LABORATORY DIAGNOSIS OF SYPHILIS
•
• 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
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 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
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
Recommended
