بسم الله الرحمن الرحيم. Rapid Plasma Reagin (RPR) Test and Venereal Disease Research Laboratory (VDRL) Assay Serological Diagnostic Tests for Syphilis.

الله بسمالرحيم الرحمن

Rapid Plasma Reagin (RPR) Test and

Venereal Disease Research Laboratory (VDRL) Assay

Serological Diagnostic Tests for Syphilis

Prof. Dr. Ezzat M HassanProf. of ImmunologyMed Res Inst, Alex UnivE-mail: [email protected]

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 TestsIn 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:

1. the body's tissue lipids (non-specific or non-treponemal)

2. 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

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++

Agglutination+ RPR Agglutination -   

Results of the Test

The test results should be reported as:

1. Reactive (even if minimally reactive)

or

2. 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.

Reading Slide Results

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

سعد - - – محمد نادية رشدي رغدة الكردى عرابى نيهال - 1امال كمال

ابراهيم - - ريوان القادر عبد عبد- – 2خلود زينب الزهراء فاطمةالمنعم

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