Enteric fever occurs when pathogenic microorganisms like S. typhi, S. parathypi A, S. parathypi B infect the human body. During the course of disease, the body responds to this antigenic stimulation by producing antibodies whose titre rises slowly in early stages, to a maxima and than slowly falls till it is undetectable. However in endemic areas, antibody titres upto 1:60 may be detectable in normal population. Antibodies to Salmonella organism may be detected in the patient serum from the second week after on set of infection. Information regarding the titres and whether or not they are rising or falling can be obtained by performing serological tests using WIDAL antigen suspension.

REAGENT

TYDAL contains ready to use concentrated, colour coded, smooth antigen suspensions of the bacilli; S. typhi O, S. typhi H, S. parathypi AH, S. parathypi BH, along with a polyspecific positive control reactive with these antigens. Each batch of reagents undergoes rigorous quality control at various stages of manufacture for its specificity and performance.

REAGENT STORAGE AND STABILITY
a) Store the reagent at 2-8°C. DO NOT FREEZE
b) The shelf life of the reagents is as per the expiry date mentioned on the

PRINCIPLE
When the coloured, smooth attenuated WIDAL antigen suspensions are mixed/incubated with patient serum, anti-salmonella antibodies present in the patient serum react with the antigen suspensions to give an agglutination. Agglutination is a positive test result, indicating he presence of anti-salmonella antibodies in the patient sample. No agglutination is a negative test result indicating absence of anti-salmonella antibodies.

NOTE :
In Vitro diagnostics reagent for laboratory and professional use only. Not for medicine use. The reagent contain 0.5% Phenol / 0.3% Formaldehyde as preservative. Avoid contact with skin and mucosa. On disposal flush with large quantities of water. Extreme turbidity may indicate microbial contamination / reagent deterioration. Such reagents should be discarded.

SAMPLES COLLECTION AND STORAGE
1. No special preparation of the patient is required prior to sample collection by approved techniques. Do not use haemolysed samples.
2. Clean and dry glassware free from detergents must be used for sample collection.
3. Do not heat inactivate the serum.
4. Though freshly collected serum is preferable, store samples at 2-8°C in case of delay in testing.

MATERIAL PROVIDED WITH THE KIT
a) Reagent Pack
Antigen suspension S. typhi 'O'
Antigen suspension S. typhi 'H'
Antigen suspension S. parathyphi 'AH'
Antigen suspension S. parathyphi 'BH'
Polyspecific positive control (Goat)

Accessories Pack
Glass slide with 6 reaction circles
Mixing sticks
Disposable sample dispensing pipettes with rubber teats

ADDITIONAL MATERIAL REQUIRED
Slide Test Method : Stop Watch
Quantitative Method : Timer, Kahn Tubes, Test Tubes, Pipettes (0.1 ml, 1ml) Isotonic saline, Incubator (37°C), Test Tube rack.

SLIDE TEST METHOD
1. Place one drop of positive control onto the reaction circle (PC) of the glass slide with the disposable droppers provided with the kit.

2. Place one drop o isotonic saline onto the next reaction circle (NC) of the glass slide.

3. Place one drop of patient serum to be tested onto the remaining four reaction circles.

4. Add one drop of WIDAL antigen suspension 'H' to the first two reaction circles (PN, NC)

5. Add one drop of WIDAL antigen suspensions 'O','H', 'AH', 'BH' to the corresponding reaction circle. with separate mixing sticks.

6. Mix contents of each circle uniformly over the entire circle with separate mixing sticks.

7. Rock the slide gently, back and forth, and observe for agglutination macroscopically at one minute.

QUANTITATIVE METHOD

TUBE TEST PROCEDURE
1. Take 4 sets of 8 Kahn tubes/test tubes and label them 1 to 8 for O, H, AH and BH, antibody detection.

2. Pipette into the tube no. 1 of all sets1.9 ml of isotonic saline.

3. To each of the remaining (2 to 8) add 1.0 ml of isotonic saline.

4. To tube no. 1 of all the sets add 0.1ml of serum sample to be tested and mix well.

5. Transfer 1.0 ml of the diluted sample from tube no. 1 to tube no. 2 and mix well.

6. Transfer 1.0 ml of the diluted sample from tube no. 2 to tube no. 3 and mix well. Continue this serial dilution till tube no.7 in each set.

7. Discard 1.0 ml of the diluted serum from tube no. 7 of each set.

8. Tube no 8 in all sets, serves as saline control. Now the dilution of the serum samples achieved in each set is as follows.

9. To all tubes (1 to 8) of each sets add one drop of the respective TYDAL antigen suspension (O,AH, and BH) from the reagents vials and mix well.

10. Cover the tubes and indicate incubate at 37°C overnight (approximately 18 hours).

11. Dislodge the sedimented button gently and observe for agglutination.

INTERPRETATION OF RESULTS

SLIDE TEST METHOD
Agglutination is a positive test result and indicates presence of clinically significant levels of the corresponding antibody in the patient serum. No agglutination is a negative test result and indicates the absence of clinically significant levels of the corresponding antibody in the patient serum.

QUANTITATIVE METHOD
The titre of the patient serum using WIDAL antigen suspensions is the highest dilution of the serum sample that gives a visible agglutination.

REMARKS
1. TAB vaccinated patients may show high titre of antibodies to each of the antigens.

2. 'O' being a somatic antigen brings about coarse, compact, granular agglutination whereas 'H' being a flagellar antigen brings about larger, loose, flocculant agglutination.

3. 'H' antigen, being species specific, is more reliable in determining the type of infection.

4. Turbid and contaminated sera should not be used for testing.

5. Generally antibody titres 1:80 or more are considered clinically and diagnostically significant.

6. It is recommended that results of the tests should be correlated with clinical findings to arrive at the final diagnosis.

7. since techniques and standardization vary from lab to lab one tube difference in tube titres can be expected.