Syphilis Serology serological test for medical laboratory.ppt

Chapter three
Syphilis Serology

Learning objective
 At the end of this chapter, the students
should be able to:
 Describe the etiology and sign and symptoms of
primary, secondary, latent and late (tertiary)
syphilis
 Discuss the principle and clinical applications of
the qualitative and quantitative VDRL procedure
and RPR card test
 Describe specific and non specific Treponemal
antibodies

Outline
3.1. Introduction
3.2. The stage of syphilis
3.3. Immune response
3.4. Diagnosis of syphilis
3.5. Serological technique

Definition
 Syphilis a systematic infection caused by the
spirochate Treponema pallidium.
 It is a chronic systemic disease, which leads to
lesions on the body.
 It is derived from a Greek word "syphilos"
meaning crippled, maimed (heart victim).
3.1. Introduction

 Reported in the medical literature as early as
1495.
 In 1905 it was discovered that syphiluis was
caused by a spirochete type of bacteria,
 T. pallidum (originally called spirochaeta pallida).
 The first diagnostic blood test, the Wassermann
test, was developed in 1906.
3.1. Introduction

 Transmitted by:
 Mainly Sexual contact (Venereal syphilis)
 Less commonly via the placenta (congenital
syphilis) OR
 By accidental inoculation from infectious
material
e.g. fresh blood transfusion
3.1. Introduction

 Hours to days after penetrates intact mucosa or
abraded skin travel via lymphatic's to general
circulation and disseminates throughout body (all
organs including CNS)
 Incubation period directly proportional to size of
inoculums
 Host has immune response resulting inflammation
responsible for clinical manifestation
3.1. Introduction

3.2. Stages of syphilis
Primary Syphilis
Secondary Syphilis
Latent Syphilis
Tertiary Syphilis Persistent Asymptomatic
Relapse Trans placental
Transmission
Congenital Syphilis
40%
60%
Primary Chancre
Secondary lesions
Infection with T. pallidium
Gumma
(5 Yrs)
Cardiovascular
Syphilis
(10-15Yrs)
Tabes Dorsalis
(20 Yrs)

Primary chancre
 Appears at the site of inoculation, initially painless
papule that quickly erodes and becomes indurated
 Appears as a hard erythematus nodule about 1cm in
diameter with regional lymph node enlarged.
 Persists for some weeks and heals
 In women it commonly develops in the vulva or
cervix
 In HIV-infected patients, may see multiple or
unusual chancres, or no primary lesion

Chancres of primary syphilis

Secondary syphilis
 Always wide spread erythematus skin about 1cm in
diameter with regional lymph node enlarged.
 Ulcerates with a clear rim
 The ulcer is painless, persists for some weeks and
heals
 In women it commonly develops in the vulva or
cervix

 Secondary syphilis (2-8 weeks after primary
inoculation)
 Inconsistent symptoms, may include:
 Rash
 Generalized lymphadenopathy
 Constitutional symptoms (fever, malaise,
anorexia , headache)
 CNS symptoms

 Symptoms last days-weeks
 In advanced HIV infection, may be more
severe or progress more rapidly

Secondary syphilis
Rash of secondary syphilis

Secondary syphilis
Rash and ulcerations
of secondary syphilis

Latent syphilis
 no overt signs/symptoms, though relapse of
manifestations of secondary syphilis may occur
Tertiary syphilis (Late syphilis)
 Appears irregularly over succeeding years and may cause
series and permanent damage by means of chronic
inflammation.
 If untreated about 25% died directly by late syphilis
 neurosyphilis, cardiovascular syphilis, gummatous syphilis; or
slowly progressive disease in any organ system

3 basic forms of late syphilis
 Gumma- necrotic masses appear in skin, liver,
testes and bones
 Cardiovascular lesions- lesions on the veins,
valves and muscles of the heart.
 Neurosyphilis - meningo vascular
- general paralysis
- tabes dorsalis
–degeneration of posterior
column of the spinal cord

Congenital syphilis
 Pregnancy does not alter the course of syphilis in
adults
 Transmission and adverse outcomes highest with
early syphilis
 Syphilis may increase risk of perinatal HIV
transmission to infants = congenital syphilis
 Screening:
 At first prenatal visit in all women; in high-
prevalence areas or high-risk women, repeat at
28 weeks and at delivery

 Infection with T. pallidum involves both CMI and
humoral immune response.
 Antigens
Wasserman Ag- phospholipid diphosphatidyl
glycerol = cardiolipin
Is a normal constituent of host tissue
 Antibodies
Wasserman Antibody=Anticardiolipin=Reagin
 Is an Ab to Ags of treponemal proteins as carriers
and cardiolipin as immunogenic determinant.

Treponemal Antigens
From one or more pathogenic species
Shared by many/different strains, spps, sub
spps or specific to spps or sub spps
Produce anti-treponemal Abs = Abs to
components of treponems
Treponemal Antibodies could be:
- Non specific directed against proteins common
to pathogenic/non-pathgenic treponems or
- Specific directed to pathogenic treponems only

1.Tests that detect the etiologic agent(directly)
2. Serologic tests for syphilis
1.Tests that detect the etiologic agent
 Dark field (Dark ground)
 Indian ink (Negative stain)
 Phase contrast microscopy
 Electron microscopy
 Silver stain
 Fluorescent stain (DF)

Morphological x-ics may be studied microscopically.
Usual methods:
 Dark field (Dark ground)
 Indian ink (Negative stain)
 Phase contrast microscopy
 Electron microscopy
 Silver stain
 Fluorescent stain (DF)
Wet preparation
Dry preparation

 Dark field microscopy
 For symptomatic patients with primary syphilis,
dark field microscopy is the test choice.
 A dark field examination is also suggested for
immediate results in cases of secondary syphilis
with a VDRL titer follow-up test.

 More than 200 tests developed and only few are
used currently.
 Generally grouped into TWO, based upon the type
of Ag used and Ab detected
A. Reagin tests for syphilis (Non-
treponemal/ Non-specific tests)(indirect
method)
B. Treponemal tests for syphilis (Specific
tests)(direct method)
3.5. Serologic tests for syphilis

A. Non- Treponemal Tests for Syphilis
 A nontreponemal test employs an antigen (E.g.,
cardiolipin-lecithin),
 Are used to detect an antibody like substances or
“reagin” antibody,
 They detect biomarkers that are released during
cellular damage that occurs from the syphilis
spirochete.
 Are not 100% specific for syphilis antibodies, but are
highly sensitive for syphilis

 Nontreponemal tests are screening tests, very rapid
and relatively simple, but need to be confirmed by
treponemal tests.
 Syphilitic infection leads to the production of
nonspecific antibodies that react to cardiolipin.
 This reaction is the foundation of “nontreponemal”
assays
 All nontreponemal tests measure immunoglobulins G
(IgG) and M (IgM) anti-lipid antibodies formed by the
host in response both to lipoidal material released
from damaged host cells early in infection and to lipid
from the cell surfaces of the treponeme itself.

 These nontreponemal tests are widely used for
qualitative syphilis screening. However, their
usefulness is limited by decreased sensitivity in
early primary syphilis and during late syphilis, when
a large number of untreated patients will be
negative by these methods.
 With nontreponemal tests, false-positive reactions
can occur for a large number of reasons, the most
common of which is other infections, both viral and
bacterial.

 Advantages of being practical, inexpensive and
widely available.
 Basically of two types:
I. Flocculation (tube or slide) and
II. Complement fixation tests.

I. Flocculation Tests
a. Slide flocculation tests,
 needs small amount of clinical specimen
and antigen suspension
 are rapidly performed
 results are usually read microscopically
 It utilizes cardiolipin, lecithin, cholesterol
antigen and heat inactivated serum.
 Performed on slide or tube
E.g., VDRL

b. Tube flocculation tests
 are performed in test tubes
 requires large quantities of specimen and
antigen suspension
 are more complicated
 are read with or without magnification.
Eg.,
 Kliane flocculation Test
 Khan flocculation Test
 VDRL
 Mazzini test
 Hinton (serum) test

C. Card flocculation tests (Rapid reagin tests):
 RPR (rapid plasma reagin)
 RPR (Teardrop) card test
 RPR (18-mm circle) card tests

II. Complement fixation Test (CFT)
 Complement components are thermo labile
proteins found in normal serum.
 It is also found in other animals.
 It is destroyed at 56C for 30 minutes.
 But up on standing at 7-370C, it may regain part
of its activity.
 Therefore, previously heated serum must be
reheated for 10 min. at 560C before a test can be
performed.

Complement Fixation
Serum without Abs
Serum with
Antibodies
Antigen binds
to antibodies
Unbound Antigen
Complement
binds to Ag/Ab
complex
Unbound Complement
No lysis Positive Lysis Negative
Hemolysin sensitized
RBCs serve as an
indicator
Hemolysin sensitized
red blood cells
serve as an indicator
Day
1
Day
2

RPR (Rapid reagain card test for syphilis)
Principle
 Destructive syphilitic lesions cause tissue damage.
Circulating antibodies called reagain are produced
against some of the tissue components. The rapid
regain card test uses a modified form of the VDRL
(Vernal Disease Research Laboratory) antigen
called cardiolipin in suspension with carbon
particles. When cardiolipin antigen reacts with
reagain antibody in patient’s serum the carbon
particles in the suspension clump together.

Materials
 The following are provided in the test kits:
 Reagin antigen suspension
 Reagin positive control serum
 Reagin negative control serum
 Reagin test card
 Dispensing bottle and needle
 Dropper tubes
 Mixing sticks

Qualitative RPR test method
Procedure
 Let the reagents and specimens warm up to room
temperature.
 Dispense one drop of negative control serum on to
one circle on the test card using a disposable
dropper tube.
 Repeat step two with the positive control serum
using a clean dropper tube.
 Dispense on drop of each sample serum or plasma
on to one circle on the card using a clean dropper
tube for each specimen.

Procedure
 Spread all the drops to cover the whole area of the
circles using the mixing sticks.
 Mix with reagain antigen suspension in the
dispensing bottle. Hold the bottle vertically and
dispense one drop on to each test sample. Do not
mix again.
 Place the card on the rotor for 8 minutes at
100rpm.

Reading the results
 Negative result:
The carbon particles remain in an even
suspension = Non reactive
 Positive result:
The carbon particles clump together
= Reactive

For the test to be valid the negative control must be non-
reactive and the positive control must be reactive

Reporting results of a qualitative test
 Qualitative results should be reported as reactive
or Non-reactive

Quantitative RPR test method
1. Dispense 1 drop of 0.85% saline on to circles 1 to
5 on the test card
2. Dispense 50µl of patient serum or plasma on to
the first circle and mix by filling and discharging
the pipette at least 6 times (do not make
bubbles). You now have a 1 in 2 dilution in the
first circle.
3. Transfer 50µl from the first circle to the second
circle and repeat the mixing procedure.
4. Continue the dilution procedure to circles 3, 4
and 5 and discard the last 50µl.

You know have the following dilutions:
Circle 1 2 3 4 5
Dilution ½ ¼ 1/8 1/16 1/32

5. Starting at circler number 5 uses a mixing stick to
spread all the drops to cover the whole area of the
circles.
6. Mix the regain antigen suspension in the
dispensing bottle. Hold the bottle vertically and
dispense one drop on to each test sample. Do not
mix again.
7. Place the card on the rotor for 8 minutes at
100rpm.

Reporting the results of a quantitative test
 The titer reported in a quantitative test is the
highest sample dilution to show a reactive
(positive) result.
 In the example below the result would be
reported as:
 Reactive to 1/8

Limitation of the test
 The rapid regain card test is a non-specific test
for syphilis. A positive reaction indicates tissue
damage such as that caused by destructive
syphilitic lesions.
 False negative reactions can occur in the early
and later stages of syphilis when there is no a lot
of tissue damage.

 False positive reactions can occur due to other
diseases which result in tissue damage such as
malaria, leprosy, viral infections, autoimmune
diseases and many other conditions including
pregnancy.
 It is very important that reactive specimens are
checked by another test procedure which is
specific for syphilis.

VDRL TEST
Slide Qualitative VDRL Test
Principle
 During the period of infection with syphilis,
reagin, a substance with the properties of an
antibody, appears in the serum affected patients.
Reagin has the ability to combine with a colloidal
suspension extracted from animal tissue and
clump together to form visible masses, a process
known as flocculation.

Procedure:
1. Pipette 0.05ml or 1drop of inactivated serum into
one ring of the ringed glass slide.
2. Add one-drop (1/60ml) antigen suspension onto
each serum.
3. Rotate slide for 4 minutes. (If rotated by hand on
a flat surface, this movement should roughly
circumscribed a 2 inch/5mm diameter circle).
4. Tests are read immediately after rotation
microscopically with a 10x ocular and a 10x
objective.

Reading and reporting of results
 Tests are read microscopically with low power
objective at 10x magnification, which appears short
rod forms. Aggregation of these particles into large
or small clumps is interpreted in degrees of
reactivity.
Reporting system
No clumping or very slight roughness : Non-reactive (NR)
Small clumps : Weakly reactive (WR)
Medium and large clumps : Reactive (R)

B. Treponemal tests for syphilis
Standard Treponemal tests for syphilis
1. T. pallidum immobilization test (TPI)
2. RPCF test ( Reiter protein complement fixation
test)
3. Fluorescent Treponnema antibody Absorption test
(FTA-ABS-test)
4. Treponoma pallidum Methylene Blue tests.
5. Treponomal pallidum agglutination test.
6. Treponemal pallidum complement fixation test.

1. T. pallidum immobilization test (TPI)
Principle:
Treponema pallidum immobilization test is
the most specific and extremely valuable test
for syphilis. It becomes positive after the
second week of infection. The test is however
quite complicated and relatively costy to
perform. The test, where available, is used
for reference and to rule out false-positive
sero reactors of other tests.

Method:
 Patient’s serum is placed in a test tube with living
spirochetes and complement.
 After incubation in an atmosphere free of 02, slide
preparations are made and examined by dark field
illumination.
 The spirochetes will be immobilized by syphilitic
serum but will be actively motile in normal serum.
The TPI test has its greatest value in confirming
syphilis or ruling out biological false positive
reaction.

 It requires live treponemas from infected animals
and is difficult to perform.
 It does not distinguish the various
treponematoses (i.e. yaws, pinta, bejel)
 It fails to detect early syphilis
 It cannot be used as an index of therapeutic
response.
 It is ineffective when the patient is on antibiotics.

Advantage:
 On the positive side, the test is the one of choice
for spinal fluids, especially for detecting
neurosyphilis when reagin tests give non-reactive
results.

Fluorescent Treponnema antibody Absorption
test (FTA-ABS-test)
 A modified form of fluorescent treponemal
antibody test (FTA-Test) with treponemal antigen
employing indirect immunofluorescence
 FTA-used for diagnosis of syphilis
 It is a specific and sensitive test

Principle
 The FTA-ABS test is a direct method of
observation. Although not recommended for
screening, it is the most sensitive serologic
procedure in the detection of primary syphilis.
 This test is recommended as a confirmatory test
for syphilis.
 It is recommended for screening test.
 A reagin test such as the VDRL or RPR is not
recommended for screening.

Stage
Primary Secondary Late
Non Treponemal (Reagin tests)
VDRL 70% 99% 1%
RPR 80% 99% 0%
Specific Treponemal test
FTA-ABS 85% 100% 95%
TPHA-TP 65% 100% 95%
TPI 50% 97% -

Non standard non treponemal and treponemal
test
 ELISA
 CAPTIA-syphilis G test
 CAPTIA-syphilis M test
 Syphilis Rapid test device

ELISA
 The ELISA immuno assays is available for both
non-treponemal and treponemal tests.
 The ELISA non-treponemal assaya uses the VDRL
antigen affixed to a micro titer plate.
 At least two different treponemal ELISA assay are
commercially available in kit form.
 These are the CAPTIA-syphilis G and the CAPTIA-
syphilis M tests (trinity Bio tech).

 CAPTIA-syphilis G test
 used to detect anti-treponemal antibody
 used to measure IgG of Treponemal infection
 CAPTIA-syphilis M test
 used to detect anti-treponemal antibodies
 This test is particularly useful for diagnosis of
congenital syphilis.
 Babies whose mothers are infected with syphilis
cannot be diagnosed using the tests that
measure IgG antibodies.
 IgM antibodies do not cross the placenta, the
identification of anti-T. pallidum antibodies in
the newborn sera indicates congenital syphilis

 Syphilis Rapid test device
 it is a rapid qualitative chromatographic
immunoassay that uses the affinity of protein A
for IgG antibodies to test for treponemal
antibodies
 Protein A binds to the Fc region of most
subclasses of IgG.
 One of the advantages of this test is that
dilutions are not required and the prozone
phenomenon is not an issue as it is for tests
whose end points are flocculation or
agglutination.

Test for Syphilis
Non Standard
Standard
Treponemal and
Non treponemal
Standard
ELISA
EIA
Western Blot
SRTD (syphilis rapid
test device)
Non specific Specific
Non treponemal
TP- complement test
TPI
TPH ABS
RPCF
TPA agglutination
TP. Methylene blue tests
FTA-ABs
Flocculation
Kolimer
Wassermann
Tube
VDRL
Kliane
Khan
Mazzini
Hinton
Card test
RPR
USR
PCT-plasma Crit
Slide
VDRL
CF
Treponemal
Summary

Review question
 Explain the stages of syphilis
 Discuss the difference between RPR and VDRL.
 Write non serological test for syphilis
 Explain specific and non-specific serologic test for
syphilis

Reference
1. Tizard. Immunology an introduction,4th edition
,Saunders publishing,1994
2. Naville J. Bryant Laboratory Immunology and
Serology 3rd edition. Serological services
Ltd.Toronto,Ontario,Canada,1992
3. Mary Louise .Immunology and Serology in
Laboratory medicine 3rd edition

Syphilis Serology serological test for medical laboratory.ppt

Recommended

Recommended

More Related Content

Similar to Syphilis Serology serological test for medical laboratory.ppt

Similar to Syphilis Serology serological test for medical laboratory.ppt (20)

More from CherenetToma

More from CherenetToma (7)

Recently uploaded

Recently uploaded (20)

Syphilis Serology serological test for medical laboratory.ppt