How to Identify and Isolate Salmonella Species Using Widal Test

The Widal test is a serological technique which tests for the presence of Salmonella antibodies in a patient’s serum. When facilities for culturing or antigen testing are not available, the Widal test if performed reliably and interrupted with care (with clinical findings), can be of value in diagnosing typhoid and paratyphoid in endemic areas. It is of no value in the investigation of Salmonella food-poisoning.

When investigating typhoid, the patient’s serum is tested for O and H antibodies (agglutinins) against the following antigen suspensions (usually strained suspensions):

1. typhi O antigen suspension, 9, 12
2. typhi H antigen suspension, d

Testing for Paratyphoid, A, B, or C: The following Antigens Suspensions are Required:

• paratyphi A O antigen suspension, 1, 2, 12
• paratyphi A H antigen suspension, a
• paratyphi B O antigen suspension, 1, 4, 5,12
• paratyphi B H antigen suspension, b. phase 1
• S, paratyphi C O antigen suspension, 6, 7
• paratyphi C H antigen suspension, c, phase 1

Salmonella antigen suspensions are widely available.* Most can be used as sliide and tube techniques, with manufacturers providing details for both slide (screen) and tube tests. Before use, the antigen suspensions must be allowed to warm to room temperature and be well-mixed. The test must be adequately controlled.

Availability of Antigen Suspensions (febrile antigen tests)

There are several suppliers by prices vary greatly (£5 to £34 for 5ml of each suspensions, 1999). Economically, priced products are available from BSL Global and Plasmatec, e.g. stained S. typhi O antigen suspension (code FAT 1010) and S. typhi  H antigen suspension (code 1002) from BSL Global each costs £5.66 (1999). Antigen O and H suspensions are also available for paratyphoid, A, B, C at the same price. 

Specimen: Sufficient serum for the Widal test can be obtained from 3 – 5ml of patients venous blood collected into a clean dry tube and allowed to clot. The serum must be free from red cells. It must not be heated. Whenever possible, collect a second specimen 7 – 10 days later to test for a rise in antibody titre. This, however, may not be possible to demonstrate.

Reporting Widal Reactions

The Widal test is reported by giving the titre for both O and H antibodies. The antibody titre is taken as the highest dilution of serum in which agglutination occurs. If no agglutination occurs the test should be reported as:

1. typhi O titre less than 1 in 20
2. typhi H titre less than 1 in 20

Note: The type of agglutination seen with O reactions is granular while that seen with H reactions is a more uneven type of clumping. Both slide and tubes are more easily read against a dark background.

Interpretation of Widal Test Results

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 a diagnostic value when judged with clinical findings and a knowledge of the ‘normal’ O and H agglutinin titres in the local population (‘baseline titres’). Of shared course concern, however, is how to avoid misuse and misinterpretation of the Widal test. Information received from tropical and developing countries where typhoid is endemic suggest that active typhoid is associated with:

• Significantly raised H or O agglutinins or both, e.g. titres greater than 1 in 180 or 1 in 200. Raising the ‘diagnostic’ titre, e.g. to 1 in 320 increases specificity but may significantly reduce the sensitivity of the test (depending on local O and H agglutinin titres, see later text).

• An early rise in antibody titre. Up to 70% of adult patients show a rise in antibody titre in the first week of infection. Some workers report that in a non-vaccinated patient the H titre is elevated earlier and more frequently than the O titre. Other workers report a rise on agglutinins as having slightly greater diagnostic value.

• Only a two or three-fold rise in one or both agglutinins when the Widal test is repeated 7 – 10 days later. A four-fold rise rarely occurs, possibly due to the fact that titres are already significantly raised when a patient’s serum is fist tested.

Note: Up to 10% of patients with active typhoid show no rise in O or H titres. This may occur in some patients due to severe hypoproteinaemia.

O and H Titres found in Local People

O and H agglutinin titres of most healthy persons have been found to be below those that occur in patients with active typhoid. A small percentage of patients with non-typhoid febrile (fever) illnesses may show significantly raised O and H titres.

Causes of Raised O and H Titres Other than Active Typhoid

These include previous Salmonella infections, chronic salmonellosis associated with schistosomal infection, vaccination with TAB or typhoid vaccine, current infection with other Salmonella species, chronic liver disease associated with raised globulin levels, and disorders such as rheumatoid arthritis, rheumatic fever, multiple myeloma, nephrotic syndrome, and ulcerative colitis. Following vaccination, H antibody titres remain elevated for 6 months or longer.

Important: A knowledge of local ‘normal’ O and H agglutinin titres is essential before attempting to interpret Widal test results.

Example of the Findings of a Survey from a Typhoid Endemic Area

Antimicrobial Sensitivity

Antimicrobials with activity against S. typhi include chloramphenicol, co-trimoxazole, and ampicillin. Chloramphenicol resistant strains, however, have been reported from developing countries and in recent years major typhoid epidemics caused by strains showing resistance to several antibiotics have occurred in Latin America, Mexico, the Middle East and Southeast Asia.

1. typhimurium multi drug resistance is causing a major public health problem in several developing countries and other parts of the world where the incidence of salmonellosis (transmitted from animals to humans) has increased greatly.