Chapter three

Syphilis Serology

Lecture For MLS students

By: Muluneh T.(MSc in Medical Microbiology)

 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

Muluneh T.(MSc) 2
 Outline

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

Muluneh T.(MSc) 3
 3.1. Introduction
 Syphilis 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.
 Syphilis 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).

Muluneh T.(MSc) 4
 Etiology and Transmission
Genus Treponema
General characteristics
- Helically coiled, corkscrew-shaped cells
- Motile, rapid rotation along its longitudinal axis
- Gram negative, but stain poorly
- Microaerophilic
- Multiply by binary transverse fission and with long in-vivo
generation time (30hrs)
- Not yet been cultured
Muluneh T.(MSc) 5
 Etiology and Transmission…
Order :- Spirochaetalis
Family :- Treponemataceae
Genera:- Borrelia, Treponema, Leptospira
Genus Treponema include
T. pallidium ssp pallidium = Sexually acquired syphilis
T. pallidium ssp pertenue =Yaws
T. pallidium ssp endemicum =endemic syphilis
T. carateum =Pinta
All are morphological identical and this diseases are collectively
known as other treponematoses.

Muluneh T.(MSc) 6
 Normal inhabitants
• T. pallidium ssp and T. carateum are obligate human pathogens
• Non pathogenic treponemes are part of the normal flora of
intestinal tract, oral cavity or the genital tract
Virulence factors
• Outer Membrane Protein,
• enzymes (hyaluronidase)
• Perivascular inflammation
• Granulomatous changes: tertiary syphilis
Antigenic structure
 Non – specific antigen = A non-specific (reagin)
 Species – Specific treponemal antigen

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 Syphilis
 Is a systematic infection caused by the spirochate Treponema
pallidium
 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

Muluneh T.(MSc) 8
 Syphilis
 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

Muluneh T.(MSc) 9
 Stages of syphilis
Infection with T. pallidium

Primary Syphilis Primary Chancre

Secondary Secondary
Syphilis 60% lesions
40% Relapse Trans
placental
Latent Syphilis Transmission

Tertiary Syphilis Persistent Congenital

Asymptomatic Syphilis

Cardiovascular
Gumm Tabes Dorsalis
Syphilis
a (20 Yrs)
(10-15Yrs)
(5 Yrs) Muluneh T.(MSc) 10
 Stages of syphilis

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 atypical chancres, or
no primary lesion
Muluneh T.(MSc) 11
 Stages of 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

Muluneh T.(MSc) 12
 Stages of syphilis
Secondary syphilis (2-8 weeks after primary inoculation)
– Protean symptoms, may include:
 Rash (macular, maculopapular, or pustular, or condyloma lata)
 Generalized lymphadenopathy
 Constitutional symptoms (fever, malaise, anorexia,
arthralgias, headache)
 CNS symptoms
 Symptoms last days-weeks
 In advanced HIV infection, may be more severe or progress
more rapidly
 Distinguish from primary HIV infection
Muluneh T.(MSc) 13
 Stages of syphilis

Secondary syphilis
Ch
s y a nc
ph re
ili
s of S
ec
on
d ar
y

Rash of secondary syphilis

Muluneh T.(MSc) 14
 Stages of syphilis

Secondary syphilis

Rash of secondary syphilis

Muluneh T.(MSc) 15
 Stages of syphilis

Secondary syphilis

Rash and ulcerations

of secondary syphilis
Muluneh T.(MSc) 16
 Stages of syphilis

Tertiary 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

Muluneh T.(MSc) 17
 Stages of syphilis
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

Muluneh T.(MSc) 18
 Stages of syphilis

 Latent syphilis: no overt signs/symptoms, though relapse of

manifestations of secondary syphilis may occur
 Late syphilis: neurosyphilis, cardiovascular syphilis,
gummatous syphilis; or slowly progressive disease in any organ
system

Muluneh T.(MSc) 19
 Stages of syphilis
Neurosyphilis
 Neurologic complications or neurosyphilis may occur earlier or
progress more rapidly in HIV-positive patients
 Meningitis, meningovascular, or parenchymatous disease similar
in HIV-uninfected patients
 Concomitant uveitis and meningitis more common in HIV-
positive patients
 Asymptomatic neurosyphilis (CSF with elevated protein,
lymphocytosis, or positive serologic test, in absence of symptoms):
not a late complication or manifestation
Muluneh T.(MSc) 20
 Stages of syphilis

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

Muluneh T.(MSc) 21
 Immune response
 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.

Muluneh T.(MSc) 22
 Immune response
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
- Specific directed to pathogenic treponems only

Muluneh T.(MSc) 23
 Diagnosis of syphilis
1.Tests that detect the etiologic agent

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)

Muluneh T.(MSc) 24
 Diagnosis of syphilis
Morphological x-ics may be studied microscopically. Usual
methods:
– Dark field (Dark ground)
– Indian ink (Negative stain)
– Phase contrast microscopy Wet
preparation

– Electron microscopy
– Silver stain
– Fluorescent stain (DF) Dry preparation

Muluneh T.(MSc) 25
 Diagnosis of syphilis
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.

Muluneh T.(MSc) 26
Diagnosis of syphilis

Muluneh T.(MSc) 27
Diagnosis of syphilis

Muluneh T.(MSc) 28
 Serologic tests for syphilis

 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)
B. Treponemal tests for syphilis (Specific tests)

Muluneh T.(MSc) 29
 Serologic tests for syphilis
A. Non- Treponemal Tests for Syphilis
 A non- treponemal test employs an antigen (E.g., cardiolipin-
lecithin),
 Are used to detect an antibody like substances or “reagin” antibody,
 Are not 100% specific for syphilis antibodies, but are highly
sensitive for syphilis

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

Muluneh T.(MSc) 31
 Serologic tests for syphilis

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
Muluneh T.(MSc) 32
 Serologic tests for syphilis
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

Muluneh T.(MSc) 33
 Serologic tests for syphilis

C. Card flocculation tests (Rapid reagin tests):

 RPR (rapid plasma reagin)
 PCT- plasma CriT
 RPR (Teardrop) card test
 RPR (18-mm circle) card tests

Muluneh T.(MSc) 34
 Serologic tests for syphilis
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.

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 Complement Fixation

Serum with Serum without Abs

Antibodies

Antigen binds Unbound Antigen

Day 1

to antibodies

Complement
Unbound Complement
binds to Ag/Ab
complex

Hemolysin sensitized Hemolysin sensitized

red blood cells RBCs serve as an
indicator
serve as an indicator
Day 2

No lysis Positive Lysis Negative

Muluneh T.(MSc) 36
 Serological technique
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.
Muluneh T.(MSc) 37
 Serological technique
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

Muluneh T.(MSc) 38
 Serological technique
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.
Muluneh T.(MSc) 39
 Serological technique
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.

Muluneh T.(MSc) 40
 Serological technique

Reading the results

 Negative result:

The carbon particles remain in an even

suspension = Non reactive

 Positive result:

The carbon particles clump together

= Reactive

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 Serological technique

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

Reporting results of a qualitative test

 Qualitative results should be reported as
reactive or Non-reactive

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 Serological technique
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.

Muluneh T.(MSc) 44
 Serological technique

You know have the following dilutions:

Circle 1 2 3 4 5

Dilution ½ ¼ 1/8 1/16

1/32
Muluneh T.(MSc) 45
 Serological technique
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.

Muluneh T.(MSc) 46
 Serological technique

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

Muluneh T.(MSc) 47
Serological technique

Muluneh T.(MSc) 48
 Serological technique
If the highest dilution (1 in 32) is reactive proceed as follows:
8. Prepare a 1in 16 dilution of the sample by adding 0.1ml of
serum to 1.5ml of 0.85% saline and mix well.
9. Dispense 1 drop of saline on to circles 6 to 10 on the test card
using a dropper tube.
10.Dispense 1 drop of the 1 in 16 dilution on to circle number 6
and proceed as before (step 3 and 4) making doubling
dilutions up to well number 10

Muluneh T.(MSc) 49
 Serological technique
You know have the following dilutions:

Circle 6 7 8 9 10
Dilution1/32 1/64 1/128 1/256 1/512

11. starting at well number 10 spread all the drops as before

12. Mix the reagin antigen suspension in the dispensing bottle.
Hold the bottle vertically and dispense one drop on to each
circle. Do not mix again

Muluneh T.(MSc) 50
 Serological technique

13. place the card on the rotor for 8 minute at 100rpm

14. report the titer as the highest dilution to show a positive
result

Muluneh T.(MSc) 51
 Serological technique

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 not a lot of tissue damage.

Muluneh T.(MSc) 52
 Serological technique
 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.
 In the CPHL this is done by the Wellcosph HA test which is
specific for antibodies to T. pallidum, the bacterium which
causes syphilis.
Muluneh T.(MSc) 53
 Serological technique
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.

Muluneh T.(MSc) 54
 Serological technique
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.
Muluneh T.(MSc) 55
 Serological technique
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)

Muluneh T.(MSc) 56
 Serological technique
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.

Muluneh T.(MSc) 57
 Serological technique
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.

Muluneh T.(MSc) 58
 Serological technique

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.

Muluneh T.(MSc) 59
 Serological technique
Limitation of the test
 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.

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 Serological technique

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.

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 Serological technique

2.RPCF test ( Reiter protein complement

fixation test)
 RPCF is a test with treponemal antigen
 is performed for the diagnosis of syphilis
 less specific and sensitive than TPI
 the test is simple to perform and quite cheap

Muluneh T.(MSc) 62
 Serological technique

3. 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

Muluneh T.(MSc) 63
 Serological technique
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.
Limitation of the test
 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.

Muluneh T.(MSc) 64
 Serological technique
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% -
Muluneh T.(MSc) 65
 Serological technique

Non standard non treponemal and

treponemal test
 ELISA
 CAPTIA-syphilis G test
 CAPTIA-syphilis M test
 Syphilis Rapid test device

Muluneh T.(MSc) 66
 Serological technique
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).
Muluneh T.(MSc) 67
 Serological technique
 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
Muluneh T.(MSc) 68
 Serological technique
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.

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Summary
Lesion

Yes No
Negative
DAF Non
treponemal
Reactive Non-reactive

Positive Titer Repeat

Treat

Treponemal test
Non reactive
Reactive

False
Treat Positiv
e
Muluneh T.(MSc) 70
 Test for Syphilis
Summary
Standard Non Standard

Non specific Specific

Treponemal and

Non treponemal
Non treponemal Treponemal

TP- complement test

Standard
Flocculation CF TPI
ELISA
TPH ABS
Kolimer EIA
Wassermann
RPCF
Western Blot
TPA agglutination
Tube Slide Card test SRTD (syphilis rapid
TP. Methylene blue tests test device)
VDRL VDRL RPR
FTA-ABs
Kliane USR

Khan PCT-plasma Crit

Mazzini

Hinton

Muluneh T.(MSc) 71
 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

Muluneh T.(MSc) 72
 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

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