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• Lab diagnosis is essential because of the asymptomatic phase in the disease.
• And also to asses the cure after treatment.o IT is done mainly by demonstration of• Spirochetes under microscope• Antibodies in serum or CSF
MICROSCOPY
• Specimens collected –infectious.so care requiredPROCEDURE:• 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
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 antigenVDRL diluantVDRL slideMicroscopeMicropipette16 guage syringeWater bathTips
• 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.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.
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
particlesAdvantages: • Evident to naked eye• Time accessible since serum collected does not
require heatingNOTE:CSF is not recommended testing with the help of this method
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…
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