The Rapid Plasma Reagin (RPR) test is primarily used to screen for syphilis, a sexually transmitted disease caused by the bacteria Treponema pallidum. The RPR test, like the Venereal Disease Research Laboratory (VDRL) test, is a non-treponemal test that detects non-specific antibodies in the patient’s blood that can mean a syphilis infection.
What are plasma reagins?
Unlike treponemal tests, which directly target antibodies against the causal bacterium, the RPR test concentrates on finding IgM and IgG antibodies that react to lipoprotein-like structure produced from T. pallidum-damaged host cells. These antibodies, known generally as “regains“, act as markers of a possible syphilis infection.
The RPR test, also known as RPR titer, is a fast diagnostic test used to detect non-specific antibodies associated with syphilis or other non-venereal treponematoses. Along with the Wassermann and VDRL tests, the RPR test is one of a set of non-treponemal assays used to diagnose syphilis. The word “reagin” suggests that this test does not explicitly target antibodies against Treponema pallidum itself, but rather chemicals produced by injured cells, such as cardiolipin and lecithin, when the bacterium is present.
Specialty of PRP
The RPR test makes use of an antigen suspension made from a modified VDRL antigen. Because this suspension contains choline chloride, there is no need of heat to inactivate the serum. It also contains EDTA, which improves solution stability, as well as finely divided charcoal particles, which act as a visualizing agent.
Traditionally, syphilis serologic testing is done in two steps. As a screening tool, non-treponemal test (NTT) such as the RPR or VDRL test done firstly. Positive NTT results are then validated with a treponemal test (TT) such as the TPPA or FTA-ABS. This algorithm, which has been approved by the Centers for Disease Control and Prevention (CDC), is frequently used. It is vital to note that the NTT titer can also be used to track the disease’s progression and response to treatment over time.
Although the RPR test is an excellent syphilis screening tool, the classic two-step technique of an NTT followed by a TT remains the usual procedure in many parts of the world.
What symptoms leads to RPR test?
This test is needed if someone have ever been active sexually and show syphilis symptoms. The syphilis symptoms are:
Small and painless sores at the area of body where syphilis infection occurs, Fever, Sometimes on the palms of hands or at the bottom of the feet skin rash appears, Hair loss, lymph gland may get swell, Headaches, loss of weight, fatigue or tiredness and muscle aches. If infection remain for years without proper treatment, symptoms may lead to final stages of the disease that involves blindness, paralysis, numbness, and dementia or death.
Principle of RPR test
The Rapid Plasma Reagin (RPR) test works on the idea of detecting antibodies generated 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 uses a specific antigen to identify these antibodies.
On a plastic-coated card, the RPR antigen is mixed with either unheated or heated serum, or unheated plasma. The detection antigen is made up of several components, including 0.03% cardiolipin, 0.21% lecithin, and 0.9% cholesterol. The antigen also contains choline chloride, EDTA, and charcoal particles.
If there are antibodies against syphilis in the patient’s sample, they will interact with the lipid particles in the antigen. This contact causes the lipid particles to agglutinate, or cluster together. Furthermore, the antibodies bind to the charcoal particles, forcing them to coagglutinate with them.
On the plastic-coated card, the visual result of the RPR test is visible. When antibodies are present, the agglutinated lipid and charcoal particles create black clumps on the card’s white background, making them clear to see. This signifies that the test was positive.
If no anti-syphilis antibodies are present in the sample, the test mixture remains uniformly grey, indicating a negative test result.
It is very important to understand that the RPR test is a screening tool, not a definitive diagnosis means. Positive RPR test results necessitate additional confirmation testing with specialized treponemal tests to establish a confirmed diagnosis of syphilis.
Figure 1: this figure represents principle of RPR test
Requirements of RPR test
Serum or Plasma of patient: A sample of the serum or plasma is required for the RPR test, which is collected through a blood draw. This sample contains antibodies that could indicate syphilis infection.
RPR Cards with Plastic Coating: The RPR test is performed on plastic-coated cards. These cards provide a stable surface for the test and make it easier to see agglutination reactions. The antigen, patient samples, and control samples are normally added to predefined wells on the cards.
RPR Antigen Suspension: The most important part of the test is the RPR antigen suspension. It’s a prepared mixture including antigens such lecithin, cardiolipin, cholesterol, EDTA, choline chloride, and charcoal particles. The antigens interact with patient antibodies, resulting in agglutination reactions.
A mechanical rotator or shaker: it is required to completely mix the RPR patient samples, antigen suspension, and control samples. This promotes reliable results from tests by ensuring adequate interaction between antigens and antibodies.
Control Serum Samples: For the quality control, control serum samples with known RPR test outcomes are required. These samples serve as reference points to confirm the test’s correctness and reliability.
Pipettes: these are used to precisely dispense the patient samples, RPR antigen suspension, and control samples on to the proper sections of the plastic-coated RPR cards; which enables uniform and standardized testing techniques.
Figure 1: this figure represents kit for rapid plasma reagin test (RPR test) (Sethi et al. 2007)
Procedure for RPR test
Qualitative method for RPR testing
- One drop (50 µl) of the sample to be tested, as well as positive and negative controls, is properly pipetted onto the RPR card’s reaction circles. The test specimen is the patient’s serum or plasma, whereas the controls are utilized for quality control and comparison.
- Following the smooth distribution of the test specimen and controls on the card, a drop of diluted antigen suspension is added to each reaction circle holding the measured volume of the test specimen, negative control and positive control. It is essential not to transfer the antigen suspension once it has been added.
- The card is placed gently on an automatic rotator and rotated constantly at a predetermined speed of 100 rotations per minute (rpm) for 8 minutes. This rotation ensures that the test components are thoroughly mixed and also supports interaction between the patient’s antibodies and antigen.
- Following the completion of the automatic rotation, a quick hand rotation and bending of the card is conducted. Three or four vibratory motions are used to distinguish nonreactive (or negative) results from slightly reactive results.
- After that, the card is viewed macroscopically with a high-intensity light source. The goal is to look for any apparent flocculation, such as clumps or precipitates in reaction circles. These flocculation patterns indicate agglutination, which indicates the presence of antibodies against syphilis in the test samples.
Quantitative method for RPR testing
- All serum specimens that gave rough nonreactive responses in the qualitative test should be diluted. Each specimen should be tested undiluted (1:1) as well as in dilutions of 1:2, 1:4, 1:8, and 1:16.
- Fill the RPR card with 50 µl of 0.9% saline in the circles numbered 2 through 5. Avoid pouring the saline solution.
- Place 50 µl of serum in circle 1 and another 50 µl of serum in circle 2 with a pipette.
- In circle 2, combine the serum and saline by moving the mixture up and down in the pipette eight times to ensure no bubbles emerge.
- 50 µl from circle 2 (1:2) to circle 3 (mix). Transfer 50 µl (1:4) from circle 3 to circle 4, mix. 50 µl from circle 4 (1:8) to circle 5 (1:16), mix thoroughly and then discard the remaining 50 µl.
- In each circle, place one free-falling drop (17 µl) of the antigen solution. Mixing is not required in this step.
- Place the card on the rotator under a damp cover and rotate it for 8 minutes at 100 rotations per minute (rpm).
- Remove the card from the rotator quickly and spin or tilt it by hand in three or four vibratory motions.
Assessment of reactivity
If the highest dilution tested (1:16) is reactive, the following procedures are followed:
- To make 1:32 and higher dilutions of the sample to be analyzed, make a 1:50 dilution of nonreactive serum in 0.9% saline.
- Make a 1:16 dilution of the test specimen by combining 0.1ml of serum with 1.5ml of 0.9% saline and do thoroughly mixing.
- Fill circles 2–5 of a new RPR card with 50 µl of the 1:50 nonreactive serum diluent.
- Place 50 µl of the 1:16 dilution of the test sample in circle 1 and 50 µl in circle 2 using a safety pipetting equipment with a disposable tip.
- Using the same pipette and tip, perform repeated twofold dilutions. Complete the test in the method given above.
Interpretation of result
- A positive RPR test is indicated by the presence of distinctive antigen-antibody clumps (black) in the centre or boundaries of the test circle. These aggregates are visible under a microscope and serve as proof of the patient’s antibodies interacting with the RPR antigen.
- The absence of antigen-antibody clumping indicates a negative test. The test circle is slightly rough, but there are no obvious aggregates or clumps.
How to measure Antibody Titer in RPR testing?
To determine the antibody titer, all reactive serum samples must be serially diluted. The titer is expressed as the reciprocal of the maximum dilution yielding a positive test result. If the maximum dilution at which clumping occurs is 1:32, the reported titer is 32.
How to Monitor Disease Progression and Treatment Response?
When the quantitative RPR test is used on syphilis patients, changes in titers over time can provide useful information. A titer increase of fourfold in a repeat samples may indicate a continued infection, reinfection, or treatment failure. A fourfold fall in titer following treatment for early syphilis, on the other hand, usually shows that the medication was appropriate and effective in decreasing antibody levels.
Applications of RPR test
- The RPR test is routinely used as a syphilis screening test. It aids in the identification of those who could be infected with the bacterium Treponema pallidum, causing syphilis. The RPR test serves as a first step in the diagnostic procedure by detecting the presence of non-specific antibodies associated with syphilis.
- Syphilis testing is now a standard part of early pregnancy screenings. Pregnant women are frequently tested for syphilis using the RPR test to identify any possible infection. Early detection and therapy of syphilis during pregnancy is essential for avoiding complications and ensuring the health of both the pregnant woman and the fetus.
- When paired with specific antibody testing, the RPR test plays an essential role in verifying the diagnosis of active syphilis infection. Following a positive RPR test result, particular treponemal tests, like the TPPA test (Treponema pallidum particle agglutination) and FTA-ABS test (Fluorescent Treponemal Antibody Absorption), are conducted to establish the existence of antibodies against Treponema pallidum. This confirmation is vital for successful diagnosis and treatment.
- The RPR test is also used in some cases, such as when a person is being treated for another sexually transmitted infections (STIs), such as gonorrhea, or if there is a known HIV infection. Individuals who participate in high-risk sexual activities, i.e. without protection or several partners, may also be subjected to the RPR test as part of routine screening to discover syphilis early.
Advantages of RPR test
- The bacterium Treponema pallidum, which causes syphilis, cannot be cultivated in artificial medium. As a result, serological assays like the RPR test are becoming increasingly important for screening and diagnosing syphilis.
- The RPR test is easily available in kit form, which simplifies the testing process and provides consistent and reliable results.
- The RPR test is a simple and effective syphilis screening test. It produces consistent results and is relatively simple to use, making it accessible to healthcare practitioners in a variety of clinical situations.
- Unlike other diagnostic procedures that necessitate the use of a microscope, the RPR test allows for direct inspection of results with the naked eye.
- The RPR test serves as a non-treponemal test, which means it does not detect Treponema pallidum. Antibodies related with other treponematoses, such as Yaws and Pinta, can also be detected using this test. As a result, it can be used as a comprehensive diagnostic tool for a variety of treponemal illnesses.
- One of the benefits of the RPR test is its ability to follow syphilis growth over time and assess response to medication. Healthcare providers can measure the efficacy of treatment and make sensible choices about patient management by measuring the titer, or level of antibodies.
- The RPR test has proved to be more efficient in diagnosis for syphilis than the Venereal Disease Research Laboratory (VDRL) test. It has higher sensitivity and specificity, which leads to greater accuracy in identifying the presence of syphilis antibodies.
Disadvantages of RPR test
- A positive RPR test does not ensure the presence of Treponema pallidum infection. A specific or treponemal test, i.e. the TPHA (Treponema pallidum hemagglutination assay) or FTA-ABS (Fluorescent Treponemal Antibody Absorption) test, is required to confirm the results. These tests offer more specific proof of syphilis infection.
- Antibodies developed as a result of a syphilis infection might remain in the body long after therapy is completed. As a result, a positive RPR test may not always indicate active syphilis infection, especially in those who have previously been treated.
- The RPR test does not always yield trustworthy results. False-negative observations can occur in people who suffered from syphilis for less than three months because the body takes time to build antibodies. Furthermore, as antibody levels fall in late-stage syphilis, the test becomes less accurate.
- The antibodies found by the RPR test are not limited to syphilis and other treponemal diseases. They can also be generated in response to a variety of non-treponemal acute and chronic illnesses that cause tissue damage. As a result, a positive RPR result may not always be the reason of syphilis infection.
- Nonreactive results of RPR test can be seen in cases of early primary syphilis, secondary syphilis (because of the prozone reaction), and late syphilis. Despite clinical indications of syphilis, the RPR test may fail to detect antibodies in many cases.
- False-positive results of RPR test can arise in a variety of illnesses, including pneumonia, IV medication usage, Lyme disease, systemic lupus erythematosus, HIV, malaria, and tuberculosis. These circumstances can result in the formation of non-specific antibodies that may react with the RPR test.
- Individuals from endemic areas for Pinta, Yaws, or non-venereal syphilis may develop antibodies which react with the RPR test. This can result in false-positive results, emphasizing the significance of taking into account local epidemiology when evaluating the test.
- Sethi S, Sharma K, Dhaliwal LK, Banga SS, Sharma MJSti. 2007. Declining trends in syphilis prevalence among antenatal women in northern India: a 10-year analysis from a tertiary healthcare centre. 83(7): 592-592.