Retrospective Audit of the Widal Test for

Diagnosis of Typhoid Fever in Pediatric

Patients in an Endemic Region

Microbiology Section

DOI: 10.7860/JCDR/2014/7819.4373

Original Article

Ralte Lalremruata1, Sanjim Chadha2, Preena Bhalla3

ABSTRACT
Introduction: Although typhoid fever is confirmed by culture
of Salmonella Typhi, Widal test is widely used in India but little
information exists about its reliability.
Materials and Methods: We examined the performance of Widal
test in our hospital for diagnosis of typhoid fever in children.
Hundred consecutive pediatric in-patients for whom, the Widal
test was requested were grouped into four categories: widal
positive and clinically consistent with typhoid fever (Group 1;
n=42), widal negative but clinically consistent (Group 2, n=12),
widal positive but not clinically consistent (Group 3, n=12) and
widal negative and also not clinically consistent (Group 4, n=34).
The results were analyzed by the test performance criteria,

namely, sensitivity, specificity, positive predictive value (PPV), and

negative predictive value (NPV) using culture-confirmed typhoid
fever cases as the true positives.
Results: We found that 7/100 patients had culture-proven
typhoid fever. Using a cut off 50 for O agglutinins or 100
for H agglutinins, the Widal test gave a sensitivity of 71.43%,
specificity of 47.31%, and a positive predictive value of 09.25%
and a negative predictive value of 95.65%.
Conclusion: The Widal test is an easy, inexpensive and relatively
non-invasive but is not reliable in our set up because of a low
PPV. There is a need for a more efficient rapid diagnostic test for
typhoid fever.

Keywords: Salmonella Typhi, Serology, Diagnosis

INTRODUCTION
Typhoid fever is endemic in India and the Widal tube agglutination
test which is almost 100 years old, has been widely used in the
serological diagnosis of typhoid fever in India [1]. While the Widal
test has played a major role in the diagnosis of typhoid fever in the
past, recent technical developments have revealed several pitfalls
in its use and interpretation of its result. Classically, a fourfold rise
of antibody in paired sera is considered diagnostic of typhoid fever
[2]. However, paired sera are often difficult to obtain and specific
antimicrobial therapy is instituted on the basis of clinical suspicion
alone [3]. A single Widal test results in an unvaccinated or unexposed
child may have some diagnostic relevance. However, the result of
a single test has no diagnostic significance in an endemic region; in
part due to difficulty in establishing a steady-state or baseline titer of
Widal agglutination test as repeated exposures to Salmonella Typhi
in endemic regions is a common occurrence [4,5]. Furthermore,
due to the possibility of fever from other infectious causes, false
positive reactions may occur because of cross-reactivities with
other non-Salmonella organisms [6,7]. Widespread use of typhoid
paratyphoid vaccine may also cause erroneous interpretation of test
results [8].
This leads to an over diagnosis of typhoid fever and limits the
usefulness of Widal test as a reliable diagnostic indicator of the
disease process and its management in endemic countries. The
diagnosis of typhoid fever on clinical grounds is difficult, as the
presenting symptoms are diverse and similar to those observed
with other febrile illnesses. The definitive diagnosis of typhoid fever
requires the isolation of Salmonella Typhi from the patient.
In our country patients often receive antibiotics prior to laboratory
testing due to which bacteria can be isolated from the blood cultures
only in a small fraction and culture facilities may not be freely available.
Evidence-based laboratory medicine tries to combat this problem
of inappropriate utilization of laboratory services by combining

22

methods from epidemiology, biostatistics, clinical and social

sciences with basic sciences to evaluate the role of investigations in
clinical decision making and outcomes for patients [9].
Clinical audit is an important tool for reviewing and improving
the quality of service in clinical laboratories [10, 11]. Taking into
considerations the above facts we present here a retrospective audit
of Widal test requests in our teaching hospital which will help us in
better understanding of the usefulness of Widal test as a diagnostic
indicator in our hospital, which will also have a deep impact on the
management of the patients.

MATERIALS AND METHODS

This is a retrospective audit of the Widal tube agglutination test carried
out from August 2012 to June 2013 at the Serology Laboratory
of Department of Microbiology, Maulana Azad Medical College,
Delhi, India after taking ethical clearance from Institutions ethical
clearance committee. This laboratory receive samples requested for
Widal test from all Departments of Lok Nayak Hospital, Delhi, India
which is the associated teaching hospital of the college. Hundred
consecutive pediatric (upto 12 years of age) in-patients for whom,
the Widal test was requested were followed up in their respective
wards in the Pediatrics Department of LN Hospital, regardless of
their test results. After taking informed consent from parents we
reviewed their epidemiological, clinical and bacteriological data
from our previous records. All these cases were being investigated
for their febrile illness and information concerning the duration of
illness before admission, presenting complaints, clinical signs and
symptoms and personal details of each case was recorded on a
performa. Clinical signs and symptoms defined by the Centers for
Disease Control and Prevention (CDC), Atlanta, Georgia, USA in the
case definition of typhoid fever were used for clinical diagnosis of
typhoid fever cases [12].

Journal of Clinical and Diagnostic Research. 2014 May, Vol-8(5): DC22-DC25

www.jcdr.net

Ralte Lalremruata et al., Retrospective audit of the Widal test for Diagnosis of Typhoid Fever in Pediatric Patients in an Endemic Region

Blood samples from all cases were received in plain vaccutainer

without anticoagulant in the Serology Laboratory. Blood was
allowed to clot in the plain vaccutainer and serum was separated
by centrifugation at 2500 rpm for 10 minutes, within one to three
hours of collection. Specimens were then processed by using
the commercial kit (Febrile Antigen Set; Span Diagnostic Ltd.)
according to the manufacturers instructions. Appropriate positive
and negative control sera were also included. The readings
for the test were taken after overnight incubation at 37C by a
clinical microbiologist with three years experience. The positivity
cut-off adopted for the diagnosis of enteric fever by Widal test
in paediatric patients in our laboratory (1:50 for O and 1:100 for
H agglutinins) was used for this audit as well. Blood samples
collected aseptically from all study participants in pediatric blood
culture bottles containing Brain Heart Infusion broth were sent
for culture to the Bacteriology laboratory of our Department on
the day of admission to the hospital before they were started
on any treatment. The blood culture bottle was incubated at
37oC aerobically for 24 hours and was then sub-cultured on 5%
sheep blood agar and MacConkey agar media. After 24 hours
of aerobic incubation of plates at 37oC i.e., third day, the plates
were examined for the growth of bacteria. The plates showing no
growth of bacteria after 48 hours were again sub-cultured on the
above 2 media on the fifth day. Cultured organisms were identified
by biochemical and serotyping tests which is the standard protocol
of our bacteriology laboratory. Enteric fever was confirmed by the
isolation of Salmonella Typhi from the blood culture.
All pediatric study subjects were started on empirical therapy with
intravenous monocef (ceftriaxone) at a dose of 100 mg/kg body
weight in two divided doses per day. Their response to this drug was
noted. All the study subjects were then accordingly grouped into four
categories: widal positive and clinically consistent with typhoid fever
(Group 1; n=42), widal negative but clinically consistent with typhoid
fever (Group 2, n=12), widal positive but not clinically consistent
with typhoid fever (Group 3, n=12) and widal negative and also not
clinically consistent with typhoid fever (Group 4, n=34).
The results were analyzed by the test performance criteria, namely,
sensitivity, specificity, positive predictive value (PPV), and negative
predictive value (NPV). Sensitivity (true-positive rate) was defined
as the probability that the Widal test result would be positive when
blood culture confirmed that typhoid fever was present. Specificity
(true-negative rate) was the probability that the Widal test result
would be negative when typhoid fever was not present. The positive
predictive value was the probability that typhoid was present when
the test was positive, and the negative predictive value was the
probability that typhoid was not present when the test was negative.
The values were calculated using culture-confirmed typhoid fever
cases as the true positives and those cases from which Salmonella
Typhi were not isolated from blood culture as the true negatives.

Samples from Group 2 children showed O and H antibody titres<50.

Eleven samples (out of 12) were sent within 7 days of fever and one
(out of 12) was sent after the 7th day. Two cases exhibited blood
culture positivity for Salmonella Typhi after 48 hrs of incubation.
All the patients from this group became afebrile within 5 days of
ceftriaxone administration.
[Table/Fig-2]shows the widal results for Group 3 cases and the final
diagnosis arrived at in these cases considering all the laboratory

All
children

Group 1
(Widal +ve
& clinically
consistent)

Group 2
(Widal -ve
& clinically
consistent)

Group 3
(Widal +ve
but not
clinically
consistent)

Group 4
(Widal ve &
not clinically
consistent)

n=100
No. (%)

n= 42
No. (%)

n= 12
No. (%)

n=12
No. (%)

n= 34
No. (%)

08(8)

07(16.67)

00(0)

01(8.34)

00(0)

=200

08(8)

08(19.04)

00(0)

00(0)

00(0)

=100

10(10)

10(23.8)

00(0)

00(0)

00(0)

=50

28(28)

17(40.47)

00(0)

11(91.67)

00(0)

<50

46(46)

00(0)

12(100)

00(0)

34(100)

25(25)

23(54.76)

00(0)

02(16.67)

00(0)

Titers
Anti TO
400

Anti TH
400
=200

05(5)

04(9.52)

00(0)

01(8.34)

00(0)

=100

24(24)

15(35.71)

00(0)

09(75)

00(0)

=50

01(1)

00(0)

01(8.34)

00(0)

00(0)

<50

45(45)

00(0)

11(91.67)

00(0)

34(34)

[Table/Fig-1]: O and H agglutinins in the study groups

S. No.

TO

TH

1.

=50

=100

Pulmonary kochs

2.

>400

>400

Chronic liver disease

3.

=50

=100

Chronic liver disease

4.

=50

=100

Pancytopenia

5.

=50

=200

Malaria

6.

=50

=100

Malaria

7.

=50

=100

Pneumonia

8.

=50

=100

Pneumonia

9.

=50

=100

Chronic liver disease

10.

=50

>400

Kerosene ingestion

11.

=50

=100

Liver abscess

12.

=50

=100

Chronic liver disease

[Table/Fig-2]: Widal results and final diagnosis of Group 3 cases(Widal +ve

but not clinically consistent) n=12

Culture positive
(Enteric fever positive)

Culture negative
(Enteric fever
negative)

Widal positive

05
(True positives)

49
(False positives)

54

Widal negative

02
(False negatives)

44
( True negatives)

46

07

93

RESULTS
A total of 100 febrile children were enrolled for this study. The mean
age of our study subjects was 5.9 3.36 years (Age range 6 months
to 12 years). The male to female ratio was 1.27:1 (56 males and 44
females). .
[Table/Fig-1] shows the widal titers for the four study groups. In group
1 cases no convalescent phase sample was received for paired
sera tests. One sample demonstrated positivity for AH (01%).Twenty
samples (out of 42) were sent within 7 days of fever and twenty-two
(out of 42) were sent after the 7th day. No sample showed positivity
for Salmonella Typhi on blood culture after 48hrs of incubation but
five (out of 42) showed positivity on the 5th day subculture. Thirtyfive (out of 42) patients became afebrile within 5 days of ceftriaxone
administration and four patients showed response to the same
beyond the 5th day. Three patients were treated with an additional
antibiotic namely azithromycin to which they responded.
Journal of Clinical and Diagnostic Research. 2014 May, Vol-8(5): DC22-DC25

Clinical diagnosis

[Table/Fig-3]: Distribution of cases with respect to Blood Culture and Widal test
results
Sensitivity

5/7(71.43%)

Specificity

44/93(47.31%)

Positive predictive value (PPV)

5/54(9.25%)

Negative predictive value (NPV)

44/46(95.65%)

[Table/Fig-4]: Performance of widal test for enteric fever diagnosis

23

Ralte Lalremruata et al., Retrospective audit of the Widal test for Diagnosis of Typhoid Fever in Pediatric Patients in an Endemic Region

investigations. None of these cases showed blood culture positivity

for Salmonella Typhi. Four samples (out of 12) were sent within 7
days of fever and eight (out of 12) were sent after the 7th day. All of
them were started on ceftraixone and five of these patients became
afebrile within 5 days of the drug treatment.
Group 4 (Widal ve & not clinically consistent) consisted of patients
whose clinical diagnoses were various non-typhoidal infectious
diseases.
[Table/Fig-3] shows the distribution of our study participants with
respect to blood culture and Widal test results.
[Table/Fig-4] shows the sensitivity, specificity, positive predictive value
(PPV), and negative predictive value (NPV) of the Widal test using
culture-confirmed typhoid fever cases as the true positives and
all other febrile children with blood culture negative for Salmonella
Typhi as the true negative

DISCUSSION
Widal test has been used for over a century in developing countries
for diagnosis but it has been reported to have low sensitivity,
specificity and positive predictive value [13,14]. We present here an
assessment of the utility of Widal test in diagnosing typhoid fever in
pediatric patients of New Delhis one of the largest and the busiest
tertiary care hospital.
Fifty four cases out of the hundred enrolled in our study fulfilled
the requirements for CDCs case definition of typhoid fever. Out of
these only 42 cases showed a positive Widal test (Group 1). Twelve
cases were Widal test positive but not clinically compatible (Group
3). This serpositivity in clinically incompatible cases may be due
to presence of pre existing antibodies from previous exposures to
Salmonella Typhi infection, subclinical infection or the presence of
cross reacting antibodies due to some other non typhoidal fevers.
But, still all of them were started on Ceftriaxone before receiving the
results of the Widal test. Blood samples for cases with a positive
result in the Widal test (n=54) were sent on the day of admission
to the hospital; 44.45 % (24 samples) of which were sent within
7 days of fever and 55.56 % (30 samples) beyond the 7th day of
fever. The traditional view that the test is more likely to be positive
in the second week of the illness is not supported by our data,
although positivity for the level of TH (p =0.0243) was more in the
second week of the illness. Statistically significant differences were
not observed for TO (p =0.0912) and AH (p =1.00) titers between
the second and the first week of illness. This finding supports the
conclusions of other researchers that in endemic areas the H and
0 agglutinins appear earlier in the course of illness [15,16] and is
most probably attributable to an already immunologically sensitized
population.
All the cases in our study were initiated on Ceftriaxone. Such an action
promotes the emergence and spread of drug resistant strains in the
community. Salmonella Typhi is showing resistance to Ceftriaxone,
Azithromycin and Ciprofloxacin from different parts of the world; also
Ciprofloxacin resistance is so common that it cannot be routinely
used [17]. Although it would be best to initiate specific antimicrobial
only after laboratory confirmation of the disease, antibiotic therapy
may be initiated among patients with high suspicion of enteric fever
on clinical grounds alone. Therefore the benefits of antibiotic therapy
need to be carefully weighed against the disadvantage of spread
of antibiotic resistant strains. Other interesting observations to be
noted in our study are that the Widal test was requested in cases
without clinical evidence of the disease (Group 3 & 4 cases) and
that the Widal test report had no impact on patient management
as the therapy was not changed in Group 2 (Widal -ve & clinically
consistent) cases after the test report.
Using a cut off 50 for O agglutinins or 100 for H agglutinins, the
Widal test gave a sensitivity of 71.43%, specificity of 47.31%, and a
positive predictive value of 09.26% and a negative predictive value
24

www.jcdr.net

of 95.65%. This is in marked contrast to the findings of other investi

gators, who have reported a high PPV of the Widal test in children
[3, 18]. However a study from Tanzania has reported similar results
with low PPV and high NPV of Widal test in children [19]. It has been
argued that PPV is the most important measure of a diagnostic
method since it represents the proportion of patients with positive
test results that are correctly diagnosed [20]. In our study a negative
result had a good predictive value for the absence of disease, but a
positive result had a very low predictive value for typhoid fever.
In summary the Widal test in our set up performed fairly well in
terms of sensitivity and specificity but the test was only useful for
excluding the disease. Considering the low cost of Widal testing
and the absence of comparably cheap tests, Widal testing is likely
to remain the test of choice in many developing country settings.

CONCLUSION
The diagnosis of typhoid fever on clinical grounds is difficult, as the
presenting symptoms are diverse and similar to those observed
with other febrile illnesses.
The definitive diagnosis of typhoid fever requires the isolation of
Salmonella Typhi or paraTyphi from the patient. However, since
patients often receive antibiotics prior to a laboratory diagnosis,
bacteria are isolated from the blood cultures in very few cases.
Besides this, the unavailability of microbiologic facilities and the long
waiting time for culture results (7 to 10 days) have been identified as
reasons for the preference for the Widal test.
A high rate of false positives results in over diagnosis of typhoid
fever leading to a worsening of antibiotic resistance in the country.
Therefore the results of this test must be interpreted with caution
taking into account the patients clinical details and history of
vaccination etc. thus emphasizing the importance of laboratory
clinic communication. However a negative Widal test result in
a child with clinical symptoms consistent with typhoid fever is
useful for excluding the presence of disease and exempting them
from unnecessary specific antimicrobial therapy as the negative
predictive value of the test is high. Some other important questions
which need to be addressed are that what should be the basis for
requisition of the Widal test in an endemic region clinical criteria
laid down by the CDC or fever alone and What should be the basis
of starting empirical specific antimicrobial therapy- clinical suspicion
or laboratory confirmation and How does the Widal test report alter
patient management in Widal test negative cases who have already
been started on empirical Ceftriaxone based on clinical grounds?
A highly specific and sensitive diagnostic test is therefore urgently
required to contribute to better health in endemic resource poor
settings where access to highly trained laboratory workers with
adequate time is rare.

ACKNOWLEDGEMENT
The authors would like to thank Mr. Sukhbir and Mrs. Seema for
their technical assistance.

REFERENCES
[1]

[2]

[3]
[4]
[5]

[6]

Pal S, Prakash R, Juyal D, Sharma N, Rana A, Negi S. The baseline widal

titre among the healthy individuals of the hilly areas in the garhwal region of
uttarakhand. India. J Clin Diagn Res. 2013; 7(3):437-40.
House D, Chinh NT, Diep TS, Parry CM, Wain J, Dougan G, et al. Use of paired
serum samples for serodiagnosis of typhoid fever. J Clin Microbiol. 2005; 43(9):
4889-90.
Kulkarni ML, Rego SJ. Value of single widal test in the diagnosis of typhoid fever.
Indian Journal of Paediatrics. 1994 Nov; 31(11): 1373-6.
Olopenia LA, King AL. Widal agglutination test-100 years later: still plagued by
controversy. Postgrad Med J. 2000; 76: 804.
Levine MM, Grados O, Gilman RH, Woodward WE, Solis-Plaza R, Waldman W.
Diagnostic value of the Widal test in areas endemic for typhoid fever. Am J Trop
Med Hyg. 1978; 27(4):795-800.
Reynolds DW, Carpenter L, Simon WH. Diagnostic specificity of Widals reaction
for typhoid fever. JAMA. 1970; 204: 2192-3.
Journal of Clinical and Diagnostic Research. 2014 May, Vol-8(5): DC22-DC25

www.jcdr.net
[7]
[8]
[9]
[10]

[11]
[12]

[13]

[14]

Ralte Lalremruata et al., Retrospective audit of the Widal test for Diagnosis of Typhoid Fever in Pediatric Patients in an Endemic Region

Pang T, Puthucheary SD. Significance and value of the Widal test in the diagnosis
of typhoid fever in an endemic area. J Clin Pathol. 1983; 36(4): 471-5.
Wicks ACB, Holmes GS. Davidson L. Endemic typhoid fever: a diagnostic pitfall.
Q J Med. 1971; 40: 341-54
Horvath AR. Evidence-based laboratory medicine. Orv Hetil. 2003; 144(18): 869-76.
Ally SH, Ahmed A, Hanif R. An audit of serological tests carried out at clinical
laboratory of Ayub Teaching Hospital, Abbottabad. J Ayub Med Coll Abbottabad.
2005; 17(2): 75-8.
Bonini P, Plebani M, Ceriotti F, Rubboli F. Errors in laboratory medicine. Clin Chem.
2002; 48(5): 691-8.
Case definitions for infectious conditions under public health surveillance. Centers
for Disease Control and Prevention. MMWR Recomm Rep. 1997; 46(RR-10):
1-55.
Sherwal Bl, Dhamija RK, Randhawa VS, Jais M, Kaintura A, Kumar M. A
comparative study of Typhidot and Widal test in patients of Typhoid fever. JIACM.
2004; 5:244-6.
Begum Z, Hossain Md A, Shamsuzzaman AKM, Ahsan Md M, Musa AKM,
Mahmud Md C, et al. Evalutaion of Typhidot(IgM) for Early Diagnosis of Typhoid
fever. Bangladesh J Med Microbiol. 2009; 3:10-3.

[15] Saha SK, Ruhulamin M, Hanif M, Islam M, Khan WA. Interpretation of the Widal
test in the diagnosis of typhoid fever in Bangladeshi children. Ann Trop Paediatr.
1996; 16(1): 75-8.
[16] Senewiratne B, Senewiratne K. Reassessment of the Widal test in the diagnosis
of typhoid. Gastroenterology. 1977; 73: 233-6.
[17] U. S. Department of Health and Human Services, Centers for Disease Control
and Prevention. Antibiotic resistance threats in the United States 2013, Available
from:http://www.cdc.gov/drugresistance/threat-report-2013/pdf/ar-threats2013-508.pdf.
[18] Noorbakhsh S, Rimaz S, Rahbarimanesh AA, Mamishi S. Interpretation of the
Widal test in Infected Children. Iranian J Publ Health. 2003; 32(1); 35-7.
[19] Ley B, Mtove G, Thriemer K, Amos B, von Seidlein L, Hendriksen I, et al.
Evaluation of the Widal tube agglutination test for the diagnosis of typhoid fever
among children admitted to a rural hospital in Tanzania and a comparison with
previous studies. BMC Infect Dis. 2010;10:180.
[20] Altman DG, Bland JM. Diagnostic tests 2: Predictive values. BMJ. 1994; 309:10.

PARTICULARS OF CONTRIBUTORS:
1. Student, Department of Microbiology, Maulana Azad Medical College, New Delhi, India.
2. Senior Resident, Department of Microbiology Maulana Azad Medical College, New Delhi, India.
3. Director Professor, Department of Microbiology, Maulana Azad Medical College, New Delhi, India.
NAME, ADDRESS, E-MAIL ID OF THE CORRESPONDING AUTHOR:
Dr. Preena Bhalla,
Director Professor, Department of Microbiology, Maulana Azad Medical College,
Bahadur Shah Zafar Marg, New Delhi-110002, India.
Phone: +91-9810677106, Email: preenabhalla@gmail.com
Financial OR OTHER COMPETING INTERESTS: None.

Journal of Clinical and Diagnostic Research. 2014 May, Vol-8(5): DC22-DC25

Date of Submission: Oct 05, 2013

Date of Peer Review: Dec 24, 2013
Date of Acceptance: Mar 10, 2014
Date of Publishing: May 15, 2014

25