Venereal Disease Research Laboratory (VDRL) Test

What is Venereal Disease Research Laboratory (VDRL) Test?

• The Venereal Disease Research Laboratory (VDRL) test is a commonly used screening test for syphilis, a sexually transmitted infection caused by the spirochete bacterium Treponema pallidum. This test is categorized as a non-treponemal test, meaning it does not directly detect the presence of the bacterium but instead identifies specific antibodies produced by the immune system in response to the infection.
• The VDRL test primarily detects the presence of IgM and IgG antibodies directed against lipoidal material that is released from damaged host cells, as well as antibodies targeting lipoprotein-like material and potentially cardiolipin released by the treponemes. These antibodies, often referred to as “reagins,” serve as indicators of the infection.
• The VDRL test is performed as a slide microflocculation test, where the patient’s blood sample is mixed with a specific antigen derived from the bacterium. If the person has been infected with syphilis, their blood will contain the aforementioned antibodies, which will react with the antigen and cause visible clumping or flocculation under a microscope. This reaction indicates a positive result for the VDRL test.
• The timing of the VDRL test is crucial for its accuracy. It usually becomes positive approximately one to two weeks after the appearance of the primary syphilis lesion, known as a chancre. During the late phase of primary syphilis, the test shows a reactive result in about 50-75% of cases. In secondary syphilis, which occurs after the primary stage, the test becomes highly reactive, showing positive results in nearly 100% of cases. However, as the infection progresses to later stages, the reactivity of the test tends to decrease, with approximately 75% of cases still yielding positive results.
• It is important to note that while the VDRL test is a useful screening tool, it is not sufficient for diagnosing syphilis on its own. If the VDRL test shows a positive result, further, more specific tests are usually conducted to confirm the presence of the infection and determine the stage of syphilis. These additional tests may include treponemal tests, such as the Treponema pallidum particle agglutination (TPPA) test or the fluorescent treponemal antibody absorption (FTA-ABS) test, which directly detect antibodies targeting the treponemes.
• In conclusion, the Venereal Disease Research Laboratory (VDRL) test is a slide microflocculation test used for screening syphilis. By detecting the presence of specific antibodies, it provides valuable information about the infection, particularly in the early and secondary stages. However, it should be used in conjunction with other tests for a comprehensive diagnosis of syphilis.

Principle of Venereal Disease Research Laboratory (VDRL) Test

The VDRL test operates on the principle of detecting antibodies that are produced in response to antigens released by damaged host cells in individuals with syphilis. This test utilizes a specific antigen known as VDRL antigen, which consists of 0.03% cardiolipin, 0.21% lecithin, and 0.9% cholesterol. The antigen is suspended in a buffered saline solution.

During the VDRL test, a drop of the VDRL antigen is placed onto a slide, followed by the addition of a drop of serum from the patient being tested. The slide is then rotated to thoroughly mix the contents. If the patient’s serum contains antibodies against the lipoidal antigens present in the VDRL antigen, flocculation or clumping will occur.

Under a microscope, the presence or absence of flocculation is observed. If flocculation or agglutination is observed, it indicates a reactive specimen, suggesting the presence of antibodies against the lipoidal antigens in the patient’s serum. On the other hand, if the specimen appears as a homogeneous suspension without any clumping, it is considered non-reactive.

Flocculation in the VDRL test signifies the presence of specific antibodies targeting the lipoidal antigens contained in the VDRL antigen. These antibodies are produced in response to the infection by the spirochete bacterium Treponema pallidum, which causes syphilis. The damaged host cells release lipoidal material, including cardiolipin, lecithin, and cholesterol, which are incorporated into the VDRL antigen.

It is important to note that a positive VDRL test indicates the presence of antibodies, but it does not necessarily confirm an active syphilis infection. Further testing, such as treponemal tests, is often required to confirm the diagnosis and determine the stage of the disease.

In summary, the VDRL test relies on the principle of detecting antibodies produced against lipoidal antigens released by damaged host cells in individuals with syphilis. The test involves mixing the patient’s serum with the VDRL antigen and observing for flocculation under a microscope. Flocculation indicates a reactive specimen, suggesting the presence of antibodies against the lipoidal antigens associated with syphilis, while a non-reactive specimen appears as a homogeneous suspension.

Requirements of Venereal Disease Research Laboratory (VDRL) Test

The VDRL test requires several specific components and equipment to ensure accurate and reliable results. The following are the requirements for conducting the VDRL test:

1. Patient’s Serum: The VDRL test necessitates obtaining a blood sample from the patient. The serum, which is the clear liquid portion of the blood after it has coagulated and the cells have been separated, is used for testing.
2. Water Bath: A water bath is required to maintain a controlled temperature during the test. The temperature is typically set at 37°C (98.6°F) to mimic the body’s conditions and optimize the antigen-antibody interaction.
3. Freshly Prepared Cardiolipin Antigen: The VDRL antigen is a crucial component of the test. It contains cardiolipin, lecithin, and cholesterol suspended in a buffered saline solution. The antigen is prepared freshly and should be of high quality to ensure accurate results.
4. VDRL Slide: The VDRL slide is a specialized slide designed for the test. It usually contains multiple small wells or depressions to hold the reagents and facilitate mixing.
5. Mechanical Rotator: A mechanical rotator, also known as a shaker or rotator, is used to mix the VDRL antigen and the patient’s serum thoroughly. The rotation facilitates the interaction between the antigens and antibodies, promoting flocculation if present.
6. Pipettes: Pipettes are necessary for accurately measuring and transferring small volumes of reagents during the test. Different sizes of pipettes may be required depending on the specific volumes needed.
7. Hypodermic Syringe with Unbeveled Needle: A hypodermic syringe with an unbeveled needle is used to draw the patient’s blood sample for serum separation. The unbeveled needle minimizes damage to the red blood cells, ensuring a clear serum.
8. Microscope: A microscope is essential for observing the VDRL test results. The microscope allows for the visualization of flocculation or clumping, indicating a reactive specimen.
9. Known Reactive and Non-Reactive Serum Controls: To validate the accuracy of the test, known reactive and non-reactive serum controls are necessary. These controls, which contain either positive or negative antibodies against syphilis, are tested alongside the patient’s specimen for comparison.

By meeting these requirements, the VDRL test can be conducted effectively, ensuring proper mixing, accurate measurements, and appropriate controls for reliable results. These components and equipment play a vital role in facilitating the detection of antibodies and the interpretation of the test outcome.

Procedure of Venereal Disease Research Laboratory (VDRL) Test

The test can be run in both qualitative and quantitative modes. Qualitatively reactive assays are submitted to quantitative testing to identify antibody titres.

Qualitative Method of VDRL Test

The qualitative method of the VDRL test involves a series of steps to determine the presence or absence of flocculation, indicating a reactive or non-reactive specimen for syphilis. The following describes the procedure in detail:

1. Inactivation of Patients’ Serum: The patients’ serum is first inactivated to remove non-specific inhibitors, such as complement, which could interfere with the test results. This is achieved by heating the serum at 56°C (132.8°F) for 30 minutes in a water bath.
2. Preparation of VDRL Antigen and Controls: The VDRL antigen suspension, which is a colloidal suspension of tissue cardiolipid or chemically synthesized cardiolipin, is brought to room temperature. Similarly, the positive and negative controls, which contain known reactive and non-reactive antibodies, are also brought to room temperature.
3. Pipetting of Specimen and Controls: One drop (50 µl) of the test specimen (patients’ serum), as well as the positive and negative controls, is pipetted onto separate reaction circles of a disposable slide. Each specimen and control is placed in its designated area.
4. Addition of Antigen Suspension: A drop of the diluted antigen suspension is added to the measured volume of the specimen, positive control, and negative control. The antigen and the specimens/controls should be added in the correct proportions.
5. Mixing of Specimen and Antigen: Using a mixing stick, the test specimen and the VDRL antigen suspension are thoroughly mixed to ensure uniform spreading over the entire reaction circle. The mixture should be well-distributed within the designated area on the slide.
6. Rotation of the Slide: The slide is gently rotated either manually or using a mechanical rotor at a speed of 180 revolutions per minute (r.p.m.). This rotation facilitates the interaction between the antibodies in the specimen and the antigens, enhancing the possibility of flocculation.
7. Microscopic Observation: After approximately 8 minutes of continuous rotation, the slide is examined microscopically. A 10X objective lens is typically used, along with the eyepiece, to check for the presence or absence of flocculation. Flocculation appears as clumps or aggregates within the reaction circles.

Quantitative Method of VDRL Test

The quantitative method of the VDRL test involves diluting serum samples to determine an endpoint titer. The following steps outline the procedure for conducting the quantitative VDRL test:

1. Dilution of Serum Samples: Serum samples are diluted to an endpoint titer. Typically, quantitative tests for three serum specimens can be performed on a single slide, starting from a 1:8 dilution. This means that the initial dilution is 1 part serum to 7 parts diluent.
2. Preparation of Circles: In circles numbered 2 through 4 on the VDRL slide, 50 µl of 0.9% saline is placed without spreading the saline.
3. Serum Placement: Using a safety pipette device, 50 µl of the serum sample is placed in circle 1, and another 50 µl is placed in circle 2.
4. Mixing of Saline and Serum: The saline and the serum in circle 2 are mixed by drawing the mixture up and down in the safety pipette eight times. This ensures thorough mixing.
5. Transfer of Serum: 50 µl of the mixture from circle 2 (1:2 dilution) is transferred to circle 3 and mixed. Then, 50 µl from circle 3 is transferred to circle 4, mixed, and the last 50 µl is discarded.
6. Resuspension of Antigen: The antigen suspension is gently re-suspended to ensure homogeneity.
7. Addition of Antigen: Exactly one freefalling drop (17 µl) of the antigen suspension is added to each circle on the slide.
8. Rotating the Slide: The slide is placed on a mechanical rotator and rotated for 4 minutes at a speed of 180 ±2 revolutions per minute (rpm).
9. Reading the Test: Immediately after rotation, the test is read. The results are observed for flocculation or clumping within the circles.

If the highest dilution tested (1:8) is reactive, further steps are taken:

10. Preparation of Additional Dilutions: A 1:8 dilution of the test specimen is prepared in a test tube. This is done by adding 0.1 ml of serum to 0.7 ml of 0.9% saline and mixing thoroughly. Additional serial dilutions are prepared for strongly reactive specimens.
11. Placement of Saline: 50 µl of 0.9% saline is placed into paraffin rings 2, 3, and 4. Serial twofold dilutions are then prepared, starting with ring 2.
12. Completion of the Test: The test is completed as described above, including the addition of antigen and rotation of the slide.

Result Interpretation of VDRL Test

The interpretation of VDRL test results is essential in determining the presence or absence of syphilis antibodies in the patient’s serum. Here is how the results of the VDRL test are interpreted:

1. Positive Test: A positive VDRL test result indicates the presence of antigen-antibody clumps in either the center or the periphery of the test circle. These clumps appear as aggregates or flocculation under microscopic examination. The presence of such clumps confirms the reactivity of the specimen, suggesting the presence of syphilis antibodies.
2. Negative Test: A negative VDRL test result is indicated by the absence of antigen-antibody clumps. The test circle appears as a smooth, even light gray without any noticeable aggregates. A negative result suggests the absence of specific antibodies against syphilis in the tested specimen.
3. Reactive and Weakly Reactive Serum: If a specimen shows reactivity or weak reactivity in the VDRL test, further steps are taken to estimate the antibody titer. This involves performing serial dilutions of the serum to determine the highest dilution that still produces a positive test result. The titer is reported as the reciprocal of this highest dilution. For example, if the highest dilution showing a positive test is 1:32, the reported titer would be 32.

False-Positive Test of VDRL Test

The VDRL test, despite its usefulness, can sometimes yield false-positive results. A false-positive result indicates that the test indicates the presence of syphilis when the individual is actually not infected with the disease. Several conditions and infections can cause false-positive VDRL test results. Here are some examples:

1. HIV: Individuals infected with the human immunodeficiency virus (HIV) may show false-positive results on the VDRL test. This cross-reactivity occurs due to similarities in the immune response and the production of antibodies that can react with the antigens used in the VDRL test.
2. Lyme Disease: Lyme disease, caused by the bacterium Borrelia burgdorferi, can lead to false-positive VDRL test results. This tick-borne illness can trigger an immune response that produces antibodies that may react with the VDRL antigens, leading to a positive result.
3. Certain Types of Pneumonia: Specific types of pneumonia, such as pneumonia caused by Mycoplasma pneumoniae, can result in false-positive VDRL test results. The immune response generated by these infections may produce antibodies that cross-react with the VDRL antigens.
4. Malaria: Malaria, a parasitic infection transmitted through mosquito bites, has been associated with false-positive VDRL test results. The presence of the malaria parasite can trigger an immune response that produces antibodies that may react with the VDRL antigens.
5. Systemic Lupus Erythematosus: Systemic lupus erythematosus (SLE), an autoimmune disease, can lead to false-positive VDRL test results. The immune system’s abnormal response in individuals with SLE can cause the production of antibodies that cross-react with the VDRL antigens.

It is important to note that a false-positive VDRL test result does not indicate the presence of syphilis. Confirmatory tests, such as specific or treponemal tests like TPHA or FTA-ABS, are necessary to differentiate true syphilis infection from these conditions. These tests help in accurately diagnosing syphilis and ruling out false-positive results caused by other factors.

To ensure accurate interpretation of VDRL test results, healthcare providers consider the individual’s medical history, clinical symptoms, and results from additional tests to confirm or rule out syphilis infection in cases where false-positive results are suspected.

Applications of VDRL Test

The VDRL test finds extensive applications in various aspects related to the screening, diagnosis, and follow-up of syphilis infection. Here are the key applications of the VDRL test:

• Screening Test for Syphilis: The VDRL test is primarily employed as a screening tool for detecting syphilitic infections. It is used to identify individuals who may have been exposed to Treponema pallidum, the bacterium responsible for syphilis. Screening for syphilis is often incorporated as a routine part of pregnancy tests to ensure early detection and appropriate management if necessary.
• Pregnancy Screening: The VDRL test plays a crucial role in the prenatal care of pregnant women. It is commonly included in the battery of tests performed during pregnancy to screen for syphilis. Early detection and treatment of syphilis during pregnancy are vital to prevent transmission of the infection from mother to fetus, as syphilis can have severe consequences for the developing baby.
• Comorbidity with Other STIs: When individuals are being treated for other sexually transmitted infections (STIs) such as gonorrhea or are infected with HIV, the VDRL test may be performed as part of a comprehensive evaluation. This is because individuals with one STI may have an increased risk of acquiring or transmitting other infections, including syphilis. The test helps identify syphilis infection and facilitates appropriate treatment and management.
• High-Risk Sexual Activity: Individuals engaged in high-risk sexual behaviors, such as having multiple sexual partners or engaging in unprotected sexual intercourse, may undergo VDRL testing. This aids in detecting syphilis infection early on, allowing for prompt treatment and prevention of further transmission.
• Follow-Up Testing: After receiving treatment for syphilis, follow-up testing is often recommended by the Center for Disease Control and Prevention (CDC). The VDRL test is used in these follow-up evaluations to monitor the effectiveness of treatment and ensure that the infection has been adequately treated. It helps determine if there is a sustained or reduced antibody response, aiding in assessing the patient’s progress and guiding further management decisions.

Advantages of VDRL Test

The VDRL test offers several advantages in the screening and diagnosis of syphilis. Here are the key advantages associated with the VDRL test:

• Widely Used and Rapid Test: The VDRL test is one of the most widely used screening tests for syphilis. It is known for its simplicity and rapidity, allowing for quick results. This makes it highly efficient, especially in situations where timely diagnosis is crucial.
• Easy to Perform: The VDRL test is relatively easy to perform, making it accessible to healthcare professionals in various settings. The procedure involves simple steps, such as mixing the serum sample with the VDRL antigen and observing for clumping or flocculation. Its straightforward nature facilitates its widespread use and integration into routine screening protocols.
• Excellent Screening Capability: The VDRL test is highly effective as a screening tool. It has demonstrated excellent sensitivity in detecting the presence of syphilis antibodies, even in individuals who may not exhibit symptoms of the infection. This characteristic is particularly advantageous as it allows for the identification of asymptomatic cases and aids in early intervention and treatment.
• Diagnostic Value for Congenital Syphilis: The VDRL test is also valuable in diagnosing congenital syphilis, a condition where syphilis is transmitted from an infected mother to her unborn child. By testing the serum samples of infants born to syphilis-positive mothers, the VDRL test can contribute to the early identification of congenital syphilis cases, enabling prompt treatment and appropriate follow-up care.
• Overcoming Culturing Limitations: Treponema pallidum, the bacterium responsible for syphilis, cannot be cultured in artificial media. This limitation necessitates the use of serological tests, such as the VDRL test, to screen for syphilis. By detecting the antibodies produced in response to the infection, the VDRL test effectively fills this diagnostic gap, providing valuable information for syphilis diagnosis.
• Investigation of Other Treponematoses: As a non-treponemal test, the VDRL test is not specific to syphilis alone. It can also be employed to investigate other treponematoses, such as Yaws and Pinta. This versatility allows for broader applications of the VDRL test in the detection and differentiation of various treponemal infections.

Limitations of VDRL Test

The VDRL test, despite its usefulness, has certain limitations that need to be considered. Here are the key limitations associated with the VDRL test:

• Lack of Confirmation: A reactive non-treponemal test, such as VDRL, does not confirm a Treponema pallidum infection without additional evidence. To confirm syphilis infection, a specific or treponemal test, such as TPHA (Treponema pallidum hemagglutination assay) or FTA-ABS (Fluorescent Treponemal Antibody Absorption) test, should be conducted. These tests help differentiate between true syphilis infection and other conditions that may produce reactive VDRL results.
• Non-Specific Antibodies: The VDRL test detects anti-lipoidal antibodies, which are not exclusive to syphilis and other treponemal diseases. These antibodies can also be produced in response to tissue damage caused by various non-treponemal acute and chronic diseases. Therefore, a positive VDRL test does not necessarily indicate syphilis and requires further evaluation and confirmation.
• Limitations in Accuracy: The VDRL test may not always provide accurate results. False-negative results can occur, especially in the early stages of syphilis when the body has not yet produced sufficient antibodies. Late-stage syphilis can also yield unreliable results. Conversely, false-positive results can be observed in conditions such as HIV, Lyme disease, malaria, pneumonia, systemic lupus erythematosus, intravenous drug use, and tuberculosis. These factors highlight the need for careful interpretation and confirmation of results.
• Persistence of Antibodies: Even after successful treatment for syphilis, the antibodies produced in response to the infection can remain detectable in the body. This means that a person who has been treated for syphilis may still have positive VDRL test results, even though the infection has been effectively eradicated.
• Prozone Reaction: In some cases, a prozone reaction may occur, where undiluted serum inhibits reactivity in the VDRL test. This can result in weakly reactive or nonreactive results, leading to potential misinterpretation if not recognized. Dilution of the serum can help overcome this phenomenon.
• Cross-Reactivity with Yaws: In regions where yaws is endemic, the VDRL test may produce reactive results. However, the titers are usually lower (<1:8) compared to those seen in syphilis. Differentiation between syphilis and yaws requires further investigation and consideration of clinical symptoms and epidemiological factors.

In summary, the VDRL test has limitations that must be taken into account. It requires confirmation with specific or treponemal tests, as a reactive result alone does not confirm syphilis infection. The test can produce false-positive or false-negative results, and the presence of non-specific antibodies can complicate interpretation. Additionally, the persistence of antibodies after treatment and the occurrence of prozone reactions should be considered. Awareness of these limitations ensures appropriate use and accurate interpretation of VDRL test results.

FAQ

References

• Nayak S, Acharjya B. VDRL test and its interpretation. Indian J Dermatol. 2012 Jan;57(1):3-8. doi: 10.4103/0019-5154.92666. PMID: 22470199; PMCID: PMC3312652.
• https://www.ucsfhealth.org/medical-tests/vdrl-test
• https://www.orangehealth.in/vdrl-test?utm_source=seo_search&utm_medium=google&utm_campaign=seo
• https://www.rxlist.com/test_venereal_disease_research_laboratory/definition.htm
• https://www.metropolisindia.com/blog/preventive-healthcare/vdrl-test-purpose-procedure-and-results/
• https://gpnotebook.com/simplepage.cfm?ID=-1664090047
• https://redcliffelabs.com/vdrl
• https://www.microrao.com/micronotes/vdrl.pdf
• https://www.apollohospitals.com/patient-care/health-and-lifestyle/understanding-investigations/vdrl-test/
• https://pharmeasy.in/diagnostics/tests/vdrl-test-153
• https://labpedia.net/syphilis-part-2-v-d-r-l-venereal-disease-research-laboratory-test/
• https://www.medicalnewstoday.com/articles/vdrl-test#procedure
• https://www.mountsinai.org/health-library/tests/vdrl-test