- 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:
a)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.
b)Local titre is upto 80 in Calcutta and 60 in Siliguri.
c)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.
d)The past enteric infection or in vaccinated cases, transient anamnestic reaction may occur during unrelated fever like malaria, influenza, etc.
e)Nonspecific antigens (fimbrial antigens) may produce false positive result.
f)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.
g)Widal tests may be positive in many healthy carriers and some have to be detected by Vi antiserum