Human Health and Diseases (Annexe Slides)

1. Widal Test
Shubhadeep Bhattacharjee (PGT Biology)
St. Xavier’s School, Harmutty

2. Introduction:
•Widal test is aserological method to diagnose
enteric or typhoid fever that is causeby the infection
with pathogenic microorganisms like Salmonella
typhi, Salmonella paratyphi A, BandC.
•Themethod of this diagnostic test is based upon a
visible agglutination (clumps) reaction either inatest
tube or strip test or on aslide between antibodies of
patient serum and antigens specificallyprepared
from Salmonella sp.

3. Introduction:
•Antibodies appears after aperiod (2-3 weeks) in theserum
of the patient.
•Typhoid fever is an acute, life threatening and fevercausing
illness transmitted by the ingestion of food or water that
contaminated with the feces of aperson infected with the
bacteria Salmonella typhi or Salmonella paratyphi A, B
& Crespectively.

4. Introduction:
•Thetests measure agglutinating antibodies directed against
aSalmonella O somatic surface antigen and/or a
Salmonella H flagella antigen of the suspectedorganism.
•TheWidal test detects antibodies against Oand Hantigens.
•Typeof techniques is usedirect agglutination.
•Widal test, developed by FernandWidal in 1896.

5. Typhoid fever symptoms:
• Hightemperature
• feel week
• Headache
• Gastroenteritis
• Diarrhea

6. Laboratory diagnosis of typhoid:
Time
Consuming
No. Methods
1 Wi d a l t e s t b y R a p i d S l i d e ( s c r e e n i n g ) t e s t 1 m in
2 W i d a l t e s t b y Tu b e A g lu t in a t i on t e st 2-4 h ou r s
3
Ty ph idot Te st s( Ig G/ Ig M r a p i d t e s t ) b y
C h r oma t og r a ph y
1 5 m i n u t e s
4 Tu bex TF 1 0 m i n u t e s
5
E L I S A ( e n z y m e - l i n k e d i m m u n o s o r b e n t a s a y )
( I g G / I g M )
45 m in u tes – 2
h o u r s
6 P C R ( Poly me r a se C h a in Re a c t i o n ) 7 - 1 0 d a y s
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.

7. Principle of Widal test:
•Antibody in the serum produced in the response to
Salmonella organism, the kit contains antigen
suspensionsthat are killed bacteria and they were
stained to enhance the reading of agglutination
tests.
•Theblue stained antigens are specific to thesomatic
antigens (O-Ag), while the red stained antigens are
specific to the flagella antigens(H-Ag).

8. Materials and reagents provided with
the Widal Kit:
•Antigen suspensions(specifically prepared from
Salmonella sp.,) 8Antigens: O,H,AO,AH,BO,BH,
CO,CH.(EXCLUDEthese types of antigens
suspension especiallyAO,AH,CO,CHbecause these
serotypes is not present in Kurdistanregion).
•Positive control (Vial).
•Negative control (Vial).

9. Widal Test
•Typically, patient serum is diluted into aseries
of tubes or wells on aslide and reacted with
bacterial antigens specific for the suspected
disease.
•Thetests measure agglutinating antibodies
directed against aSalmonella Osomatic
surface antigen and/or an Hflagella antigen
of the suspectedorganism.

10. Widal controls
-Widal negative control (-):
• Contains no antibodies against the specificbacteria.
-Widal positive control (+):
• Thewidal positive control contains ready to usestandardized goat
antiserum with polyspecific antibodies having specific reactivity
towards S.typhi Oand Hantigens, S.paratyphi AHand BH,S.
paratyphi AOand BO,S.paratyphi COand CHantigens and is useful in
the validation of the performance of Widal reagents.

11. Notes:
1Apositive and negative control shouldbe
run with eachtest.
2If the reagent fails to agglutinate with the
positive control, or dose agglutinate withthe
negative control, it should bediscard.

12. Additional materials and reagents
required for the febrile agglutinins test
•Applicator sticks (glassrod or wooden).
•Glassplate or card plastic slide or disposabletest
slide or reaction slides withwhite background.
•Pipettes or plasticdroppers.
•Sterile saline (0.85%).
•Test-tubes and test-tube racks.
•Timer or clock.
•Water-bath, temperature-controlled.

13. 1-Rapid Slide (screening)
Test:
Producers:
1Place1 drop (or transfer 50 µl) of the serum
patient into each of circleslide.
2Add 1 drop of well shakeAg.O,H,B(O) & B(H)
respectively into eachcircle.
3Spread the contents to fill the whole circle area.
4- Mix & rotate the slide for 1 minute & observe for
agglutination.
5- Report the result.

14. Observation and Result:
Complete agglutination =1:640
Approximately 75%of the cells are clumped =1:320
Approximately 50%of the cells are clumped =1:160
Approximately 25%of the cells are clumped =1:80
No agglutination =Negative
*Patient history should be taken into account before giving the result.
*This test is a screening test only for the detection of Brucella agglutinins. If result
is positive it must be confirmed by other serological tests forBrucellosis.

15. Interpretation:
•Salmonella typhi O(+ve mean recentinfection)
•Salmonella typhi H(+ve mean old infection)
•Salmonella paratyphi b (+ve mean carrier can infectother)
*Patient history should be taken into account beforegiving
the result.

16. Summary:
• TheWidal test hasavery 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.
• While more specific tests are now available like Typhidot Tests, but the
Widal test is still considered useful in diagnosing typhoid fever in
developing countries because of its low cost, and it remains in usein
many areas throughout the world.
• 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.

17. AnyQuestions ??!!!