LABORATORY DIAGNOSIS OF SYPHILIS

Lab diagnosis is essential because of the asymptomatic phase in the disease. And also to asses the cure after treatment. IT is done mainly by demonstration of Spirochetes under microscope Antibodies in serum or CSF

MICROSCOPY Specimens collected –infectious.so care required PROCEDURE: Lesion first cleaned with gauze soaked in warm saline & margins-gently scraped so that superficial epithelium is abraded. Gentle pressure applied at base of lesion & serum that exudes is collected Wet mount is prepared & observed under DARK GROUND MICROSCOPE

SLENDER SPIRALS

CONTD.. Treponema pallidum is identified by its slender SPIRAL structure with spiral ends & pointed ends. IMPORTANCE: Useful in primary , secondary and congenital syphilis. NOTE: Negative results don’t exclude diagnosis of syphilis because of its low sensitivity.

CONTD.. DFA-TP: Direct fluorescent antibody test-better & safe for diagnosis. Smears fixed with acetone & sent to laboratory Requires fluorescent tagged anti –Treponemal antiserum. More reliable-Specific monoclonal antibody

SEROLOGICAL TESTS The serological tests that are in practice are: Standard test for Syphilis – test for antibodies reacting with cardiolipin antigen. Tests for antibodies reacting with group specific Treponemal antigen Tests for specific antibodies to pathogenic Treponema

REAGIN ANTIBOBY TESTS Antigen – CARDIOLIPIN (or) LIPOIDAL antigen Wassermann complement fixation test(1906) Modificated method by PANGBORN(1945) Tube flocculation test of KAHN Venereal disease research laboratory test(VDRL) Rapid plasma reagin test(RPR)

VDRL TEST Slide flocculation test Term-REAGIN Principle: patients suffering from syphilis produce antibodies that react with antigen CARDIOLIPIN to produce flocculation that is read by microscope.

Requirements: VDRL antigen VDRL diluant VDRL slide Microscope Micropipette16 guage syringe Water bath Tips

VDRL antigen: alcoholic solution of composed of 0. 03% cardiolipin,0 VDRL antigen: alcoholic solution of composed of 0.03% cardiolipin,0.21% lecithin,0.9% cholesterol. VDRL slide: glass slide having eight depressions.

SAMPLE PREPARATION: serum is separated from patient’s blood that is collected & is inactivated . Sample is allowed to reach room temperature. ANTIGEN PREPARATION: 4.5ml VDRL diluant is taken & added to vial drop by drop by thorough mixing. Use limited to 18-24 hours.

Procedure: VDRL slide is taken. To test well,serum sample is added.(0.05ml) Positive & negative controls added to their respective wells.(50microL) With the help of 16 guage syringe,1ml of prepared antigen is added to all wells drop by drop

Antigen & specimen mixed thoroughly using separate tips. Now,slide is placed on VDRL rotator & rotated for 4min,observed under microscope Negative & positive controls are observed first to verify the quality of antigen. No flocculation -negative test well Flocculation – positive well

If flocculation is observed,screening test is considered REACTIVE If flocculation is observed,screening test is considered REACTIVE.accordingly,it is termed reactive,weakly reative & non-reactive. further confirmed by semiquantitative assay To say it reactive,minimum of 1/8 titre is required. Non-reactive- less than 1/2 titre.

NOTE:CSF is not recommended testing with the help of this method CONTD.. RPR test : Advantages: NOTE:CSF is not recommended testing with the help of this method Antigen – VDRL antigen with fine CARBON particles Evident to naked eye Time accessible since serum collected does not require heating

CONTD.. RPR test : Antigen – VDRL antigen with fine CARBON particles Advantages: Evident to naked eye Time accessible since serum collected does not require heating NOTE:CSF is not recommended testing with the help of this method

Automated VDRL-ELISA test CONTD… Automated RPR For large scales Automated VDRL-ELISA test To measure IgM & IgG separately & suitable for large scales

CONTD… BFP TESTS: biological false positive Reason :cardiolipin is present in mammalian tissue too Positive in about 1% individuals

CONTD… BFP REACTIONS: Acute-only for few weeks or months Due to acute infections,injuries,inflammation Chronic-greater than 6months Seen in SLE,leprosy,malaria,relapsing fever,infectious mononucleosis,hepatitis,tropical eosinophilia

CONTD… sensitivity titre Primary stage-60.75% Low-8 REAGIN ANTIBODY : detectable 7-10 days after appearance of primary chancre sensitivity titre Primary stage-60.75% Low-8 Secondary stage-100% High-16 to 128 or more

CONTD… Reagin tests are preferred mostly because they become negative on treatment.

GROUP SPECIFIC TREPONEMAL TESTS Tests using cultivable treponemes as antigen Reiter protein complement fixation test Antigen-lipopolysaccharide protein complex derived from treponeme Sensitivity & specificity-low