Adhikari et al.

BMC Infectious Diseases (2015) 15:523

DOI 10.1186/s12879-015-1248-6

RESEARCH ARTICLE Open Access

Evaluation of sensitivity and specificity of

ELISA against Widal test for typhoid
diagnosis in endemic population of
Kathmandu
Anurag Adhikari1,4, Ramanuj Rauniyar4, Pramod Prasad Raut2, Krishna Das Manandhar3,4
and Birendra Prasad Gupta3,4*

Abstract
Background: Widal test, which has poor predictive outcomes in predominant typhoid population, is not standard
enough to predict accurate diagnosis. This study aims to compare the diagnostic accuracy of Widal test to ELISA
using blood culture as gold standard.
Methods: The blood samples were collected in Capital Hospital, Kathmandu, Nepal from febrile patients having ≥48 h
fever in 3 years study period for blood culture, Widal test and IgG-IgM ELISA.
Results: Amongst 1371 febrile cases, 237 were Salmonella typhi positive to blood culture and 71.4 % typhoid fever
patient were of 46–60 years old with male to female ratio of 2:1. Blood culture confirmed patients had ≥1:40 anti-TH
and anti-TO titre in 45.56 % (n = 108) and 43.88 % (n = 104) patients respectively. The sensitivity and specificity of IgG
(0.96 and 0.95) and IgM (0.95 and 0.94) at 95 % confidence level were significant compared to Widal anti-TH (0.72 and
0.58) and TO (0.80 and 0.51) test (p value, 0.038) at titre level ≥1:200. Further the PPV of Widal TH and TO (0.38 and
0.23) was low compared to IgG and IgM ELISA (0.78 and 0.77) (p value, 0.045).
Conclusion: Widal test is not sensitive enough for an endemic setting like Nepal and thus should be either replaced
with more accurate test like ELISA or follow an alternative diagnostic methodology.
Keywords: Typhoid Fever, Salmonella typhi, Widal test, ELISA, Sensitivity, Specificity, Nepal

Background Outbreaks are more common in summer season affect-

Typhoid fever is becoming a key health problem in de- ing mainly the children in Kathmandu [5–7]. Diagnosis
veloping countries. Lack of safe and clean drinking water is done by culture and immunological tests, however,
supplies to the people and adequate sewage disposal are isolation of the etiological agent, the Salmonella enterica
the major reasons [1]. The fever has been one of the (serovar typhi) from bone marrow culture is an ideal
leading diagnosed fever ailment among the fever related gold standard [8]. Invasive procedure and unavailability
caseses in most of the hospitals of Nepal [2]. It is popu- of culture facilities in rural health centers are chief limit-
larly known as ‘Bisham Joro’ in local language meaning ing factors. Besides, the widely used blood culture has
the ‘fever with poison’. Typhoid is prevalent in mid-hills, poor sensitivity of 40–60 % making room for false nega-
valleys and southern belts as an endemic disease with tive results to around half of the typhoid patients [9]. In
peak incidence in between April and August [3, 4]. addition, the requirement of 3 days time duration for
diagnosis delays the line of treatment loosing applicabil-
ity of early diagnosis. Further, usage of the antibiotics
* Correspondence: [email protected]
3
Central Department of Biotechnology, Tribhuvan University, Kirtipur, prior to hospital admittance, which is a common prac-
Kathmandu, Nepal tice in Nepal, cause poor sensitivity to the culture base
4
Everest Institute of Virology and Immunology, Kathmandu, Nepal diagnosis [10]. In spite of the all, blood culture technique
Full list of author information is available at the end of the article

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Adhikari et al. BMC Infectious Diseases (2015) 15:523 Page 2 of 7

is still the gold standard in the febrile cases of typhoid. Rou- standard (Blood Culture). The control populations with
tine Widal test is alternatively adopted second most negative blood culture reports were also included in the
popular choice for diagnosis as blood culture remains con- study, so as to make self evaluation of reference standard.
troversial due to its biased diagnosis [11]. Enzyme-linked The sample population inclusive of blood culture positive
immunosorbent assay (ELISA) based diagnosis has also was grouped into five different groups (Fig. 1).
been studied previously with good diagnostic accuracy [12–
15]. This study aims to compare diagnostic accuracy of Blood collection and bacterial culture for pathogen
Widal test and ELISA in febrile patients taking blood cul- isolation
ture as gold standard tool for the diagnosis of typhoid fever. Blood were collected in BACTEC Peds Plus™/F culture
vials and immediately (within 10 min) transferred to la-
Material and methods boratory to be loaded in Bactec 9240 (Becton Dickinson,
Patients, inclusion/exclusion criteria and data analysis USA) culture instrument for growth. Blood isolates which
Patients attending Capital Hospital, a centrally located were found culture positive were reconfirmed for Salmon-
hospital at Kathmandu, during the months January 2011 ella by slide agglutination, using monospecific anti sera
to December 2013 with complaint of fever over 72 h with- (Sifin, Germany) as described by the Kauffmann-White
out obvious focus of infection and clinical suspicion of scheme [16–18].
typhoid fever (high fever, malaise, headache, constipation
or diarrhoea) were prospectively enrolled in this study. Quantitative Widal test
Patients were divided into five age groups i.e. 1–15 The Widal tube agglutination test was performed using
(children), 16–30 (young), 31–45 (young adults), 46–60 Wellcolex®(Remel, UK) in the group A (n = 1371) as well
(adults) and >60 years (olds). Pregnant women were ex- as for group B, C D and E according to the manufactur-
cluded from the study. The ELISA and Widal tests were er's instructions parallel to the blood culture procedure
performed by a single specialized clinical researcher who of individual groups. Briefly, serum remained after keep-
was blind to the patient's diagnosis in reference to stand- ing for blood culture was diluted in 0.86 % saline solu-
ard throughout the study period. The patients with febrile tion starting with 1:100. Salmonella ‘O’ and ‘H’ antigens
cases were screened when found positive to reference from the standard preparation were added and the tubes

Fig. 1 Consort chart for patients in study. Group A: Febrile patients with suspected typhoid fever (n = 1371), Group B: Blood culture positive
patients (n = 237), Group C: Post treated patients from group A (>Day 30) (n = 237), Group D: Endemic healthy population (n = 237), Group E:
Other diseased patients (infection from; Mycobacterium tuberculosis, Staphylococcus aureus, Cryptococcus neoformans, Vibrio cholera, HIV-1, Neisseria
meningitidis, Listeria monocytogenes, Haemophilus influenza, Streptococcus pneumonia and Rabies virus): Negative to Salmonella tests (n = 237)
Adhikari et al. BMC Infectious Diseases (2015) 15:523 Page 3 of 7

were incubated at 37 °C for 1 h. After incubation, the Ethical statement

tubes were centrifuged for 5 min, and respective aggluti- The study was approved by the Capital Hospital Ethical
nations were observed. The Widal TO/TH titre were Review Board (CHRB). The adult participants gave writ-
taken as the highest dilution of serum with visible agglu- ten informed consent while children below 18 year were
tination. The cut off titre set for Salmonella typhi anti O enrolled only after the written informed consent from
was >1:80 while it was >1:160 for anti H in this study [11]. their guardian was received.

Enzyme Linked Immunosorbent Assay (ELISA) Results

Similar to the Widal test, Anti-IgM and anti-IgG sandwich Demographic and geographical distribution
ELISA (MyBioSource, Inc. CA, USA) was done in all the The 1371 patients, enrolled in the study held between
groups. Briefly, 100 μl of coating antigen (1 μg/ml) diluted January 2011 and December 2013, had representation
in antigen coating buffer (Immunochemistry, MN, USA) from all the three geographical regions of Nepal viz.
were dispensed in Nunc-Immuno 96 MicroWell solid Mountain, Hill and Terai, as Kathmandu, the capital
plates (Thermo Fisher Scientific, USA) along with negative city, hosts a broad range of people from all over the
control (Only coating buffer) according to plan. The plates country. The prevalence of typhoid cases in Terai region
were incubated at 4 °C overnight and the wells were was highest (69 %; p value, 0.03) in comparison to mid
blocked using 1 % bovine serum albumin (BSA) prepared hill regions around Kathmandu valley (30 %) and the
in phosphate buffer saline (PBS). The plates were washed least in mountain region (1.39 %) (Table 1). There were
by 125 μl washing buffer (0.1%BSA with Tween20). Hun- 400 females and 971 males who consented for this study.
dred microliter of serially diluted sera (1:200 to 1:3200) in The infection was found more in males of 16–30 years
PBS-BSA was dispensed to each well and incubated at (median age, 21.5 years) age group with male–female ra-
25 °C for 4 h. After washing, 100 μl detector antibody con- tio of 2:1, among the positive cases (n = 237) of typhoid
jugated with horse reddish peroxidase (HRP) was added in as confirmed by blood culture, and was followed by age
dilutions (1:500 anti IgA, 1:5000 anti IgG, 1:2500 anti groups of 1–15, 31–45, 46–60 and >60 year.
IgM) and incubated for 30 min at 25 °C. After that, 100 μl
trimethyle benzidine (TMB) substrate was added and in- Pathogen distribution among febrile cases
cubated for 15 min at dark. The reaction was stopped by Out of 1371 cases, only 237 samples (17.28 %) showed
addition of 1 N H2SO4 to measure optical density (OD) at positive growth of S. typhi in the blood culture and
450 nm in ELISA plate reader (Bio-Rad). Cut off values 120 patients were admitted in the hospital for treat-
were assessed following the mean ± SD of the OD from ment. The cultures also had co-infection of Actinobac-
healthy endemic controls of group D which was 0.3 for teria (n = 3, 2.5 %) and Klebsiella pneumonia (n = 2,
IgG and 0.2 for IgM. 1.67 %) in the admitted inpatients of the typhoid fever
(Table 2). In 237 patients, Actinobacteria (0.21 %) was
Statistical analysis found only in male while Klebsiella pneumoniae
The positive predictive value is the principle finding of (0.14 %) were found only in female. Pathogen other
this study which elucidates the efficacy of current diag- than Salmonella was isolated from 49 patients (Fig. 1).
nostic assays. The sensitivity, specificity and predictive
values were calculated as described earlier [19]. Re- Clinical symptoms of the disease
ceiver operating Characteristics (ROC) used to establish Different symptoms were found in the typhoid positive
cut off titre, was determined by plotting specificity (x- cases. The absolute symptom was fever (100 %; p value,
axis) versus sensitivity (y-axis) at all tested cut off titres. 0.04) however, other major symptoms observed were
The highest number of true positive and the lowest
false positive results were used to represent cut off
values by comparing the points with maximum Youden Table 1 Distribution of Typhoid patients according to regions
index (J = maxc [Se (c) + Sp (c) – 1]). Collected data and sex
were analyzed and interpreted statistically using graph- Geographical Regions Patients
Pad prism version 6.0 and SPSS 17.0. Normal distribu- Male Female Total
tion of data sets were analysed by Kolmogorov-Smirnov Terai Region 621 (65.92 %) 321 (34.07 %) 942 (68.71 %)*
test. All the values are expressed as mean ± SD and are (<2297 ft)
analyzed using Student’s t test which is parametric as Hill Region 340 (82.92 %) 70 (17.07 %) 410 (29.91 %)
well Mann–Whitney test wherever applicable. A value (2000–10000 ft)
(p value, <0.05) was considered significant unless stated Mountain Region 10 (52.63 %) 9 (47.36 %) 19 (1.39 %)
otherwise. Sensitivity, specificity and predictive values (>10000 ft)
were calculated as described elsewhere [20, 21] *p value, <0.05 while comparing the patients from Hill region
Adhikari et al. BMC Infectious Diseases (2015) 15:523 Page 4 of 7

Table 2 Culture report of S.typhi positive cases and co-infection Table 4 Number of anti TH and anti TO levels in blood isloates
with other pathogens among inpatient and outpatient of capital Titration Record Total Patients Culture positive Culture negative
hospital (n = 1371) patients (n = 237) patients (n = 1134)
Inpatients Outpatients Anti TH
Salmonella typhi 115 (95.83 %) 122 (100 %) Aggutinition 311 (22.68 %) 108 (45.57 %) 203 (17.92 %)
Actinobacteria 3 (2.5 %)* 0 (0 %) ≥1:640 78 (25.08 %) 36 (33.33 %) 42 (20.69 %)
*
Klebsiella pneumonia 2 (1.67 %) 0 (0 %) 1:320 156 (50.16 %) 55 (50.93 %) 101 (49.75 %)
Co-infection with S.typhi
*
1:160 176 (56.59 %) 89 (82.41 %) 87 (42.86 %)
1:80 201 (64.63 %) 98 (90.74 %) 103 (50.74 %)
headache (97.47 %; p value, <0.034), loss of appetite 1:40 311 (100 %) 108 (100 %) 203 (100 %)
(90.3 %; p value, 0.028) and chill (74.26 %; p value, No 1060 (77.32 %) 129 (54.43) 931 (82.14 %)
0.034). Abdominal discomfort, myalgia, vomiting, consti- Agglutination
pation, rigor, diarrhea, and dysuria were other symptoms Anti TO
observed in less than half of culture confirmed typhoid pa- Aggutinition 298 (21.74 %) 104 (43.88 %) 194 (17.11 %)
tient. Hence, fever, headache and loss of appetite were sig- ≥1:640 71 (23.83 %) 29 (27.88 %) 42 (21.65 %)
nificant symptoms for the fever ailment (Table 3).
1:320 147 (49.33 %) 45 (43.27 %) 102 (52.58 %)

Qualitative slide agglutination Widal test 1:160 164 (55.03 %) 84 (80.77 %) 80 (41.24 %)
Widal test used as the primary screening assay by typ- 1:80 198 (66.44 %) 91 (87.5 %) 107 (55.15 %)
ing O and H antigen of Salmonella showed overall 1:40 298 (100 %) 104 (100 %) 194 (100 %)
positivity rate of 21.74 % and 22.68 % respectively at No 1073 (78.26 %) 133 (56.12 %) 940 (82.89 %)
the titre ≥1:40. (Table 4). Among culture confirmed pa- Agglutination
tients, group A (n = 237), the number of TH (n = 108)
and TO (n = 104) positive at titre ≥1:40 was signifi-
cantly lower (p value, 0.04) than from the culture nega- after ≥3 days of reported fever case, which were un-
tive patients (n = 1134) group. Though blood culture detectable in initial 3 days of feverish condition. Pa-
showed negative to typhoid test, Widal test was found tients of group C (n = 237), who were enrolled in
positive to TH antigen (n = 203, 17.92 %) and TO anti- medication and recovered, showed significant level of
gen (n = 194, 17.11 %) test at same titre (Table 4). serum IgG (p value, 0.046) but not IgM, when com-
pared to previous data from same patients before medica-
Assessment of ELISA for the diagnosis of typhoid fever tion. For the endemic healthy group D (n = 237), the titre
The OD value for IgM in the culture confirmed group B for IgG was 400 but with no significant IgM titre value. In
(n = 237) was significantly higher (p value, 0.041) than that case of other disease patients of group E (without
of control groups C, D and E. There was no significant dif- Salmonella but other infections), the ELISA results
ference between the control groups D and E (p value, were negative for both Salmonella anti IgM and IgG
0.039). Titre value of 3200 for IgM and 200 for IgG, (Fig. 2). Serology based typhoid diagnostic tests using
was observed for group B patients (193 of 237) only sera from the culture confirmed typhoid patients and

Table 3 Clinical sign and symptoms distribution

S.N. Symptom expressed by patient Salmonella typhi (n) Percentage
1 Fever 237 100.00
2 Headache 231 97.47
3 Loss of appetite 214 90.30
4 Chills 176 74.26
5 Abdominal Discomfort 109 45.99
6 Myalgia 99 41.77
7 Vomiting 89 37.55
8 Constipation 64 27.00
9 Rigor 55 23.21
10 Diarrhea 45 18.99
11 Dysuria 42 17.72 Fig. 2 IgG and IgM occurrence in group B, C, D and E
Adhikari et al. BMC Infectious Diseases (2015) 15:523 Page 5 of 7

the control subjects showed that both IgG and IgM case definition for suspected and probable cases of ty-
based ELISA tests were superior to the Widal TH and phoid fever (Table 6).
TO tests. When sensitivity, specificity, positive predict-
ive value (PPV) and negative predictive value (NPV) of Discussion
ELISA was compared among group A (n = 1371) pa- Typhoid is a major public health problem in third world
tients against Widal test, ELISA (IgG/IgM) had higher countries [1,2]. In Nepal, typhoid fever is endemic and
PPV at ≥1:400 (Table 5). The IgM/IgG titre ≥1:200 had the major factors for high prevalence rate include, but
a high sensitivity (95.50 %/96.85 %) and specificity are not limited to, illiteracy, poverty, poor sanitation and
(94.69 %/94.95 %). The diagnostic sensitivity of Widal inadequate facilities for safe drinking water supply. The
TO test at titre ≥1/400 was 84.09 % and specificity was Widal test based on TO and TH titre values were ob-
52.65 %. There was a significant difference between served higher in healthy subjects relating to the endemic

Table 5 Sensitivity, Specificity, PPV and NPV for typhoid fever of ELISA and Widal test in different cut off titers
Test Titer Sensitivity Specificity PPV NPV
95 % CI 95 % CI 95 % CI 95 % CI
IgM ≥1:200 95.50 % 94.69 % 77.66 % 99.09 %
91.87 % to 97.82 % 93.23 % to 95.92 % 72.24 % to 82.46 % 98.33 % to 99.56 %
≥1:400 81.23 % 98.29 % 91.77 % 95.70 %
75.95 % to 85.78 % 97.34 % to 98.97 % 87.45 % to 94.98 % 94.36 % to 96.80 %
≥1:800 61.97 % 96.48 % 78.38 % 92.50 %
55.41 % to 68.21 % 95.24 % to 97.48 % 71.74 % to 84.08 % 90.85 % to 93.93 %
≥1:1600 51.89 % 93.79 % 60.44 % 91.42 %
44.94 % to 58.78 % 92.24 % to 95.11 % 52.94 % to 67.60 % 89.68 % to 92.95 %
≥1:3200 47.44 % 92.73 % 54.84 % 90.46 %
40.61 % to 54.34 % 91.08 % to 94.16 % 47.39 % to 62.13 % 88.65 % to 92.08 %
IgG ≥1:200 96.85 % 94.95 % 78.75 % 99.36 %
93.61 % to 98.72 % 93.52 % to 96.14 % 73.42 % to 83.45 % 98.69 % to 99.74 %
≥1:400 85.95 % 98.76 % 93.69 % **
97.04 %
80.92 % to 90.07 % 97.93 % to 99.32 % 89.65 % to 96.51 % 95.89 % to 97.94 %
≥1:800 62.61 % 97.09 % 81.87 % 92.51 %
56.12 % to 68.77 % 95.93 % to 97.99 % 75.49 % to 87.18 % 90.87 % to 93.95 %
≥1:1600 51.89 % 94.48 % 63.22 % 91.48 %
44.94 % to 58.78 % 93.00 % to 95.72 % 55.59 % to 70.39 % 89.75 % to 93.00 %
≥1:3200 47.44 % 92.73 % 54.84 % 90.46 %
40.61 % to 54.34 % 91.08 % to 94.16 % 47.39 % to 62.13 % 88.65 % to 92.08 %
TO ≥1:100 90.32 % 52.41 % 44.22 % 92.12 %
87.25 % to 92.47 % 49.10 % to 53.91 % 41.18 % to 50.73 % 88.71 % to 94.83 %
≥1:200 80.95 % 51.77 % 23.29 % 93.76 %
74.98 % to 86.03 % 48.85 % to 54.68 % 20.27 % to 26.53 % 91.60 % to 95.50 %
≥1:400 84.09 % 52.65 % 25.34 % 94.54 %
78.58 % to 88.66 % 49.72 % to 55.57 % 22.22 % to 28.66 % 92.49 % to 96.17 %
TH ≥1:100 80.32 % 53.22 % 48.40 % 90.87 %
75.28 % to 85.91 % 46.42 % to 55.36 % 42.83 % to 50.27 % 87.14 % to 92.84 %
≥1:200 72.23 % 58.33 % 38.13 % 89.37 %
68.24 % to 79.72 % 52.93 % to 64.22 % 31.23 % to 43.10 % 83.11 % to 94.07 %
≥1:400 62.47 % 55.23 % 39.48 % 90.62 %
59.23 % to 66.82 % 47.21 % to 59.99 % 31.34 % to 45.83 % 86.92 % to 95.27 %
**
Highest PPV value
Adhikari et al. BMC Infectious Diseases (2015) 15:523 Page 6 of 7

Table 6 Sensitivity, specificity, PPV and NPV for typhoid fever of WHO case definition
Suspected case of Sensitivity Specificity PPV NPV
Typhoid fever
95 % CI 95 % CI 95 % CI 95 % CI
Isolation in Blood culture of;
S. typhi 76.81 % 99.22 % 97.07 % 92.71 %
71.99 % to 81.16 % 98.47 % to 99.66 % 94.31 % to 98.73 % 91.01 % to 94.18 %
Probable case of Typhoid fever
TH(1:160) 45.22 % 82.32 % 34.23 % 87.83 %
41.27 % to 49.17 % 79.91 % to 87.82 % 29.46 % to 38.96 % 81.59 % to 91.31 %
TO(1:80) 43.45 % 82.31 % 34.11 % 87.14 %
39.29 % to 48.97 % 78.96 % to 84.78 % 31.41 % to 38.85 % 83.72 % to 92.91 %

setting of typhoid fever in Nepal. Even the typhoid re- Abbreviations

lapsed subject group B had significant titre value of TH BACTEC: Bactenecin; ELISA: Enzyme Linked Immunosorbent Assay;
Peds: Pediatrics; TH: Titer of Salmonella H- Flagellar antigen; TO: Titer of
≥1:160, describing the persistent anti H in blood sample Salmonella H- Somatic antigen; BSA: Bovine Serum Albumin; PBS: Phosphate
[11] which also had higher serum IgG but lower IgM Buffer Saline; HRP: Horse Reddish Peroxidase; TMB: Trimethyle Benzidine;
antibodies suggesting the late class switching from IgM OD: Optical Density; SD: Standard Deviation; ROC: Receiver Operating
Characteristics; CHRB: Capital Hospital Ethical Review Board; PPV: Positive
to IgG in response to the infection well after elimination Predictive Value; NPV: Negative Predictive Value; NATRC: Aayurveda Training
of pathogen [22]. The sensitivity and specificity of the and Research Center; UGC: University Grant Commission.
IgG/IgM ELISA were higher than that of Widal test
Competing interests
showing Widal’s incompetence in accurate diagnosis. The authors declare that they have no competing interests.
The cut off titre is more in developing countries com-
pared to developed countries due to sanitary and en- Author contributions
demic reasons [23], thus the titre value from Widal test BPG, AA and RR designed the experiment. BPG, AA, RR, KDM performed the
research study. BPG, AA, RR, PPR & KDM analyzed the data. BPG, AA, PPR &
is not accurate enough to picture the complete diagno- KDM wrote the manuscript. All authors have read and approved the final
sis, since the PPV value of TO and TH were too low version of the manuscript.
(0.44 and 0.48 respectively). In developing country like
Acknowledgement
Nepal, the haphazard usage of antibiotic is common thus The authors would like to thank the technical staff of Capital Hospital for
providing false negative in blood culture, though there their contribution in the study and National Aayurveda Training and
were no patients who admitted taking antibiotic within Research Center (NATRC) as well University Grant Commission (UGC)
for providing partial grants in support of this research.
two weeks before enrollment in this study. The false
negative Widal test results were probably due to early Author details
1
blood collection before the adequate antibody produc- Asian Institute of Technology & Management, Lalitpur, Nepal. 2Capital
Hospital and Research Center, Kathmandu, Nepal. 3Central Department of
tion [24]. In case of Widal test, which when compared Biotechnology, Tribhuvan University, Kirtipur, Kathmandu, Nepal. 4Everest
with the WHO case definition, didn’t perform well. The Institute of Virology and Immunology, Kathmandu, Nepal.
sensitivity dropped by >40 % compared to gold standard
Received: 4 February 2015 Accepted: 28 October 2015
blood culture showing that Widal test alone can’t be
used as diagnostic approach, though is widely used in
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