Chapter 32: Common Serological Tests, Widal test, Rheumatoid factor (RA factor)

COMMON SEROLOGICAL TEST

Some of the standard serological tests are discussed.

Widal test

Principle: This test is advised for antibodies in patients’ serum against Enteric bacilli to diagnose enteric or typhoid fever.

Sample

The serum of patients is needed.

Procedure:

1. Qualitative
2. Quantitative

This can be done by:

1. Slide method
2. Tube method
   

   Widal test dilution

Antigens

Salmonella groups A, B, C, D, and E and Paratyphi A, B, C.

Salmonella Typhi  antigens are:

1. O-Antigen (Somatic Antigen):- These are 0-17 groups. 95% fall in group A, B, C, D, and E.
   1. O-Ag rises in 50% of the cases by the first week. It disappears in 6-12 months. This is diagnostic for acute infection.
2. H-Antigen (Flagellar Ag).
   1. H-Ag rises slowly and disappears after many years.
3. V1-Virulence antigen. This is also a surface antigen. this is used to find the carrier state.

Precautions

1. No value if done before 7 days of the onset of fever. Two or preferably more tests are done every 3-5 days to see a rising titer.
2. Many people without the disease have agglutinin in low titer.
3. The immunized person will also show agglutinin (antibodies).
4. There will be a significant rise after 7-10 days.
5. The anamnestic reaction will show negative results in low dilution and will be positive in high dilution.
6. The test has to be done with a battery of antigens.
7. The slide test is suitable for screening, but confirmation should be done by tube method.

Interpretation

1. The history of patients and discussions with physicians will be helpful.
2. A single test is not diagnostic.
3. In a vaccinated case, antibodies may be present. So a significant level is a four-fold rise in O-Ag or at least O Ag is > 1:80.
4. Antibiotic use prevents the rise in antibody levels.

Negative Result

1. When there is no agglutination
2. Blood was drawn early before 7 days.
3. A negative test does not rule out an enteric fever.

Positive Result

1. Indicate infection.
2. In-person with a history of vaccination.
3. A high titer of H-Ag indicates recent/past disease or vaccine.
   

   Widal test result

Rheumatoid Factor (RA Factor)

1. Pathogenesis:
2. The etiology of rheumatoid arthritis remains unknown. Related factors are:
   1. Genetic factors are important.
   2. Hormonal factors.
   3. Psychosomatic factors.
3. There are pieces of evidence of immunologic factors involved in the articular and extraarticular manifestation of rheumatoid arthritis.
4. Some belief in infections but is not established.
5. In the USA, RA is among the most prevalent chronic conditions.
6. In most of the studied population is 1% to 2%.
7. This disease (RA) has three distinct stages:
   1.  The development of synovitis by the primary etiological factors.
   2. This is followed by immunologic manifestations that will initiate the initial inflammatory reaction.
   3. There is a transient inflammatory reaction in the synovium which, leads to the proliferative destructive process.
      

      Rheumatoid Arthritis swan neck deformity

8. Age:
   1. This may be seen at any age.
   2. Initially, this disease was seen among the ages of 30 to 50 years.
   3. Older age groups and overweight people are prone to arthritis.
9. Sex:
   1. Females are 2 to 3 times more prone than males to develop RA.
10. Signs and symptoms:
    1. This is a chronic, usually progressive inflammatory disorder of the joints.
    2. This disease may be:
       1. Mild illness of brief duration.
       2. This may be progressive destructive polyarthritis associated with systemic vasculitis.
    3. RA usually starts with fatigue, anorexia, weakness, generalized aches, and pain.
    4. Stiffness of the joints appears after weeks to months.

Mechanism of damage to cartilage and bone: The damage to the cartilage takes through the immune complexes.

Rheumatoid factor is IgM and IgG.

The outcome of rheumatoid arthritis

Mechanism of damage in the rheumatoid arthritis

Diagnosis:

1. Rheumatoid factor is used to diagnose Rheumatoid Arthritis.
2. This factor consists of a group of immunoglobulin reacting with the Fc region of IgG. This may be IgM, or IgG, or IgA.
3. RA factor is present in:
   1. Blood
   2. Joint fluid
4. It first appears in the joint fluid then in the blood.

Principle

1. These antibodies “RA” factor is identified by agglutination reaction.
2. The rheumatoid arthritis agglutination test is based on the reaction between patients’ antibodies in the serum. This is known as a rheumatoid factor that will react with the antigens derived from gamma globulins.
   

   Rheumatoid factor agglutination test principle

3. Haemagglutination: When sheep RBC are coated with IgG-Ab are used. Now add serum of patients, and this will give agglutination.
   

   Fig 174 Rheumatoid factor

2. Latex particles are coated with IgG, and then serum is added, and this will show agglutination.

Fig 175: Rheumatoid Factor Agglutination

Limitations

1. 70-90% of the cases are positive for rheumatoid arthritis.
2. A negative result does not rule out rheumatoid arthritis.
3. The false-positive test may be seen in:
   1. 71% Rheumatic fever.
   2. 30-40% SLE.
   3. 12% Gout
4. RA factor may be positive in tuberculosis & syphilis.
5. False Positive may also be seen in old age, liver disease, SBE, chronic lung disease & syphilis.