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

Bone Marrow

Blood

Faeces Urine

90%

Isolation rates

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 2/52 3/52 4/52 5/52

Blood Highest rate

Start to drop

Found only in 50% of 1/52 cases

Isolation from blood is infrequent

Feaces Found only 50% of cases

Rate ↑ more than blood

Highest at the end of 2/52 to 3/52

Bacteria does not disappear from faeces for long time, Chronic carriage > 1yr

Urine May present in urine for variable time after 3rd week

Bone Marrow aspirate

If properly performed highest isolation sensitivity, Even after 4/52 week possibility of having isolation rates up to 90%

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 anti-Salmonella 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 10–14 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 non-endemic 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 rise…thus a single acute-phase widal may be useful in endemic settings (with a strong baseline titer)

18 10/4/2011 Tshokey/CC 2011