WIDAL TEST

principles of common

Latex investigations methods and reporting 

Resources Needed:

·         Flip chart, Marker pens

·         Black/White board and chalk

·         Handout with different types of diagrams

·         Dispensing bottles with needles

·         Disposable test cards

·         Disposable dropper pipettes

·         Rubber teats

·         Disposable mixing sticks

·         O-Antigens

·         H-Antigens

·         Controls – positive and negative

·         Instruction manual

·         Disinfectant

·         Gloves

·         Rotator

·         Timer clock/stop watch

·         Saline, 0.85%

·         Disposal bucket

 Describe specific Immunology / Serology  investigations(Diagnostic procedures 

 List common specific immunology/serology investigation

·         VDRL/RPR,

·         HBs Ag,

·         Cryptococcus serum test,

·         Widal test,

·         pregnancy test,

·         rheumatoid factor test,

·         ICT

·         ASOT

Explain the principle and reporting of each common  immunology/serology investigations according to SOP

SIGNIFICANCE OF INVESTIGATING TYPHOID FEVER

·         To confirm diagnosis of typhoid fever

·         To determine the carrier status who has recovered from typhoid fever.

·         Pyrexia of unknown cause(persistent high temperature)

WIDAL TEST  BY TILE METHOD,

Reagents

·         O-Antigens

·         H-Antigens

·         Controls – positive and negative

Demonstration of the widal test  by tile method according to the SOP 

·         Demonstrate of the pregnancy test according to the SOP.

 Performance of the widal test by tile method(Qualitative analysis)

• Principle

Killed salmonella suspensions with O and H Ag reacts with corresponding Ab in the serum of the patient with infection and shows agglutation.

·         Procedure

o   Bring the test kit reagents to room temperature.

o   Mix the reagents before use

o   Pippette one  drop of O-Ag and H-Ag on  the circles on the tile or test cards.

o   Using a rubber teat and dropper pipette, dispense 1 drop (0.05ml) of sample on one of the test card circles labeled O and H.

o   Mix using the applicator stick

o   Dispense the positive and negative control sera in the same way and mix.

o   use your hands to rotate the test card for 1 minute..

o   Read the results macroscopically in a good light and check for agglutination.

o   Place all the materials in the bucket marked “INCINERATION”. 

NB: Each laboratory must determine the time necessary to perform the procedure (turnaround time).

·         Reporting results

o   Visible Agglutination indicates presence of salmonella Antibodies. Proceed to quantitative test( tube titration method)

o   No visible agglutination indicates absence of salmonella antibodies. Report as negative or non-reactive.

·         Limitations:

o   The test reagent must be stored at 2 – 8^oC and when in use place at room temperature before use.

o   Check carefully the expiry dates on the test kits.

·         Quality control

o   Always run positive and negative controls when performing the test, and make sure the control samples are reading correctly.

points for the widal test by tile method

·         Store reagents at the correct temperature according to the manufacturer’s instructions. 

·         Check carefully the expiry dates on the test kits.

·         Check that the catalogue numbers on the reagent containers match the numbers on the kit procedures in the package insert.

 

 Evaluation 

 QUANTITATIVE WIDAL TEST BY TUBE METHOD

This is done to all reactive patients sample after tile method to determine the degree of antibodies(infection)

materials

·         Disposable dropper pipettes

·         Rubber teats

·         Disposable mixing sticks

·         O-Antigens

·         H-Antigens

·         Controls – positive and negative

·         Instruction manual

·         Disinfectant

·         Test tubes

·         Incubator or water bath(37⁰c)

·         Gloves

·         Rotator

·         Timer clock/stop watch

·         Saline, 0.85%

·         Disposal bucket

 

Method

1.Arrange a total of 16 test tubes, each row 8 tubes as shown below

2.Label the 2 rows as O and H

Test Tube no.	1	2	3	4	5	6	7	8
Saline (ml)	1.9	1.0	1.0	1.0	1.0	1.0	1.0	1.0
Patient serum	0.1(neat)	-	-	-	-	-	-	-
Mix& transfer(ml)	1.0	1.0	1.0	1.0	1.0	1.0	1.0	Discard
Dilution	1:20	1:40	1:80	1:160	1:320	1:640	1:1280	Saline control

3.Add one drop of O and H antigen in to the each of the row of test tube.

4.Mix well and incubate at 37⁰c for 16-20 hrs.

5.check for agglutination. Titre is the highest dilution of the at which agglutination/flocculation is observed

Interpretation of results

• A titre of 1:80 or more is considered significant and rise in titre after a few days will confirm the diagnosis.
• Individuals with previous infection or with TAB vaccine will show H positive reaction. Culture the specimen to the diagnosis
• Chronic liver disease can also result to high O or H more than active typhoid fever.

References:

·         Ivan M. Roitt & Peter J.Delves ROITTS ESSENTIAL IMMUNOLOGY, 10th Edition, (2004) Replika press  Pvt.ltd ,india

·         Monica Cheesbrough – MEDICAL LABORATORY MANUAL FOR TROPICAl Countries, part 2, Edition (2003) Cambridge University press,international sales department.

·         Monica Cheesbrough – MEDICAL LABORATORY MANUAL FOR TROPICAL COUNTRIES, volume 2, Microbiology, Edition (1984) Cambridge University press.

·         Warren Levinson. MD,PHD– Medical Microbiology & Immunology edition (2004) Mc Graw –HillCompanies

Satish Gupte THE SHORT TEXT BOOK FOR MEDICAL MICROBIOLOGY(including parasitology) 10th Edition, (2004) Replika press  Pvt.ltd ,india