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SYPHILIS

A SEXUALLY TRANSMITTED DISEASE WHICH CAN BE TRANSMITTED CONGENITALLY OR BY VENEREAL ROUTE


-IS

DOLLY D. BAARES, RMT NATIONAL BLOOD CENTER

Causative Agent

Treponema pallidum Slender, tightly coiled, pointed-end, 6-10 axial filamentous gram negative rod

Microaerophilic, obligate parasite Requires mammalian cells for survival Sheath cover aids its pathogenicity by reducing or limiting antigenic stimulation of the hosts immune system Does not survive in citrated blood at 4C for more than 72 hrs.

Treponema pallidum sub speciation

T. pallidum subspecies pallidum - syphilis T. pallidum subspecies pertenue - yaws T. pallidum subspecies endemicum - nonvenereal (endemic) syphilis/bejel T. carateum - pinta

Species- indistinguishable serologically and morphologically Differentiation- based on clinical & epidemiological characteristics

Diagnostic Evaluation

Nontreponemal Methods (Screening):


Treponemal Methods (Confirmatory):

Venereal Disease Research Laboratory (VDRL) Rapid Plasma Reagin (RPR)

Fluorescent Treponema pallidum-absorbed (FTA-ABS) Microhemagglutination Treponema pallidum (MHA-TP) Passive Agglutination Treponema pallidum test (TP-PA)

Clinical Limitations Associated with Serological Testing

Infectious disease (False +s):


Measles Chicken pox Hepatitis Infectious mononucleosis Leptospirosis Malaria Leprosy Rickettsial disease Trypanosomiasis Lymphogranuloma venereum (LGV)

Clinical Limitations Associated with Serological Testing

Noninfectious conditions:

Autoimmune disorders Drug addiction Old age Pregnancy Recent immunization

RPR test
The RPR test is a non treponemal testing procedure for the serologic detection of syphilis

Principle of RPR

The RPR Card antigen suspension


Reagin

A carbon particle cardiolipin antigen Detects and quantify reagin

The reagin (Ag) binds to the test antigen which consists of cardiolipin-lecithin coated particles macroscopic flocculation

An antibody like substance present in serum or plasma from individuals with syphilis

Antibodies

associated with syphilis

Begin to appear in blood 4 to 6 weeks after infection IgG and IgM antibodies

Materials for RPR


RPR

Test Cards RPR Control Cards RPR Antigen Distilled Water Dispenstirs Rotator

RPR Test Background


White

plastic coated card that consist of several circles that are 18 mm in diameter Controls (strongly reactive, weakly reactive, and non-reactive) are contained on each kit

Reactions for Controls

Reactive control - characteristic strong clumping

Weakly Reactive control - moderate clumping

Non-reactive control - smooth, grayish appearance of unclumped particles

The test results should be reported as reactive (even if minimally reactive) or nonreactive

Procedure for Test


Label rings on test card with number of samples to be tested Use Dispenstir to draw up serum sample Hold Dispenstir in a perpendicular position directly over the test circle to which the specimen is to be delivered Squeeze Dispenstir to allow 1 drop to fall on to each circle

Procedure for Test


Invert Dispenstir,and using the sealed end spread the specimen in the confines of the circle Reconstitute the antigen bottle, by shaking. Holding the bottle in a straight vertical position drop one or two drops in the upper corner of each test circle, then place one free falling drop on each test area

Rotate at 100rpm for 8 mins

rotated manually by hand 3 to 4 rotations and then read immediately macroscopically in the wet state under a high intensity lamp

Explanation of Results
A negative RPR test may indicate one of the following: 1. The patient does not have syphilis 2. The infection is too recent for antibodies to be produced. (Repeated tests should be administered at 1 week, 1 month, and 3 month intervals to establish presence or absence of disease) 3. The syphilis is latent or inactive 4. Faulty immunodefense mechanism 5. Faulty lab techniques

Explanation of Results
A positive reaction is not conclusive for syphilis. Several conditions produce biologic false positive results for syphilis (False positive means that the test revealed a positive reaction when it was actually negative) False positives may reveal the presence of other serious diseases

Non-syphilitic Conditions Giving Biologic False-Positive Results

Malaria Leprosy Relapsing fever Infectious Mononucleosis Atypical pneumonia

Viral pneumonia Lupus erythematosus Measles Pregnancy Drug abuse

Resolving False Positive RPR Tests


False

positive RPR tests may be resolved by testing the patients serum with a specific treponemal antigen test

Confirmatory Tests for Syphilis


I. FTA-ABS Flourescent Treponemal Antibody Test II. TP-PA Particle Agglutination T. pallidum Test III. MHA-TP MicroHemagglutination Assay - T.

pallidum

TP-PA
Passive Particle Agglutination Test for Detection of antibodies to Treponema pallidum

Principle of TP-PA
The

kit is manufactured using gelatin particle carriers sensitized with purified pathogenic T. pallidum (Nichols strain) Sensitized particles are agglutinated by the presence of antibodies to T. pallidum in human serum / plasma

A. Reconstituting solution B. Sample Diluent ( liquid ) C. Sensitized particles ( lyophilized ) D. Positive control

( liquid )
E. Droppers (25ul)

QUALITATIVE TEST PROCEDURE


WELL NO
Sample Diluent (ul) Test Specimen (ul)

1
100 25 1:5

2
25 25 1:10

3
25 25 1:20 25

4
25 25 1:40

DISCARD

Test Specimen Dilution Unsensitized Particle (ul) Sensitized Particle (ul) Final Dilution

25 1:40 1:80

MIX CONTENT USING A PLATE MIXER ( 30 SECONDS )

Incubate for 2 hours

INTERPRETATION OF RESULTS
SETTING PATTERNS OF PARTICLE READING INTERPRETATION

Particles concentrated in the shape of a button in the center of the well with a smooth round outer margin.
Particles concentrated in the shape of a compact ring with a smooth round outer margin. Definite large ring with a rough multiform outer margin and peripheral agglutination. Agglutinated particles spread out covering the bottom of the well uniformly.

( - )

NEGATIVE

( + / - )

INDETERMINATE

( + )

POSITIVE

( ++ )

NEGATIVE (-)

INDETERMINATE (-/+)

POSITIVE ( + / ++)

CRITERIA FOR INTERPRETATION

WELL Unsensitized Particles Sensitized Particles

FINAL DILUTION

REACTION

1:40 1:80

(-) (+)

POSITIVE

CRITERIA FOR INTERPRETATION

WELL Unsensitized Particles Sensitized Particles

FINAL DILUTION

REACTION

1:40 1:80

(-) (-)

NEGATIVE

ARCHITECT Syphilis TP

ARCHITECT

Syphilis TP assay

Chemiluminescent Microparticle Immunoassay (CMIA) for the qualitative detection of antibody to Treponema pallidum in human or plasma as an aid to diagnosis of syphilis.

REAGENTS:
1. Microparticles 2. Conjugate 3. Assay Diluent

4. Pre-trigger Solution
5. Trigger Solution 6. Wash Buffer

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