4. To the tube No. 1 of all sets, add 0.1 mL of serum

sample to be tested and mix well.

5. Transfer 1.0 mL of the diluted serum from the tube

No. 1 to tube No. 2 and mix well.

6. Transfer 1.0 mL of the diluted serum from the tube

No. 2 to tube No. 3 and mix well. Continue this serial

dilution till tube No. 7.

7. Discard 1.0 mL of the diluted serum from tube No. 7.

8. This will give final dilutions in tubes 1 to 7 as 1:20,

1:40, 1:80, 1:160, 1:320, 1:640, 1:1280.

9. Tube No. 8 serves as saline control.

10. To all the tubes (1 to 8) of each set add one drop of

well mixed Vital Widal suspension of the respective

specificity. Mix well.

11. Incubate at 37°C overnight (approximately 18 ± 2

hours).

12. Dislodge the sedimented button gently and observe

for agglutination macroscopically.

Interpretation of Results

The titer of the patient serum using Vital Widal is the

highest dilution of serum that gives a visible agglutination.

Serology/Immunology 631

Generally antibodies having titers of 1:80 or more are

considered diagnostically significant.

Apart from the pattern of sedimented antigens, in the

tube test method a decrease in opacity as compared to the

saline control must also be considered while judging the

degree of agglutination.

Remarks

1. Serum from individuals vaccinated with TAB may also

show moderately elevated titers of ‘H’ agglutinin.

2. ‘O’ being a somatic antigen, brings about coarse,

compact and 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 serum should not be used

for testing.

5. It is recommended that results of the tests should be

correlated with clinical findings to arrive at the final

diagnosis.

6. Do not interchange vial top squeeze dropper cap.

POSITIVE CONTROL FOR WIDAL TEST

Summary

Enteric fever occurs when pathogenic microorganisms like

S. typhi, S. paratyphi A, S. paratyphi B, S. paratyphi C infect

the human body. During the course of disease, the body

responds to this antigenic stimulus by producing antibodies.

Antibodies to Salmonella organisms may be detected in the

patient serum from the second week after onset of infection.

Information regarding the titers and whether or not they are

rising or falling can be obtained by performing serological

tests using Widal antigen suspensions. The performance

of the Widal antigen suspensions can be validated with the

help of positive control for Widal test.

Reagent

The Widal Positive Control contains ready-to-use

standardized goat antiserum with polyspecific antibodies

having specific reactivity towards S. typhi ‘O’ and ‘H’ antigens,

S, paratyphi ‘AH’ and ‘BH’ antigens, S paratyphi ‘AO’ and

‘BO’ antigens and S. paratyphi ‘CO’ and ‘CH’ antigens and is

useful in the validation of the performance of Widal reagents.

Reagent Storage and Stability

1. Store the reagent at 2 to 8°C. Do not freeze.

2. The shelf-life of the reagent is as per the expiry date

mentioned on the reagent bottle label.

Each batch of reagent undergoes rigorous quality control

at various stages of manufacture for its specificity, sensitivity

and performance.

Principle

The positive control is mixed with the Widal antigen

suspensions to be tested and allowed to react. Specific

reactivity of Salmonella antigens if present in the antigen

suspensions will produce an agglutination reaction. No

agglutination indicates the deterioration of the performance

of the antigen suspensions.

Note

1. In vitro diagnostic reagent for laboratory and professional use only. Not for medicinal use.

2. The reagent contains sodium azide 0.1% as preservative.

Avoid contact with skin and mucosa. On disposal flush

with large quantities of water.

Additional Material Required

Stopwatch, isotonic saline, glass slide with clear/white

background, appropriate pipettes/micropipettes, mixing

sticks and a high intensity direct light source.

Procedure

a. Bring reagent to room temperature before testing.

b. Shake and mix the positive control for Widal test well

before dispensing.

Slide Test Method

1. Place one drop of positive control onto the reaction

circle of the glass slide.

2. Place 50 µL of saline onto the next reaction circle of

the glass slide.

3. Add one drop of test reagent (Salmonella antigen

suspensions) in each of the above circles.

4. Mix contents of each circle uniformly over the entire

circle with separate mixing sticks.

5. Gently rock the slide back and forth, observe for

agglutination macroscopically at one minute against

a white background.

Interpretation of Results Slide Test Method

Agglutination is a positive test result and indicates that the

test reagent (Salmonella antigen suspension) are performing

satisfactorily.

No agglutination is a negative test result and indicates

the deterioration of the test reagent (Salmonella antigen

suspension).

632 Concise Book of Medical Laboratory Technology: Methods and Interpretations Troubleshooting

Problem: False positive results

Possible causes Solutions

1. Past history of immunization, inapparent infection or prior disease Positive reaction will be seen in all three ‘H’ antigens in such cases

whereas in case of infection, antibodies will be seen only against the

infecting species. Also check the patient’s history

2. Anamnestic response to other vaccines and unrelated fevers in the

case of persons who have had a prior infection or immunization

The anamnestic fever can be differentiated from enteric fever by repetition of the test after a week

3. If reagents have been exposed to excessively high temperatures,

precipitates could be formed

The anamnestic response shows only a transient rise, while in enteric

fever the rise is sustained

Ensure that the reagents are not exposed to high temperatures and

are stored properly at 2–8°C

4. Contamination of serum could lead to false positives Ensure that clean and dry glassware free from detergents are used for

sample collection. If samples are not tested immediately, store them

at 2–8°C. Turbid serum should not be used for testing

Possible causes Solutions

1. Infection is in very early stages, when antibody titre is very low In enteric fever specific agglutinins are detectable in the patient’s blood

only after the first week hence blood taken earlier for testing may give a

negative result. Testing should be repeated after a week in such cases.

Demonstration of a rise in titer of antibodies by testing two or more

serum samples is more meaningful than a single test

2. Patients on antibiotic therapy during the testing phase Check the history of the patient for administration of the antibiotics

3.  Insufficient serum could give postzone effect Perform the dilutions as mentioned in the pack insert

4. Hemolyzed samples may be have been used Do not use hemolyzed samples for testing

5. Reagents not brought to room temperature.