Comprehensive Guide to the Rapid Plasma Reagin (RPR) Test: Syphilis Screening, Procedure, and Interpretation

Table of Contents

• Introduction to RPR Test
• Principle of RPR Test
• Requirements for RPR Test
• Procedure of RPR Test
• Result Interpretation of RPR Test
• Applications of RPR Test
• Advantages of RPR Test
• Limitations of RPR Test
• References

Introduction to Rapid Plasma Reagin (RPR) Test

• Rapid Plasma Reagin (RPR) is a screening test for syphilis, similar to the Venereal Disease Research Laboratory (VDRL) test.
• Syphilis is a sexually transmitted infection caused by the spirochete bacterium Treponema pallidum.
• RPR is a rapid non-treponemal test that detects non-specific antibodies in the patient's blood which may suggest a syphilis infection.
• The test does not detect antibodies against T. pallidum directly, but instead identifies IgM and IgG antibodies produced against lipoprotein-like material released from damaged host cells due to T. pallidum infection.
• It may also detect antibodies against cardiolipin that could be released from the treponemes.
• These antibodies are collectively referred to as ‘reagins’.
• The test antigen used is a modified VDRL antigen suspension.
• This suspension includes choline chloride to eliminate the need for heat inactivation of the serum.
• Ethylenediaminetetraacetic acid (EDTA) is added to enhance the stability of the suspension.
• Finely divided charcoal particles are included in the antigen suspension to act as a visualizing agent.

Principle of RPR Test

• The Rapid Plasma Reagin (RPR) test is a macroscopic, non-treponemal flocculation card test.
• It detects antibodies produced against antigens released from damaged host cells in patients with syphilis.
• During the test, RPR antigen is mixed with either unheated or heated serum, or with unheated plasma, on a plastic-coated card.
• The antigen mixture used for detection contains 0.03% cardiolipin, 0.21% lecithin, and 0.9% cholesterol, along with choline chloride, EDTA, and charcoal particles.
• If antibodies are present in the sample, they bind with the lipid particles in the antigen suspension, leading to agglutination.
• The charcoal particles coagglutinate with these antibodies and appear as black clumps on the white card.
• If antibodies are absent, the mixture remains uniformly gray, indicating a negative result.

Requirements for RPR Test

• Patient’s serum or plasma
• RPR antigen suspension
• Control serum samples
• Plastic-coated RPR cards
• Mechanical rotator
• Pipettes

Procedure of RPR Test

The RPR test can be carried out in both qualitative and quantitative formats. Specimens that test reactive in the qualitative test are further evaluated using the quantitative method to determine antibody titers.

Qualitative Method

• Using a pipette, add one drop (50 µl) of the patient’s specimen, as well as positive and negative control samples, onto separate reaction circles on the RPR card.
• Spread each drop evenly and smoothly within the circle.
• Without mixing or spreading, add one drop of diluted RPR antigen suspension to each sample (including controls).
• Place the card on an automatic rotator set to 100 ± 2 rpm and rotate for 8 minutes.
• After mechanical rotation, manually tilt and rotate the card gently three to four times back and forth to help distinguish nonreactive results from weakly reactive ones.
• Observe the test results under a high-intensity light source. Look for macroscopic flocculation (black clumps) to indicate a reactive sample.

Quantitative Method

• For samples that show weak or reactive results in the qualitative test, perform serial dilutions to determine endpoint titers.
• Test the serum undiluted (1:1) and at dilutions of 1:2, 1:4, 1:8, and 1:16.
• Add 50 µl of 0.9% saline to reaction circles 2 through 5 (do not spread).
• Add 50 µl of the serum to circle 1 and also to circle 2.
• Mix the contents of circle 2 by aspirating and dispensing with a pipette eight times, avoiding bubbles.
• Transfer 50 µl from circle 2 (1:2 dilution) to circle 3 and mix.
• Then transfer 50 µl from circle 3 (1:4 dilution) to circle 4 and mix.
• Continue by transferring 50 µl from circle 4 (1:8 dilution) to circle 5 (1:16 dilution), mix, and discard the final 50 µl.
• Add exactly one free-falling drop (17 µl) of RPR antigen suspension to each circle without mixing.
• Place the card on a rotator under a humidifying cover and rotate at 100 ± 2 rpm for 8 minutes.
• Immediately remove the card, and gently rotate and tilt it by hand (three or four to-and-fro motions).

If the sample remains reactive at the highest dilution tested (1:16), continue with further dilutions:

• Prepare a 1:50 dilution of nonreactive serum in 0.9% saline to use as a diluent.
• Make a 1:16 dilution of the test specimen by mixing 0.1 ml of serum with 1.5 ml of 0.9% saline. Mix thoroughly.
• Dispense 50 µl of the 1:50 nonreactive serum diluent into circles 2 through 5.
• Add 50 µl of the 1:16 diluted test specimen to circle 1 and also to circle 2.
• Perform serial twofold dilutions by transferring and mixing as before.
• Complete the test using the same steps as described in the earlier procedure.

Result Interpretation of RPR Test

• Presence of characteristic antigen-antibody clumps (black) in the center or the periphery of the test circle indicates a positive RPR test.
• Absence of antigen-antibody clumps, indicated by slight roughness and no aggregates, suggests a negative test.
• All reactive serum requires serial dilution to estimate antibody titer.
• The titer is reported as the reciprocal of the highest dilution which shows a positive test result.
• When the quantitative RPR card test is performed on patients with syphilis, a fourfold rise in titer in a repeat specimen may suggest an infection, a reinfection, or a treatment failure.
• A fourfold decrease in titer following treatment for early syphilis usually indicates that therapy was adequate.

Applications of RPR Test

• It is used mostly as the screening test for syphilitic infection.
• Combined with specific antibody testing, the RPR test allows confirming the diagnosis of active infection and starting treatment.
• Screening for syphilis is a routine part of pregnancy tests.
• The 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.

Advantages of RPR Test

• The RPR test is an effective, easy to perform, and fast screening test.
• It is readily available in kit form for purchase.
• Results are observed without the use of a microscope.
• In addition to screening for syphilis, a titer can be used to track the progress of the disease over time and its response to therapy.
• Since the organism Treponema pallidum cannot be cultured in artificial media, the screening of syphilis via serological testing such as RPR becomes important.
• The patient need not have the symptoms of syphilis for this test to be accurate. It can detect syphilis very effectively in patients without symptoms.
• Since it is a non-treponemal test (non-specific test), it is used to investigate syphilis along with other treponematoses, such as Yaws and Pinta.
• It offers more effective screening than the VDRL test.

Limitations of RPR Test

• Without some other evidence for the diagnosis of syphilis, a reactive non-treponemal test such as RPR does not confirm Treponema pallidum infection. Any reactive RPR test must be confirmed with a specific or treponemal test such as TPHA or FTA-ABS test.
• The anti-lipoidal antibodies detected are not only produced as a consequence of syphilis and other treponemal diseases, but also may be produced in response to non-treponemal diseases of an acute and chronic nature in which tissue damage occurs.
• The RPR test isn’t always accurate. For example, false-negative results may arise if an individual had syphilis for less than three months, as it could take longer for the body to make antibodies. The test is also unreliable in late-stage syphilis.
• False-positive results can be seen in conditions like HIV, Lyme disease, malaria, pneumonia, systemic lupus erythematosus, IV drug use, and tuberculosis.
• The antibodies produced as a result of a syphilis infection can stay in the body even after syphilis has been treated.
• A nonreactive RPR card test with clinical evidence of syphilis can be seen in early primary syphilis; in secondary syphilis, as a result of the prozone reaction; and in some cases of late syphilis.
• The RPR card test cannot be used to test spinal fluids.
• The RPR card test may be reactive in persons from areas where yaws, pinta, or nonvenereal syphilis is endemic.

References

• Jameson, J. L., Kasper, D. L., Braunwald, E., Fauci, A. S., Hauser, S. L., & Longo, D. L. (2005). Harrison’s Principles of Internal Medicine (16th ed.). New York: McGraw-Hill Medical Publishing Division.
• Centers for Disease Control and Prevention (CDC). (1998). Syphilis Testing Manual: Chapter 10 - Nontreponemal Tests. Retrieved from: https://www.cdc.gov/std/syphilis/manual-1998/chapt10.pdf
• Matthews, H. M., Yang, T. K., & Jenkin, H. M. (1979). Unique lipid composition of Treponema pallidum (Nichols virulent strain). Infection and Immunity, 24(3), 713–719.
• University of Rochester Medical Center (URMC). Rapid Plasma Reagin (RPR) Syphilis Test. Retrieved from: https://www.urmc.rochester.edu/encyclopedia/content.aspx?contenttypeid=167&contentid=rapid_plasma_reagin_syphilis