MODULE 4: SEROLOGIC TEST FOR SYPHILIS

• Test for Syphilis

OUTLINE
I Syphilis 1. Non-specific or non-treponemal serological tests
A Antigens ▪ For screening of syphilis
B Antibodies ▪ Serological tests that detect reagin
C Spinal Fluid Tests in Syphilis - Reagin: substance present in the serum of patients with certain diseases
i Biological False Positive Antibody (BFP) Reagin Antibody including syphilis
▪ Become positive 1-4 weeks after the appearance of initial symptoms
▪ Other Non-treponemal tests:
I. SYPHILIS - Toluidine Red Unheated Serum Test (TRUST)
• Causative agent: Treponema pallidum - Unheated Serum Reagin (USR)
• Gram (-) negative spirochetes, obligate intracellular parasite - Reagin Screen Test (RST)
• Venereal disease 2. Specific Treponemal antibody tests
• Two types of transmission: ▪ Do this after tested (+) positive in Non-specific or non-treponemal serological tests
1. Acquired: transmission through body fluids (VDRL or RPR)
▪ Sexual contact ▪ Confirmatory test
▪ Sharing of contaminated needles (skin lesion) ▪ Uses Treponema pallidum
▪ Direct contact - Antigen
2. Congenital: Mother to baby transmission - To detect specific antibodies produced by the patients suspected with syphilis
▪ Transplacental ▪ Other Serological Tests For Syphilis
▪ During Childbirth - Anti-Treponemal antibody (ATA)
• Stages of Syphilis - Anti-Treponemal ABs group detected by Reiter Protein Complement Fixation
1. Primary Stage: Hard Chancre/Hunterian Chancre Test (RPCFT)
2. Secondary Stage: Condylomata lata, may involve CNS, eyes, bones, liver ✓ Appears later than specific ABs
3. Latent Stage: No sign asymptomatic/non-reactive ✓ Some syphilis patient do not produce the form of ABs
4. Tertiary Stage: Gummas, involve deep organs ✓ Use is limited
Non-specific or Non-treponemal Serological Tests
Venereal Disease Research Laboratory (VDRL) Rapid Plasma Reagin (RPR)
Old method Modified version
• Read Microscopically using Microscope • Read Macroscopically
○ Cannot be read macroscopically because it will be hard to see the flocculation due o due to charcoal
to its color (White) o Clumping of cells is seen immediately Non-reactive Weakly reactive Strongly reactive
Non-reactive Reactive
Examination

From Left to Right: Reactive (with flocculation), non-reactive (without flocculation)

• Heated serum (common) - heated at 56C for 30 minutes • Unheated Serum
Specimen
• CSF (only if the baby is suspected with neurosyphilis) *Not recommended for CSF specimens
Method Microflocculation method Macroflocculation method
composed of rings which are 15mm in diameter and 1.75mm deep
Slide

• 0.03% Cardiolipin: serves as antigen • Original VDRL reagent • Phosphate: enhances solution
Reagent • 0.21% Lecithin: enhances sensitivity of the cardiolipin • Disodium EDTA: enhances the suspension • Thimerosal: preservative
• 0.90% Cholesterol: enhances the whole reaction • Charcoal: visualization • Choline Chloride: eliminates heating process
1. Add 1 drop of VDRL reagent using an
• Methods:
appropriate needle gauge depending on the
o Qualitative: 18-gauge needle • Add 1 drop of each reagent
Procedure method used (refer below)
o Quantitative: 19-gauge needle • Place in the ROTATOR at 100 rpm for 8 mins
2. Place it in ROTATOR at 180 rpm for 4 mins
o CSF: 22/23-gauge needle
o >4 mins = cannot be read
• Reactive (+): with flocculation • Reactive (+) o HIV • Weakly Reactive
Manner of Reporting/Result
• Non-Reactive (-): without flocculation o Syphilis: w/ warts o HPV: if w/ warts • Non-Reactive (-)
• False (+) positive result o Rheumatic fever o Malaria • False (+) positive result ▪ TB o Pregnancy
False result o SLE o Infectious Mononucleosis (IM) o Pregnancy o Collagen disease ▪ Chickenpox o Old age
▪ Arthritis ▪ Hepatitis • False (-) negative result

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 MODULE 4: SEROLOGIC TEST FOR SYPHILIS

▪ Lupus Erythematosus ▪ Measles o Technical errors

o Infections ▪ Leprosy ▪ wrong specs of agitation
▪ Infectious Mononucleosis (IM) o Autoimmune disease ▪ expired reagents
▪ Malaria ▪ Gonorrhea o Low antibody titer
▪ Prozone

Specific Treponemal Antibody Tests

Treponema Pallidum Immobilization Fluorescence Treponemal Antibody Absorption Treponema Pallidum Microhemagglutination T. Enzyme Linked
Test
Test (TPI) Test (FTA-ABS) Hemagglutination Test (TPHA) Pallidum Test (MHA-TP) Immunosorbent Assay (ELISA)
• In Other books: Most specific is FTA-ABS • Less sensitive
o If there are TPI and FTA-ABS in multiple choices, • Disadvantage: Less reliable in
Description Most specific test for syphilis
answer FTA-ABS diagnosing primary syphilis (causes
o UPDATED false (+) results)
• Neutralization
o Specific T. pallidum antibodies from • Indirect fluorescence
Principle Indirect agglutination Hemagglutination Indirect ELISA
the patient are mixed with actively o Increase the specificity of the reiter strain
motile T. pallidum (Nichols strain)
• T. pallidum reiter strain • T. pallidum antigen
• T. pallidum nio\chols strain • Glutaraldehyde stabilized either with • Enzyme-labeled antibodies
• Nichols strain: live actively motile T.
pallidum organism extracted from • Fluorescent-labeled antibodies animal RBC (refer below) coated with
Tanned formalin sheep RBC
o Enzyme-labeled used:
Reagent and Composition tissues/lesions of infected rabbits o Composition: Dead T. pallidum. Dried and fixed treponemal antigen
coated with treponemal
Alkaline phosphatase
o Absorbent: Reiter teponemes o Sheep RBC o Substrate used: Para-
• Complement: Guinea pig o Chicken RBC
antigen
o Conjugate: Fluorescent-labeled AHG nitrophenyl phosphate
complement
▪ Fluorescein isothiocyanate: Dye used • Turkey RBC ▪ White turns to yellow when
o Dilution: 1:15 alkaline PO is added
1. Patient serum is diluted and mixed with reiter strain 1. Tanned sheep RBCs are coated with
to remove nonspecific T. pallidum antibodies T. pallidum antigen from Nichol’s strain
Procedure 2. The absorbed serum is then added on a slide 2. If specific T. pallidum antigen is
x\containing nichols strain of T. pallidum present in the sample, agglutination
3. Fluorescent-labeled antibodies are then added will occur
(+) Positive: Fluorescence
• (+) Positive: immobilization of
treponemas
(+) Positive:
Result • Reactive (+): >50% (+) Positive: hemagglutination
hemagglutination
• Doubtful: 20-50%
• Negative: <20%

Antigens Antibodies Tests

Non-Treponemal or Non-specific Wasserman antigen (CARDIOLIPIN) REAGIN and anti-cardiolipin 1. VDRL 2. RPR
1. Treponemal group antigens - REITER CHON 1. Group Abs 2. Specific T. pallidum Abs 1. TPI 3. MHA-TP
Treponemal or Specific
2. Specific T. pallidum Antigens (pathogenic) ○ Anti-reiter CHON ○ Anti T. pallidum abs 2. FTA-ABS 4. TPHA

A. ANTIGENS • Treponemal Antibodies

• Wasserman Antigens o Directed against pathogenic T. pallidum and closely related strains
o Cardiolipin (Wasserman Ag) o Group Antibodies: directed group antigens
▪ A hapten and must be bound to a suitable carrier to be antigenic o Specific Treponemal Antibodies: specific for each treponemal antigen
▪ A phospholipid
▪ Microbial Cell Treponemes: serves as the foreign carrier C. SPINAL FLUID TESTS IN SYPHILIS
▪ Bound Phospholipid (cardiolipin): serves as the antigenic determinant
• Treponemal Antigens • Testing of Cerebrospinal Fluid (CSF) is an important part of patient monitoring as well
o Reiter CHON as diagnostic test.
▪ A protein found in most treponemes (both in saprophytic and pathogenic • Neurosyphilis
treponemes) o Abnormally high WBC count
▪ Specific Treponemal Antigens: specific for each treponeme species o Elevated protein levels in the CSF
o (+) VDRL result
B. ANTIBODIES Note: FTA abs IgG and IgM detection continues to be a confirmatory test in diagnosis of
• Non-Treponemal Antibodies syphilis
o Reagin: an anti-cardiolipin Ags o Currently, most hospitals use ELISA, Western blot and PCR

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 MODULE 4: SEROLOGIC TEST FOR SYPHILIS
▪ Methods that are being studied as an additional diagnostic test especially for o Pneumonia
congenital syphilis and neurosyphilis o Vaccination with live attenuated viruses
o Malaria
i. BIOLOGICAL FALSE POSITIVE ANTIBODY (BFP) REAGIN ANTIBODY o Pregnancy
• Chronic
• Associated with other diseases (BFP)
o Leprosy: reagin titer lowers immediately after treatment
• Acute
Comparison of Precipitation Techniques
Technique Application Principle
Nephelometry Igs, Complement, CRP, other serum proteins Light that is scattered at an angle is measured, indicating the amount of antigen or antibody present
Antigen diffuses out into gel that is infused with antibody.
Radial Immunodiffusion Igs, Complement
Measurement of the radius indicates concentration of the antigen
Both antigen and antibody diffuse out from wells in a gel.
Diffusion Complex antigens such as fungal antigens
Lines of precipitate formed indicate the relationship of antigens
Immunoelectrophoresis Differentiation of serum proteins Electrophoresis of serum followed by diffusion of antibody from wells
Immunofixation Electrophoresis Over – or – under production of antibody Electrophoresis of serum followed by direct application of antibody to the gel

Comparison of Agglutination Reactions

Type Of Reaction Principle Results
Direct Agglutination Antigen is naturally found on a particle
Indirect (Passive) Agglutination Particles coated with antigens not normally found on their surfaces Agglutination indicates the presence of patient antibody
Reverse-Passive Particles are coated with reagent antibody
Haptens attached to carrier particle.
Agglutination Inhibition Lack of agglutination is a positive test, indicating the presence of
Particles compete with patient antigens for a limited number of antibody sites
patient antigen
Hemagglutination Inhibition Red blood cells spontaneously agglutinate if viral particles are present

Comparison of Tests Used for the Diagnosis of Syphilis

Type of Reaction Principle Results Comments
Direct Agglutination Antigen is naturally found on a particle
MICROSCOPY
Indirect (Passive)
Particles coated with antigens not normally found on their surfaces Agglutination indicates the Requires active lesion. Must have good specimen, experienced technologist;
Agglutination
presence of patient antibody inexpensive
Requires active lesion.
Reverse-Passive Particles are coated with reagent antibody
More specific than darkfield; specimen does not have to be live
Haptens attached to carrier particle.
Agglutination Inhibition
Particles compete with patient antigens for a limited number of antibody sites Lack of agglutination is a positive test,
indicating the presence of patient antigen Flocculation; good for screening tests, treatment monitoring, spinal fluid testing;
Hemagglutination Inhibition Red blood cells spontaneously agglutinate if viral particles are present
false positives
RPR Modified VDRL with charcoal particles, more sensitive than VDRL in primary SY
Cardiolipin Reagin
TRUST Uses red particles to visualize the reaction; similar to RPR
Treponemal
FTA-ABS Nichol’s strain of T. pallidum Confirmatory; specific, sensitive, may be negative in primary stage
EIA Treponemal or recombinant Anti-treponemal Simple to perform; can be automated; not as sensitive as FTA-ABS
MHA-TP or SERODIA TP-PA Sheep’s RBCs or gel particles sensitized with T. pallidum sonicate Anti-treponemal Not as sensitive as FTA-ABS
PCR Non-treponemal DNA in patient sample is amplified None Highest sensitivity is in primary stage of SY; availability is limited

Characteristics of Acute Phase Reactants

Protein Response Time (hr) Normal Conc. (mg/dl) Increase Function
CRP 6-10 0.5 1000x Opsonization, complement activation
Serum Amyloid A 24 3.0 1000x Removal of cholesterol
Alpha-Antitrypsin 24 200-400 2-5x Protease inhibitor
Fibrinogen 24 110-400 2-5x Clot formation
Haptoglobin 24 40-200 2-10x Binds hemoglobin
Ceruloplasmin 48-72 20-40 2x Binds copper, oxidizes iron
C3 48-72 60-140 2x Opsonization, lysis
Mannose-Binding Protein 0.15-1.0 Complement activation

• CRP, Serum Amyloid: 1000x increase in inflammation

REFERENCES
• Procalcitonin: biomarker of sepsis Notes from the discussion by Mrs. Maria Redora R. Esteban, RMT
• Albumin, Transferrin, Prealbumin: negative acute-phase reactants CANVAS Notes

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