IMMUNOLOGY AND SEROLOGY LAB

LECTURE 4: SEROLOGIC TESTS FOR

SYPHILIS
PROF. BENJO A. DALUPAN, RMT
AUGUST 30, 2021
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SYPHILIS ○ Primary chancre appear on hands, mouth, skin

● Caused: Spirochete Treponema pallidum subsp. pallidum and other parts of the body.
● A sexually transmitted disease that affects the skin and ○ Typically heal on their own over a few months
mucous membranes of the external genitalia and also ● Some spirochetes go to nearby lymph nodes where they
sometimes the mouth. caused by lymphadenopathy (enlargement of lymph
● Gram negative bacteria that look like corkscrews. nodes)
● Possess 6 thin endoflagella called the axial filaments ● If it is acquired thru a blood transfusion, there may not be
which renders them move in a unique spinning motion. early localized stage at all or no primary chancre.
● It cannot be cultured in an ordinary media. ● Primary syphilis
● Too small to be seen in the light microscope that's why ○ Can usually be found in the chancre
special procedures and serologic tests are required to ○ Treponyms can usually be found in the
help screen the Treponema pallidum. chancres
■ We can view or observe them in dark
Mode of Transmissions: field microscope (appear white
1. Acquired Syphilis against a black background)
● Happens when T.pallidum enters the body through body ● People with primary syphilis tend to have a non reactive
fluids. result to serologic tests
○ Cuts/Breaks
○ Sexua lContact. 2. Secondary Syphilis
■ Including oral, anal and vaginal sex. ● Dissemination stage
○ Contaminated Needles ● Appears 6-8 weeks
○ Direct Contact’ ● Generalized rash and secondary lesions (eyes, joints or
■ With a skin lesion of an infected CNS)
person. ● Condylomata lata
■ Lesion - covered with rich of ○ smooth , white, painless wartlike lesions
spirochetes ○ Appear on most areas like the genitals, around
2. Congenital the anal region, and armpit
● When a pregnant person has syphilis and T.pallidum ● (+) dark field microscopy
enters a baby either in the uterus or while the baby exits ● Reactive to serologic tests
thru the vagina at birth.
● Also called as vertical transmission 3. Latent Syphilis (Hidden syphilis)
● Disease enters a dormant or asymptomatic phase
4 CLINICAL STAGE OF SYPHILIS ● 2nd year of infection
● No clinical symptoms
1. Syphilis Primary (early) ● Serologically reactive
2. Secondary ● Spirochetes can mostly be found in the tiny capillaries of
3. Latent various body organs and tissues
4. Tertiary (late) ● Therefore, the results will be serologically reactive

1. Primary/ Early Syphilis 4. Tertiary/Late Syphilis

● Appears 2-8 weeks ● Gumma/ Gummata
○ After it lands on the skin or mucous ● 80% - CNS involvement “neurosyphilis”
membranes. ● 10% - heart involvement “cardiovascular syphilis”
● HARD CHANCRE - painless (chancUred) ● Serologically reactive
● lasts for 1-5 weeks ● Type IV hypersensitivity reaction
● (+) Darkfield microscopy ○ Immune response lead by the t cells
○ Typically appear white against a dark ○ Recruit phagocytes and causes release of
background pro-inflammatory cytokines such as the tumor
● Nonreactive to Serologic tests necrosis factor, IL-1 and IL-6
● Destroy the soft tissue and skin→ formation of ulcers ○ All of these immune responses leads to local
called as syphilitic chancres swelling or edema, redness, warm and
○ Painless systemic symptoms like fever
○ Has a hard base ● In some cases, the immune cells start to huddle around
○ Usually covered by fluids rich in spirochetes. and form a granulomatous lesion called
○ This can spread to other parts of the body and Gumma/Gummata
other individuals. ○ Has lots of different types of immune cells that
● Soft chancre get surrounded by the outermost layer
○ Caused by another bacteria Haemophilus fibroblast
ducreyi. ○ There aren't any spirochetes in gumma/
● In individuals who acquire syphilis thru sexual contact, gummata, its just immune cells getting excited
primary chancre develops around external genitalia. for no apparent reason
● Physical touch, lesion and in some other way ○ The tissues at the center often ends up without

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 oxygen leading to coagulative necrosis NONTREPONEMAL TEST
● in tertiary syphilis, various organs like the heart and ● Venereal disease research laboratory (VDRL) test
blood vessels are damaged --- Cardiovascular syphilis ● Rapid plasma reagin (RPR)
○ Around 10% of the patients develop ● These non-treponemal tests both use cardiolipin or the
cardiovascular problems which results in aortic wasserman antigen
aneurysm ● They detect the reagin or the anti-cardiolipin
● Brain and spinal cord can also get damaged, called as
Neurosyphilis
○ Around 80% patients experience CNS
involvement which can result in paralysis or
dementia
● Other organs may also get damaged, like the liver, joints
Venereal Disease Research Laboratory (VDRL) Test
and testes which haven't earned their unique names yet
● Patients with tertiary syphilis will be serologically reactive ● Principle: Flocculation
○ Flocculation occurs when the precipitate floats
4 Clinical Stages of Syphilis instead of sedimentation
1. Primary (early) - Hard chancre ● Equipment:
2. Secondary - Condylomata lata ○ Antigen Needles
3. Latent - Asymptomatic ■ Qualitative - 1/60 mL (60 drops/mL) of
4. Tertiary (late) - Gumma antigen
■ Quantitative - 1/75 mL (75 drops/mL)
Darkfield Microscopy of antigen
○ Saline Needles - Quantitative - 1/100 mL (100
drops/mL) of saline
○ Mechanical rotator - 180 rpm
○ Glass Slides
○ Serological Pipets

● Diagnosis of acquired syphilis starts with identifying the

spirochetes in the fluid from chanre which can be done
using darkfield microscopy or dark field microscope
● A darkfield microscope shines thin slivers of light on a
slide so that the background appears dark while the
extremely thin spirochetes lights up
● Spirochetes appears to be white against the black ● Ex. Patients with syphilis develop an antibody known as
background reagin to a tissue derived substance which is known as
cardiolipin or antigen
Nontreponemal Treponemal ● So when the cardiolipin, the nontreponemal antigen is
Antigens Wasserman antigen Nichols added to heated serum of patient containing the reagin or
(Cardiolipin) Reiter the antibody, there will be antigen and antibody reaction
Antibodies Reagin Anti-treponemal resulting in flocculation
(Anti-cardiolipin) antibodies ● Positive result of this test: Flocculation
● The diagnosis is confirmed with serological tests which ● Reagent:
look for antibodies against treponema pallidum antigens ○ Antigen’
● 2 type of antigens: Alcoholic solution of
○ Nontreponemal antigens “Cardiolipin-cholesterol-lecithin”
■ Wasserman (Cardiolipin) ■ Cardiolipin
● is a normal constituent of ● Serves as an antigen which
host tissue and it can be is a phospholipid in nature
released when there is isolated from the beef heart
damage in the cells ■ Cholesterol
● Not specific for it ● Center of absorption of
○ Treponemal antigens tissue lipids to increase the
■ Nichols strain size of the antigen
● Strain of treponema ■ Lecithin
pallidum ● Produce standard reactivity
● Pathogenic & virulent ○ 1% buffered saline, pH 6.0
■ Reiter strain
● Comes from the treponemes ● Sample Preparation:
but not from the treponema ○ INACTIVATION
pallidum ■ Serum- heated at 56 degrees Celsius
● Non pathogenic for 30 minutes
● For the antibodies, ■ Heating the serum at 56 degrees
○ Nontreponemal celsius for 30 minutes inactivate the
■ Anti-cardiolipin complements in the serum
● Anti-cardiolipin antibodies = ■ Compliments interfere with tests
called as reagin in the blood ○ RE-INACTIVATION
○ Anti- treponemal antibodies ■ Serum- heated at 56 degrees Celsius
■ Detects antibodies directed against 10 minutes
the treponema pallidum or detects the ■ Serum should be re-inactivated if
anti-treponemal antibody there will be an interval of 4 hours or
more between inactivation and testing

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Qualitative Quantitative
● We need to inactivate the complement by heating the ● Recall the “dilution Lesson”
serum at 56 degrees Celsius for 30 minutes ● Perform on “reactive and Minimally Reactive Sera”
● Serum + 1 drop (1/60mL) antigen ● Twofold dilutions serum ranging in 0.9% saline
○ Serum:Antigen Ratio (3:1) ● Report the highest dilution giving reactive results
○ Antigen: Cadiolipin-Cholesterol-Antigen solution
● Rotate on a mechanical rotator for 4 minutes at 180 rpm
● Observe MICROSCOPICALLY for the presence of
flocculation IMPORTANT NOTES TO REMEMBER

False Positive
● VDRL - SLE, RF, IM, Malaria, and Pregnancy
● RPR - IM, Leprosy, RF

POSITIVE RESULTS (VDRL/RPR) ⟶ MORE SPECIFIC

TREPONEMAL TEST
● Interpretation ● All positive results in VDRL or RPR must be confirmed
○ Reactive when there are medium to large by a more specific treponemal test
clumps
○ Weakly reactive if there are small clumps TREPONEMAL TEST
○ Non-reactive- no clumps
● All sera with reactive or weakly reactive results in TREPONEMAL TEST
qualitative VDRL must be tested with quantitative VDRL 1. Treponema pallidum immobilixation (TPI) test
2. Fluorescent treponema pallidum antibody absorbed
Quantitative (FTA-ABS) test
● Recall the “Dilution Lesson” 3. Microhemagglutination-treponema pallidum (MHA-TP)
○ Do two-fold serum dilution ranging from 1:2 to test
1:32
● Perform on “Reactive and Weakly Reactive Sera” These treponemal tests use treponemal antigens like:
● Twofold dilutions serum ranging from 1:2 to 1:32 ● Nichols (pathogenic and virulent)
● Reporting: ● Reiter (Not T. pallidum and not pathogenic)
○ WR - Weakly Reactive ○ These stains are used to detect
○ Reactive Anti-treponemal antibodies present in the
● Highest dilution showing a positive result serum of the px
● TITER (reactive in (1:2 dilutions)
● Reciprocal of dilution (Reactive 2 dilution) TREPONEMA PALLIDUM IMMOBILIZATION (TPI) TEST
● Principle:
RAPID PLASMA REAGIN (RPR) ○ Antibodies against T. pallidum - immobilize live
● Nontreponemal treponemes
○ WASSERMAN Antigen (Cardiolipin) ● Antigen:
○ Detect nontreponemal antibody ○ MOTILE T. pallidum (NICHOLS STRAIN)
■ Reagin (Anti-Cardiolipin) ● Preparation:
● Principle: Flocculation ○ Diluted patient’s serum + antigen suspension
○ Advantage ⟶ DARKFIELD ILLUMINATION
■ No need to inactivate serum ● Result:
■ No heating ○ HIGHEST serum dilution - immobilizes the
○ Equipment treponemes ⟶ IMMOBILIZING ANTIBODY
■ Plastic coated slide TITER
○ Reagent
■ Cardiolipin with added charcoal
● Charcoal particles form FLUORESCENT TREPONEMA PALLIDUM ANTIBODY
larger particles that are ABSORPTION (FTA-ABS) TEST
visible as clumps when
● Preparation:
aggregated by an antibody
○ Pre-absorption of serum with Reiter strain (non
● Help the test to be read
pathogenic treponemes)
macroscopically
■ Enhances the specificity of FTA-ABS
test
○ Antigen - Nichols strain is fixed (slide)
■ The virulent strains came from the
rabbit testicles
○ Addition of serum
○ Addition of conjugate (fluorescent-labeled AHG)
○ POSITIVE - FLUORESCENCE observed
Qualitative
against a black background
● Spread 0.05mL unheated serum to fill the circle
● +1 drop (1/60mL) antigen without mixing
● Rotate on a mechanical rotator for 8 minutes at 100rpm
● Observe macroscopically for clumping
● Result:
○ Reactive - medium to large clumps
○ Minimally reactive - small clumps
○ Nonreactive - no clumps

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MICROHEMAGGLUTINATION-TREPONEMA PALLIDUM
(MHA-TP) TEST
● PRINCIPLE: Simple Passive Hemagglutination

○ The sheep RBCs are coated with Treponemal

antigen, resulting to sensitized sheep RBCs
● ANTIGEN: Tanned formalin sheep RBC coated with
treponemal antigen
● POSITIVE RESULT: HEMAGGLUTINATION
● Use as substitute for FTA-ABS
● TPI, MHA-TP, FTA-ABS are highly sensitive and specific
tests but not routinely used
● However, currently there are many treponemal tests
available in the market
● RESULTS INTERPRETATION

RPR & FTA-ABS Reactive POSITIVE FOR SYPHILIS

RPR; Reactive; NEGATIVE FOR SYPHILIS
FTA-ABS Non Reactive
RPR; Non Reactive; LATE/LATENT/PREVIOUS
FTA-ABS Reactive

Recall:
● RPR is a non treponemal test and nonspecific test; there
are a lot of diseases that could give a false positive result
in RPR
● So, in order to confirm the positive RPR result, we need
to proceed to a more specific treponemal test (FTA-ABS)
● If the FTA-ABS is non reactive, we can conclude that the
RPR positive result is a false positive
● The RPR will eventually become non reactive if the
patient is treated
● The FTA-ABS test remains reactive since Treponemal
antibodies persists for life