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Syphilis

You are here : Home/ Blood Bank Zone/ Trasfusion Transmitted Diseases/ 8. Syphilis

8. Syphilis

Syphilis is basically a sexually transmitted disease caused by Treponema pallidum. Post-transfusion syphilis was a big problem in the past, however as the incidence of syphilis has remarkably come down after the advent of antibiotics, the problem of post-transfusion syphilis has also reduced.

T.pallidum can be transmitted by fresh blood and platelets. It is not transmitted by plasma products fractionated from pooled plasma such as Factor VIII.

The incubation period of transfusion syphilis is 4 weeks to 4-5 months. It is usually not transmitted by stored blood as refrigeration at 4�C inactivates the organisms within 27 hours.

The incidence of transfusion syphilis has also been reduced due to :
1. Careful selection of donors and exclusion of high risk donors practising promiscuous behaviour.
2. Concomitant antibiotic therapy in most patients needing transfusion therapy.

However, there is still a possibility of transmitting syphilis due to increasing demand for fresh blood, blood components such as platelets and blood for exchange transfusion in newborns. it is still mandatory toscreen the donated blood units for syphilis as :
1. Screening for spirochaetes helps to exclude potential high risk donor groups who are likely to suffer from HBV & HIV infections.
2. Incidence of syphilis is on the rise again in certain regions.
3. Increasing demand for fresh blood components.
4. Cost of screening by VDRL/TPHA is relatively low.
5. Recipients of infected blood may acquire syphilis due to incomplete antibiotic therapy.
6. Spirochaetes may be present in blood in the early latent seropositive stage i.e. when the antibody just becomes detectable.

It may be important to remember that the positive test in the recipient may also be due to passively acquired antibodies from the donor.

Infectious agents

Syphilis is caused by spirochaetes which have flexible gram-ye type cell wall. Outer membrane of the bacterium cell wall contains the antigenic protein of infectious agent.

Diagnosis

Serology is the major diagnostic method. There are two groups of tests available;

Nonspecific: Using cardiolipin, a normal component of tissues as the antigen, to detect nonspecific antibodies (reagian) found in the patients with syphilis. 19% normal adults may give false-positive reactions.

Specific Antigen used is derived from T. pallidum to detect antibodies specific for pathogenic treponema. A number of particle agglutination tests have been developed which are cheap, specific and sensitive.

Non Specific Tests

Commonly employed nonspecific tests are of two types flocculation (VDRL) and complement fixation. These tests are relatively easy to perform, rapid and inexpensive and are primarily used for routine screening. VDRL test is the most widely used. It generally becomes positive 4-6 weeks after infection or 1-3 weeks after appearance of the primary lesion. It is invariably positive in the secondary stage and shows a high titre.

False positive tests are encountered in a variety of siutations (Biological False Positive-BFP). These can be transient or chronic.

Transient BFPS occur in
- Viral infection
- Vaccination
- Malaria
- Pregnancy

Chronic BFPs seen in
- Collagen disorders e.g. SLE
- Infectious mononucleosis
- Drug addiction
- Other autoimmune disorders

VDRL Test

Serological test for syphilis using VDRL carbon antigen.

Principle
A qualitative test for screening donors for syphilis. The antigen suspension used in the test must be prepared meticulosly prior to each run of test from a VDRL antigen stock.

The VDRL antigen is composed of a colourless alcoholic solution of beef cardiolipin, cholesterol and lecithin. Patients with syphilis produce IgM and IgG antibodies to lipid material released from damaged tissue and from the organism. The antilipid antibodies react with the VDRL antigen suspension producing microflucculation.

Equipment & Reagent

1. VDRL glass slides having 12 ring cavity each
2. Micropipette (O.05m1), disposable tips and graduated pipette
3. Tuberculin syringe along with 22 gauge regular bevel needle for 1/60 ml Ag drop.
4. VDRL carbon antigen with diluent
5. Mechanical rotator with timer
6. Microscope
7. Positive control
8. Negative control

Sample

Clotted blood/serum sample. Heat inactivate complement at 56�C for 30 minutes and test within 4 hours.

Procedure

1. Place 0.05 ml (50 ul) of patient�s serum/plasma in each cavity of VDRL glass slide. Also dispense one positive and one negative control.
2. Add 1 drop (1/60 ml) of prepared VDRL carbon antigen to each of the well using the tuberculin syringe.
3. Rotate the slide for 8 minutes on ashaker/rotator at 100 rpm.
4. Read the test immediately under low magnification (lOx) in a microscope.

Microscopic evaluation	Interpretatlon
Smooth suspension	Non-reactive
Slight roughness	Non-reactie
Small clumps	Weakly reactive
Medium or large clumps	Reactive

False negative results may occur in the or very late stage of the disease.

Treponema Pallidum Haemagglutinatlon Assay.

It is a specific test for detection of syphilis.

Principle

The test is used to detect human serum antibody to T.pallidum by an indirect haemagglutination (IHA) method. Preserved avian erthrocytes are coated with antigenic components of pathogenic T. pallidum. These test cells agglutinate in the presence of specific antibodies to T. pallidum and show characteristics patterns in microtitration plates.

Non-specific reactions are detected by using uncoated avain RBCs as negative control.

Reagents

1. Test cell-preserved avian erythrocytes sensitised with T. pallidum antigen.
2. Control cells-preserved avian erythrocytes
3. Diluent
4. Reactive control serum
5. Non-reactive control serum

Equipment

1. Accurate and properly maintained pipettes for delivering 10,25,75 and 190 microlitres.
2. U-well microtitration plates

Procedure (Qualitative/Screening Test)

Arrange samples according to laboratory worksheet

Each test requires 3 wells of a microtitration plate.

1. Add 190 ul of diluent to well 1.
2. Add 10 ul of serum to well 1.
3. Using a micropipette, mix the contents of well 1 and transfer 25 ul to wells 2 and 3.
4. Ensure that the test and control cells are throughly resuspended. Add 75 ul of control cells to well 2. Add 75 ul of test cells to well 3.
5. Tap the plate gently to mix the contents throughly.
6. Incubate 45-60 minutes at room temperature
7. Read results.

Results

Reactive and non-reactive controls (supplied in the kit) should be tested in parallel with each batch of tests. The non-reactive control should result in no agglutination and a tight button of cells being formed. The reactive control should show agglutination.

Interpretation

Carpet of agglutinated cells at the bottom of the well shows a positive reaction.

Prevention of post transfusion syphilis
1. Avoid use of fresh blood unless really indicated.
2. Recruit voluntary donors.
3. Prophylactic treatment of recipients of fresh blood to avoid post-transfusion syphilis.
4. Mandatory screening of all donated units for syphilis.

Although spirochactes get inactivated in blood stored that 4�C for 72 hours, it is still mandatory to screen all blood units for syphilis. A positive PPHAJVDRL test suggests promiscuous behaviour of the donor and the corresponding units should not be used for transfusion to prevent transmission of other blood borne infections.

VDRL/TPHA test therefore acts as a surrogate or non-specific marker of promiscuity.

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