syphilis annual tech

7/31/2019 SYPHILIS Annual Tech

http://slidepdf.com/reader/full/syphilis-annual-tech 1/41

-

DOLLY D. BAÑARES, RMT

NATIONAL BLOOD CENTER



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 it’s pathogenicity by reducing or limiting

antigenic stimulation of the host’s immune system

Does not survive in citrated blood at 4ºC for more than 72hrs.



T. pallidum subspecies pallidum - syphilis

T. pallidum subspecies pertenue - yaws

T. pallidum subspeciesendemicum - non-

venereal (endemic)syphilis/bejel

T. carateum - pinta

Species- indistinguishableserologically andmorphologically

Differentiation- based onclinical & epidemiologicalcharacteristics



Nontreponemal Methods (Screening): Venereal Disease Research Laboratory (VDRL) Rapid Plasma Reagin (RPR)

Treponemal Methods (Confirmatory): 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)



RPR test The RPR test is a non treponemal

testing procedure for the serologicdetection of syphilis



Principle of RPR

The RPR Card antigen suspension A carbon particle cardiolipin antigen Detects and quantify reagin

Reagin An antibody like substance present in serum or plasma from

individuals with syphilis

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



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 AntigenDistilled Water

Dispenstirs

Rotator



RPR Test Background

White plastic coated card that consistof several circles that are 18 mm indiameter

Controls (strongly reactive, weaklyreactive, and non-reactive) arecontained 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 bereported as reactive (even ifminimally reactive) or non-

reactive



Procedure for Test

Label rings on test card with number ofsamples to be tested

Use Dispenstir to draw up serum sample

Hold Dispenstir in a perpendicular positiondirectly over the test circle to which thespecimen is to be delivered

Squeeze Dispenstir to allow 1 drop to fall onto each circle



Procedure for Test

Invert Dispenstir,and using the sealed endspread the specimen in the confines of thecircle

Reconstitute the antigen bottle, by shaking.Holding the bottle in a straight verticalposition drop one or two drops in the uppercorner of each test circle, then place one

“free falling” drop on each test area


http://slidepdf.com/reader/full/syphilis-annual-tech 22/41Rotate at 100rpm for 8 mins



rotated manually by hand 3 to 4 rotations and thenread immediately macroscopically in the “wet” state

under a high intensity lamp



Explanation of Results

A negative RPR test may indicate one ofthe following:

1. The patient does not have syphilis2. The infection is too recent for antibodies

to be produced. (Repeated tests should beadministered at 1 week, 1 month, and 3month intervals to establish presence orabsence of disease)

3. The syphilis is latent or inactive4. Faulty immunodefense mechanism5. Faulty lab techniques



Explanation of Results

A positive reaction is not conclusive forsyphilis. Several conditions produce biologicfalse positive results for syphilis

(False positive means that the test revealed apositive reaction when it was actuallynegative)

False positives may reveal the presence ofother 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



False positive RPR tests may beresolved by testing the patient’s serum

with a specific treponemal antigen test



Confirmatory Tests for Syphilis

I. FTA-ABSFlourescent Treponemal Antibody Test

II. TP-PAParticle Agglutination T. pallidum Test

III. MHA-TP

MicroHemagglutination Assay - T. pallidum



TP-PAPassive Particle Agglutination Test

for Detection of antibodies toTreponema pallidum



Principle of TP-PA

The kit is manufactured using gelatinparticle 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

solutionB. Sample Diluent

( liquid )

C. Sensitized

particles

( lyophilized )

D. Positive

control( liquid )

E. Droppers

(25ul)



QUALITATIVE TEST PROCEDURE

WELL NO 1 2 3 4Sample Diluent (ul)

Test Specimen (ul)

100

25

25

25

25

25

25

25

Test Specimen

Dilution 1:5 1:10 1:20 1:40

Unsensitized

Particle (ul) 25

Sensitized

Particle (ul) 25

Final Dilution 1:40 1:80

DISCARD

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 abutton in the center of the well with a

smooth round outer margin.( - ) NEGATIVE

Particles concentrated in the shape of acompact ring with a smooth round outer

margin.( + / - ) INDETERMINATE

Definite large ring with a rough multiform

outer margin and peripheral agglutination. ( + )POSITIVE

Agglutinated particles spread out coveringthe bottom of the well uniformly. ( ++ )



CRITERIA FOR INTERPRETATION

WELL FINALDILUTION

REACTION

UnsensitizedParticles 1:40 ( - )SensitizedParticles

1:80 ( + )POSITIVE



CRITERIA FOR INTERPRETATION

WELL FINALDILUTION

REACTION

UnsensitizedParticles 1:40 ( - )

SensitizedParticles

1:80 ( - )NEGATIVE



1. Microparticles

2. Conjugate

3. Assay Diluent

4. Pre-trigger Solution

5. Trigger Solution

6. Wash Buffer

syphilis annual tech

Documents