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7/31/2019 SYPHILIS Annual Tech
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DOLLY D. BAÑARES, RMT
NATIONAL BLOOD CENTER
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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.
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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
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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)
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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)
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RPR test The RPR test is a non treponemal
testing procedure for the serologicdetection of syphilis
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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
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Antibodies associated with syphilis Begin to appear in blood 4 to 6 weeks
after infection IgG and IgM antibodies
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Materials for RPR
RPR Test Cards
RPR Control Cards
RPR AntigenDistilled Water
Dispenstirs
Rotator
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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
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Reactions for Controls
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Reactive control - characteristic
strong clumping
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Weakly Reactive control - moderate
clumping
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Non-reactive control - smooth, grayish
appearance of unclumped particles
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The test results should bereported as reactive (even ifminimally reactive) or non-
reactive
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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
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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
7/31/2019 SYPHILIS Annual Tech
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rotated manually by hand 3 to 4 rotations and thenread immediately macroscopically in the “wet” state
under a high intensity lamp
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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
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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
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Non-syphilitic Conditions Giving
Biologic False-Positive Results
Malaria
Leprosy
Relapsing fever Infectious
Mononucleosis
Atypical pneumonia
Viral pneumonia
Lupus erythematosus
Measles Pregnancy
Drug abuse
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False positive RPR tests may beresolved by testing the patient’s serum
with a specific treponemal antigen test
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Confirmatory Tests for Syphilis
I. FTA-ABSFlourescent Treponemal Antibody Test
II. TP-PAParticle Agglutination T. pallidum Test
III. MHA-TP
MicroHemagglutination Assay - T. pallidum
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TP-PAPassive Particle Agglutination Test
for Detection of antibodies toTreponema pallidum
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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
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A.Reconstituting
solutionB. Sample Diluent
( liquid )
C. Sensitized
particles
( lyophilized )
D. Positive
control( liquid )
E. Droppers
(25ul)
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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 )
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Incubate for 2 hours
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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. ( ++ )
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CRITERIA FOR INTERPRETATION
WELL FINALDILUTION
REACTION
UnsensitizedParticles 1:40 ( - )SensitizedParticles
1:80 ( + )POSITIVE
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CRITERIA FOR INTERPRETATION
WELL FINALDILUTION
REACTION
UnsensitizedParticles 1:40 ( - )
SensitizedParticles
1:80 ( - )NEGATIVE