Widal test is a milestone invention in medicine. This test was devised by Frank Widal in 1896. Widal originally described the test to diagnose Salmonella enterica serotype Paratyphi B infection.
Patients infected with S. enterica serotype Typhi and Paratyphi produce serum antibodies to the O and H antigens of these pathogens, and the detection of these specific antibodies forms the basis of this test, the standardized protocol of which was established in 1950 (Felix, 1950).
Widal test is the most widely used diagnostic test for typhoid fever in developing countries. The Widal test has been in use for more than a century as an aid in the diagnosis of typhoid fever. It measures agglutinating antibody levels against O and H antigens. The Widal test is positive after the tenth day of the disease and may be false positive if an individual previously received a TAB vaccine.
Unfortunately, S. enterica serotype Typhi shares these antigens with other Salmonella serotypes and shares cross-reacting epitopes with other Enterobacteriaceae. The levels are measured by using serial dilutions of sera. Tube agglutination method is the recommended method of performing the Widal test; where serial two-fold dilutions of the subject’s serum from 1:20 to 1:1280 are tested.
After its development, the rapid slide test became the most commonly used technique in local laboratories because of its convenience.
Ideally, the Widal test should be run on both acute and convalescent-phase sera to detect an increase in the agglutination titre but patient management cannot wait for results obtained with a convalescent-phase sample. For practical purposes, a treatment decision must be made on the basis of the results obtained with a single acute-phase sample. It is, therefore, important to establish the antibody level in the normal population in a particular locality in order to determine a threshold above which the antibody titre is considered significant.
In a situation where second sample collection is not feasible, knowledge of the agglutinin levels in the sera of normal subjects from the patients’ community can form the baseline on which a diagnosis can be made. For practical purposes, titres occurring in more than 5% of the subjects under study were not diagnostically significant and should be regarded as normal in that population (Collard et al., 1959).
Widal test is simple and inexpensive, so it has gained widespread use despite shortcomings of both sensitivity and specificity that compromise its utility as a diagnostic test.
Serological response with Widal agglutinins
The earliest serological response in acute typhoid fever is a rise in the titre of the O antibody, with an elevation of the H- antibody titre developing more slowly but persisting longer than that of the O- antibody cutoff titre. Usually, O antibodies appear on days 6-8 and H antibodies on days 10-12 after the onset of the disease. The O antibody concentrations fall about 6 months after previous exposure to typhoid. Patients from communities where typhoid is endemic have higher H-antibody titres than do those not previously exposed to the antigens.
Some authors have reported that the level of H agglutinins is unhelpful in the diagnosis of typhoid, mentioning that the H-agglutinin titre remains elevated for a longer period than the O-agglutinin titre after an episode of typhoid fever and also may rise as a nonspecific response to other infections. Others, however, have proposed that the H-agglutinin titre is as useful as or more useful than the O-agglutinin titre. Some of the studies found O titre to be of greater diagnostic significance.
Although simple to perform, the Widal test is difficult to interpret, requiring detailed knowledge of the patient’s medical, travel and vaccination history. The interpretation of a Widal test is greatly affected by the nature and extent of the patient’s previous contact with typhoid antigens, whether the contact depends on a clinical or subclinical infection with typhoid or related organisms or is from TAB vaccination. The less the degree of the previous contact, the greater the possibility that the findings of a Widal test may be usefully interpreted. In countries where typhoid is endemic and prevalent the test can be interpreted only if the reporting laboratory has information about the basic level of O and H agglutinin in the population. Figure well in excess of the known titres especially if the amount of antibody rises during the illness are highly suggestive of infection.
The value of the Widal test in diagnosing enteric fever in endemic areas remains controversial. Some express the view that the test lacks standardization and adequate sensitivity and specificity to be clinically useful, while others consider the test to have diagnostic value when judged with clinical findings and knowledge of the ‘normal’ O and H agglutinin titres in the local population (‘baseline titres’). The results from a single sample are difficult to interpret because high background rates of circulating antibodies to Salmonella enterica serotype Typhi or other Salmonella serotypes may produce a false-positive result. If paired sera are not available a single serum may be valuable if it yields an antibody concentration significantly in excess of the community norm for the test.
The sensitivity and specificity of the Widal test are known to vary widely, and a specific cutoff value should be established for each specific situation.
Sensitivity and Specificity of Widal test in Different Endemic Countries
|Country||Agglutinin||Cut-off titre||Sensitivity (%)||Specificity (%)||Reference|
|Philippines||O||1:20||61||88||(Aquino et.al., 1991)|
|Philippines||O||1:80||64||100||(Buck et al., 1987)|
|Jordan||O and H||1:160||92||–||(Shehabi,1981)|
|Ceylon||O and H||1:160||85.7||88||(Senewiratne and Senewiratne, 1977)|
|Philippines||O||1:160||72.5||57.5||(Roxas et al., 1989)|
|Ethiopia||O and H||1:160||82||–||(Abraham et al., 981)|
|S.Africa||O and H||1:200||75||92.5||(Sommerville et al., 1981)|
- Widal test kit (killed colored suspension of S. enterica serotype Typhi O antigen, S. enterica serotype Typhi H antigen and S. enterica serotype Paratyphi AH antigen and S. enterica serotype Paratyphi BH antigen).
- Normal saline
- Applicator stick
- Graduated pipette
The Widal agglutination test was performed using standardized suspension of S. enterica serotype Typhi ‘O’ and ‘H’ and S. enterica serotype Paratyphi A ‘H’ and S. enterica serotype Paratyphi B ‘H’ antigen.
Before use, the antigen suspensions were allowed to warm to room temperature and were well-mixed.
Rapid screening test
- Using a micropipette dispense one drop (80 µl) of undiluted serum was onto 4 different circles.
- Dispense one drop of O, H, AH, and BH antigens on these circles respectively.
- Mix it thoroughly with the aid of the applicator stick and rotate the slide gently.
- Agglutination was observed within a minute (+ve test).
- No agglutination (-ve test)
Rapid slide titration needs to perform for the samples which showed positive titre during rapid screening.
- Using a micropipette, dispense 40, 20, 10 and 5µl of undiluted serum onto a row of 3 cm diameter circles.
- Shake the reagent bottle rigorously shaken and add a drop (0.03ml) of the undiluted antigen suspension to each serum aliquot.
- Mix it thoroughly mixed with the aid of a stirring stick and rotate the slide gently.
- Observe the reactions after a minute.
- Agglutination was observed within a minute (+ve test).
- No agglutination (-ve test)
Reporting Widal test
The Widal test was reported by giving the titre for both O and H antibodies. The titre of each serum was read as the highest serum dilution giving visible agglutination.
The agglutination observed in any circle was indicative of the following results in a test tube.
Reporting of Widal test
|Serum volume||80 µl||40 µl||20 µl||10 µl||5 µl|
|Amount of antigen||1 drop||1 drop||1 drop||1 drop||1 drop|
|Equivalent tube titre||1:20||1:40||1:80||1:160||1:320|
Quality control was done using the positive polyspecific control of the same dilutions as the test sample. Normal saline was used for a negative control.
Significance of Widal test in typhoid endemic and non endemic areas
Epidemiologic studies in an endemic country have shown that at least seven subclinical cases of typhoid fever occur for each clinical case. Therefore a positive Widal test may be seen in apparently healthy persons from an endemic area as a result of previous subclinical infection.
Factors affecting Widal agglutinin titre (Limitation of Widal Test)
- Previous typhoid vaccination may contribute to elevated agglutinins in the non-infected population.
- Cross reaction between malaria parasites and salmonella antigens may cause false-positive Widal agglutination test
- False-positive Widal tests have also been reported for patients with non-enteric salmonella infection, for example, Typhus, immunological disorders, chronic liver disease, and cryptococcal meningitis
- Prior use of antibiotics can dampen antibody response giving a low titre in the Widal test even in the face of bacteriologically confirmed typhoid fever resulting in misdiagnosis
- The Widal agglutination titre varies with the geographic location based on the endemicity of the enteric fever, the prevalence of non-typhoid salmonellae infection and other infections which cross-react with salmonella antigen.
- Past infection with serotype Typhi or another nontyphoidal Salmonella serotype that shares common antigens gives false positive Widal test.
The value of the Widal test in diagnosing enteric fever in endemic areas remains controversial. But is still useful and widely available test in endemic areas. Most of the researchers reported that Widal test has a diagnostic value when judged with clinical findings and knowledge of the baseline O and H agglutinin titres in the local population. Although there was no consensus on the diagnostic titre for a single Widal test, all the studies reported a “positive” Widal test based on a fourfold rise in O agglutinins in repeated tests or a titre of >1:80 or greater in a single test. Some researchers found that the level of H agglutinins is more helpful than the level of O agglutinins.
Study carried out by Punia et al (2003) recommended the baseline titres for anti-O and anti- H for Salmonella enterica serotype Typhi and anti-H for Paratyphi as 160 for anti-O and 320 for anti-H to be diagnostic for enteric fever in Chandigrah India. A study done by Roxas et al (1989) showed O agglutinins of 1:80 in 65% of the apparently normal individuals by the slide agglutination technique. A study by Willke et al (2002) in Turkey; a typhoid endemic country, concluded that O and H agglutinin titres of ≥1:200 are of diagnostic significance.
To curb the overdiagnosis and misdiagnosis of Typhoid fever, Adeleke and Ihesiulor, 2008 recommended a single Widal titre of >1:160 or a four-fold increase in titre between two samples taken at least 10 – 14 days apart, as diagnostic. Acharya T et al reported that; both O and H agglutinin titre >1:160 could be diagnostically significant in the presumptive diagnosis of enteric fever in Nepal.
Elevated levels of both O and H agglutinin titre are more helpful than either of them alone, in making a presumptive diagnosis. When blood cultures are not available or impractical, a single Widal test can still have diagnostic significance, if the results are interpreted with relevant clinical findings and prevailing O and H agglutinin titres in the local population.