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Serological Diagnostic Tests for Syphilis Rapid Plasma Reagin (RPR) Test and Venereal Disease Research Laboratory (VDRL) Assay Prof. Dr. Ezzat M Hassan Prof. of Immunology Med Res Inst, Alex Univ E-mail: elgreatlyem@hotmail.com

Objectives To know the causative agent of Syphilis To know diagnostic evaluation of the disease To define different serological tests for syphilis To describe the basics and procedure of RPR To describe the basics and procedure of VDRL To know the difference between RPR & VDRL To describe the basics & procedure of FTA-ABS test To know how to interpret the results of different tests

Syphilis: Causative Agent and Disease Description The causative agent for syphilis is the bacterium Treponema pallidum . Initially, T. pallidum penetrates intact mucous membranes or enter the body through tiny defects in the epithelium. Upon entrance, the microorganism is carried by the circulatory system to every organ of the body. Untreated, syphilis is a chronic infectious disease with many clinical manifestations that occur in four distinct stages: Primary, Secondary, Latent and Tertiary (Late) stages, separated by asymptomatic intervals.

Diagnostic Evaluation The diagnosis of syphilis is based on Clinical picture Darkfield Microscopy Demonstration of microorganisms in the lesion by Serological tests for specific and non-specific Antibodies

Serological Tests In patients with Syphilis 2 types of antibodies are produced: Nonspecific antibodies- produced against Lipoidal antigen present in both host tissues and T. pallidum and are known as nontreponemal (Reagin) antibodies. Detected by nontreponemal Screening serological tests – Venereal Disease Research Laboratory (VDRL) and Rapid Plasma Reagin (RPR) procedures Specific antibodies- produced against T. pallidum. Detected by Treponemal Confirmation serological tests – Fluorescent Treponema pallidum-absorbed (FTA-ABS) and Microhemagglutination Treponema pallidum (MHA-TP)

Tests For Reagin Antibody They measure the presence of Reagin, which is an antibody formed in reaction to syphilis A large numbers of tests for Reagin: VDRL (Venereal Diseases Reference Laboratory). RPR (Rapid Plasma Reagin) ART (Automated Reagin Test) Good sensitive screening Tests Reagin titre falls rapidly with treatment.

RPR test (Rapid Plasma Reagin Test ) The RPR is a nontreponemal test for the serologic detection of syphilis.

Theory of the Reaction Syphilis infection starts the breakdown of the patient's own tissue cells. Fatty substances which are released, combine with protein from T. pallidum to form an antigen which stimulates the body to produce antibodies against both: the body's tissue lipids (non-specific or non-treponemal) the T. pallidum protein (specific or treponemal). The RPR Card test detects the nonspecific antilipid antibody and is referred to as a non-treponemal test for syphilis. .

Principle of RPR The RPR antigen suspension consists of Cardiolipin, lecithin, and cholesterol bound to charcoal particles which make the reaction visible The serum of a person with syphilis contains a non-specific anti-lipid antibody (termed Reagin), which is not found in normal serum. The reagin binds to the test antigen particles that cause macroscopic flocculation with the resulting aggregation of the carbon particles. The flocculation appears as black clumps against the white background of the plastic coated card.

Materials for RPR RPR Test Cards (white plastic coated card that consist of several circles that are 18 mm in diameter.) RPR Control Cards (strongly reactive, moderately reactive, and non-reactive controls are contained on the control card in a dried form.) RPR Antigen (consists of Cardiolipin, lecithin, and cholesterol bound to charcoal particles.) Distilled Water Dispenstirs Rotator

Samples not suitable for Testing Type of samples used in the Test Serum Plasma (centrifuge to remove fibrin before testing) Samples not suitable for Testing Haemolysed Lipemic High conc. Of Billirubin

Procedure for Test Label rings on test card with numbers of samples to be tested Place one drop of sample into separate circles on the test card and one drop of water to each +ve and –ve controls on control test card Swirl the RPR-carbon reagent gently before using. Add one drop (20 µl) of this reagent next to the controls and the samples to be tested. Mix the drops with a dispenstir, spreading them over the entire surface of the circle. Use different dispenstirs for each sample. Place the slide on a mechanical rotor (shaker) at 80-100 r.p.m. for 8 minutes. After rotating mechanically, the test card should be rotated manually by hand 3 to 4 rotations and then read immediately macroscopically in the “wet” state under a high intensity lamp.

Reactions for Controls The following reactions should be observed to compare against the test results: Reactive control - characteristic strong clumping. Reactive moderate control - moderate clumping. Non-reactive control - smooth, grayish appearance of unclumped particles

Agglutination+++ RPR Agglutination++

Results of the Test The test results should be reported as: Reactive (even if minimally reactive) or Non-reactive.

The VDRL Test (Venereal Disease Research Laboratory Test)

Principles VDRL Antigen is a nontreponemal antigen composed of cardiolipin, cholesterol and lecithin. The nontreponemal tests measures anti-lipid antibodies, which are formed by the host in response to lipids released from damaged host cells early in infection with T. pallidum, and lipid-like material frmm the treponemal cell surface.

Required Materials VDRL Antigen with buffered saline solution composed of cardiolipin, cholesterol and lecithin. Control sera : Reactive, weakly reactive and nonreactive 0.9% saline slide cards (serum) or concavity slides(CSF) Stirrers Rotator

Antigen Suspension Preparation Pipette 0.4ml of VDRL buffered saline to the bottom of a round 30 ml glass Stoppard bottle with a flat inner-bottom surface. Gently tilt bottle so that VDRL buffered saline will cover the entire inner-bottom surface of the bottle. Add 0.5 ml of VDRL Antigen drop by drop at a rate that allows about 6 sec for 0.5 ml of antigen while continuously but gently rotating the bottle. Add 4.1 ml of VDRL buffered saline. Do not drop saline directly on antigen; allow it to flow down the side of the bottle.

Antigen Suspension Mix by gentle inversion. Allow to stand at least 5 minutes but no more than 2 hours. Remix suspension by swirling only

Procedure: Step 1 Wells should be labeled R, WR, NR, and PS for reactive, weakly reactive, and nonreactive, respectively.

Procedure: Step 2 Pipette .05ml of specimen into one of an agglutination slide.

Procedure: Step 3 Add one drop (.01 ml) of sensitized antigen suspension to each specimen

Procedure: Step 4 Rotate slides for 8 minutes on a mechanical rotator at 180 rpm. Note: when the tests are performed in a dry climate, the slides may be covered with a box lid to prevent evaporation.

Procedure: Step 5 Read test immediately after rotation with a 10x objective.

Reading Slide Results R WR NR PS

Non reactive and Reactive VDRL Tests

Results Qualitative Testing - Medium to large clumps (Reactive); Small clumps (Weakly Reactive); No clumping or very slight roughness (Nonreactive). Quantitative Testing is Performed to endpoint on all serum samples that produce reactive results in the qualitative slide test. Report the titer as the highest dilution that produces a Reactive (not weakly reactive) results

VDLR vs RPR 1. RPR is the more advanced VDRL and can detect syphilis more effectively than a VDLR. 2. RPR Ag is just the VDRL Ag, but it contains carbon particles. 3. The specimen for VDLR should be heated before testing, unlike for RPR specimen 4. A microscope is not needed in RPR; the result can be seen by naked eye. In contrast, a VDLR requires a microscope to read the result of the test. 5. RPR, is easy to use and can be readily purchased in kit form in contrast to a VDLR test. 6. A VDLR test is the only test that can be performed using cerebrospinal fluid (CSF)

Confirmatory Tests for Syphilis (Treponemal specific Tests) Treponemal tests confirm syphilis when a reactive non-treponemal result is obtained. These tests Include: I. FTA-ABS : Flourescent Treponemal Antibody-Absorption test II. TP-PA : T. pallidum Particle Aggluntination test III. TP-HA: T. pallidum HemAgglutination test

The fluorescent treponemal antibody absorption (FTA-ABS) Test It measures a specific antibody made against Treponema pallidum It should always be followed to confirm a positive RPR and/or VDRL test for syphilis. The ABS suffix refers particularly to a processing step used to remove nonspecific antispirochetal antibodies present in normal serum. Once a person tests positive, he or she will usually test positive for life. Therefore, the test cannot be used to measure disease activity or differentiate past from present infection. very sensitive and highly specific for syphilis, this test tends to be expensive, subjective, and time-consuming, as it requires interpretation by an experienced technician.

FTA-ABS Test Procedure The antigen for the FTA-Abs test is whole bacteria (lyophilized suspension of T. pallidum ) . This is spread over and fixed to a slide. Preheated Patient serum is mixed with an absorbent (the "ABS" part of the test) containing an extract of a non-pathogenic treponeme,  The purpose of the absorbent is to remove anti-treponemal antibodies that are not specific for the syphilis bacteria. The pre-adsorbed patient serum is then added to the slide; if the patient has been infected by syphilis, their antibodies will bind to the bacteria.  FITC (a fluorophore)-labeled anti-human antibodies are added as secondary antibodies. The spirochete location is identified using flourescent microscope FITC staining is +ve if the patient has anti-T. pallidum antibodies.

FTA-ABS Test Result

Interpretation Results of the serum RPR &VDRL tests must be confirmed by a treponemal specific test. Several conditions produce biological false positive results (FBR) for syphilis. (False positive means that the test revealed a positive reaction when it was actually negative). Syphilis is diagnosed when both the screening and confirmatory test are reactive. A reactive treponemal test confirms an old or new diagnosis of syphilis A non-reactive treponemal test suggests that the reactive non-treponemal test was a FBR.

Serological diagnosis for Syphilis

STUDY QUESTION: Write a brief account on : The Principle of RPR Test

Assignment Write shortly on: 1- Screening tests for Syphilis 2- Confirmatory tests for syphlis امال عرابى الكردى - رغدة رشدي - نادية محمد سعد – 1- نيهال كمال خلود عبد القادر - ريوان ابراهيم - 2- فاطمة الزهراء – زينب عبد المنعم

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