1. Serological Diagnosis of Bacterial Infection Experiment six

2. Serological Identification of Salmonella Select the specimen React with A-Z polyvalent antiserum React with several individual types of anti-O and anti-H antiserum React with anti-Vi antiserum Agglutination reaction ( ＋ ) ( － ) ( ＋ ) ( － ) Identify its group and phage

3. Serological diagnosis Classification: Classification: agglutination agglutination precipitation precipitation Samples: Samples: ASO test ------ Streptococcus ASO test ------ Streptococcus Widal’s test ------ S. typhi and paratyphi Widal’s test ------ S. typhi and paratyphi Weil-Felix Test ------ Richettsia Weil-Felix Test ------ Richettsia

4. Widal’s test

5. Introduction  It is a serological approach for the diagnosis of typhoid and paratyphoid fever in clinical laboratory.  This is a test for the measurement of H and O agglutinins of typhoid and paratyphoid bacilli in patient’s sera.  To assist to diagnosis typhoid fever or other salmonella infection.

6. Materials Antigens: 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 Antibody: serum of suspected patient Normal saline Normal saline Test tubes and pipettes Test tubes and pipettes

7. 1 2 3 4567 O H PA PB PROCEDURE

8. PROCEDURE Make the mark Make the mark Add reagents as the following: Add reagents as the following: Reagent (ml) 1 2 3 4 5 6 7 Reagent (ml) 1 2 3 4 5 6 7 Saline 0.5 0. 5 0.5 0. 5 0. 5 0. 5 0. 5 Patient serum 0.5 0.5 0.5 0.5 0.5 0. 5 discard Serum dilution 1:20 1:40 1:80 1:160 1:320 1:640 - Bacteria suspension 0. 5 0. 5 0. 5 0. 5 0. 5 0. 5 0. 5 Final serum 1:40 1:80 1:160 1:320 1:640 1:1280 - dilution Shaking several times, put it in 37 0 C water bath for 16-18 hours.Then let it stand at room temperature over night. Bacteria suspension: O H PA PB

9. Observation: 1. Control tube: no agglutination (-) 1. Control tube: no agglutination (-) 2. Lowest titer tube: absolutely agglutination (++++) 2. Lowest titer tube: absolutely agglutination (++++) 3. Other tubes: 3. Other tubes: three quarters agglutination(+++) half agglutination (++) three quarters agglutination(+++) half agglutination (++) one quarter agglutination (+) no agglutination (-) one quarter agglutination (+) no agglutination (-)Judgment: Agglutination titer: the highest dilution of serum Agglutination titer: the highest dilution of serum which appears (++) bacteria agglutination. which appears (++) bacteria agglutination.

10. Interpretation of results O<1:80,TH<1:160, PH<1:80 Normal value O ≥1:80 & TH ≥1:160 or O ≥1:80 & PH ≥1:80 Typhoid fever Paratyphoid fever O ≥1:80 & H <1:160 or O ≥1:80 & PH <1:80 Early infection or other salmonella infections O <1:80 & H ≥1:160 or O <1:80 & PH ≥ 1:80 Vaccination or nonspecific memory reaction

11. Interpretation of results  Dynamic observation – A gradual rise of antibody titers – A gradual rise of antibody titers – Titer of convalescence serum ≥4 times of that of early specimen – Titer of convalescence serum ≥4 times of that of early specimen  False negative – Preantibiotic – Preantibiotic – Immunosuppressed – Immunosuppressed

12. Conclusion 1 2 3 4 5 6 7 O ++++ ++ ++ + - - - H ++++ +++ ++ ++ - - - PA ++ + - - - - - PB - - - - - - - 1:40 1:80 1:160 1:320 1:640 1:1280 Negative control

13. Precautions Make the mark Make the mark Make sure the procedure Make sure the procedure carefully use the pipettes carefully use the pipettes Correctly Judge the results Correctly Judge the results