This document provides information about the Widal test, which is used to diagnose typhoid fever. It discusses that typhoid fever is caused by Salmonella typhi bacteria transmitted through contaminated food or water. The Widal test detects antibodies against Salmonella antigens in the patient's blood and is most reliable in the second and third week of illness. It describes how to perform the Widal test, noting that it detects antibodies against O and H antigens and that positive results are indicated by agglutination reactions. The document also covers limitations and factors affecting the accuracy of the Widal test results.

1. WIDAL TEST
Dr. Iman M. Fawzy; MD, MSc, PhD
Clinical Pathology
Mansoura, Egypt

2. caused by Salmonella typhi and less commonly by
Salmonella paratyphi.
The infection always comes from another human,
either an ill person or a healthy carrier of the
bacterium.
Transmitted through the ingestion of food or drink
contaminated by the feces or urine of infected people
The bacterium can withstand both drying and
refrigeration.

3. Countries endemic for typhoid(U.S. CDC 2006)

4. Salmonella typhi
flagellated, aerobic, Gram
-ve bacilli.

5. Socio-economic factors: It is a disease of
poverty as it is often associated with
inadequate sanitation facilities and
unsafe water supplies.
Environmental factors: Though the
cases are observed through out the
year, the peak incidence of typhoid
fever is reported during September. This
period coincides with Summer season
and a substantial increase in fly
population.
Social factors: pollution of drinking
water supplies, open air defecation, and
urination, low standards of food and
personal hygiene, and health ignorance
Risk factors

6. Incubation period :
Usually 10-14 days but it may be as
short as 3 days or as long as 21
days depending upon the dose of
the inoculums.
Reservoir of infection :
Man is the only known reservoir
of infection - cases or carriers.
Period of communicability:
A case is infectious as long as
the bacilli appear in stool or
urine.

7. Diagnosis of Typhoid Fever
oClinical examination
oBlood, bone marrow, or stool cultures for S. typhi
oSerological Tests

8. 8
Duration of
diseaseS
Specimen
examination
%positivity
1st
week Blood culture 90%
2nd
week Blood culture,
Faeces culture
Widal test
75%
50%
Low titre
3rd
week Widal test
Blood culture
Faeces culture
80-100%
60%
80%
Diagnosis of Typhoid Fever

11. Salmonella
Gram negative bacilli
SS agar: Non lactose fermenter, pale
colonies, H2S production
Selenite: for
stool
enrichment

12. TSI: alkaline red
slant, acid yellow
butt, H2S, Gas
production
MIO: indole negative,
ornithine negative,
motile
Citrate negative
Urease negative

13. 1.Detection of Antibodies in serum:
1.Widal test (Tube or Slide), 2.Typhidot assay
3.Tubex system, 4. Dipstick assay.
2. Detection of Antigens in serum:
1. Tubex system 2. Immunoelectrophoresis (CIE).
3. Co-agglutination test. 4. ELISA
3. Detection of Antigens in urine:
1.Tubex system 2. CIE,
3. Latex agglutination 4. Co-agglutination
Serodiagnosis of Typhoid :

14. Antigenic structure of Salmonella
H( flagella ) antigens
O (somatic) antigens
Vi (Virulence) capsular
polysaccharide antigens

15. Tube and slide agglutination test.
Detects anti O and H antibodies in serum
Diagnosis of Typhoid and Paratyphoid cases
Carriers of typhoid bacilli possess antibody against
the Vi antigen of S. typhi. (Vi tires seem to correlate
better with the carrier state than do O or H titres).
For this reason, the use of Vi agglutination for
detection of carriers was suggested .
WIDAL Test

16. Significance
 I st week negative.
Titers raise in 2nd week
Raise of titers is diagnostic
N.B.
Single test not diagnostic
Paired samples tests with rising titer
H agglutinins appear first

17. Typical Serological Profile After Acute
Infection
Note that during Reinfections, IgM may be absent or present at a low level transiently

18. Materials
Antigens:
◦Suspension of S. typhi "O" antigen, O
◦Suspension of S. typhi "H" antigen; H
◦Suspension of S. paratyphi A "H" antigen, PA
◦Suspension of S. schottmuelleri "H" antigen, PB
Antibody: serum of suspected patient
Normal saline
Test tubes and pipettes

19. 11 22 33 44 55 66 77
OO
HH
PAPA
PBPB
PROCEDURE
Shake several times, put it in 37Shake several times, put it in 3700
C water bath for 16-18 hours.C water bath for 16-18 hours.
Then let it stand at room temperature over nightThen let it stand at room temperature over night..

21. Observation:
**Do not shake tubes before reading the results
 Control tube (Tube No. 7): no agglutination (-)
 Lowest titer tube: absolutely agglutination (++++)
Interpretation:
Agglutination titer: the highest dilution of serum
which appears bacteria agglutination.

22. 1 2 3 4 5 6 7
O ++++ ++ ++ + - - -
H ++++ +++ ++ ++ - - -
PA ++ + - - - - -
PB - - - - - - -
1:20 1:40 1:80 1:160 1:320 1:640
Negative
control

23. Agglutination how it appears after
reactivity
O
Felix tube
Round bottom
O agglutination
Compact
granular
agglutination
H
Dreyer’s tube
Conical bottom
H agglutination
Loose
Cotton woolly
clumps
Observed for agglutination:
H : Loose , cotton woolly clumps;
 O : Compact Fine granular agglutination;
Supernatant should be clear;

24. O (somatic) antigens H (flagella) antigens
LPS in the cell wall;
Heat stable
Less immunogenic
Agglutination with antisera:
Fine, compact, granular
chalky clumps
 Present in flagella;
 Heat labile;
 Strongly immunogenic;
Induce rapid & High Ab titres;
 Agglutination with antisera:
Large, loose, cotton
wool clumps

25. Vi (virulence) antigen
Capsular polysaccharide expressed on certain serotypes
Heat labile;
Poorly immunogenic, BUT antibodies are protective:
1. Detection of Vi antibody not helpful in diagnosis
2. Absence in a case of typhoid  poor prognosis;
3. Persistence of Vi antibody : carrier state

27. Pozone phenomenon in
Agglutination tests
Prozone effect - Occasionally, it is observed that
when the concentration of antibody is high (i.e.
lower dilutions), there is no agglutination and then,
as the sample is diluted, agglutination occurs.
Lack of agglutination in the prozone is due to
antibody excess resulting in very small complexes
that do not clump to form visible agglutination

28. Limitation of Widal Test
The Widal test is time consuming and often times
when diagnosis is reached it is too late to start an
antibiotic regimen.
In spite of several limitation many Physicians depend
on Widal Test

29. False Positive Reactions with WIDAL Test
patients who have had previous vaccination or infection
with S typhi.
Cross-reaction with non – typhoidal Salmonella.
in association with some autoimmune diseases.
Infection with malaria

30. False Negative Reactions with WIDAL Test
Early treatment,
Relapses of typhoid fever.
Occasionally the infecting strains are poorly
immunogenic.

31. Slide Widal
test:

32. Prevention
And
Treatment

33. Antibiotics
Antibiotics, such as ampicillin, chloramphenicol,
fluoroquinolone
trimethoprim-sulfamethoxazole, Amoxicillin and
ciprofloxacin etc used to treat typhoid fever.

34. THANK YOU