Widal Test: Principle, Procedure, Results, Limitation

Widal test is the most widely used diagnostic test for typhoid fever in developing countries. The Widal test has been in use for more than a century as an aid in the diagnosis of typhoid fever. The Widal test is positive after the tenth day of the disease and may be falsely positive if an individual previously received a typhoid vaccine.

The Widal agglutination test is performed using a standardized suspension of S. enterica serotype Typhi ‘O’ and  ‘H’ and S. entericaserotype Paratyphi A ‘H’ and S. enterica serotype Paratyphi B ‘H’ antigen. It measures agglutinating antibody levels against O and H antigens using serial dilutions of sera. Tube agglutination method is the recommended method of performing the Widal test; where serial two-fold dilutions of the patient’s serum from 1:20 to 1:1280 are tested. After its development, the rapid slide test became the most commonly used technique in local laboratories because of its convenience.

Figure: Agglutination reactions of O, H, AH, and BH antigen in Widal test

Widal test was devised by Frank Widal in 1896. Widal originally described the test to diagnose Salmonella entericaserotype Paratyphi B infection.

Principle

Patients infected with S. entericaserotype Typhi and Paratyphi produce serum antibodies to the O and H antigens of these pathogens, and the detection of these specific antibodies forms the basis of Widal test.

Antigens specifically prepared from Salmonella are used in the Widal agglutination test to detect the presence of antibodies in patients’ serum. Four specific antigen suspensions are used in Widal test; they are O, H, AH, and BH. Unfortunately, S. entericaserotype Typhi shares these antigens with other Salmonella serotypes and shares cross-reacting epitopes with other Enterobacteriaceae which increases the chance of false-positive results.

The earliest serological response in acute typhoid fever is a rise in the titer of the O antibody, with an elevation of the H- antibody titer developing more slowly but persisting longer than that of the O- antibody. Usually, O antibodies appear on 6 to 8 days and H antibodies on 10-12 days after the onset of the disease. Patients from communities where typhoid is endemic have higher H-antibody titers so some researchers claim that the level of H agglutinins is unhelpful in the diagnosis of typhoid.

Figure: Widal Test: Sample showing H positive in screening test

Salmonella Antigens Used in the Widal Test

Understanding the antigens used in the Widal test is essential for interpreting results correctly. Four antigen suspensions are used:

O Antigen (Somatic antigen)

The O antigen is a somatic (cell body) antigen located on the outer membrane lipopolysaccharide (LPS) of the bacterial cell wall. Its specificity is determined by the repeating units in the outer O-polysaccharide chain.

Key properties:

• Heat stable and alcohol resistant
• Forms compact, granular clumps when mixed with O antisera — the clumps are small and granular, distinctly different from H agglutination
• O antibodies appear earlier in disease — typically 6–8 days after onset of illness
• O antibody titers decline faster after recovery or treatment
• More specific for active/current infection than H antibody, but cross-reacts with other Enterobacteriaceae

H Antigen (Flagellar antigen)

The H antigen is a flagellar antigen — it is a protein component of the bacterial flagella.

Key properties:

• Heat labile — inactivated by boiling and alcohol
• Forms large, fluffy, loose clumps when treated with corresponding antisera — easily distinguishable from O agglutination
• H antibodies appear later — typically 10–12 days after illness onset
• H antibodies persist longer after recovery — sometimes for years after infection or vaccination
• In endemic populations, high H titers may reflect past infection or vaccination rather than current disease

AH antigen — flagellar antigen of S. enterica serotype Paratyphi A BH antigen — flagellar antigen of S. enterica serotype Paratyphi B

Vi Antigen (Virulence antigen)

The Vi antigen is a capsular polysaccharide that overlays the O antigen on the cell surface.

Key properties:

• Heat labile
• Increases bacterial infectivity by reducing complement-mediated killing and phagocytosis
• Can interfere with O antigen testing — if Vi antigen is present in large quantities, it may block O antibody detection, giving a false-negative O result
• Vi antigen is NOT routinely included in standard Widal test kits but is detected using specific Vi antisera
• A positive Vi agglutination may suggest chronic typhoid carriage

Antigen	Type	Stability	Agglutination appearance	Antibody timeline
O	Somatic (LPS)	Heat stable, alcohol resistant	Compact, granular, small clumps	Appears day 6–8; declines early
H	Flagellar (protein)	Heat labile, alcohol labile	Large, fluffy, loose clumps	Appears day 10–12; persists long
AH	Flagellar (Paratyphi A)	Heat labile	Large, fluffy clumps	Indicates Paratyphi A infection
BH	Flagellar (Paratyphi B)	Heat labile	Large, fluffy clumps	Indicates Paratyphi B infection
Vi	Capsular polysaccharide	Heat labile	—	Chronic carrier detection

Materials required

• Widal test kit (killed colored suspension of S. enterica serotype Typhi O antigen, S. enterica serotype Typhi H antigen and S. enterica serotype Paratyphi AH antigen, and S. entericaserotype Paratyphi BH antigen).
• Incubator
• Normal saline
• Refrigerator
• Applicator stick
• Graduated pipette

Before use, bring all reagents to room temperature and mix well.

Procedure of Widal Test

Rapid screening test

1. Mark the circles of slides as PC (positive control), NC (negative control), O, H, AH, and BHas per antigen solutions used for testing.
2. Add 1 drop of positive control (25μL) into the circle marked as PC.
3. Then add 1 drop of negative control (25μL) into the circle marked as NC.
4. Add 1 drop of the test sample (25μL) into each circle labeled as O, H, AH, and BH.
5. Add 1 drop of antigen solution of Salmonella typhi ‘H’ into PC and NC circle each. Mix well by using a new mixing stick for each circle.
6. To circles labeled as O, H, AH, BH in which test samples have been added, add antigen solutions of Salmonella typhi ‘O’, Salmonella typhi ‘H’, Salmonella paratyphi ‘AH’ and Salmonella paratyphi ‘BH’, respectively.
7. Mix the content of each reaction circle uniformly with a separate mixing stick.
8. Rock the glass slide gently (approximately for one minute) and observe for agglutination.

Result:

• Positive Widal test: Agglutination was observed within a minute.
• Negative Widal test: No agglutination

Figure: Widal Test Result: Sample (6) showing BH positive in screening test

Slide Titration

Rapid slide titration needs to perform for the samples which showed positive titer during rapid screening.

• Using a micropipette, dispense 40, 20, 10, and 5µl of undiluted serum onto a row of 3 cm diameter circles.
• Shake the reagent bottle rigorously shaken and add a drop (0.03 ml) of the undiluted antigen suspension to each serum aliquot.
• Mix it thoroughly mixed with the aid of a stirring stick and rotate the slide gently.
• Observe the reactions after a minute.

Result:

• Positive test: Agglutination was observed within a minute.
• Negative test: No agglutination

Reporting Widal test

Figure: Widal Test: Quantitative test for H antibody (Serial double dilution order from 1-2-3-6-5)

Circle	1	2	3	4	5
Serum volume	80 µl	40 µl	20 µl	10 µl	5 µl
Amount of antigen	1 drop	1 drop	1 drop	1 drop	1 drop
Equivalent tube titre	1:20	1:40	1:80	1:160	1:320

The Widal test is reported by giving the titer for both O and H antibodies. The titer of each serum is read as the highest serum dilution giving visible agglutination. The agglutination observed in any circle was indicative of the following results in a test tube.

Quality control

Quality control was done using the positive polyspecific control of the same dilutions as the test sample. Normal saline was used for negative control.

Importance of Baseline Titer in Widal Test

Ideally, the Widal test should be run on both acute and convalescent-phase sera to detect an increase in the agglutination titer but patient management cannot wait for results obtained with a convalescent-phase sample. For practical purposes, a treatment decision must be made on the basis of the results obtained with a single acute-phase sample. It is, therefore, important to establish the antibody level in the normal population in a particular locality in order to determine a threshold above which the antibody titer is considered significant.

In a situation where second sample collection is not feasible, knowledge of the agglutinin levels in the sera of normal subjects from the patients’ community can form the baseline on which a diagnosis can be made.

Interpretation of Widal Test Results

What titer is significant?

There is no universal consensus on a single diagnostic titer, because the significant titer varies with the local endemicity of typhoid fever and the baseline agglutinin levels in the population. This is the most important concept in Widal test interpretation and the most commonly misunderstood.

Interpreting a single acute-phase sample:

Setting	Suggested significant O titer	Suggested significant H titer
Non-endemic area (traveller)	≥1:40	≥1:80
Low-endemic area	≥1:80	≥1:160
Endemic area (general recommendation)	≥1:160	≥1:160
Nepal (established local baseline)	>1:80 for anti-O	>1:160 for anti-H

Interpreting paired acute and convalescent samples (ideal):

A fourfold or greater rise in titer between acute (first week) and convalescent (3–4 weeks later) samples is the most reliable criterion for diagnosis, regardless of the absolute titer. This is the gold standard approach but is impractical in most clinical settings where treatment decisions cannot be delayed.

O vs H titer — what does each tell you?

Result pattern	Most likely interpretation
Elevated O titer only	Active/recent infection — O antibody rises first
Elevated H titer only	Past infection or previous typhoid vaccination
Elevated both O and H	Active infection — most diagnostically significant combination
Elevated AH only	Salmonella Paratyphi A infection
Elevated BH only	Salmonella Paratyphi B infection
Elevated O + H + AH/BH	Mixed infection or cross-reaction

**Nepal-specific baseline titer data**

Our research (Acharya T, Tiwari BR, Pokhrel BM. Baseline Widal Agglutination Titre in Apparently Healthy Nepalese Blood Donors. JHAS. 2013;3(1):27–30) established the baseline Widal agglutination titres among 490 apparently healthy blood donors across five developmental regions of Nepal. The study found that a significant proportion of healthy, uninfected individuals already carried anti-O titers ≥1:80 and anti-H titers ≥1:160, reflecting background exposure in an endemic population.

Based on these findings, the appropriate diagnostic cutoffs for Nepal are:

• Anti-O titer: >1:80 (titers at or below this level may be seen in healthy individuals)
• Anti-H titer: >1:160 (titers at or below this level may reflect past infection or vaccination)

This is important because applying a lower threshold — such as ≥1:20 or ≥1:40, which may be used in non-endemic settings — in Nepal leads to significant overdiagnosis, as many healthy individuals from endemic communities already carry titers at these levels due to past subclinical infection or vaccination.

This highlights the fundamental principle: a Widal test result must always be interpreted against locally established baseline titers, not against a universal fixed threshold.

Limitations of Widal Test

The value of the Widal test in diagnosing enteric fever in endemic areas remains controversial but is still a useful and widely available test in endemic areas. Epidemiologic studies in an endemic country have shown that at least seven subclinical cases of typhoid fever occur for each clinical case. Therefore a positive Widal test may be seen in apparently healthy persons from an endemic area as a result of previous subclinical infection.

• Previous typhoid vaccination may contribute to elevated agglutinins in the non-infected population.
• Cross reaction between malaria parasites and salmonella antigens may cause false-positive Widal agglutination test
• False-positive Widal tests have also been reported for patients with non-enteric salmonella infection, for example, typhus, immunological disorders, chronic liver disease, and cryptococcal meningitis.
• Prior use of antibiotics can dampen antibody response giving a low titer in the Widal test even in the face of bacteriologically confirmed typhoid fever resulting in misdiagnosis
• The Widal agglutination titer varies with the geographic location based on the endemicity of the enteric fever, the prevalence of non-typhoid salmonellae infection, and other infections which cross-react with salmonella antigen.
• Past infection with serotype Typhi or another nontyphoidal Salmonella serotype that shares common antigens gives a false positive Widal test.

Sensitivity and Specificity of the Widal Test

The Widal test's sensitivity and specificity vary widely depending on the cut-off titer used, the endemicity of the region, and the population studied. This variability is the central reason why the test remains controversial despite over 125 years of use.

Region	Cut-off titer	Sensitivity	Specificity
Philippines (tube agglutination)	O ≥1:80	64%	100%
Philippines (slide agglutination)	O ≥1:160	72.5%	57.5%
Ethiopia	O and H ≥1:160	82%	—
South Africa	O and H ≥1:200	75%	92.5%
Nepal (Kathmandu)	O and H ≥1:160	See Acharya T et al., 2015	—

The wide variation in sensitivity (47–92%) and specificity (50–100%) across studies reflects the difficulty of using a fixed titer threshold across different endemic settings. A titer that is diagnostically significant in a non-endemic country may be entirely within the normal background range in a highly endemic region like Nepal, India, or the Philippines.

Widal Test vs Newer Diagnostic Tests for Typhoid Fever

The Widal test remains the most widely used typhoid diagnostic tool in developing countries due to its low cost and wide availability. However, several newer tests have been developed with improved sensitivity and specificity:

Diagnostic test	Principle	Sensitivity	Specificity	Cost	Availability
Widal test	Tube/slide agglutination of O and H antibodies	47–77%	50–92%	Very low	Universally available
Blood culture	Gold standard — isolation of S. typhi	40–80% (depends on timing and prior antibiotics)	~100%	Moderate	Limited in resource-poor settings
Typhidot (IgM/IgG ELISA)	Detects IgM and IgG against a 50 kDa outer membrane protein of S. typhi	70–80%	75–90%	Low-moderate	Increasingly available
Typhidot-M	Detects IgM only (eliminates IgG cross-reactivity)	70–85%	85–96%	Low-moderate	Available in endemic areas
Tubex TF	Inhibition agglutination detecting anti-O9 IgM antibody	56–78%	87–95%	Moderate	Available in some endemic areas
PCR	Detects S. typhi DNA	High	High	High	Reference laboratories only
Vi serology	Detects anti-Vi IgG	70–80%	80–90%	Moderate	Useful for carrier detection

Key points:

• Blood culture remains the gold standard but has low sensitivity in the second and third weeks of illness and after antibiotic use — exactly when the Widal test is most positive
• Typhidot and Typhidot-M are better suited for early diagnosis (detects IgM from day 3–4 of illness)
• In settings where blood culture is unavailable or impractical, the Widal test combined with clinical judgment and local baseline titers remains a reasonable diagnostic tool
• No single test is perfect — the clinical picture, local epidemiology, and laboratory results must be interpreted together

→ Full article: Salmonella — Disease, Properties, and Laboratory Diagnosis

→ Full article: Culture Media for Salmonella → Full article: SS Agar

→ Full article: Hektoen Enteric Agar

References

1. Winn, W. C., & Koneman, E. W. (2006). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (Color Atlas & Textbook of Diagnostic Microbiology). Lippincott Williams & Wilkins
2. Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
3. Olopoenia, L. A., & King, A. L. (2000). Widal agglutination test – 100 years later: still plagued by controversy. Postgraduate medical journal, 76(892), 80–84. https://doi.org/10.1136/pmj.76.892.80
4. Adhikari, A., Rauniyar, R., Raut, P. P., Manandhar, K. D., & Gupta, B. P. (2015). Evaluation of sensitivity and specificity of ELISA against Widal test for typhoid diagnosis in endemic population of Kathmandu. BMC infectious diseases, 15, 523. https://doi.org/10.1186/s12879-015-1248-6
5. Acharya T, Tiwari BR, Pokhrel BM. Baseline Widal Agglutination Titre in Apparently Healthy Nepalese Blood Donors. Journal of Health and Allied Sciences. 2013;3(1):27–30.