- It is a type of agglutination test.
- This test is used to detect presence of serum agglutinins (H and O) in patient’s serum with typhoid and paratyphoid fevers.
- At the end of first week, the antibody for Salmonella starts to appear in serum.
- During the third week, the antibody for Salmonella rises sharply in enteric fever.
- Two specimens of the sera are preferred for testing purposes at an interval of 7 to 10 days.
- This is done to demonstrate a rising antibody titre.
- Convalescent sera in case of Salmonella enteritis often agglutinate a suspension of the causal serotype which is helpful in retrospective diagnosis.
- Two types of tubes were used in Widal test. Dreyer’s tube which is narrow tube with conical bottom for H agglutination.
- Similarly, Felix tube was used which is short round bottomed tube for O agglutination as second type of tube.
- Nowadays, Kahn tubes are used which is of size 3 x 0.5 ml for both types of agglutination.
- Eight small tubes (8×0.5ml) are used where a serial two fold dilution of patient’s serum in normal saline is prepared in the ratio of 1:20, 1:40 and so on up-to 1280 or more.
- Seven tubes are used for serum dilutions and 5th for non-serum control.
- Equal volume (0.4cc) of antigen suspension (TH, TO, AH and BH) are added to the diluted serum and control saline.
- These mixtures are mixed thoroughly by shaking the rack and incubating at 370C for 4 hours.
- The reading is taken after overnight refrigeration at 40C.
- In some cases, the incubation is done in water bath at 370C overnight.
- Loose and cotton-wooly clumps are formed in H agglutination and a disc like granular deposit in O agglutination at the bottom of tube.
- Compact deposit can be noticed in case of control tubes.
- The maximum dilution of serum where agglutination occurs indicates the titre of antibodies.
- H and O of typhi, H of S. paratyphi A and B are routinely used antigens.
- O antigen of paratyphoid cross reacts with the typhoid O antigen due to sharing of factor 12 by them.
- For this reason paratyphoid O antigens are not used.
Preparation of Widal antigen
- 1% formalin is added to a 24 hours broth culture of saline suspension of an agar culture for preparing H suspension of bacteria.
- The organisms are cultured on phenol agar (1:800) if we have to prepare O suspension of bacteria.
- Standard smooth strains of the organisms are used i.e. S typhi 901, O and H antigens are employed for this purpose.
Interpretation of Widal test
- By the end of first week, agglutination appears in serum which rises sharply in 2nd and 3rd week.
- The titre remains steady till 4th week after which it starts declining.
- Demonstration of rising titre of four-fold or greater of H and O agglutinins at an interval of 4 to 7 days is the most important diagnostic criterion.
- Active infection is present if a titre of 100 of O or more and titre of 200 of H agglutinins are read.
- But, this is interpreted by following various factors like:
- Local titre can be read as positive reaction due to sub-clinical infection of salmonellosis in endemic area in which low titre of agglutinins is already present in the serum of normal individuals.
- Local titre is upto 80 in Calcutta and 60 in Siliguri.
- TAB vaccine immunization is also responsible to show high titres of antibody (H antibody titre 160 or more) in vaccinated individuals to each of the Salmonellae.
- The past enteric infection or in vaccinated cases, transient anamnestic reaction may occur during unrelated fever like malaria, influenza, etc.
- Nonspecific antigens (fimbrial antigens) may produce false positive result.
- Before the appearance of agglutinins if treatment is started with chloramphenicol, they are unlikely to appear subsequently; if the antibody is already present, no further rise in titre is expected.
- Widal tests may be positive in many healthy carriers and some have to be detected by Vi antiserum.
Widal test: Procedure, Interpretation