Immunology and Serology Lab - 04 Serologic Test for Syphilis

IMMUNOLOGY AND SEROLOGY LABORATORY Serologic Test For Syphilis VENEREAL DISEASE RESEARCH LABORATORY (VDRL) • • OUTLINE • Serologic Test for Syphilis • o Syphilis o Non-Treponemal Test o False Reactions o Treponemal Tests • • SEROLOGIC TEST FOR SYPHILIS • • Syphilis is a sexually transmitted disease caused by the spirochete, Treponema pallidum. There are 2 main type of serologic assays used to detect patient antibodies in the diagnosis of syphilis: 1.) Non-Treponemal Antigen testutilizes beef heart (cardiolipin) that reacts with nontreponemal antibody (reagin). 2.) Treponemal Antigen testutilizes antigen from the treponemes to detect specific antibodies. The non- treponemal test is done for rapid screening, while the treponemal test is a confirmatory test. VDRL and RPR are screening tests whereas FTA- Abs is an example of confirmatory test for syphilis. SYPHILIS • • • • • • Venereal Caused by Treponema pallidum o Gram negative spirochete, Obligate parasite o Targets endothelial lining Has two types of transmission: o Acquired o Congenital Transmission o Acquired – Transmission through body fluids ▪ Sexual contact (Anal, Vaginal, Oral) ▪ Sharing of contaminated needles ▪ Direct contact with skin lesions o Congenital – Mother to baby transmission ▪ Transplacental ▪ During childbirth Laboratory diagnosis o Non-specific or non-treponemal serological tests o Specific Treponemal antibody tests Symptoms o Primary Syphilis ▪ Chancre (primary/initial symptom) ▪ Lymph adenopathy ▪ Lesions in penis (men), cervix (female) o Secondary Syphilis ▪ Infection fever RAPID PLASMA REAGIN (RPR) • • • • • • NON-TREPONEMAL TESTS • • • • • For screening of syphilis. Serological tests that detect reagin. Become positive 1 to 4 weeks after the appearance of initial symptoms. VDRL and RPR Initial symptom (Chancre) Microflocculation method VDRL Slide: Composed of rings which are 16mm in diameter and 1.75mm deep. VDRL Reagent: o 0.03% Cardiolipin – Serves as the antigen. o 0.21% Lecithin – Enhances the sensitivity of your antibody the cardiolipin. o 0.90% Cholesterol – Enhances the whole reaction Sample is serum or csf – Hence, useful in monitoring of neurosyphilis. o Methods: ▪ Qualitative ▪ Quantitative ▪ CSF o Sample is heated first at 56degC for 30mins ▪ Qual, Quan, CSF – 0.05mL sample ▪ Qual – 1 drop of VDRL reagent from a gauge 18 needle (1/60mL), put on an agitator set at 180RPM for 4 minutes. ▪ Quan – 1 drop of VDRL reagent from a gauge 19 needle (1/75mL), put on an agitator set at 180RPM for 4 minutes. ▪ CSF – 1 drop of VDRL reagent from a gauge 22/23 needle (1/100mL), put on an agitator set at 180RPM for 4 minutes. • Macroflocculation method Performed on unheated serum Recommended for screening Reagent: o Original VDRL reagent o Disodium EDTA o Charcoal o Phosphate o Thimerosal o Choline Chloride Reagent Components o Reagent – 1 drop of reagent from a gauge 20 needle (1/60mL) o Agitator – 100RPM for 8 minutes. o EDTA – Enhances suspension o Charcoal – Visualizing agent o Phosphate – Buffer o Thimerosal – Preservative o Choline Chloride – Eliminates the need of heating RPR (Rapid Plasma Reagin) Card test is a nontreponemal testing procedure for the serologic detection of syphilis. The test is performed on unheated serum or plasma with modified VDRL Antigen. There are various modifications of these tests. The teardrop card test uses plasma from a finger puncture blood. The Circle Card is recommended when various blood collections are used. The RPR Card Antigen suspension is a carbon- particle, cardiolipin antigen which detects “regain”- an antibody – like substance. When a specimen contains antibody (Ab), flocculation occurs with a coagulation of the carbon particles of the RPR card antigen, which appear as black clumps against the white background. Non- reactive specimen appears light gray. Both can be read macroscopically. • RPR Procedures o Materials ▪ RPR card reagin kit ▪ Dispenstirs ▪ Diagnostic cards ▪ RPR card antigen ▪ Clinical rotator o Procedure 1. Hold a dispenstirs device between thumb and forefinger near the stirringN or sealed end. Squeeze and do not release finger pressure until open end is below surface of specimen. Release pressure to draw up a sample. 2. Holding in a vertical position directly over the card test area to which the specimen is to be delivered (not touching card surface); squeeze dispenstirs device allowing one drop to fall onto card. 3. Invert dispenstirs device and with string end; spread the specimen covering the entire surface of the circle. Discard dispenstirs device. Repeat procedure for number of specimens to be tested. 4. Gently shake antigen dispensing bottle use. Holding in a vertical position, place one “free falling” drop onto each test area. Mixing of antigen and specimen is accomplished during rotation. 5. Rotate for 8mins, at 100 rpm, on mechanical rotator. 6. To help differentiate Non- reactive from Minimally reactive results, make a brief rotating and tilting of the card by hand. o Interpretation ▪ Reactive result shows a characteristic clumping ranging from slight but definite (minimal- tomoderate) clumping indicates the presence of syphilis whereas non-reactive result shows slight roughness or no clumping that indicates the absence of syphilis. OTHER NON-TREPONEMAL TESTS • • • • • • For confirming the presence of syphilis. Methods: o Treponema Pallidum Immobilization Test (TPI) o Fluorescence Treponemal Antibody Absorption Test (FTA-ABS) o Treponema Pallidum Hemagglutination Test (TPHA) o Enzyme Linked Immunosorbent Assay (ELISA) TREPONEMA PALLIDUM IMMOBILIZATION TEST (TPI) • • • • • Most specific test for syphilis Principle: Specific T. pallidum antibodies from the patient are mixed with actively motile T. pallidum (Nichols strain) o Neutralization Results: o >50% Reactive o 20-50% Doubtful o <20% Negative/Non-reactive Rarely used bec: Expensive, Highly technical, Time consuming. Nichols strain – Extracted from tissues of infected rabbits. FLUORESCENCE TREPONEMAL ANTIBODY ABSORPTION TEST (FTA-ABS) • • • • • • • Principle: Indirect fluorence Reagents: o T. pallidum reiter strain o T. pallidum nichols strain o Fluorescent-labeled antibodies Patient’s serum is diluted and mixed with reiter strain to remove non-specific T. pallidum antibodies. The absorbed serum is then added on a slide containing nichols strain of T. pallidum. Fluorescent-labeled antibodies are then added. Increased specificity due to the use of reiter strain Dilution: 1:15 Fluorescent = Fluorescein Isothiocyanate Anti-antibodies = Antibodies that bind to other antibodies. Toluidine Red Unheated Serum Test (Trust) Unheated Serum Reagin (USR) Reagin Screen Test (RST) FALSE REACTIONS • TREPONEMAL TESTS False positive o Collagen diseases such as Arthritis and Lupus Erythematosus o Infections such as Infectious mononucleosis, malaria, TB, chickenpox, hepatitis, measles and leprosy o Others: Pregnancy, Old age, Autoimmune disease False negative o Technical errors - Wrong specs of agitator, expired reagents. o Low antibody titer o Prozone • TREPONEMA PALLIDUM HEMAGGLUTINATION TEST (TPHA) • • • Principle: Indirect agglutination Tanned Sheep RBCs are coated with T. Pallidum antigen from Nichols strain. If Specific T. pallidum antigen is present in the sample, agglutination will occur. Antibody is coated in the tube. ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) • • • • • • Principle: Indirect ELISA Reagents: o T. pallidum antigen o Enzyme-labeled antibodies Tubes are coated with T. pallidum antigen. Antibody in the patient’s serum will bind to the antigen. Enzme-labeled antibodies are then added after washing. Finally, substrate is added following a second washing. Enzyme – Alkaline Phosphatase Substrate - p-Nitrophenyl Phosphate (Colorless solution that turns yellow if theres a presence of alkaline phosphatase)