Online - 2455-3891

Vol 10, Issue 7, 2017 Print - 0974-2441

Research Article

USE OF WIDAL TEST IN DIAGNOSIS OF TYPHOID FEVER IN NORTH INDIAN POPULATION BY

ESTIMATING BASELINE TITER IN CONTROL GROUP

SHETTY JEEVAN*
Department of Microbiology, G.S Medical College and Hospital (Chaudhary Charan Singh University), Pilkhuwa, Uttar Pradesh, India.
Email: [email protected]
Received: 30 March 2017, Revised and Accepted: 13 April 2017

ABSTRACT

Objective: This study was undertaken to establish a cutoff significant titer for Widal test using healthy volunteers as control group. Utilizing the
baseline titer obtained from the control group, a diagnosis of typhoid fever was made in the test group comprising outpatients and inpatients.

Methods: Blood samples were collected from healthy volunteers and patients attending G.S Medical College and Hospital, Pilkhuwa, over a period of
6 months from September 2016 to March 2017. Antibodies to Salmonella typhi (TO, TH) and Paratyphi A (AH) and Paratyphi B (BH) are determined
by this tube agglutination test. A total of 124 healthy controls and 303 patients having clinical suspicion of typhoid fever were subjected to Widal test.

Results: In healthy control group, titer TO ≥20 was observed in 43 (34.7%), TO ≥40 in 48 (38.7%), TO≥80 in 25 (20.2%), and titer TO ≥160, TO ≥320
was observed in none of the control group. Titer TH ≥40 in 58 (46.8%), AH ≥40 in 7 (5.6%), and BH ≥40 in 13 (10.5%) were observed in the control
group. Among the test group, 96 (31.7%) sera were positive out of 303 clinically suspected enteric fever by the Widal test. Among different age group
studied, 34 (46.6%) patients belonged to the age group of 11-20 years which formed the highest followed by the age group of 21-30 years (33.3%).

Conclusion: Based on the study, a cutoff titer of ≥160 for anti-O and anti-H antibodies and titer of ≥80 for anti-AH and anti-BH antibodies be considered
as significant titer in diagnosis of enteric fever in this region. The baseline titer helps in early recognition and treatment of this serious health problem.

Keywords: Titer, Widal, Antibodies, Typhoid fever, Agglutination.

© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.
org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2017.v10i7.18789

INTRODUCTION rise is not demonstrable even in blood culture-confirmed cases so

prompting baseline titer estimation of population a necessity [8].
Enteric fever is endemic in the Indian subcontinent, and population-
based surveillance studies in selected regions add considerably in There are no studies about the baseline titer been estimated earlier in
estimating the global burden of enteric fever. The incidence rate of this region. In studies done previously in other parts of the country, the
typhoid is more than 100 cases per 100000 people per year [1]. Enteric antibody titer was found to be unusually high though it can vary from
fever caused by Salmonella typhi is called as typhoid fever and Salmonella region to region [9]. In the background of such reports, it is necessary
paratyphi causes the paratyphoid fever. Typhoid is characterized by to perform baseline titer in healthy control group before conducting the
malaise, fever, abdominal discomfort, relative bradycardia, transient test on patient sera.
rash, and organomegaly and may end up in complications such as
intestinal perforation, hemorrhage, and perinephric abscess [2,3]. METHODS
Human beings are the only known reservoir host for typhoid fever, and
infected person or carrier transmits the organism in his feces. Ingestion The study was conducted in the Department of Microbiology,
of food or water contaminated by such feces is the source of infection [4]. G.S Medical College and Hospital, which is a tertiary care hospital
Drug resistance has been posing major problems in the treatment of with 350 beds situated in Pilkhuwa, Uttar Pradesh, for 6 months from
typhoid fever and continues to be a serious problem for public health September 2016 to March 2017. Consent and the approval of the
authorities [5]. Studies are being carried out to use plant constituents Institutional Ethical Committee was obtained for carrying out this
as potential antimicrobial agents in future for treatment [6]. Newer study. People with underlying respiratory infections, malaria, dengue,
vaccines are being developed to overcome poor immunogenicity of hepatitis, hematological, or other systemic disorders were excluded
some available vaccines, and research work on better adjuvant delivery from the study.
systems is being carried out [7].
Control group
The definitive diagnosis of enteric fever is by culture of blood, stool, The control group consisted of 124 healthy people who are free of signs
urine, and intestinal secretions. However, in many countries, where and symptoms and not having significant ill health within the past
injudicious use of antibiotics and inadequate culture facilities is 2 months or typhoid fever in the past 6 months. Those who gave history
common, Widal test is still the most convenient way for serological of vaccination in the preceding 3 years were excluded from the study.
diagnosis of typhoid fever. Moreover, for effective diagnosis of typhoid Among the control group, male constituted 41.13% and female 58.87%
fever, baseline titer of the population has to be assessed. from age group of 1 to 65 years.

Widal test is a classical tube agglutination test which measures Test group
agglutinating antibodies against lipopolysaccharide O and protein The test population consisted of 303 clinically suspected typhoid
flagellar H antigens of Salmonella. The diagnosis relies on demonstrating fever cases attending the hospital as outpatients (O.P) or admitted as
rising antibody titer in paired samples 10-14 days apart. However, this inpatients (I.P). Patients previously treated with antibiotics such as
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Asian J Pharm Clin Res, Vol 10, Issue 7, 2017, 251-253

Table 1: Antibodies titer against Salmonella antigens in the control group (N=124)

Antibody titer N (%) ≥20 N (%) ≥40 N (%) ≥80 N (%) ≥160 N (%) ≥320 N (%)
S typhi O 116 (93.5) 43 (34.7) 48 (38.7) 25 (20.2) 0 (0) 0 (0)
S typhi H 108 (87.1) 39 (31.5) 58 (46.8) 11 (8.9) 0 (0) 0 (0)
S paratyphi AH 08 (6.5) 01 (0.8) 07 (5.6) 00 (0) 0 (0) 0 (0)
S paratyphi BH 19 (15.3) 06 (4.8) 13 (10.5) 00 (0) 0 (0) 0 (0)
N: Total number of positive sera in the control group, N: Number of positive sera against different antibody titer

chloramphenicol or had recent typhoid illness or having chronic active Table 2: Prevalence of typhoid fever in test group after
liver disease were excluded from the study. Significant titer determined estimating baseline titer
from control group was applied to make a clinical diagnosis of enteric
fever. Age group (years) N M F O.P I.P N (%)
<10 59 31 28 34 25 12 (20.3)
A volume of 3-5  ml of venous blood was collected into a sterile test 11‑20 73 40 33 35 38 34 (46.6)
tube and centrifuged for 5 minutes to separate the serum from blood. 21‑30 60 31 29 41 19 20 (33.3)
Commercially available antigen containing Salmonella enterica serovar 31‑40 46 21 25 29 17 14 (30.4)
Typhi O and H antigens and Paratyphi AH and BH antigen were used 41‑50 30 08 22 20 10 08 (26.7)
(Arkray Healthcare Pvt., Ltd., Surat, India).The test used two-fold serially ˃50 35 22 13 19 16 08 (22.9)
diluted sera of control and test group, dilution being from 1:20 to 1:320. Total 303 153 150 178 125 96 (31.7)
All serum samples were first diluted in a 1:20 ratio with isotonic normal N: Total number of patients examined in different age groups (N=303), M: Male,
saline in such a way that the final volume amounts to 1 ml. Dilutions of F: Female, O.P: Outpatient, I.P: Inpatient, N: Number of patients positive by
1:40, 1:80, 1:160, and 1:320 each were made serially in 4 rows. Add a Widal test by utilizing baseline titer
drop of the appropriate antigen to all the corresponding tubes in each
row. A known positive control, negative control, and antigen control
were also set up in each row. All the tubes are mixed well and incubated ≥80 for TH antibodies. Positive agglutinins for paratyphi AH and BH
at 37°C for 16-20 hrs and examined for agglutination. The antibody titer were found in only 3.04% and 1.3%, respectively. In this study, 8 sera
is the highest dilution of serum showing distinct agglutination. The O (6.5%) and 19 sera (15.3%) showed positive agglutination titer for
somatic antigen being the somatic antigen gives compact, granular AH and BH antibodies. The baseline titer for paratyphi AH and BH
agglutination whereas H antigen, a flagellar antigen, gives large loose groups was found to be ≥20 each in their study [10]. Similar baseline
fluffy agglutination. titer was also noticed in Thiruvananthapuram in South Kerala [11].
The baseline titer determined was 40 for anti-O and anti-H antibodies
Data were entered and analyzed using SPSS (Statistical Package for and <20 for anti-AH and anti-BH antibodies in another study done in
Social Science) program version 24, and statistical significance was North Kerala where public health facilities and hygienic conditions are
considered when p value was less than 0.05. better [12]. Another study done in Raichur recommended TO and TH
titer of ≥320 as diagnostic of typhoid fever, and for AH and BH titers, it
RESULTS
is ≥40 and ≥160, respectively [13]. Baseline Widal titer study done in
A total of 124 healthy controls and 303 patients having clinical suspicion rural Pondicherry recommended ≥160 for O and H agglutinins while
of typhoid fever were subjected to Widal test. In healthy control group, for AH to be ≥80 and BH to be ≥40 to be considered a significant titer
titer TO ≥20 was observed in 43(34.7%), TO ≥40 in 48(38.7%), TO ≥80 in diagnosing typhoid [14]. A study done in Dehradun on apparently
in 25(20.2%), and titer TO ≥160, TO ≥320 was observed in none of the healthy population found agglutination titer for TO ≥20 in 28%, TO ≥40
control group. Titer TH ≥40 in 46.8%, AH ≥40 in 5.6%, and BH ≥40 in in 24.6%, TO ≥80 in 10.3%, TH ≥80 in 7.6%, AH ≥20 in 6.6%, and
10.5% were observed in the control group. Out of the 124 sera tested, BH ≥20 in 4.6% [15].
only 8 sera (6.5%) and 19 sera (15.3%) showed positive agglutination
titer for S. paratyphi AH and BH antibodies, respectively. Male In this study, TO antibodies (93.5%) agglutinated more compared with
constituted 41.13% (52 out of 124) of the control group. TH (87.1%), AH (6.5%), or BH (15.3%). Even infected patients’ sera
agglutinated more with somatic antigen of S. typhi than the flagellar
Among the test group, 96  (31.7%) were positive by Widal test and antigen. These findings have been noted in several studies [16].
34  (46.6%) patients belonged to the age group of 11-20  years which Furthermore, several studies highlight the increasing cases of enteric
formed the highest group whereas age group <10 years constituted the fever due to paratyphi, but this study differs from such observation.
least (20.3%). Male constituted 50.49% and female 49.50% and O.P Widal test helps the physician in making a rapid diagnosis instead of
accounted for 58.74% and I.P accounted for 41.25% in the test group relying on blood or stool culture which is time-consuming. Added to
(Tables 1-3). this is self-medication by the patients and injudicious prescription of
antibiotics by local doctors make culture tests less reliable.
The present study has found the paratyphi BH titer ≥40 which is found
to be higher than other studies done on healthy volunteers (Table 4). Among the patients who attended the hospital with clinical suspicion
of typhoid, 31.7% showed significant titer to S. typhi antibodies and
DISCUSSION none to paratyphi. Age group of 11-20 years showed highest prevalence
The baseline titer in Widal test among control group for TO, TH (46.6%) followed by the age group of 21-30 years (33.3%). A similar
antibodies was found to be ≥80 and AH and BH to be ≥40 each. The study done in Aba, Nigeria, showed 24.5% positive Widal test, and
high titers noticed against the salmonella antigens in the study may in age-related prevalence, the age bracket 31-40  years showed the
be due to the exposure to the cross-reacting organisms in the region. highest prevalence rate by both Widal and blood culture method. Sex-
Based on this, the significant titer was determined as a cutoff titer related prevalence showed that more males were affected than female
of ≥160 for anti-O and anti-H antibodies and titer of ≥80 for anti-AH population [17]. Study done in Turkey recommended agglutinin titers
and anti-BH antibodies to make a diagnosis of enteric fever [9]. A study of ≥200 having diagnostic significance and Widal test performed
done in determining baseline titer in Garhwal region of Uttarakhand 7-10 days after hospitalization gave the most reliable results with high
has recommended the baseline titer of ≥40 for TO antibodies and specificity, sensitivity, and positive and negative predictive value [18].

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Asian J Pharm Clin Res, Vol 10, Issue 7, 2017, 251-253

Table 3: Distribution of the positive cases according to the gender‑age

Sex Number Mean±SD SEM Chi‑square Df Asymptotic significant

Male 44 21.090±13.738 2.071 10.045 28 0.999
Female 52 26.423±17.744 2.460 20.500 0.846
p<0.05 considered significant, Df: Degree of freedom, SD: Standard deviation, SEM: Standard error mean

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