Widal test

(A short presentation to stimulate a long discussion!!)

Dr. Tshokey, MD Clinical Microbiologist JDWNRH

Typhoid fever & the Widal test in Bhutan

Background: - Bhutan is a (hyper?)endemic country, but no laboratory proven data - about 2000 cases reported annually - 2009 - 1811 cases, with 640 admissions, 1 death - 2010 Widal tests JDW - 5365. - ERRH-615 - CRRH 942 - Almost all the district hospitals also do the test. - Culture ?........

Diagnosis
Isolation of organism Demonstration of the genome Antibody response The definitive diagnosis of the disease requires the isolation of Salmonella typhi from the blood, faeces, urine or other body fluids.

Specimens for culture

Blood Faeces Urine Intestinal aspirate/vomitus BMA Bile culture

Isolation rates

90%
Blood

Bone Marrow

Faeces

Urine

Time in weeks after onset of symptoms

Clot culture
Recommended for rural setting where no clinical microbiology facilities are available Blood allowed to clot Clot and serum sent to lab Clot digestion by streptokinase or ground in Ox bile broth Incubated and subcultured on to routine media Serum is used for serology Very high rates of contamination Blood culture is superior but may be an option for our setting

Isolation rates
1/52 Blood Highest rate 2/52 Start to drop 3/52 4/52 5/52 Found only in 50% of 1/52 cases Found only Rate Highest at 50% of more than the end of cases blood 2/52 to 3/52 Isolation from blood is infrequent Bacteria does not disappear from faeces for long time, Chronic carriage > 1yr

Feaces

Urine

May present in urine for variable time after 3rd week Bone If properly performed highest isolation sensitivity, Even after Marrow 4/52 week possibility of having isolation rates up to 90% aspirate

The Widal test

Since late 19th century, Widal test developed by Widal and Sicard. Principle: Patients suffering from enteric fever would possess antibodies in their sera which can react & agglutinate Salmonella antigens. - S.typhi O and S.typhi H antigens. - O antigens for S.paratyphi A and S.paratyphi B are not taken as they cross-react with S.typhi O antigen. Status Lost/losing importance in many developed countries

Serological tests
Has many disadvantages Culture Gold standard Useful in
non endemic setting and for retrospective diagnosis (confirm the clinical diagnosis) epidemiologically Specially useful for atypical presentations

Both H and O are equally elevated and diagnostic or one of the two have been found to be more positive and diagnostic in different endemic areas and population.

Serological/Molecular tests for Typhoid

Widal agglutination test Counter-immuno electrophoresis(CIEP) Haemagglutination Enzyme linked immunosorbent assay (ELISA) Bactericidal antibody test Adherence test for detection of IgM antibodies Radio immunoassay (RIA) Co-agglutination test Latex agglutination test Polymerase chain reaction Diazo test of Urine

General interpretation of Widal Test

Timing is important, antibodies begin to arise during end of 1st wk. The titers increase during 2nd, 3rd and 4th wk after which it gradually declines. The test may be negative in early part of first week. Single test is usually of not much value. A rise in titer between two sera specimens is more meaningful than a single test. If the first sample is taken late in the disease, a rise in titer may not be demonstrable. Instead, there may be a fall in titer.

General interpretation.
Baseline titer of the population must be known before attaching significance to the titers. The antibody levels of healthy individuals in population of a given area give the baseline titer. A titer of 100 or more for O antigen is considered significant and a titer in excess of 200 for H antigens is considered significant generally. Patients already treated with antibiotics may not show any rise in titer, instead there may be fall in titer. Patients treated with antibiotics in the early stages may not give positive results.

General interpretation
Patients who have received vaccines against Salmonella may give false positive reactions. This can be differentiated from true infection by repeating the test after a week. True untreated infection results in rise in titer. Those individuals with past infections may develop antiSalmonella antibodies during an unrelated or closely related infection. anamnestic response ..differentiated from true infection by lack of any rise in titer on repetition after a week.

Problem with the test

Serological diagnosis relies classically on the demonstration of a rising titer in paired samples at an interval of 1014 days. However, a 4 fold rise is not always demonstrable, even in blood culture confirmed cases probably due to: the acute phase sample was obtained late high levels of background antibodies in endemic areas antibody response blunted by the early administration of antibiotics So, treatment decision are mostly made on the basis of results obtained with a single acute phase sample

The baseline
A study in Malaysia (endemic)showed, antibody titers up to 1/160 for both H and 0 antigens normal population In Sri Lanka, another endemic area, normal population had titers of 1/80 for both Any interpretation as to the significance of a Widal test result must be made against this "baseline" information. For example, titers of 1/50 and 1/100 on a single specimen, which are considered significant in nonendemic areas, are of no diagnostic significance in areas where S. typhi is endemic. We need to set our diagnostic criteria on the basis of our baseline (already planned to work on this)

Why the Widal test still survives?

Widely available & remains the only practical test available in most centers in developing countries Easy to perform and convenient Culture the gold standard not widely available and positivity rate is very low (<50% even in ideal situations) Good negative predictive value Still useful as a presumptive diagnosis in strong clinical settings (not confirmatory) Not other better options except slight modifications of the same test

Widal in our setting

In endemic areas, high titers could be demonstrated at an early stage in the illness, often during the first week. This suggests that, in an endemic area with frequent exposure to S. typhi and antigenically related salmonellae, the immune response may often not be a primary one. About 70% of patients express high antibody titers in the first week and rarely express a 4 fold risethus a single acute-phase widal may be useful in endemic settings (with a strong baseline titer)

10/4/2011

Tshokey/CC 2011

18