any reactive VDRL test result must be confirmed with a treponemal test, such as TPHA, FTA-ABS, or TP-EIA. These tests are specific for syphilis and can detect antibodies to the T. pallidum bacterium.

The VDRL test can also be used to measure the antibody titers in the blood. The titer is the reciprocal of the highest dilution that shows a positive reaction. For example, if a 1:8 dilution is positive but a 1:16 dilution is negative, the titer is 8. The titer can indicate the stage and activity of the infection. Generally, higher titers are associated with more active infection and lower titers are associated with less active or treated infection.

The VDRL test can also be used to monitor the response to treatment. The antibody titers should decrease after effective treatment and become nonreactive within 6 to 12 months for primary or secondary syphilis and within 12 to 24 months for latent or tertiary syphilis. If the titers do not decrease or increase after treatment, it may indicate treatment failure or reinfection.

The VDRL test has several applications in the diagnosis and management of syphilis and other treponemal diseases. Some of the main applications are:

• 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). The VDRL test can help monitor the response to therapy and detect any relapse or reinfection.
• The VDRL test can also be used to detect if syphilis has involved the central nervous system (Neurosyphilis) by testing cerebral spinal fluid. The test can help diagnose neurosyphilis in patients with neurological symptoms or signs of late-stage syphilis.
• The VDRL test is also helpful in the diagnosis of congenital syphilis. The test can detect antibodies in the blood of newborns or infants born to mothers with syphilis. The test can help prevent complications such as stillbirth, prematurity, low birth weight, or birth defects.

VDRL test is one of the most widely used and simple tests for syphilis. It has several advantages, such as:

• It is fast and easy to perform, and excellent for screening of large numbers of samples.
• It is helpful in the diagnosis of congenital syphilis, which is the infection of the fetus or newborn by the mother who has syphilis.
• It can be quantitated, which means that the titer (the concentration of antibodies) can be measured and used to monitor the disease activity and response to treatment.
• It can detect syphilis in patients without symptoms, as it checks for antibodies produced as a result of the infection.
• It can also be used to investigate other treponemal diseases, such as yaws and pinta, which are caused by closely related bacteria.

The VDRL test has some limitations that may affect its accuracy and reliability. Some of these limitations are:

• The VDRL test is not specific for syphilis. It can detect antibodies produced in response to other conditions that cause tissue damage, such as leprosy, hepatitis B, infectious mononucleosis, and various autoimmune diseases. These conditions can cause false-positive results, meaning that the test shows a positive reaction even when the person does not have syphilis.
• The VDRL test may not detect syphilis in the early or late stages of the infection. It can take up to three months after exposure for the body to produce enough antibodies to be detected by the test. This means that the test can miss syphilis in people who have been recently infected. On the other hand, the test can also become negative over time as the antibody levels decline, especially after treatment. This means that the test can miss syphilis in people who have had the infection for a long time or who have been cured.
• The VDRL test may be affected by a prozone phenomenon. This occurs when the antibody concentration is too high and interferes with the formation of clumps with the antigen. This can cause a false-negative result, meaning that the test shows a negative reaction even when the person has syphilis. To avoid this, the test should be performed with serial dilutions of the serum sample.
• The VDRL test may cross-react with antibodies from other treponemal infections, such as yaws and pinta. These infections are caused by bacteria that are closely related to T. pallidum, the causative agent of syphilis. This can cause false-positive results, especially in areas where these infections are endemic.

Because of these limitations, the VDRL test cannot confirm or rule out syphilis by itself. It should be used as a screening test, followed by a confirmatory treponemal test, such as TPHA or FTA-ABS. These tests are more specific and sensitive for syphilis and can distinguish between active and past infections.

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