SEROLOGICAL TESTS FOR

DIAGNOSING SYPHILLIS
Tuesday, March 13, 2018 1:51 PM

SYPHILIS
CHANCRE FORMATION : 20 DAYS AFTER, THE FF ANTIBODY APPEARS
­NONTREPONEMAL ANTIBODIES
ƒ NON­SPECIFIC
ƒ REAGIN ­
- PRODUCT OF THE INTERACTION OF VARIOUS TISSUE PHOSPHOLIPID PRODUCTS
EITHER DERIVED FROM THE HOST OR FROM THE SPIROCHETE REACTING WITH THE
IMMUNE SYSTEM OF THE HOST
○ TREPONEMAL ANTIBODY
ƒ DUE TO THE ORGANISM
TEST FOR SYPHILLIS IS CATEGORIZED INTO TWO
a. NONTREPONEMAL TESTS
ƒ SCREENING TESTS SUCH AS RAPID PLASMA REAGIN TEST
ƒ REAGIN ANTIBODY TESTS
ƒ ALWAYS PERFORMED FIRST
ƒ IF DETECTED = PROCEED TO QUANTITATIVE NT TEST IF TITER IS TOO HIGH PERFORM
CONFIRMATORY TEST
ƒ TWO MOST POPULAR
□ VDRL
 VENEREAL DISEASE RESEARCH LABORATORY
 THE VERY FIRST SLIDE TEST FOR SYPHILLIS
 WAS THE GOLD STANDARD SLIDE TEST FOR SYPHILLIS
 PRINCIPLE : FLOCCULATION
 USED FOR DIAGNOSIS OF NEUROSYPHILLIS
◊ JUST CENTRIFUGE CSF ; USE SUPERNATANT ; COMPLEMENTS ARE ABSENT
 REQUIREMENTS
◊ COLLECT SERUM; INACTIVATE THE SAMPLE (56C FOR 30 MINS; 60­62C
FOR 2­3 MINS DO NOT EXCEED 65C AND 5 MINS = DENATURATION OF
PROTEIN / GELATINIZATION OF SERUM)
` DESTROY NATIVE COMPLEMENTS
` REMOVE ANTI­COMPLEMENTARY SUBSTANCES
` STABILIZE SEROLOGIC PROPERTIES OF SERUM
◊ PREPARE ANTIGEN SUSPENSION
` CARDIOLIPIN ­ FROM BEEF'S HEART
– COLORLESS FLUID ; ALCOHOLIC EXTRACT
– FROM MITOCHONDRIAL MEMBRANES WHICH ARE
CHEMICALLY PHOSPHOLIPIDS (PHOSPHATIDYL GLYCEROL)
` LECITHIN : DISSOLVING MEDIUM FOR CARDIOLIPIN
– NEUTRALIZE ANTI­COMPLEMENTARY PROPERTY OF
CARDIOLIPIN
` CHOLESTEROL : CENTER OF ABSORPTION
– ENHANCE THE EFFECTIVE REACTING SURFACE OF
CARDIOLIPIN
– ENHANCE COMPLEMENT FIXING CAPACITY OF CARDIOLIPIN
` MIX VDRL ANTIGEN WITH VDRL BUFFER DLUENT (5.9 ­ 6.1)
TRANSFER IN AMBER BOTTLE = MILKY WHITE
` STORE IN AMBER BOTTLE (PHOTOREACTIVE)
` ONLY FOR 24 HOURS
◊ CALIBRATE DELIVERY NEEDLES USED TO DISPENSE ANTIGEN
` QUALITATIVE
– 18 GAUGE BEVEL
– 1 ML = 60 DROPS +/­ 2 (58­62)
– 1 DROP = 0.017 ML
` QUANTITATIVE
– 19 GAUGE ­ USE SENSITIZED ANTIGEN
◊ CARRY OUT TEST AT 23­29C (73­75F)
◊ 14 MM DIAMETER OF RING FOR SERUM TESTS
◊ CSF : 16 MM; DEPTH OF 1.75 (BOERNER SLIDE)
 PROCEDURE
◊ 50 UL SERUM SAMPLE + 1 DROP ANTIGEN
◊ CONTROL : NSS (NEG C)
◊ MIX.
◊ ROTATE AT 180 RPM FOR 4 MINS
 ◊ OBSERVE PRESENCE OF FLOCCULES MICROSCOPICALLY
` FLOCCULES : SMALL RODS IN CLUMPS
 REPORTING
◊ REACTIVE | WEAKLY R | BORDERLINE R | NONREACTIVE
□ RPR
 RAPID PLASMA REAGIN TEST
 USES RPR CARD ANTIGEN
 CSF CANNOT BE USED
 PRINCIPLE : Flocculatiom
 USE 20 GAUGED NEEDLE (delivery needle) = 0.017 ML
◊ FITTED INTO A DISPENSING BOTTLE
◊ ANTIGEN SUSPENSION : BLACK DUE TO CHARCOAL
` CARDIOLIPIN COATED WITH CHARCOAL PARTICLES
` CHARCOAL AS VISUALIZING AGENT
◊ + AND ­ CONTROLS ARE PROVIDED
 18 MM CIRCLE CARD (ADAPTED FOR HUMIDIFIED MECHANICAL ROTATOR)
 CHEMICAL PRESRVATIVE AND PHOSPHATES STABILIZA THE RGT (LAST FOR
YEARS)
 BREWER SLIDE : ANTICOAGULANT AND LECTIN ; FOR FIELD WORKS
 TEAR­DROP CARD : ALTERNATIVE ; NO NEED FOR MECHANICAL ROTATOR;
ADAPTED FOR MANUAL ROTATION
ƒ WASSERMAN COMPLEMENT FIXATION TEST
 THE VERY FIRST SEROLOGIC TEST FOR SYPHILLIS
 USES EXTRACTS DERIVED FROM SYPHILLITIC LESIONS (CHANCRE)
b. TREPONEMAL TESTS
ƒ CONFIRMATORY TESTS
ƒ Tests
□ TPI (T. pallidum IMMOBILIZATION TEST)
 FIRST STANDARD COINIFRMATORY TEST FOR SYPHILLIS
 HANDLE LIVE TREPONEMES
 TREPONEMES CAMOUFLAGE AS FIBRONECTIN IN VIVO
□ FLUORESCENCE TREPONEMAL ANTIBODY ABSORPTION TEST
 RECOMMENDED STANDARD BY CDC
 INDIRECT FLUORESCENCE METHOD
 SLIDE COATED WITH SYPHILLITIC LESIONS (SMEARS FROM SYPHILLITIC LESIONS)
 ADD PATIENT'S SERUM
 ADD ANTIBODY TAGGED WITH FLUORESCENT DYE
 ILLUMINATED SPIROCHETES (COMPLEXED WITH THE ANTIBODY)
□ HEMAGGLUTINATION PROCEDURES (INDIRECT = uses carriers)
 Uses microtiter plate
 Diluent : TRIS buffer
 Control cells : Avian RBC with Reiter Strain ­ Non­Pathogenic
 Test cells : Avian RBC with Nichol Strain ­ Pathogenic
Well 1 : dilution Well 2: control cells W3: test cells
Result : Cell button + = smooth mat of agglutinated cell
Hole : Saprophytic ­ = button of non­agglutinated cell
Treponemes remove by
using absorbing diluent
(Sorbent ­ phosphate

buffer saline containing
sonicated red cell
membranes of ox or
goat / reiter
treponemes ) =
Indeterminate
 T. pallidum Hemagglutination test (TPHA)
◊ Avian erythrocytes (Glutaraldehyde­Fixed)
` Glutaraldehyde : will promote cross­linking of antigen to the carrier
◊ Before : formalinized tanned sheep rbc (tannic Acid)
` Tannic Acid or Chromic chloride will promote cross­linking
 Hemagglutination test for T. pallidum (HATP)
◊ Avian erythrocytes (Glutaraldehyde­Fixed)
 Microhemagglutination test for Treponema pallidum (MHATP)
◊ Use sheep's rbc as carrier