Widal test

Mr. Parag Chauhan

Introduction

  1. The Widal test is a serological diagnostic method widely used to identify typhoid and paratyphoid fevers, collectively known as enteric fever. 
  2. The bacteria Salmonella typhi and Salmonella paratyphi cause these diseases.
  3. The test detects specific agglutinating antibodies—O (somatic) and H (flagellar)—produced in response to these antigens in the patient’s serum.
  4. While simple and rapid, the Widal test’s accuracy is influenced by prior vaccination, background antibody levels in endemic areas, and co-existing infections.

 

Principle

The Widal test is based on the principle of agglutination, where antigen-antibody complexes form visible clumps.

    • O Antigen: Somatic antigen, a heat-stable polysaccharide component of the bacterial cell wall. Its detection indicates acute infection.
    • H Antigen: Flagellar antigen, a heat-labile protein. It is associated with a later stage of infection or previous exposure.

Agglutination occurs when a patient’s serum containing specific antibodies is mixed with antigen suspensions. This reaction is visible to the naked eye on a slide or test tubes.

 

Requirements

  1. Samples:
    • Serum is the preferred sample. Collect 2–3 mL of venous blood in a sterile container and allow it to clot.
    • Centrifuge to separate serum.
  2. Reagents:
    • Commercially prepared Salmonella antigen suspensions, including:
      • S. typhi O antigen
      • S. typhi H antigen
      • S. paratyphi A O and H antigens
      • S. paratyphi B O and H antigens
    • Physiological saline (if serial dilution is needed).
  3. Equipment:
    • Clean glass slides.
    • Applicator sticks or mixing rods.
    • Micropipettes and tips for accurate measurement.

 

Procedure

Slide Agglutination Method (Qualitative Test):

  1. Preparation:
    • Arrange a clean glass slide and antigen suspensions.
    • Place a drop of each antigen (S. typhi O, H; S. paratyphi A, B) separately on different slide parts.
  2. Testing:
    • Add an equal volume (1 drop) of the patient’s serum to each antigen drop.
    • Mix the antigen and serum drops thoroughly using separate applicator sticks for each mixture.
    • Gently tilt the slide back and forth for 1–2 minutes.
  3. Observation:
    • Examine for visible clumping (agglutination) using direct light.

Semi-Quantitative Method (Titration):

  • Serially dilute the patient’s serum in saline (1:20, 1:40, 1:80, 1:160, and so on).
  • Mix each dilution with an equal volume of antigen suspension.
  • Observe for the highest dilution that still shows visible agglutination (titer).

Requirements

  1. Samples
  • Serum:
    • Collect 2–3 mL of venous blood aseptically in a plain (clot) tube.
    • Allow the blood to clot, and separate the serum by centrifugation at 2,500–3,000 rpm for 10 minutes.
    • Use fresh serum or store at 2–8°C for short durations (up to 24 hours).
  1. Reagents
  • Standardized Salmonella Antigen Suspensions (commercially available):
    • S. typhi O antigen.
    • S. typhi H antigen.
    • S. paratyphi A O and H antigens.
    • S. paratyphi B O and H antigens.
    • S. paratyphi C O and H antigens (less common).
  • Physiological saline: For preparing serial dilutions.
  1. Equipment
  • Clean test tubes (12 × 75 mm or equivalent).
  • Test tube racks.
  • Micropipettes or graduated droppers.
  • Water bath or incubator (maintain 37°C).
  • Timer or clock.

Master Dilution Preparation

Objective: Create serial dilutions to determine antibody concentration.

Steps:

  1. Label a series of tubes as 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, etc.
  2. Add 1.9 mL of physiological saline to the first tube. Add 1 mL of saline to all other tubes.
  3. Mix 0.1 mL of patient serum into the first tube (1:20 dilution).
  4. Mix 1 mL from the first tube to the second tube thoroughly. Continue the serial transfer until the last tube, discarding 1 mL from the last tube to maintain equal volume.

Procedure

  1. Preparing the Test
  • Label the test tubes for each antigen type (S. typhi O, S. typhi H, S. paratyphi A O/H, S. paratyphi B O/H).
  • Prepare master dilutions of the serum as described above.
  1. Adding Antigen
  • Add one drop (0.5 mL) of the respective Salmonella antigen suspension to each tube in the series.
  • Ensure each tube has a final volume of 1 mL after adding the antigen.
  1. Mixing
  • Mix the contents of each tube gently but thoroughly. Avoid creating bubbles, as they may interfere with observation.
  1. Incubation
  • Place the tubes in a water bath or incubator at 37°C for 16–18 hours.
  1. Controls
  • Positive Control: Include a serum sample with known Salmonella antibodies.
  • Negative Control: Use physiological saline without serum to ensure the antigen suspension does not auto-agglutinate.

 

Results

Observation

  • After incubation, observe the tubes for agglutination.
  • Positive results show clumps or granules, while the remaining liquid becomes clearer.
  • Negative results appear as uniform turbidity without clumping.

Titer Determination

  • The titer is the serum’s highest dilution (lowest concentration) that shows visible agglutination.
  • Example: If the 1:160 dilution shows agglutination but the 1:320 dilution does not, the antibody titer is 1:160.

Titer Thresholds for Diagnosis

  • O antibody: Titers ≥1:160 are significant for active typhoid fever in endemic areas.
  • H antibody: High titers (>1:320) may indicate past infection, convalescence, or vaccination.

Paired Sera Testing

  • Collect a second serum sample after 7–10 days to observe a fourfold titer rise, confirming active infection.

 

Clinical Significance

  1. Diagnostic Applications
  • Acute Typhoid: High S. typhi O titers (≥1:160) suggest ongoing infection.
  • Chronic Carrier State: Persistent low H titers without O titers may indicate carrier status.
  • Paratyphoid Fevers: Agglutination with S. paratyphi antigens confirms the involvement of these strains.
  1. Monitoring Treatment
  • Falling titers during treatment indicate successful resolution of infection.
  1. Epidemiological Studies
  • Helps in understanding typhoid prevalence and immunity levels in endemic areas.

Limitations

  1. Cross-Reactivity: Antibodies against other bacteria (e.g., E. coli, Proteus) may cause false positives.
  2. Vaccination History: Recent vaccination against typhoid may yield elevated titers.
  3. Background Antibodies: In endemic areas, baseline titers may already be high due to prior exposure.
  4. Carrier State: Low-level titers may persist in asymptomatic carriers.

 

Precautions

  1. Use fresh reagents to ensure reliable results.
  2. Avoid contamination of antigen suspensions.
  3. Ensure proper incubation at 37°C for accurate agglutination.
  4. Use positive and negative controls to validate the test setup.

 

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