Rapid Plasma Reagin (RPR) Test – Principle, Procedure, Result, Applications

What is Rapid Plasma Reagin (RPR) Test?

• The Rapid Plasma Reagin (RPR) test is a screening test primarily used to detect syphilis, a sexually transmitted infection caused by the bacteria Treponema pallidum. Similar to the Venereal Disease Research Laboratory (VDRL) test, the RPR test is a non-treponemal test that identifies non-specific antibodies in the patient’s blood that may indicate a syphilis infection.
• Unlike treponemal tests that directly target antibodies against the causative bacterium, the RPR test focuses on identifying antibodies known as IgM and IgG that react to lipoprotein-like material released from damaged host cells caused by T. pallidum. These antibodies, commonly referred to as “reagins,” serve as indicators of a potential syphilis infection.
• The RPR test utilizes an antigen suspension derived from a modified VDRL antigen. This suspension contains choline chloride, which eliminates the need for heat inactivating the serum. Additionally, it incorporates ethylenediaminetetraacetic acid (EDTA) to enhance suspension stability, and finely divided charcoal particles that act as a visualizing agent.
• The RPR test, also known as the RPR titer, is categorized as a rapid diagnostic test that detects non-specific antibodies associated with syphilis or related non-venereal treponematoses. Alongside the Wassermann test and VDRL test, the RPR test belongs to the group of nontreponemal tests used for syphilis diagnosis. The term “reagin” indicates that this test does not specifically target antibodies against Treponema pallidum itself but rather against substances released by damaged cells, such as cardiolipin and lecithin, triggered by the presence of the bacterium.
• Traditionally, syphilis serologic testing follows a two-step approach. Initially, a nontreponemal test (NTT) like the RPR or VDRL test is performed as a screening tool. Positive results from the NTT are then confirmed using a specific treponemal test (TT) such as the TPPA or FTA-ABS. This algorithm, endorsed by the U.S. Centers for Disease Control and Prevention (CDC), is widely accepted. It is important to note that the NTT titer can also be used to monitor the progression of the disease and its response to treatment over time.
• Although the RPR test serves as an effective screening tool for syphilis, the traditional two-step approach using an NTT followed by a TT remains the standard procedure in many regions around the world.

Principle of Rapid Plasma Reagin (RPR) Test

The principle of the Rapid Plasma Reagin (RPR) test is based on the detection of antibodies produced in response to antigens released by damaged host cells during a syphilis infection. The RPR test is a macroscopic, non-treponemal flocculation card test that utilizes a specific antigen to facilitate the detection of these antibodies.

During the test, the RPR antigen is combined with either unheated or heated serum, or with unheated plasma, on a plastic-coated card. The antigen used for detection consists of various components, including 0.03% cardiolipin, 0.21% lecithin, and 0.9% cholesterol. In addition, the antigen contains choline chloride, EDTA, and charcoal particles.

If antibodies against syphilis are present in the patient’s sample, they will interact with the lipid particles present in the antigen. This interaction leads to the agglutination or clumping together of the lipid particles. Additionally, the antibodies also bind with the charcoal particles, causing them to coagglutinate with the antibodies.

The visual result of the RPR test is observed on the plastic-coated card. In the presence of antibodies, the agglutinated lipid and charcoal particles form black clumps against the white background of the card, making them easily visible. This indicates a positive test result.

On the other hand, if antibodies against syphilis are not present in the sample, the test mixture remains uniformly gray, indicating a negative test result.

By observing the agglutination or lack thereof, healthcare providers can interpret the results of the RPR test and determine the presence or absence of antibodies associated with a syphilis infection.

It is important to note that the RPR test is a screening test and not a definitive diagnostic tool. Positive results obtained from the RPR test require further confirmatory testing using specific treponemal tests to establish a definitive diagnosis of syphilis.

Requirements for Rapid Plasma Reagin (RPR) Test

To perform the Rapid Plasma Reagin (RPR) test, several requirements are necessary. These include:

1. Patient’s Serum/Plasma: The RPR test requires a sample of the patient’s serum or plasma, which is obtained through a blood draw. This sample contains antibodies that may indicate the presence of syphilis infection.
2. RPR Antigen Suspension: The RPR antigen suspension is a crucial component of the test. It is a prepared mixture containing specific antigens, such as cardiolipin, lecithin, cholesterol, choline chloride, EDTA, and charcoal particles. The antigens allow for the interaction with patient antibodies, leading to agglutination reactions.
3. Control Serum Samples: Control serum samples with known RPR test results are necessary for quality control purposes. These samples serve as reference points to ensure the accuracy and reliability of the test.
4. Plastic-Coated RPR Cards: The RPR test is conducted on plastic-coated cards. These cards provide a solid surface for performing the test and facilitate the observation of agglutination reactions. The cards typically have designated wells or areas for adding the antigen, patient samples, and control samples.
5. Mechanical Rotator: A mechanical rotator or shaker is required to mix the RPR antigen suspension, patient samples, and control samples thoroughly. This ensures proper interaction between the antigens and antibodies, promoting accurate test results.
6. Pipettes: Pipettes are used for precise and controlled dispensing of the RPR antigen suspension, patient samples, and control samples onto the designated areas of the plastic-coated RPR cards. This allows for consistent and standardized testing procedures.

Procedure of Rapid Plasma Reagin (RPR) Test

The examination can be conducted qualitatively and quantitatively. Those qualitatively reactive assays are subjected to quantitative testing to determine antibody titres.

Qualitative Method of RPR Test

The qualitative method of the Rapid Plasma Reagin (RPR) test involves the following steps, ensuring the accurate and reliable interpretation of test results:

1. Pipetting Test Specimen and Controls: One drop (50 µl) of the test specimen, as well as positive and negative controls, is carefully pipetted onto separate reaction circles of the RPR card. The test specimen refers to the patient’s serum or plasma, while the controls are used for quality assurance and comparison purposes.
2. Adding Antigen Suspension: After the test specimen and controls are spread smoothly on the card, a drop of diluted antigen suspension is added to each reaction circle containing the measured volume of the test specimen, positive control, and negative control. It is important not to spread or move the antigen suspension once added.
3. Rotating the Card: The card is gently placed on an automatic rotator, and it is rotated continuously at a specific speed of 100 ± 2 rotations per minute (rpm) for a duration of 8 minutes. This rotation ensures thorough mixing of the test components and promotes the interaction between the patient’s antibodies and the antigen.
4. Hand Rotation and Tilting: After the automatic rotation is complete, a brief hand rotation and tilting of the card are performed. This involves three or four to-and-fro motions to aid in differentiating nonreactive (negative) results from minimally reactive results.
5. Macroscopic Flocculation Check: The card is then examined macroscopically under a high-intensity light source. The purpose is to observe any visible flocculation, which appears as clumps or precipitates in the reaction circles. These flocculation patterns are indications of agglutination, suggesting the presence of antibodies against syphilis in the test specimen.

Quantitative Method of RPR Test

The quantitative method of the Rapid Plasma Reagin (RPR) test involves a series of dilutions and measurements to determine the endpoint titer of serum specimens. Here are the steps involved:

1. Diluting Specimens: Dilute all serum specimens that showed rough nonreactive results in the qualitative test to an endpoint titer. Test each specimen undiluted (1:1) and in dilutions of 1:2, 1:4, 1:8, and 1:16.
2. Placing Saline: Place 50 µl of 0.9% saline in circles numbered 2 through 5 on the RPR card. Do not spread the saline.
3. Adding Serum: Using a pipette, place 50 µl of serum in circle 1 and another 50 µl of serum in circle 2.
4. Mixing Serum and Saline: Mix the serum in circle 2 with the saline by drawing the mixture up and down in the pipette eight times, ensuring no bubble formation.
5. Dilution Transfer: Transfer 50 µl from circle 2 (1:2) to circle 3, mix. Transfer 50 µl from circle 3 (1:4) to circle 4, mix. Transfer 50 µl from circle 4 (1:8) to circle 5 (1:16), mix, and then discard the last 50 µl.
6. Adding Antigen Suspension: Add exactly 1 free-falling drop (17 µl) of the antigen suspension in each circle. Do not mix.
7. Rotating the Card: Place the card on the rotator under a humidifying cover and rotate it for 8 minutes at a specific speed of 100 ± 2 rotations per minute (rpm).
8. Hand Rotation and Tilt: Immediately remove the card from the rotator and briefly rotate and tilt the card by hand using three or four to-and-fro motions.
9. Reactivity Assessment: If the highest dilution tested (1:16) is reactive, the following steps are taken:

• Prepare a 1:50 dilution of nonreactive serum in 0.9% saline for making 1:32 and higher dilutions of the specimen to be tested.
• Prepare a 1:16 dilution of the test specimen by adding 0.1ml of serum to 1.5ml of 0.9% saline and mix thoroughly.
• Place 50 µl of the 1:50 nonreactive serum diluent in circles 2 through 5 of a new RPR card.
• Using a safety pipetting device with a disposable tip, place 50 µl of the 1:16 dilution of the test specimen in circle 1 and 50 µl in circle 2.
• Make serial twofold dilutions using the same pipette and tip. Complete the test as described in the steps above.

By following these steps, the quantitative method of the RPR test allows for the determination of endpoint titers for serum specimens. This method involves precise dilutions, mixing, and rotation, enabling a more detailed assessment of the patient’s antibody response to syphilis.

Result Interpretation of Rapid Plasma Reagin (RPR) Test

Interpreting the results of the Rapid Plasma Reagin (RPR) test involves the following considerations:

1. Positive Test: The presence of characteristic antigen-antibody clumps (black) in the center or periphery of the test circle indicates a positive RPR test. These clumps are visible macroscopically and serve as evidence of the interaction between the patient’s antibodies and the RPR antigen.
2. Negative Test: A negative test is indicated by the absence of antigen-antibody clumps. The test circle may show slight roughness, but there are no visible aggregates or clumps present.
3. Serial Dilution and Antibody Titer: All reactive serum samples require serial dilution to estimate the antibody titer. The titer is reported as the reciprocal of the highest dilution that shows a positive test result. For example, if the highest dilution at which clumping occurs is 1:32, the reported titer would be 32.
4. Monitoring Disease Progression and Treatment Response: In cases where the quantitative RPR test is performed on patients with syphilis, changes in titers over time can provide valuable information. A fourfold rise in titer in a repeat specimen may suggest an ongoing infection, reinfection, or treatment failure. Conversely, a fourfold decrease in titer following treatment for early syphilis usually indicates that the therapy was adequate and effective in reducing the antibody levels.

Interpreting the RPR test results requires careful observation of the presence or absence of antigen-antibody clumps. Positive results indicate the presence of antibodies associated with syphilis infection, while negative results suggest the absence of such antibodies. Serial dilution and titer determination allow for quantification and monitoring of antibody levels. This information aids in assessing disease progression, treatment response, and the need for further investigation or intervention.

Applications of Rapid Plasma Reagin (RPR) Test

The interpretation of the Rapid Plasma Reagin (RPR) test results has several important applications in clinical settings. These applications include:

1. Screening for Syphilis: The RPR test is commonly used as a screening test for syphilis. It helps identify individuals who may be infected with the bacterium Treponema pallidum, which causes syphilis. By detecting the presence of non-specific antibodies associated with syphilis, the RPR test acts as an initial step in the diagnostic process.
2. Confirming the Diagnosis: The RPR test, when combined with specific antibody testing, plays a crucial role in confirming the diagnosis of active syphilis infection. Specific treponemal tests, such as the TPPA (Treponema pallidum particle agglutination) or FTA-ABS (Fluorescent Treponemal Antibody Absorption), are performed after a positive RPR test result to confirm the presence of antibodies against Treponema pallidum. This confirmation is important for accurate diagnosis and appropriate treatment.
3. Pregnancy Screening: Screening for syphilis is a routine part of pregnancy tests. Pregnant individuals are often tested for syphilis using the RPR test to identify any potential infection. Early detection and treatment of syphilis during pregnancy are vital to prevent complications and ensure the health of both the pregnant individual and the fetus.
4. Co-infection or High-Risk Activities: The RPR test is also performed in certain situations, such as when an individual is being treated for another sexually transmitted infection (STI) like gonorrhea, or when there is a known HIV infection. Additionally, individuals engaged in high-risk sexual activities, such as unprotected sex or multiple partners, may undergo the RPR test as part of regular screening to detect syphilis early.

Advantages of Rapid Plasma Reagin (RPR) Test

The interpretation of the Rapid Plasma Reagin (RPR) test offers several advantages, making it a valuable tool in the screening and diagnosis of syphilis. These advantages include:

• Effectiveness and Ease of Use: The RPR test is an effective and straightforward screening test for syphilis. It provides reliable results and is relatively easy to perform, making it accessible to healthcare professionals in various clinical settings.
• Availability in Kit Form: The RPR test is readily available in kit form, allowing for convenient procurement. The availability of standardized kits simplifies the testing process and ensures consistent and accurate results.
• Macroscopic Result Observation: Unlike some other diagnostic tests that require the use of a microscope, the RPR test allows for the direct observation of results with the naked eye. This simplifies the interpretation process and reduces the need for specialized equipment.
• Disease Progress and Response Monitoring: One of the advantages of the RPR test is its ability to track the progress of syphilis over time and monitor the response to therapy. By measuring the titer, or the level of antibodies, healthcare professionals can assess the effectiveness of treatment and make informed decisions regarding patient management.
• Importance in Detecting Treponema pallidum: Treponema pallidum, the bacterium responsible for syphilis, cannot be cultured in artificial media. As a result, serological tests such as the RPR test become essential for screening and diagnosing syphilis. The RPR test allows for the detection of syphilis in patients even in the absence of symptoms, enabling early intervention and prevention of complications.
• Non-Specific Test for Investigating Syphilis and Related Infections: The RPR test is a non-treponemal test, meaning it is not specific to Treponema pallidum. This test can also detect antibodies associated with other treponematoses such as Yaws and Pinta. Therefore, it serves as a comprehensive screening tool for investigating a range of treponemal infections.
• Enhanced Screening Capability: Compared to the Venereal Disease Research Laboratory (VDRL) test, the RPR test has been found to be more effective in screening for syphilis. It offers improved sensitivity and specificity, leading to enhanced accuracy in detecting the presence of syphilis antibodies.

Limitations of Rapid Plasma Reagin (RPR) Test

While the interpretation of the Rapid Plasma Reagin (RPR) test offers valuable insights, it also has certain limitations that need to be considered. These limitations include:

• Confirmation with Specific Test: A reactive RPR test alone does not confirm the presence of Treponema pallidum infection. It is necessary to confirm the results with a specific or treponemal test such as the TPHA (Treponema pallidum hemagglutination assay) or FTA-ABS (Fluorescent Treponemal Antibody Absorption) test. These tests provide more specific evidence of syphilis infection.
• Production of Antibodies in Other Diseases: The antibodies detected by the RPR test are not exclusive to syphilis and other treponemal diseases. They can also be produced in response to various acute and chronic non-treponemal diseases that involve tissue damage. Therefore, a positive RPR result does not necessarily indicate syphilis infection.
• Accuracy Limitations: The RPR test may not always provide accurate results. False-negative results can occur in individuals who have had syphilis for less than three months because it takes time for the body to produce antibodies. Additionally, the test becomes less reliable in late-stage syphilis when antibody levels may decline.
• False-Positive Results: False-positive RPR results can occur in certain conditions such as HIV, Lyme disease, malaria, pneumonia, systemic lupus erythematosus, intravenous (IV) drug use, and tuberculosis. These conditions can lead to the production of non-specific antibodies that may react with the RPR test.
• Persistence of Antibodies: Antibodies produced as a result of a syphilis infection can persist in the body even after successful treatment. Therefore, a positive RPR test does not necessarily indicate active syphilis infection, especially in individuals who have been treated in the past.
• Nonreactive Results in Specific Situations: Nonreactive RPR test results may be observed in early primary syphilis, secondary syphilis (due to the prozone reaction), and certain cases of late syphilis. In these situations, despite clinical evidence of syphilis, the RPR test may not detect the antibodies.
• Inability to Test Spinal Fluids: The RPR card test is not suitable for testing spinal fluids. Alternative tests, such as the Venereal Disease Research Laboratory (VDRL) test or specific treponemal tests, are used for diagnosing syphilis in cerebrospinal fluid samples.
• Reactivity in Endemic Areas: Individuals from regions where yaws, pinta, or non-venereal syphilis are endemic may produce antibodies that react with the RPR test. This can lead to false-positive results, highlighting the importance of considering the local epidemiology when interpreting the test.

FAQ

References

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