Widal test pdf - Asst. Lec. Amer Ali Khaleel

Practical Immunology and

Serology

Hawler Medical University

College of Health Sciences

Clinical Biochemistry Dept.

Ass. Lec. Amer Ali Khaleel

(M.Sc. Clinical Immunology)

Lab.3 :

Widal test

Introductio

• Widal test is a serological method to diagnose enteric or typhoid

fever that is cause by the infection with pathogenic

microorganisms like

Salmonella typhi

Salmonella paratyphi

a, b

and c.

• The method of diagnostic test is based upon a visible to the

naked eye agglutination (clumps) reaction between antibodies of

patient serum and antigens specifically prepared from

Salmonella

sp (KIT).

• Salmonella

possess the following 3 antigens:

• Flagellar antigen or H antigen

• Somatic antigen or O antigen

• Surface antigen or Vi antigen

Introduction: (cont.)

• The tests measure agglutinating antibodies directed against

Salmonella O somatic

surface antigen and/or

Salmonella H flagella

antigen of the suspected organism.

• The Widal test detects antibodies against O and H antigens.

• Type of techniques is use direct agglutination.

• Widal test, firstly descried by

Fernand Widal

in 1896.

Clinical manifestation:

•Chest congestion

•Constipation

•Diarrhea

•Feel week

•Gastroenteritis

•Headache

•High temperature

•Loss of appetite

•Stomach pains

•Weakness

Modes of

transmission :

•Ingestion of contaminated food or water.

•Rarely , from person to person – fecal-oral route.

•Food handlers/ Carriers.

Serodiagnosis of Typhoid

No.

Methods

Time

Consuming

Widal test by Rapid Slide (Screening) test

1 min

Widal test by Tube Agglutination test

2-4 hours

Typhidot Tests (IgG/IgM rapid test) by Chromatography

15 minutes

Tubex TF

10 minutes

ELISA (Enzyme-Linked ImmunoSorbent Assay) (IgG/

IgM)

45 minutes –

2 hours

ECL (ElectroChemiluminescent immunoassays) (IgG/ IgM)

45 minutes –

2 hours

PCR (Polymerase Chain Reaction)

7-10 days

The rapid slide test is widely used in private laboratory and hospitals in Hawler. PCR an excellent tool
for the early diagnosis of typhoid, due to its very high sensitivity and specificity.

Principle of Widal

test:

•Antibody in the serum produced in the response to

Salmonella

organism, the kit contains antigen

suspensions that are killed bacteria and they were

stained to enhance the reading of agglutination

tests.

•The

blue

stained antigens are specific to the somatic

antigens (O-Ag), while the

red

stained antigens are

specific to the flagella antigens (H-Ag).

Materials And Reagents Provided With The

Widal Kit:

•Antigen suspensions (specifically prepared from

Salmonella

sp.) 8 Antigens: O, H,

, BO, BH,

• Positive control (Vial).
• Negative control (Vial).
• Instruction for use (leaflet).
• White Glass slide.
• Stirring Sticks.

Widal

controls:

Widal negative control (-)

•Contains no antibodies against the specific bacteria.

Widal positive control (+)

•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, S. paratyphi AO and
BO, S. paratyphi CO and CH antigens and is useful in the validation of
the performance of Widal reagents.

Notes

1- A positive and negative control should be run with each test.

2- If the reagent fails to agglutinate with the positive control, or
dose agglutinate with the negative control, it should be discard or
review the procedure and repeat the test with a new test device.
If the problem persists, discontinue using the test kit immediately
and contact your local distributor.

3-Before started the Widal test EXCLUDE these types of antigens
suspension especially AO, AH, CO, CH because these serotypes is
not present in Kurdistan region.

Additional materials and reagents

required for the febrile agglutinins

test:

•Applicator sticks (glass rod or wooden).
•Automatic pipettes or plastic droppers.
•Laboratory alarm clock or Timer.
•Glass plate or card plastic slide or disposable test

slide or reaction slides with white background.

Rapid Slide

(screening) Test:

• Slide Widal test is more popular as it gives rapid results.

Producers:

1- Place 1 drop (or transfer 50 µl) of the serum patient into

each of circle slide.

2- Add 1 drop of well shake Ag. O, H, B (O) & B (H)

respectively into each circle.

3- Spread the contents to fill the whole circle area.

4- Mix & rotate the slide for 1 minute & observe for

agglutination.

5- Report the result.

Observation and

Result:

• No agglutination = Negative

• Result reported as ‘

titres’

: Highest dilution where agglutination is

seen:

• If agglutination appear after 15 seconds = (1:640)
• If agglutination appear after 30 seconds = (1:320)
• If agglutination appear after 1 min. = (1:160)
• If agglutination appear after 1.30 min. = (1:80)

• This test is a screening test only for the detection of Widal agglutinins.

If result is positive it must be confirmed by other serological tests for

Widal.

Interpretatio

•Salmonella typhi O

(+ve mean recent (acute) infection).

•Salmonella typhi H

(+ve mean old (chronic) infection).

•Salmonella paratyphi bO,bH

(+ve mean carrier can infect other).

•Test results need to be interpreted carefully in the light of past history
of enteric fever, typhoid vaccination, general level of antibodies in the
populations in endemic areas of the world.

Limitations of Widal test:

• The Widal test has a very low specificity, less sensitive, confusing

and difficult to interpret for the diagnosis of typhoid f ever,

because cross-agglutinating antibodies remaining from past

infections with related salmonella serotypes give

false-positive

results

• Furthermore, in areas where fever due to infectious causes is a

common occurrence. So

false positive reactions

may occur as a

result of non-typhoid.

• In spite of several limitation many Physicians depend on Widal

Test.

Causes Of False-positive Widal

Agglutination Tests:

1. Previous immunization with Salmonella antigen.
2. Cross-reaction with non- typhoid Salmonella.
3. Variability and poorly standardized commercial

antigen preparation (kit).

4. Infection with malaria.
5. Other Enterobacteriaceae sharing the same s-LPS .

Why We Still Use Widal Test

Widal A Deleted Test In Many Nations

• The Widal test is an old serologic assay for detecting antibodies

to the O and H antigens of salmonella.

• Although the test is no longer performed in the U.S. or other

developed countries, it is still in use in many developing

countries because of its low cost and limited resources require

the use of rapid

, and it remains in use in many areas throughout the

world.

Prophyla

xis:

1. Simple hand hygiene and washing can reduce several

cases of typhoid.

2. All milk and dairy products should be pasteurized.

Summary:

• It is conclude, that even today, the Widal test remains

one of the best, easily accessible, cheap and simple

method for the diagnosis of typhoid fever.

1- Trying to drawing of blood sample from your colleagues.

2- Preparation of serum.

3- Do

Widal test

for your colleagues.

4- Report the result and interpretation.

Practical Part