1. LABORATORY DIAGNOSIS OF SYPHILIS

2. 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

3. 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

4. SLENDER SPIRALS

6. 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.

7. 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

10. 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

11. 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)

12. 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.

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

14. 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.

17. 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 hours.

18. 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

19. 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

20. 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.

21. 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

22. 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

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

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

26. 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

27. 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

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

29. 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