WIDAL AGGLUTINATION

TESTdr. Dewi Suryani, M.InfectDis

CONTENT

Principle of Agglutination Test Widal Agglutination Test Performance technique Interpretation of test results

- false positive- false negative

Basic Principle of Agglutination Test

Agglutination is a classic serologic reaction that results in clumping of a cell suspension by a specific antibody, directed against specific antigen

Source: Olopoenia & King, 2000

Image: Mayer, 2010

Image: Serum Analysis Centre, n.d

History of Widal Agglutination Test

Widal Agglutination test was first developed by Georges Fernand Isidore Widal (French physician and bacteriologist) in 1896 to utilse the diagnose of Typhoid Fever.Source: Rao, 2009

Image: APPL, 2008

Different type of Detection Methods

1. Serology Ab detection:

Widal

2. Immunochromatogrphy essay: Ag detection w/ monoclonal and polyclonalAb

Different type of Detection Methods

3. PCR to detect S. Typhi 3. Culture

Widal Agglutination Test

The Widal Test was based on demonstrating the presence of agglutinin (antibody) in the serum of an infected patients, against the Antigens of Samlomenella typhi and Salmonella paratyphi A and B: H (flagellar) Antigens O(Somatic) Antigens Vi (Virulen) Antigens

SLIDE TEST TUBE TEST

Performance technique: qulitative

Make 6 wells/circle on object glas and give the respective code: A, O, AO, AH, BH, positive control

and negative control

Place 0.02 cc of patient serum samples in each respective well

Add one drop of antigen into each well

Mix well with disposible applicator stick and rotate gently for 1 -5 minute

Check results: positive agglutination results is proceed for quantitative estimation

Performance techniques: Semiquantitative

Make circles on the glass object and Label the respective well

Circle no

Serum volume

(ml)

Antigen drop

Titre

1 0.02 1 drop 1:80

2 0.01 1 drop 1:160

3 0.005 1 drop 1:320

4 0.0025 1 drop 1:640

Performance technique: Semi Quantitative

Mix well with disposible applicator stick and rotate gently for 1 -5 minute

Interpretation of results: The last titre of the patient serum corresponds to the visible agglutination in the test circle

A significan results if there is an increase of titre after following chcek with 4-6 days interval

Performance technique

videoplayback_3.FLV

INTERPRETATION

Serologic studies are considered significant in endemic area if patients have four-fold or greater increases in O or H agglutinin titres in serum specimens obtained several days apart

Source: Olopoenia & King, 2000

INTERPRETATION

Report of test should be:- Agglutination -- Agglutination +, with titres

This is significant as there could be misleading interpretation due to false positive and false negative results.

Source: Olopoenia & King, 2000

REMARKS

A rising titre is more significant then a single high titre

There are range of cut off point for titres --> may vary between population and need to be established for each area

Always correlate clinical findings with lab findings

Interpretation of Widal Test

False Positive False Negative

• Previous immunisation with Salmonella antigen

• An inadequeate inoculum of bacetrial antigen in the host to induce antibody production

• Cross-reaction with non typhoidal Salmonella

• technical difficulty or errors in the performance of the test

• variability and poorly standarised commercial antigen preparation

• previous antibiotic treatment

• Infection with malaria or other Enterobactericaea

• variability in the preparation of commercial antigens

• other disease such as dengue

• read more then 1 minute after mixing the slide test

Source: Olopoenia & King, 2000

References

Mayer G. Immunoglobulins-Antingen-Antibody Reactions and Selected Test. 2010 [updated 12 july 2010; cited 1 Novemeber ]. Available from: http://pathmicro.med.sc.edu/mayer/ab-ag-rx.htm.

Olopoenia LA, King AL. Widal Agglutination Test - 100 Years Later: Still Plagued Controversy. Postgrad Med J. 2000; 76.

Rao S. Widal Test. Davanere; 2009 [updated 2 November 2011]. Available from: www.microrao.com.

Serum Analysis Centre. Serology. Kolkata; n.d [updated 2 November ]. Available from: http://www.indiamart.com/serum-analysiscentre-privatelimited/serology.html.