Pathogens and Global Health

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Achieving accurate laboratory diagnosis of typhoid

fever: a review and meta-analysis of TUBEX® TF
clinical performance

Reynaldo Bundalian Jr., Madonna Valenzuela & Raphael Enrique Tiongco

To cite this article: Reynaldo Bundalian Jr., Madonna Valenzuela & Raphael Enrique
Tiongco (2019): Achieving accurate laboratory diagnosis of typhoid fever: a review and
meta-analysis of TUBEX® TF clinical performance, Pathogens and Global Health, DOI:
10.1080/20477724.2019.1695081

To link to this article: https://doi.org/10.1080/20477724.2019.1695081

Published online: 28 Nov 2019.

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PATHOGENS AND GLOBAL HEALTH
https://doi.org/10.1080/20477724.2019.1695081

Achieving accurate laboratory diagnosis of typhoid fever: a review and meta-

analysis of TUBEX® TF clinical performance
a
Reynaldo Bundalian Jr. , Madonna Valenzuelab and Raphael Enrique Tiongco c

a
Center for Research and Development, Angeles University Foundation, Angeles City, Philippines,; bPublic Health Program, Graduate
School, Angeles University Foundation, Angeles City, Philippines; cDepartment of Medical Technology, College of Allied Medical
Professions, Angeles University Foundation, Angeles City, Philippines

ABSTRACT KEYWORDS
This review discusses currently available serological diagnostic methods for typhoid fever with Serological test; Widal test;
a focus on the clinical utility of TUBEX® TF as an alternative to the Widal or Typhidot test. TUBEX® TF; typhoid fever;
A literature search was conducted in PubMed for related publications written in English. Salmonella Typhi; sensitivity;
A qualitative analysis was done to determine various serological tests used for typhoid fever specificity; meta-analysis
diagnosis with emphasis on TUBEX® TF in comparison to the Widal of Typhidot test. Further,
a meta-analysis was performed to obtain a pooled estimate of diagnostic accuracy (sensitivity
and specificity) using different analysis models. A total of sixteen studies was included in the
qualitative analysis. Further screening of these studies yielded ten studies that were used for
the meta-analysis. The sensitivity/specificity range of different commonly used serological tests
in typhoid patients is between 55-100%/58-100% for TUBEX® TF, 54-67%/54-95% for Typhidot,
and 32-95%/4-98% for the Widal test. As for the pooled meta-analysis estimates, the TUBEX® TF
showed superior results when differentiating individuals with febrile illness of unknown origin
from those with typhoid fever. Overall, the results of this review and meta-analysis suggest that
the TUBEX® TF is more advantageous to use as a serological test for typhoid fever diagnosis due
its accuracy and simplicity. However, further studies are still needed to validate our results.

1. Introduction The definitive diagnosis of typhoid fever is achieved

by isolating the S. Typhi bacteria from different speci-
Typhoid fever, also known as enteric fever, is an infectious
mens such as blood, bone marrow, and other body
disease caused by Salmonella Typhi and characterized by
fluids in the laboratory. In many of the developing
clinical symptoms of high fever, fatigue, abdominal pain,
countries where trained technicians and laboratory facil-
diarrhea, headache and complications of bleeding and
ities are limited, this method remains impractical. Due to
intestinal perforation [1]. It is transmitted through the
this, laboratory diagnosis of typhoid fever in developing
ingestion of contaminated food or drinking water.
or underdeveloped countries is primarily performed
Some factors that cause typhoid fever constitute an
through the detection of serum antibodies by applying
essential health problem in developing countries and
the Widal test (agglutination latex slide test) and in
continue to be a significant public health problem world-
some cases, using bacterial culture. The Widal test is
wide [2–4]. Typhoid fever is treatable with antibiotics.
the standard serological method for diagnosis of
However, without antibiotic therapy, the illness may last
typhoid fever in many countries, due to practical rea-
up to one month, with a high fatality rate. Reported
sons. However, both culture methods and the Widal test
surveillance studies provide critical information for guid-
demonstrate relatively low sensitivity and specificity and
ing public health decisions related to typhoid fever.
are time-consuming [5–7]. Furthermore, culture meth-
Typhoid fever is currently diagnosed based on clin-
ods require good laboratory facilities, which are lacking
ical signs, symptoms, and by applying various labora-
in many developing countries, where testing for typhoid
tory tests. The diagnosis of typhoid fever based on
fever if performed. Many hospitals lack facilities for
clinical signs and symptoms is often not adequate.
blood culture, and approximately 90% of typhoid
Usually, symptoms are nonspecific and may present
patients are treated as outpatients [8,9].
similarities with other acute febrile illnesses. Because
In Asia, disease burden estimates have relied on
of this, it is often impossible to find a specific clinical
clinically diagnosed cases of typhoid fever compiled by
symptom for typhoid fever, particularly during the first
governmental facilities and hospitals, usually with
week of illness. This is an indication of the need for
uncertain denominators [10]. Accurate estimates of
laboratory investigations to confirm the clinical diag-
rates of compilation and death at the population level
nosis of typhoid fever.
are not available. Population-based estimates of blood

CONTACT Raphael Enrique Tiongco tiongco.raphael@auf.edu.ph Department of Medical Technology, College of Allied Medical Professions,
Angeles University Foundation, Angeles City 2009, Philippines
© 2019 Informa UK Limited, trading as Taylor & Francis Group
2 R. BUNDALIAN ET AL.
culture-confirmed typhoid cases are sparse. Though,
recent reports of decreased susceptibility to diagnostic
agents might suggest a reemergence of untreatable
typhoid fever and an increasing global burden [11,12].
The primary objective of this review and meta-analysis
is to define the present outlook in terms of the laboratory
confirmation of typhoid fever using serological tests, spe-
cifically the clinical performance of TUBEX® TF. Rapid
diagnostic tests with acceptable levels of sensitivity, spe-
cificity, and accuracy for the definitive diagnosis of
typhoid cases are essential for better estimates of disease
burden and prompt treatment of infection.
2. Materials and methods
2.1. Search strategy
A literature search in PubMed was carried out using
a combination of the following key search terms:
‘TUBEX’ and ‘typhoid fever,’ for related studies written
in English. The titles and abstracts of the resulting
publications were screened to filter relevant studies.
Full texts were then evaluated carefully, and references
cited were manually screened for additional eligible
articles.
2.2 Study selection for the qualitative synthesis
The following inclusion criteria were used for the initial
qualitative synthesis: (1) studies that used TUBEX-TF in
determining typhoid fever (or cultures positive for S.
Typhi); (2) studies that contain a control group either in
the form of febrile illness with known origin (any infec-
tious disease aside from S. Typhi), febrile illness of
unknown origin (maybe infectious or noninfectious in
nature), culture negative controls, and healthy con-
trols; and (3) studies that used other serological tests
such as the Widal test or Typhidot test as a comparison
to TUBEX® TF. All studies were investigated indepen-
dently for eligibility by two of the authors (R Bundalian
and RE Tiongco).
2.3. Data extraction for the qualitative synthesis
Two authors (R Bundalian and RE Tiongco) indepen-
dently extracted the data and reached an agreement
on all the items. For each study included, the following
data were obtained: (1) first author’s last name; (2) year
of publication; (3) country where the study was con-
ducted; (4) sample size; (5) method of typhoid fever
testing; (6) controls used; and (7) sensitivity and speci-
ficity of TUBEX-TF and Widal of Typhidot test.
2.4. Study selection and data extraction for the
meta-analysis of diagnostic test accuracy
Studies included in the qualitative synthesis were
further screened to obtain articles to be used for the
meta-analysis. The additional inclusion criteria used
are: (1) studies that contain a case group (individuals
with known typhoid fever) vs. control group compar-
ison (either with febrile illness of known origin aside
from S. Typhi, febrile illness of unknown origin, culture
negative, or healthy individuals); and (2) studies that
contain the total number of positive and negative
cases in both the case and control group.
2.5. Diagnostic test accuracy meta-analysis
Estimation of the overall diagnostic test accuracy was
carried out using Meta-DiSc ver. 1.4. Pooled estimates
of sensitivity, specificity, and area under the curve
(AUC) were estimated, and a summary receiving opera-
tion characteristic (SROC) curve were drafted. Meta-
analysis was performed only for categories where
enough data was available to produce average sensi-
tivity and specificity estimates. Three analysis models,
namely: (1) Model 1: healthy controls vs. typhoid fever;
(2) Model 2: febrile illness of known origin aside from
S. Typhi vs. typhoid fever; and (3) Model 3: febrile
illness of unknown origin + culture negative controls
vs. typhoid fever, was performed.
3. Results and disucssion
3.1. Characteristics of the included studies
Summary of the literature search is presented in Figure 1,
whereas the characteristics of the included studies are
presented in Table 1. Overall, a total of 16 studies were
included in the qualitative analysis, and 12 in the meta-
analysis. Year of publication of articles ranged from 1998
to 2016. Of the total number of publications retrieved, ten
were conducted in Asian countries [13–22], five in the
African region [23–27] and the remaining study in Papua
New Guinea [28]. The reported findings focused mainly
on sensitivity, specificity, positive predictive value (PPV),
and negative predictive value (NPV) of TUBEX® TF, Widal,
and Typhidot with their clinical outcome.
3.2. Results of the qualitative analysis
Different studies have been made to evaluate the clin-
ical utility of serological tests for typhoid fever diag-
nosis. In Table 1, a summary of the major findings in
different evaluation studies of TUBEX® TF in Asian
countries are highlighted. Several studies were also
made in the African region that evaluated the useful-
ness of TUBEX® TF in diagnosing typhoid fever. Details
of the clinical performance of TUBEX® TF are further
described in Table 1 as well. The sensitivity/specificity
range of different commonly used serological tests in
typhoid patients is between 55–100%/58–100% for
TUBEX® TF, 54–67%/54–95% for Typhidot, and
32–95%/4–98% for the Widal test.
 PATHOGENS AND GLOBAL HEALTH 3

Key search terms:

“TUBEX” and “typhoid fever”

Records identified through Additional records identified

PubMed searching through other sources
(n = 29) (n = 1)

Records identified after review of titles and abstracts as well as

removal of duplicates (n = 24)

Abstracts screened Records excluded (n = 2)

(n = 24) Due to incomplete or irrelevant
data

Full-text articles Full-text articles excluded (n = 6)

assessed for eligibility Deemed irrelevant to our study
(n = 22)

Studies included in the

qualitative analysis
(n = 16)

Studies included in the

quantitative analysis
(n = 10)

Figure 1. Summary of the literature search.

3.2.1. Laboratory diagnosis of typhoid fever
There are at present several strategies to diagnose for
typhoid fever in the laboratory. Approaches can be
categorized into (1) direct methods and (2) indirect
methods. Of these, isolation of S. Typhi from clinical
specimen remains as the gold standard in the diagno-
sis of typhoid fever. This section describes some of the
conventional serological diagnostic methods used for
typhoid fever.
Most enteric fevers occur in low and middle-income
countries where culture methods are seldom available,
unaffordable, or inconsistently applied. Furthermore,
several members of the genus Salmonella may share
the same antigenic composition. Thus confirmatory
testing by means of biochemical tests such as
Analytical Profile Index (API) is often necessary.
Different forms of serological procedures have been
used in diagnosing for typhoid fever. These methods
are based on the detection of specific IgM and IgG
antibodies against either the somatic O-antigen or
flagellar H-antigen of S. Typhi. Currently, the Widal
test is still used in laboratories for the diagnosis of
typhoid fever. This serological test is dependent on
the production of IgM and IgG antibodies and agglu-
tination reaction using patient’s sera. Both the O- and
H-antigens used in the Widal test are nonspecific and
may cross-react with other Salmonella species as well
as species of Plasmodium. This may lead to false posi-
tive test results for those with non-Salmonella infection
and even malaria. It may also lead to false negative
cases if tested during the early phase of typhoid fever.
However, due to its simplicity and cost-effectivity,
4 R. BUNDALIAN ET AL.

Table 1. Characteristics of the included studies and summary of the sensitivity and specificity of TUBEX® TF compared with Widal
test or Typhidot test.
First Sample Sensitivity Specificity Ref.
Author Year Location Size (%) (%) Control Group No.
Lim 1998 Hong Kong/ Total: 90 TUBEX® TF Total: 65 [14]
Malaysia Bacteriologically 100 100 Healthy individuals;
confirmed: 14 Widal Test Patients with autoimmune diseases; Typhus positive
Clinically 81 43 patients; Septicemic patients
diagnosed:11
House 2001 Vietnam Total: 127 TUBEX® TF Febrile hospitalized cases [16]
Culture 87 76
confirmed: 64 Widal Test
32 98
Olsen 2004 Vietnam Total: 79 TUBEX® TF Laboratory confirmed febrile illness other than typhoid [20]
Culture 78 94
confirmed: 59 Widal Test
64 76
Dutta 2006 India Total: 459 TUBEX® TF Paratyhoid fever cases and malaria cases [15]
Culture 55 81
confirmed: 176 Widal Test
67 85
Khanna 2015 India Total: 200 TUBEX® TF Other febrile illness and healthy controls [19]
Culture 76 98
confirmed: 50 Widal Test
68 98
Rahman 2007 Bangladesh Total: 243 TUBEX® TF Blood culture negative cases [18]
Culture 91 82
confirmed: 34 Widal Test
82 69
Dong 2007 China Total: 33 TUBEX® TF S. Paratyphi [22]
Culture 69 95 cases
confirmed: 13 Typhidot
54 91 Blood culture negative cases [17]
Kawano 2007 Philippines Total: 177 TUBEX® TF
Culture 95 80
confirmed: 75 Typhidot
55 65
Naheed 2008 Bangladesh Total: 867 TUBEX® TF Blood culture negative cases and other bacteremia [21]
Culture 60 58
confirmed: 43 Typhidot
67 54
Islam 2016 Bangladesh Total:127 TUBEX® TF Other febrile illness and healthy controls [13]
Culture 75 89
confirmed: 28 Typhidot
64 80
Ley 2011 Tanzania Total: 139 TUBEX® TF Non S. Typhi infection cases [23]
Culture 79 89
confirmed: 33 Widal Test
Not
performed Not performed
Keddy 2011 South Africa/ Total: 92 TUBEX® TF Non S. Typhi infection cases [24]
Tanzania Culture 73 69
confirmed: 28 Widal Test
95 4
Fadeel 2011 Egypt Total: 388 TUBEX® TF Healthy individuals [25]
Culture 77 85
confirmed: 67 Typhidot
63 95
Neil 2012 Uganda Total: 76 TUBEX® TF Blood or stool culture negative cases [26]
Culture 88 84
confirmed: 27 Widal Test
Not
performed Not performed
Tarupiwa 2015 Zimbabwe Total: 136 TUBEX® TF Unspecified [27]
Culture 100 94
confirmed: 131 Widal Test
Not
performed Not performed
Siba 2012 Papua New Total: 500 TUBEX® TF Malaria-negative healthy controls [28]
Guinea Culture 77 87
confirmed: 22 Widal Test
86 95

many clinical laboratories still use the test even if
considered less specific [5].
Presently, serological methods still suffer from lim-
itations of sensitivity and specificity emphasizing the
need for a quick and reliable serologic test for acute
infection of typhoid fever as an alternative to the old
Widal test and as a complement to blood culture.
A serologic assay is clinically useful if it is characterized
by high sensitivity and specificity. Advances in the
development of serological assays for the diagnosis
 PATHOGENS AND GLOBAL HEALTH 5

of typhoid fever have recently demonstrated that
a rapid test such as TUBEX® TF assay might be of
clinical value for the acute diagnosis of typhoid fever
and surveillance of treatment. Such assay provides
critical clinical information to public health decision-
makers, regarding clinical management, disease pre-
vention, and infection control strategies [1].
Serologic tests that have been applied for the diag-
nosis of typhoid fever in clinical practices are TUBEX®
TF (IgM), Typhidot (IgM/IgG), IgM/IgG ELISA, IgM dip-
stick and the Widal test (hemagglutination). Technical
characteristics of the most applied serological tests for
the diagnosis of typhoid fever (IgM antibody determi-
nation) are presented in Table 2.
3.2.1.1. Widal test. The Widal test is a whole cell
agglutination reaction carried out on serum samples
using commercially available reagents evaluated about
the saline reaction (negative control) to demonstrate
the presence of agglutinins (antibodies) in the serum
of infected patients. It can either be a slide agglutina-
tion test (using undiluted serum) and tube agglutina-
tion methods (serially diluted patient sera). Serum
samples are collected 2–10 days from the appearance
of symptoms. Several limitations of this test are nota-
ble. The result of the Widal test is difficult to interpret
unless the sensitivity and specificity for the specific
laboratory and patient population are known [29].
Also, the sensitivity of the Widal test may require rela-
tively high antibody concentrations (more than 500
ng).
Furthermore, the value of the Widal test depends
upon the standardization and maintenance of the anti-
gen to produce consistent results [25]. Applying the
Widal test in typhoid-endemic regions may also be
problematical due to patients’ repeated exposure to
S. Typhi or due to cross-reactivity to S. Paratyphi A or
malaria. This makes it difficult to establish a relevant
baseline titer for the population [5].
3.2.1.2. TUBEX® TF (inhibition magnetic binding
immunoassay). TUBEX® TF (Inhibition Magnetic
Binding Immunoassay) is a semi-quantitative in-vitro
diagnostic rapid test for the detection of acute S. Typhi.
TUBEX® TF is based on the detection of S. Typhi IgM anti-
O9 antibodies in patient serum [14,30]. The inhibition of
binding between an anti-O9 IgM monoclonal antibody
conjugated to colored latex particles and S. Typhi lipopo-
lysaccharide (LPS) O9 antigen coated to magnetic latex
particles is measured. The latex reagent and the magnetic
beads (blue and brown particles) are mixed in a specially
designed reaction well for 2 minutes together with sam-
ple or test control, and the magnetic beads are separated
from the solution on a magnet. The resultant color of the
supernatant is recorded and compared with color stan-
dards (color scale). A TUBEX® TF score between 0 (nega-
tive), and 10 (positive) is assigned, and a TUBEX® TF score
of 4–10 is positive for typhoid fever, while TUBEX® TF
scores in the range above 2 and below 4 are inconclusive,
after which the tests should be repeated.
3.2.1.3. Typhidot. Typhidot is a qualitative dot
enzyme immunoassay that detects either IgM or IgG
antibodies against a specific antigen (50 kDa) on the
outer membrane protein of serotype S. Typhi. Patient
serum and control are incubated with the strips fol-
lowed by washing. After further washing, the strips are
incubated with color reagent, and the intensity of the
sample dots are compared with dots from the positive
control strip. The detection of IgM antibodies reveals
acute typhoid disease in the early phase of infection,
while the detection of both IgG and IgM suggests
acute typhoid in the middle phase of infection. In

Table 2. Comparison of the commonly used serological test for typhoid fever.
TUBEX ® TF Typhidot ELISA IgM Dipstick Widal
Test principle One-step Dot blot EIA, multi-step Enzymatic detection in Immuno- chromato- One-step
Rapid test, magnetic microtiter plates graphic agglutination
separation test
Result Semi- Qualitative Quantitative Qualitative Semi- quantitative
quantitative titration
Specimen/ Serum, Serum, 4 drops Serum, Blood, serum Serum, 50 µL
volume 45 µL 50–100 µL
Antigen LPS O-9 50 kDa antigen O- and H- antigens LPS antigen O-and H- antigens,
detected antigen Whole organism
Antibody IgM IgM, IgG IgM, IgG IgM IgM, IgG
detected
Hands on time 20 minutes 60 minutes 3 hours 3 hours Overnight,
37°C
Result/ Color scale Color Color Staining Type of
interpretation developed, developed, of test line agglutination
compared to control compared to standard
Current Yes (IgM) Yes (IgM, IgG) Yes (IgM, IgG) Yes (IgM) Yes
infection
Instrumentation Magnetic stand Shaker Shaker, washer, reader No Incubator
Controls Positive and negative Positive and negative Positive and negative Negative control Local standard
control included control included control included line- mandatory
IgM
6 R. BUNDALIAN ET AL.

areas highly endemic for typhoid fever, the detection 3.3. Results of the meta-analysis
of typhoid specific IgG antibodies increases.
Based on the results of the pooled diagnostic accuracy
estimates (Table 3), the sensitivity and specificity of the
3.2.1.4. IgM/IgG ELISA. IgM/IgG ELISA is a solid- TUBEX® TF range between 68–86% and 84–97%,
phase immunochemical assay that measures antibo- respectively. Meta-analysis performed using Model 1
dies against defined epitopes on S. Typhi antigens. showed an average sensitivity of 76% (95% CI: 65–85)
Serum samples or controls are added to wells coated and specificity of 97% (95% CI: 92–99). No AUC were
with purified typhoid LPS antigen (S. Typhi somatic drafted for this association since there are fewer than
O-antigen and flagellar H-antigen). After blocking the three studies included. For Model 2, average sensitivity
solid-phase for unspecific binding, samples are per- and specificity are at 68% (95% CI: 63–73) and 90%
mitted to react with the immobilized solid-phase anti- (95% CI: 87–93), respectively, with an AUC of 0.8752
gen. If present, IgM or IgG specific antibodies bind to (Figure 2). On the other hand, Model 3 showed super-
the antigen. After a specified incubation period, ior results compared to the other two models with an
unbound material is washed away, and the enzyme- average sensitivity and specificity of 86% (95% CI:
conjugate is added in order to bind to the antibody- 80–91) and 84% (95% CI: 80–87), respectively with an
antigen complex. After another washing step, the AUC of 0.9205 (Figure 3).
substrate is added, and the absorbance is quantified
(proportional to the amount of IgM or IgG specific
3.3.1. TUBEX® TF studies from Asian countries
antibodies in the sample). The detection limit for
All studies conducted used the culture from either
ELISA assays applied for typhoid testing is, in general,
blood or bone marrow sample to confirm for typhoid
very high, (0.05 ng, according to data reported by
infection. The confirmed test was defined by the isola-
Fadeel et al. in 2011). However, ELISA assay is time-
tion of S. Typhi from the sample. The majority (7 out
consuming and requires several instrumentations
of 9) of the studies conducted in Asia evaluating the
such as a microplate reader making it challenging to
performance of TUBEX® TF, reported higher sensitivity
be routinely used for typhoid diagnosis.
and specificity compared with either Widal test and
Typhidot. Highest sensitivity (100%) and specificity
3.2.1.5. Typhoid IgM dipstick assay. The typhoid (100%) was reported compared with Widal test using
IgM dipstick assay is an immunochromatographic the first version of TUBEX® TF test that utilizes a one-
method for qualitative determination of IgM antibodies step procedure based upon particle separation, using
to LPS antigens released by the S. Typhi bacteria. The anti-O9 LPS IgM antibody as the detector reagent [31].
patient sample, containing S. Typhi LPS antibodies, will Among the different studies conducted, only the study
react with specific gold-labeled anti-human IgM. The performed in India [15], reported lower sensitivity for
antigen and antibody complex will migrate to the reac- TUBEX® TF compared with the Widal test. A possible
tion zone where the complex reacts with another spe- reason for this observation may be due to the selection
cific antibody forming a sandwich. The antigen-
antibody complexes are captured by anti-IgM antibo-
dies immobilized on the test strip and will be detected Table 3. Summary of the pooled sensitivity and specificity
as a visible line. The excess of gold-labeled antibodies estimates.
Analysis Model TP FP FN TN SN% (95% CI) SP% (95% CI)
will migrate further into a second reaction zone creating
Model 1 (Healthy controls vs. Typhoid fever)
a control line. The dipstick method provides a rapid and Khanna, 2015 38 1 12 99 76 (62, 87) 99 (95, 100)
simple alternative for the diagnosis of typhoid fever. Islam, 2016 21 3 7 17 75 (55, 89) 85 (62, 97)
Overall - - - - 76 (65, 85) 97 (92, 99)
Model 2 (Febrile illness of known origin aside from S. Typhi vs. Typhoid fever)
Dong, 2002 9 1 4 20 69 (39, 91) 95 (76, 100)
3.2.1.6. TPTest. TPTest or Typhoid and Paratyphoid Dutta, 2006 58 14 45 99 56 (46, 66) 88 (80, 93)
test is a recently reported platform for the serodiagno- Islam, 2016 21 1 7 14 75 (55, 89) 93 (68, 100)
Khanna, 2015 38 2 12 48 76 (62, 87) 96 (86, 100)
sis of typhoid fever. This test uses a heparinized blood Ley, 2011 26 12 7 94 79 (61, 91) 89 (81, 94)
sample from which peripheral blood mononuclear Naheed, 2008 26 3 17 21 60 (44, 75) 88 (68, 97)
Olsen, 2004 43 1 12 17 78 (65, 88) 94 (73, 100)
cells (PBMC) are separated through density gradient Overall - - - - 68 (63, 73) 90 (87, 93)
centrifugation. This procedure is then followed by cul- Model 3 (Febrile illness of unknown origin + culture negative controls vs. Typhoid
ture of the PBMCs in RPMI medium at 37°C and 5% fever)
Fadeel, 2011 50 17 17 126 75 (63, 84) 88 (82, 93)
CO2. Culture supernatants are tested for S. Typhi spe- Kawano, 2007 71 20 4 82 95 (87, 99) 80 (71, 88)
cific IgA 48 hours after incubation using ELISA. Rahman, 2007 31 37 3 172 91 (76, 98) 82 (76, 87)
Overall - - - - 86 (80, 91) 84 (80, 87)
Measurements of >10 ELISA units are considered posi-
TN: true positive; FP: false positive; FN: false negative; TN: true negative;
tive for the test based on established cutoff value. SN: sensitivity; SP: specificity; CI: confidence interval

Figure 2. SROC curve for the Model 2 comparison.
Figure 3. SROC curve for the Model 3 comparison.
of the control group, S. Paratyphi cases were chosen as
the negative control in the study. Also, the knowledge
about the high degree of cross-reactivity between S.
Typhi and S. Paratyphi A when using TUBEX® TF was
not previously known [32]. In contrary, in another
study conducted in China where S. Paratyphi cases
PATHOGENS AND GLOBAL HEALTH 7
were used as controls, higher sensitivity (69%) and
specificity (95%) was observed using TUBEX® TF when
compared with the sensitivity (54%) and specificity
(91%) of Typhidot. Looking at the different studies,
the choice of control group might be critical in evalu-
ating the clinical usefulness of different serological
8 R. BUNDALIAN ET AL.
tests for typhoid fever. The sensitivity and specificity of
the test are affected by the choice of control groups in
the studies. In one of the studies conducted, the spe-
cificity for TUBEX® TF increased from 82% to 90% when
healthy non-febrile subjects or cases with dengue
fever instead of blood culture negative cases were
used as the control group [18].
Compared with Typhidot, only the study performed
in Bangladesh [21] reported a lower sensitivity (60%)
for TUBEX® TF. However, slightly higher specificity
(58%) was observed for TUBEX® TF compared with
Typhidot (54%) in this study. One of the probable
cause of this observation is due to a large number of
indeterminate results for both tests. At least, 139
patients could not be properly read against the
TUBEX® TF color scale (indeterminate borderline
cases), and 186 patients could not be properly evalu-
ated with Typhidot (only IgG positive). The number of
inconclusive cases included in the study group will
likely have a significant influence on the calculated
sensitivity and specificity for TUBEX® TF and Typhidot.
In most studies conducted, the stage of the febrile
illness at which the blood sample was drawn for test-
ing was not specified. Only the length of febrile epi-
sodes was described before the inclusion in the study.
It has been observed that the sensitivity (55% to 57%)
and PPV (72% to 85%) of TUBEX® TF improve when
only cases with more than five days of febrile episodes
are assessed [15]. This indicates that the sensitivity and
specificity of the test are affected by the kinetics of the
antibody response in the patient. Also, it is notable that
in some of the studies made, patients who have
initiated antibiotic therapy were not excluded among
the subjects [20]. Antimicrobial therapy before blood
collection is known to affect the results of the culture.
Seven out of the nine studies performed also high-
lighted equal or better specificity when TUBEX® TF is
used compared to either the Widal test or Typhidot in
diagnosing typhoid fever. Only the studies in India [15]
and Vietnam [16] reported higher specificity for Widal
test compared with TUBEX® TF. Lower specificity for
TUBEX® TF in the study made in India [15] is mainly
a result of the use of S. Paratyphi cases as control, since
TUBEX® TF may cross-react with S. Paratyphi. In the
study performed in Vietnam [16], although higher spe-
cificity (98%) (at 1/400 titer) was observed for Widal
test, reduced sensitivity (32%) is reported when com-
pared with the sensitivity (87%) of TUBEX® TF. In sev-
eral studies, poor clinical outcome for the Widal test is
notable [5,16–18,20]. There is significant relative varia-
tion in the Widal data [20], mostly due to lack of local
standardization and difficulties in the interpretation of
generated data. When repeated in a local laboratory
(Pasteur Institute) a significant increase in specificity
(from 76% to 100 %) with maintained sensitivity [5] can
be observed indicating the necessity to establish
a local baseline and cutoff values when using this test.
3.3.2. TUBEX® TF studies from African countries
A total of five studies have been performed in the
African region evaluating the clinical usefulness of
TUBEX® TF in typhoid fever diagnosis. Similar with
previous studies in Asian countries; typhoid fever was
confirmed using blood culture in all studies reported.
The first TUBEX® TF study was performed in Tanzania,
involving children hospitalized with symptoms of feb-
rile illness [23]. In this study, controls were categorized
into two groups. The first group includes only non-
Typhi Salmonella cases, while the second group
includes all blood culture positive non-Salmonella
cases. No significant difference in the sensitivity (79%)
and specificity (89%) using the first group as control
compared with the sensitivity (79%) and specificity
(97%) when the second group of controls was used in
the analysis. Although, a notable increase in the speci-
ficity (89% to 97%) of TUBEX® TF is observed.
In another study performed, commercially available
serologic tests for typhoid fever diagnosis were ana-
lyzed for clinical efficacy. Efficacy was defined as the
ability of the test to detect IgM antibodies against S.
Typhi in patients suspected of having typhoid fever.
A total of 28 patients were confirmed for typhoid fever
using blood culture, while 52 patients with negative
blood culture for S. Typhi or other pathogens were
used as control. In this study, both Typhidot and
TUBEX® TF demonstrated lower sensitivity to the Widal
test. However, while high sensitivity (95%) for the Widal
test was reported, specificity (4%) of Widal was very low.
The reported low sensitivity (77%) and specificity (85%)
for TUBEX® TF can be attributed to the selection of
control cases that were suspected of being typhoidal
[16,17]. Furthermore, the control group may also have
included S. Paratyphi A positive patients, which may
cross-react with S. Typhi and thus be cross-detected
using TUBEX® TF [17,20,23] decreasing the specificity.
An alternative to the conventional serologic test for
typhoid, an in-house ELISA assay, measuring IgG, IgM,
and IgA antibodies against bacterial culture antigens,
was evaluated in Egypt [25]. Efficacy of an ELISA assay
was compared with TUBEX® TF, Widal test, and
Typhidot among typhoid fever patients. Higher sensi-
tivity (77%) for TUBEX® TF was reported compared with
the sensitivity of the Widal test (71%) and Typhidot
(63%). The sensitivity (88%) of the experimental IgM,
IgG, IgA ELISA assay was higher compared with all
conventional serological tests.
On another case, a laboratory surveillance study and
a community survey were performed in Uganda to
determine the origin of typhoid fever outbreak [26].
TUBEX® TF assay was used as a rapid laboratory test for
measuring IgM antibodies against S. Typhi antigens,
where blood and stool cultures were also performed.
Using TUBEX® TF in 76 of the patients with samples
collected four days after the onset of illness, sensitivity
(88%) and specificity (84%) was established. In this

study, the lack of microbiological laboratory capacity,
equipment, supplies as well as trained personnel, led
to difficulties in isolating S. Typhi consequently delay-
ing the confirmation of etiology.
More recently, TUBEX® TF and OnSite Typhoid IgM/
IgG rapid test were evaluated among patients sus-
pected of typhoid fever in Zimbabwe [27]. Using cul-
ture as the reference standard, the sensitivity (100%)
and specificity of (94%) of TUBEX® TF was comparable
with the sensitivity (100%) and specificity (94%) of
OnSite Typhoid IgM/IgG rapid test. Both test also
reported similar PPV (63%) and (100%).
3.3.3. TUBEX® TF studies from other countries
PCR-based techniques are an interesting development
in molecular diagnostics as a detection tool for patho-
gens. Coupled with Salmonella-specific PCR, this
method could be used instead of conventional sero-
typing methods to identify the presence S. enterica
serovar accurately. Real-time PCR was applied in
a study from Papua New Guinea together with the
serological methods TUBEX® TF, Typhidot, and the
Widal test on 530 outpatients (fever >2 days) from
two different hospitals [33]. Of these patients, 500
were tested negative for malaria, and these subjects
were included in the study with suspicion of typhoid
fever. Typhoid fever was diagnosed in patients using
real-time PCR and blood culture. Some of the
PCR-positive patients were blood culture negative
and further, some blood culture positive subjects
were PCR-negative, demonstrating the limited value
of applying the molecular identification of S. Typhi
infection. Blood culture and PCR testing were used as
a reference standard in 47 typhoid positive patients.
TUBEX®TF showed sensitivity (51%) similar to the sen-
sitivity (51%) of the Widal test. Sensitivity (70%) of
Typhidot was higher compared with both TUBEX®TF
and Widal test. On the other hand, the specificity (96%)
of the Widal test was higher compared to the specifi-
city (88%) of TUBEX®TF and the specificity (80%) of
Typhidot. It was concluded in the study that either
TUBEX® TF or Typhidot was sufficiently sensitive to be
used in routine clinical practice [33].
3.4. TUBEX® TF: advantages and limitations
Several advantages and limitations of the TUBEX® TF
test are notably based on this review and meta-
analysis. One of the significant advantages of TUBEX®
TF is its selectivity for S. Typhi IgM antibodies. It has
been assumed that TUBEX® TF is a selective assay that
measures only IgM antibodies in typhoid patient
serum [17,32]. This was later confirmed in a study per-
formed where IgM antibodies were separated from IgG
antibodies in a typhoid fever patient serum and tested
with TUBEX® TF [32]. Only IgM antibodies with
PATHOGENS AND GLOBAL HEALTH 9
specificity toward O9 antigens were detected, and
the addition of IgG antibodies did not influence the
detection of IgM antibodies. Thus, the direct inhibition
of IgG antibodies could not be demonstrated, and the
IgG antibodies appear to be non-reactive in the
TUBEX® TF assay. This is clinically important for any
form of serologic test in order to easily differentiate
acute infection from previous exposure. In addition,
the enhancement effect of IgG on the detection of
IgM antibodies assayed with TUBEX® TF was also illu-
strated [18].
Another main advantage of TUBEX® TF compared
with other available serologic test is its simplicity.
Compared with other serological tests, TUBEX® TF is
simple, easy to use, and requires short test time with
very minimal instrumentation and equipment
required. This allows the potential use of the test in
decentralized platforms where limited laboratory facil-
ities are available in communities.
In addition to the previous advantages mentioned
the semi-quantitative nature of the TUBEX® TF further
adds value to its clinical usefulness. The TUBEX® TF
score read against the color scale, showed some corre-
lation between the semi-quantitative TUBEX® TF score
and the stage of the disease. Low scores denote either
an early or a very late stage, while high scores generally
denote late phase or convalescence. However, more
often than not, many patients can mount a good
response early in the infection and maintain the same
response (unchanged TUBEX®TF score) for many weeks
thereafter. The TUBEX® TF scores like the Widal titer or
the ELISA titer, add value (confidence) to the diagnosis
of S. Typhi. A high score generally denotes a result
undisputable positive, whereas a low score raises the
possibility that the result might be spurious due to
technical reasons. In the latter case, repeating the
test on the same patient sample or another specimen
can help to resolve the dilemma. More often, many
patients who yielded a low TUBEX® TF score,
a subsequent specimen obtained a week or so later
produce a more definitive TUBEX®TF result. In order to
improve POC testing for typhoid fever, it has been
proposed that the use of paired samples might
improve the outcome of the laboratory testing [6].
On the other end, a limitation of the TUBEX® TF test,
which uses a colorimetric reaction, is the potential
difficulty in interpreting the results accurately, compar-
ing the color of the test solution with a color scale (gray
zone or borderline cases). Assessing the color change
in the reaction well strip and comparing the color of
the test solution with a color scale (results graded from
0 (negative) to 10 (positive) requires some experience
and good lighting conditions. Earlier studies using an
old version of TUBEX® TF showed that the gray zone is
too great when reading the color scale leading to
indeterminate results.
10 R. BUNDALIAN ET AL.
Another identified concern when using TUBEX® TF
assay is the interpretation of hemolyzed/icteric sam-
ples when reading against the color scale. Hemolyzed/
icteric samples may produce false positive signals due
to discoloration. The current manufacturer recommen-
dation for hemolyzed/icteric samples is to take a new
sample and re-test with TUBEX® TF. However, this issue
was already resolved. Recently, a new version of
TUBEX® TF assay that contains an extra wash step
with glycine buffered saline to eliminate serum disco-
loration of hemolyzed/icteric samples was presented
[33]. The introduction of wash buffer, as a complement
to TUBEX® TF assay, is a quick and simple modification
of the TUBEX® TF protocol and enables detection of
acute typhoid fever in discolored serum samples. The
wash procedure decreases the gray zone with border-
line or inclusive results and allows a more accurate
reading of the color scale. This, in turn, increases the
sensitivity of TUBEX®TF assay.
3.5. TUBEX® TF: future research perspectives
Serologic laboratory testing of typhoid fever is essen-
tial and can be a beneficial complement to blood
culture in order to increase the clinical information.
Laboratory tests play an integral role in confronting
typhoid fever infection and can help to establish the
primary diagnosis. Early detection of typhoid fever by
TUBEX® TF assay can help in the clinical management
of the disease and patient surveillance while reducing
the diagnostic confusion. This review and meta-
analysis substantiate that in comparison with other
commercially available serologic methods, TUBEX® TF
assay can be a useful laboratory tool for diagnosis of
typhoid fever infection especially, within the first three
to seven days of febrile illnesses.
TUBEX® TF test procedure has been improved dur-
ing the past few years – new type of latex particles,
a more precise reagent volume, a careful and specified
LPS (O9 antigen), a standardized coupling procedure
of LPS-antigen to the magnetic particles, improved
indicator reagents (IgM antibody-coupled particles),
and more stable color scale, a better defined TUBEX®
TF control reagent and a specific washing step to
reduce the negative influence of hemolyzed samples/
icteric samples has been integrated. The improved
version of TUBEX® TF test has made the assay more
applicable for the clinical diagnosis of typhoid fever. If
used in a proper way on a single serum sample of
a suspected case, TUBEX® TF assay has the ability to
improve the diagnostic algorithm, contributing signifi-
cantly to the clinical treatment and control of typhoid
bacterial infection. However, according to several stu-
dies, interpretation of TUBEX® TF results has been diffi-
cult in many health centers. It has been recommended
that the evaluator should be trained in interpreting
TUBEX® TF results because of variation among
individual readers (i.e. visual interpretation). This,
therefore, may represent a possible area requiring
further improvement.
As based on the review of the clinical studies per-
formed for TUBEX® TF selection of appropriate control
is clearly critical. For the evaluation of statistical para-
meters for serological assays such as TUBEX® TF, the
selection of control (negative) cases is critical. Studies
revealed that this might be one of the factors that
account for the wide range of variation in sensitivity,
specificity, PPV, and NPV of TUBEX® TF. In a recent
meta-analysis performed in Belgium, the overall accu-
racy of TUBEX® TF and Typhidot for the diagnosis of
typhoid fever was evaluated, and extensive range esti-
mates for sensitivity and specificity were noted [34].
The calculated pooled sensitivity for TUBEX® TF in the
meta-analysis ranged between 56–95% (average sen-
sitivity 69%) and the pooled specificity between
72–95% (average specificity 88%). In a recent diagnos-
tic test accuracy review, TUBEX® TF had an average
sensitivity of 78% (95% confidence interval (CI) 71%
to 85%) and specificity of 87% (95% CI 82% to 91%).
The overall diagnostic accuracy score was moderate,
with no significant difference compared with Typhidot
and Test-it Typhoid test [13].
Selection of control cases is very critical to establish
better estimates of accuracy. In some of the studies
conducted for TUBEX® TF, febrile patients with patho-
gens other than Salmonella were used as control.
Furthermore, in many reported studies blood culture,
negative patient samples are applied as negative con-
trol even if there is a possibility, that these patients are
true typhoidal, as blood culture demonstrates low
sensitivity compared to serological assays [18]. These
false negative cases might influence the calculation of
sensitivity and specificity. In addition, the utilization of
typhoid negative blood culture with the presence of
other pathogens as control generally results in low
sensitivity and specificity [17,21,24]. Protocol harmoni-
zation may be required in order to come up with the
comparable interpretation of clinical studies for sero-
logical assays.
Another area of concern in the selection of control is
the inclusion of S. Paratyphi A patients in the control
group. In reported studies where a number of S.
Paratyphi A patients were included in the control
together with cases with another type of febrile ill-
nesses, the sensitivity and specificity of TUBEX® TF
were low [15,22]. S. Typhi and S. Paratyphi A show
a cross-reactivity of about 50% with TUBEX® TF [20]
leading to false positive cases that reduce the overall
sensitivity and specificity of the test. Most authors
recommend the use of non-Salmonella cases (other
febrile illnesses or those with other bacterial diseases)
as control subjects when evaluating sensitivity, specifi-
city, PPV and NPV characteristics of TUBEX® TF which
can be consequently made in larger studies.

4. Conclusion
As compared to other existing serological test for typhoid
fever detection, the TUBEX® TF is noted to be more
advantageous due to its selectivity for S. Typhi IgM and
its simplicity. Also, the TUBEX® TF is also easy to use,
would require less time and instruments to perform. In
terms of diagnostic accuracy, the test shows reasonable
sensitivity and specificity, especially when differentiating
individuals with a febrile illness that are negative for
culture versus those with typhoid fever. It also showed
a high level of specificity when differentiating healthy
individuals from those with typhoid fever. However,
further studies are still needed, especially with the use
of an appropriate control group, are still needed to verify
our claims.
Disclosure statement
No potential conflict of interest was reported by the authors.
ORCID
Reynaldo Bundalian Jr. http://orcid.org/0000-0003-2377-
006X
Raphael Enrique Tiongco http://orcid.org/0000-0002-
7793-6558
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