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 flagellatedflagellated, 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 o Clinical examination o Blood, bone marrow, or stool cultures for S. typhi o Serological Tests

8. 8 Duration of diseaseS Specimen examination %positivity 1 st weekBlood culture90% 2 nd weekBlood culture, Faeces culture Widal test 75% 50% Low titre 3 rd weekWidal 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. 1 2 3 4567 O H PA PB PROCEDURE Shake several times, put it in 37 0 C water bath for 16-18 hours. Then 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