Treponema pallidum- Laboratory Diagnosis, Prophylaxis & Treatment

 Laboratory Diagnosis

• Laboratory diagnosis important

-because of asymptomatic periods during the disease course and to assess the cure after treatment

Laboratory diagnosis is of great responsibility because of emotional and social nature of the disease (STD)

• Mainly microscopical examination and detection of antibodies in serum and CSF

Microscopy

-In cases of primary and secondary syphilis and in cases of congenital syphilis with superficial lesions

Lesions highly infectious-sampling done with care- lesion cleaned with gauze soaked in warm saline, superficial epithelium broken and serum exudate (without blood mixing in it) collected by applying slight pressure on the base of the lesion

Wet films of exudate prepared, covered with coverslip and observed under dark field microscopy- T. pallidum identified by slender spiral structure and slow movement- negative tests common because of its low sensitivity (104/ml number of organisms required for positive result)

Direct fluorescent antibody test for T. pallidum (DFA-TP) with fluorescent tagged anti- T. pallidum antiserum- Better and safer method

 Serological Tests

Ø  Tests for antibodies reacting with cardiolipin antigen –STS- standard tests for syphilis

Ø  Tests for antibodies reacting against group specific treponemal antigen

Ø  Tests for specific antibodies against pathogenic T. pallidum

STS- standard tests for syphilis /Nontreponemal tests / Reagin antibody tests

-for screening, primarily

-low specifity

-Reagin is the antibody reacting with the antigen, cardiolipin- hence the name Reagin tests

Measure antibodies directed against lipid antigens, principally cardiolipin, derived from host tissues

Eg., (1)Complement fixation test, flocculation test (earlier)

      (2) Venereal Disease Research Laboratory (VDRL) test, Rapid Plasma Reagin (RPR) tests (used now)

Complement fixation test (Wassermann reaction)

• Formerly used for serodiagnosis of Syphilis
• Purified lipid extract of beef heart with added lecithin and cholesterol was used as antigen and reacted with suspected serum
• Initially used as the principal test for syphilis, later replaced by flocculation tests

Flocculation test

• Soluble antigen + antibody---- antigen-antibody complex form which  remain suspended as floccules
• Flocculation test of Kahn -  widely used flocculation test, now replaced by VDRL

VDRL (Venereal Disease Research Laboratory) test

Developed in Venereal Disease Research Laboratory, USA

• ·        Inactivated serum (serum heated at 56^oC for 30 minutes) is mixed with cardiolipin antigen on a special slide  and rotated for four minutes
• ·         Clumps formed on reacting with the reagin antibody indicates positive result
• ·         By testing serial dilutions, antibody titre is determined, results expressed as “reactive’” weakly reactive’ “not reactive”
• ·         VDRL test can be used for CSF but not for plasma

Rapid Plasma Reagin (RPR) tests

• ·         Modification of VDRL test is RPR
• ·         uses the VDRL antigen containing carbon particles
• ·         RPR test can be done in unheated serum but not CSF

• ·         Automated RPR is also available 
• ·         Automated VDRL-ELISA test is also developed- determine IgG and IgM antibodies-suitable for large scale testing of sera

•     BFP (Biological False Positive) reactions occur due to cardiolipin antigen present in both Treponemal and mammalian tissues, with negative results in specific Treponemal tests.

•   Acute BFP (few weeks or months- in case of acute infections/inflammations) and Chronic BFP (longer than 6 months- in cases of SLE and other serious diseases)

Group specific tests for Treponema

• To avoid BFP’s, group specific tests done using cultivable treponemes-Reiter treponemes-

• RPCF-Reiter protein complement Fixation test-using a protein-lipopolysaccharide antigen from the treponeme
• Sensitivity and specifity lower than Specific T. pallidum tests
• some biological false positives can occur

 

Specific T. pallidum tests

Treponemal Test -detect antibodies directed against protein constituents of T pallidum.

Treponema pallidum Immoblisation (TPI) test

• ·         Test serum is incubated with complement and T. pallidum maintained in a complex medium         anaerobically
• ·         If antibody is present the treponemes are immobilized i.e. non-motile when observed under         dark ground illumination
• ·         Complex procedure
• ·         Once, considered gold standard, now replaced by Fluorescent treponemal antibody (FTA test) and other tests

Fluorescent treponemal antibody (FTA test)

• ·         Indirect immunofluorescent test using Nichol`s strain as antigen-Smears prepared on slides with Nichol`s strain

• 
  
• Currently used modification is FTA-absorption (FTA-ABS) test
• ·         Test serum is pre-absorbed with heat extract from cultures of non-pathogenic Reiter strain- to eliminate group specific reactions
• ·     FITC-labelled anti human immunolobulin is added and combined with patient antibodies adhering to T. pallidum, resulting in FITC stained spirochetes
• ·         As specific as TPI testt, but require suitably equipped laboratories

Hemagglutination methods

• ·         TPHA (T. pallidum Haemagglutination assay) uses tanned erythrocytes (sensitized with sonicated extract of T. pallidum as antigen)
• ·         presence of treponemal antibodies in patient serum detected by indirect agglutination of sensitized erythocytes
• ·         The procedure now employed is MHA-TP (MicroHaemagglutination test) which can be automated
• ·         Simpler and economical to perform than flourescent antibody tests
• ·         Sensitivity and specificity similar to that of the FTA-ABS test

Particle agglutination methods

• MHA test has been modified to use gelatin particles rather than erythocytes as the antigen carrier creating T. pallidum particle agglutination
• Sensitivity and specificity similar to that of the FTA-ABS test

Latex agglutination methods-

• use cloned T. pallidum antigens bound to latex particles
• easy to perform, fast and require less than 30 min for result

Enzyme immunoassay (EIA)

• First applied in 1975 as a serology test for syphilis
• 2 types of EIA tests are available- one uses sonicated T. pallidum as antigen and other one uses cloned antigen
• Advantage of EIA are capability to automate the test and run large number of samples in short time
• TPHA and FTA-ABS can identify BFP’s and confirm/exclude the diagnosis of Syphilis. 

Detection of IgM is helpful in doubtful cases-persistent IgM antibody indicates active continuing disease

Congenital syphilis is difficult to diagnose in asymptomatically infected neonates because maternal antibodies (IgG) which pass through the placenta and enter the fetal circulation cause reactivity in both nontreponemal and treponemal tests

• In uninfected infants, such maternal antibodies disappear by 3 months

• Because of the presence of maternal antibodies in the newborn, quantitative VDRL or RPR tests should be performed monthly over the first 6 months

• If the titer increases or stabilizes and does not decrease, congenital syphilis is indicated and the baby should be treated accordingly 

Prophylaxis

• ·         Refraining from sexual contact with an individual infected with Syphilis
• ·         Using a condom
• ·         Antiseptics or antibiotics minimize the risk
• ·     Penicillin remains the drug of choice for treating syphilis - Penicillin G & Tetracycline in penicillin allergic patients
• ·         No vaccine

 Treatment

Therapy for Primary, Secondary, and Early Latent Syphilis

Early cases- Benzathine penicillin G 2.4 million units IM in a single dose

Late Syphilis - Benzathine penicillin G 7 3 doses of 2.4 million units IM each at 1week intervals

 If penicillin allergic - Doxycycline / Tetracycline

Neurosyphilis -ceftriaxone

A person who has an STI (urethral discharge or genital ulcer) runs as much as four times the risk of contracting HIV from a sexual partner, than a person who is not infected with STI - HIV virus can be enter the body more easily through the genital ulcer

 STI Clinics in Kerala - 'Pulari' or STI clinics are centres for the treatment of Sexually Transmitted Infections -free of cost to anyone who walks into the clinics