Widal test 3rd yr.pptx

Widal test 3rd yr.pptx

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  WIDAL TEST PRESENTED BY:ASMITA PRAJAPATI 3RD YEAR BSC. MLT JFIHS/ LACHS
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  INTRODUCTION  Widal testis a common agglutination test which is employed in the diagnosis of enteric fever caused by Salmonella typhi & Salmonella paratyphi A, B & C.  It detects the presence of serum agglutinins i.e. ‘O’ & ‘H’ in patients serum with typhoid & paratyphoid fever.
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   This testwas developed by Georges Ferdinand Widal in 1896.  It is reliable & can be of value in the diagnosis of typhoid fevers in endemic areas when facilities for culturing are not available.
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   Salmonella antibodystarts appearing in the serum at the end of 1st week & rise sharply during the 3rd week of endemic fever.  In acute typhoid fever, O agglutinins can usually be detected 6-8 days after the onset of fever & H agglutinins after 10-12 days.  It is preferable to test two specimens of sera at an interval of 7-10 days to demonstrate a rising antibody titre.
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  PRINCIPLE  Its mainprinciple is that if homologous Ab is present in patients serum, it will react with respective Ag in the reagent & gives visible clumping on the test card & agglutination in the tube.  The Ags used in the test are ‘H’ & ‘O’ Ags of S. typhi & ‘H’ Ag of S. paratyphi. ‘O’ is a somatic Ag & ‘H’ is a flagellar Ag.  The ‘O’ Ag of S. paratyphi is not used as it cross reacts with the ‘O’ Ag of S. typhi due to the sharing of factor 12.
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  PREPARATION OF WIDAL ANTIGENS The Ag suspensions used for the Widal test may be prepared from suitable stock cultures in the lab. But generally commercially prepared Ags are used.  S. typhi is used to prepare S. typhi ‘O’ & ‘H’ Ags.  H suspension of bacteria is prepared by treating overnight broth culture or saline suspension of Salmonella with 0.1% formalin.
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   For preparationof ‘O’ suspension of bacteria, the organism is cultured on phenol agar (1:800) to inhibit flagella. The growth is then emulsified in small volume of saline, mixed with 20X its volume of alcohol, heated at 40-50°C for 30 mins. & centrifuged.  The Ags thus prepared are treated with chloroform (preservative) & appropriate dyes are added for easy identification of Ags.
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  METHODS OF WIDALTEST  It can be performed in 2 different ways: 1. Slide agglutination Widal test a) Qualitative slide test b) Quantitative slide test 2. Tube agglutination Widal test • Tube agglutination test has more accuracy as compared to the slide agglutination technique. • However, a slide Widal test is more popular among diagnostic laboratories as it gives rapid results.
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  Slide Agglutination Test 1.Qualitative slide agglutination test Procedure:  Firstly, bring all reagents to room temperature & mix well.  Add 1 drop of test sample (25µL) into each reaction circle labeled as O, H, AH &BH according to given Ag solution.  Add 1 drop of PC into the circle marked as PC & 1 drop of isotonic saline on the next reaction circle marked as NC.  Add 1 drop of ‘H’ suspension to PC & NC and 1 drop of ‘O’, ‘H’, ‘AH’ & ‘BH’ Ag to the respective circles labeled.  Mix thoroughly with the aid of applicator stick.  Observe for agglutination.
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  Interpretation  Positive test:Agglutination within a minute.  Negative test: No agglutination.
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  2. Quantitative slideagglutination test Procedure:  Bring all reagents to room temperature to RT & mix well.  Dispense 1 drop of isotonic saline to the 1st reaction circle & then place 5, 10, 20, 40, 80µL of the test sample on the remaining circles.  Now, add 1 drop of the Ag, which showed agglutination with the test sample in the qualitative (screening) method, to each circle.  Mix the contents of each circle with the aid of applicator stick & rotate gently.  Observe for agglutination.
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  Interpretation  The Abtitre of the test sample is its highest dilution that gives a visible agglutination.  80µL corresponds to 1 in 20 dilution, 40µL to 1 in 40, 20µL to 1 in 80, 10µL to 1 in 160 & 5µL corresponds to 1 in 320 titre.  Agglutinin titre >1:80 is considered as significant infection & low titre indicate absence of infection.
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  Quantitative tube agglutinationWidal test  In Widal test, 2 types of tubes were originally used: 1. Dreyer’s tube (narrow tube with conical bottom) for H agglutination & 2. Felix tube (short round-bottomed tube) for O agglutination.  Nowadays, 3 x 0.5 mL Kahn tubes are used for both types of agglutination.
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  Procedure for Quantitativetube test:  Firstly, bring all the reagents to RT.  Prepare 4 sets of 8 Kahn tubes/test tubes & label them 1 to 8 for O, H, AH & BH Ab detection.  Pipette 1.9mL of isotonic saline to tube no. 1 of each antigen set.  Then, to tube no. 2-8 of all sets add 1mL of isotonic saline.  To the tube 1 of each set, add 0.1mL of the serum sample to be tested & mix well.  Then, transfer 1.0mL of the diluted serum from tube 1 to tube 2 & mix well.
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   Then, transfer1.0mL of the diluted serum from tube 2 to tube 3 & mix well. Continue this serial dilution till tube 7 in each set.  Discard 1.0mL of the diluted serum from tube no. 7 in each set.  Tube no. 8 in all the sets, serves as a saline control.  Now, the dilution of the serum sample achieved in each set is as follows: Tube no. 1 2 3 4 5 6 7 8(Contro ) Dilutions 1:20 1:40 1:80 1:160 1:320 1:640 1:1280
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   To allthe tubes (1 to 8) of each set add 1 drop of the respective Widal test Ag suspension (O, H, AH & BH) from the reagent vials & mix well.
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   Cover thetubes & incubate overnight at 37°C (approx. 18 hrs.).  Dislodge the sediment after incubation & observe for agglutination.  Interpretation  Agglutinin titre greater than 1:80 is considered as significant infection & low titre indicate the absence of infection.
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  LIMITATIONS OF WIDALTEST  It is done within 7 days of illness & after 4 weeks are usually negative.  The local titre of the place should be known for the results to be interpreted correctly.  The quantitative tube method of this test is highly time consuming.  Previous typhoid vaccine may contribute to elevated agglutinins in the non-infected population.  Other infections of non-enteric Salmonella infections such as typhus, immunological disorders, chronic liver disease may cause false positive result. Cross reaction between malaria parasites & Salmonella Ags may also cause false positive agglutination test.