Treponema pallidum

Haemagglutination Assay

Aasma Karki
BScMLT 3rd year
JF Institute of Health Sciences
Syphilis
Introduction
 Infectious veneral disease caused by Treponema
pallidum, sub-species pallidum

 Two types
 Acquired
 Veneral (best known form)
 Non veneral (endemic syphilis)
 Congenital
Syphilis
Laboratory diagnosis
 Direct demonstration of Treponema

 Serological tests for Ab detection

 Non-treponemal (screening)
 Treponemal (confirmatory)

 T. pallidum can’t be cultured in clinical lab

Syphilis
Laboratory diagnosis
 Table. Diagnostic tests in syphilis

Direct demonstration
a. Dark field microscopy
b. Direct immunofluorescence (DFA-TP) test
c. PCR
d. Fontana’s stain
e. Demonstration in tissue (Levadit’s stain)
Syphilis
Laboratory diagnosis
 Table. Diagnostic tests in syphilis

Serological tests
A. Non treponemal (reagin test) VDRL, RPR
B. Treponemal tests
a. Immunofluorescence FTA, FTA-Abs
b. Immobilization test TPI test
c. Haemagglutination test MHA-TP, TPHA
TPHA
Treponema pallidum
Haemagglutination Assay
Treponema pallidum haemagglutination assay
Introduction
 Treponemal test for the serological diagnosis of
syphilis
 Based on the principle of haemagglutination
 Detects anti-treponemal antibodies (IgG and IgM)
in serum or CSF
 Used as confirmatory test for diagnosis
Treponema pallidum haemagglutination assay
Principle
 Test detects human T. pallidum antibodies (IgG and
IgM) based upon indirect haemagglutination in
serum and CSF

 Fowl erythrocytes are sensitized with T. pallidum

fragments (test cells)
Treponema pallidum haemagglutination assay
Principle
 In presence of specific anti T.pallidum Ab
 Sensitized erythrocytes agglutinate
 Induce a characteristic structure on the microtiter plate
in bottom of the well
 Appearance of even layer

 Unspecific reactions are detected using controlled

cells (unsensitized fowl erythrocytes)
Treponema pallidum haemagglutination assay
Principle
Treponema pallidum haemagglutination assay
Method
 Test sample diluted in absorbing diluent
 To remove possible cross-reacting heterophile Ab
 To remove, block or absorb potentially cross-reacting
non-pathogenic treponemal Abs
Treponema pallidum haemagglutination assay
Method
 Sera containing Abs to T.pallidum react with
erythrocytes (chicken or avian) sensitized with
sonicated T.pallidum, Nichols strain (the Ag), to
form a smooth mat of agglutinated cells in the
microtiter tray well

 If Abs are not present, cells settle to bottom of the

tray well, forming a compact button of
unagglutinated cells
Treponema pallidum haemagglutination assay
Reagent
 Test cell suspension
 Preserved RBCs treated with tannic acid and coated
with T.pallidum antigen

 Control cell suspension

 Preserved RBCs (without immobilized T.pallidum Ag)
Treponema pallidum haemagglutination assay
Reagent
 Buffer
 Phosphate buffered saline solution containing Ab
absorbers (used to remove possible cross-reacting
heterophile Abs)
Treponema pallidum haemagglutination assay
Reagent
 Positive control serum
 Human serum containing Ab against T.pallidum ready
for use
 This will give an equivalent titer of 1/640 to 1/2560
with the quantitative test

 Negative control serum

 Human serum free of Abs against T.pallidum
Treponema pallidum haemagglutination assay
Procedure
 Before performing the test procedure, bring the
sample, diluent, control and test cells in room
temperature (25 – 30º C)

 For each qualitative test, a test card with three

wells is needed
Treponema pallidum haemagglutination assay
Procedure
 Dilution of serum sample 
 Add 10μL of patient’s serum in first well (say well A)
 Add 190 μL of diluent (provided by manufacturer)
 Mix the content well using a micropipette

 We will use this diluted serum later

Treponema pallidum haemagglutination assay
Procedure
 Testing of serum sample for presence of specific
antibodies 
 Add 75μL of “control cells” to well B and 75 μL of
“test cells” to well C
 Add 25μL of diluted serum on each B and C well
 Shake the plate gently to mix the contents thoroughly
 Cover the plate and protect to direct sunlight, heat and
any source of vibration
Treponema pallidum haemagglutination assay
Procedure
 Testing of serum sample for presence of specific
antibodies 
 Incubate 45-60 minutes at room temperature
 Read the test results and interpret
 Positive control and negative control should be run
along with the test serum
Treponema pallidum haemagglutination assay
Results and Interpretation 
Results Test Cells Control Cells
Strongly Full cell pattern covering No agglutination
Reactive the bottom of the well. tight button
Weakly Reactive Cell pattern covers approx. No agglutination
1/3 of well bottom tight button

Indeterminate Cell pattern shows a No agglutination

(Equivocal) distinctly open center tight button
Nonreactive Cells settled to a compact No agglutination
bottom, typically with a tight button
small clear center
Treponema pallidum haemagglutination assay
Results and Interpretation 
 If the controls (positive control and negative
control) do not give the expected result, all assays
performed in that batch are invalid and must be
tested again
Treponema pallidum haemagglutination assay
Results and Interpretation 
 Reactive (R)
 May indicate an active, past, or successfully treated
infection
 A diagnosis should be made with a careful history of
the patient and a physical examination as well as
pertinent laboratory results

 Indeterminate
 Confirmed with MHATP and FTA-ABS tests
Treponema pallidum haemagglutination assay
Results and Interpretation 
 False Positive results
 Although TPHA test is highly specific, false positive
results have been known to occur in patients suffering
from
 Leprosy
 Infectious mononucleosis
 Connective tissue disorders

 For confirmation FTA-ABS test should be used

THANK