Sensitivity and specificity of typhoid fever rapid antibody tests for laboratory

diagnosis at two sub-Saharan African sites

Karen H Keddy a, Arvinda Sooka a, Maupi E Letsoalo b, Greta Hoyland c, Claire Lise
Chaignat d, Anne B Morrissey e & John A Crump e
a. Enteric Diseases Reference Unit, National Institute for Communicable Diseases of the
National Health Laboratory Service, P/Bag X4 2131, Sandringham, South Africa.
b. Directorate of Research and Innovation, Tshwane University of Technology, Pretoria,
South Africa.
c. National Health Laboratory Service, Nelspruit, South Africa.
d. World Health Organization, Geneva, Switzerland.
e. Department of Medicine, Duke University Medical Center, Durham, United States of
America.
C. orrespondence to Karen H Keddy (e-mail: [email protected]).
(Submitted: 23 February 2011 Revised version received: 25 May 2011 Accepted: 26
May 2011 Published online: 13 June 2011.)
Bulletin of the World Health Organization 2011;89:640-647. doi: 10.2471/BLT.11.087627
Introduction
Typhoid fever remains an important cause of disease in developing countries. In 2002, it
caused an estimated 408 837 episodes of illness in Africa.1 SalmonellaTyphi, the
causative agent, is most frequently isolated from blood during the first week of illness but
can also be isolated during the second or third week of illness, during the first week of
antimicrobial therapy and during clinical relapse.2 Isolation ofSalmonella Typhi from
bone marrow is the current gold standard method for confirming a case of typhoid fever.
However, this requires equipment, supplies and trained laboratory personnel seldom found
in primary health-care facilities in the developing world.3,4 Blood culture is a more
practical albeit less sensitive alternative to bone marrow culture. However, it is not always
available and, when it is, it takes 2 to 3 days. As a result, diagnosis may be delayed or
overlooked and patients without typhoid fever may receive unnecessary and inappropriate
antimicrobial treatment. For this reason, in developing countries typhoid rapid antibody
tests can facilitate diagnosis and disease management.
New commercially available typhoid rapid antibody tests have been evaluated in Asia,
where typhoid fever is known to be highly endemic.1,5 In Asian studies the tests have
shown variable performance. While the TUBEX test was the most sensitive and specific
in the Philippines,6 neither TUBEX nor Typhidot was both sensitive and specific in two
evaluations undertaken in Viet Nam7,8 and performance was poor in a trial conducted in a
community clinic in Bangladesh9 and in a study in Egypt in which it was compared with a
new ELISA not yet commercially available.10 Rapid typhoid tests have not been
evaluated in sub-Saharan Africa, where the typhoid fever burden may be smaller than in
Asia.1113 The World Health Organization (WHO) has issued no recommendations on the
use of typhoid rapid antibody tests.14 Accurate diagnostics for typhoid fever could
provide valuable diagnostic information for patient management and make it possible to
estimate the incidence of typhoid fever in low-resource settings.
We evaluated three diagnostic kits that are commercially available internationally using
four rapid methods for detecting antibodies to Salmonella Typhi (typhoid rapid antibody
tests) and used blood culture as the standard for comparison.
Methods
Participants

Patients were recruited from two sub-Saharan African sites: Mpumalanga province, South
Africa, and Moshi, United Republic of Tanzania. They were selected to represent patients
from southern and eastern Africa, respectively.
The recruitment method differed between sites. In South Africa, we enrolled subjects
suspected of having typhoid fever; in the United Republic of Tanzania, we enrolled
patients who were participants in a study on the etiology of febrile illness.
Patients were recruited at both sites between 2007 and 2009. In the South African site we
obtained blood from suspected typhoid fever cases reporting no current use of
antimicrobials who presented to Rob Ferreira Hospital (RFH), in Nelspruit, Mpumalanga
province, or to hospitals referring patients to RFH. In the United Republic of Tanzania
site, we obtained blood from consecutive febrile inpatients admitted to Kilimanjaro
Christian Medical Centre (KCMC) and Mawenzi Regional Hospital.15,16 At both sites we
incubated the blood in a continuously monitored blood culture system (Bac-T Alert,
bioMrieux, Marcy Ltoile, France). Bottles flagged as positive by the instrument were
removed for subculture and identification by standard techniques.17
In both study sites, we enrolled patients who presented with a febrile illness suspected of
being typhoid fever. We collected data on those patients who fulfilled the clinical criteria
for suspected typhoid fever (a history of fever or demonstrated pyrexia [body temperature
> 38 C.]) before performing the index test and blood culture.
Test methods

In both study sites a typhoid fever case was defined as being a patient whose blood culture
was positive for Salmonella Typhi. Patients whose blood cultures were negative or yielded
pathogens other than Salmonella Typhi were used as controls. We drew additional blood
and separated the serum, which was stored at 20 or 80 C in cryotubes and shipped on
dry ice to the Enteric Diseases Reference Unit, National Institute of Communicable
Diseases (Sandringham, South Africa), for evaluation with typhoid rapid antibody tests.
We screened the serum using the semiquantitative slide agglutination and tube Widal tests,
TUBEX and the typhidot test. Laboratory staff were blinded to the blood culture results,
which were reviewed only after testing was completed.
Typhoid rapid antibody tests were carried out according to manufacturers instructions.
Test characteristics are summarized in Table 1. Laboratory personnel, trained in the use of
all tests, recorded information about the ease of use and non-kit consumables and
equipment required for each test. Because the cost of consumables, equipment and
personnel differed between the two study sites, we did not calculate the cost of the tests.
Table 1. Comparative characteristics of three rapid tests for the detection
ofSalmonella Typhi antibodies
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Linear Cromotest (Linear Chemicals, Barcelona, Spain)
This test, derived from Salmonella Typhi O and H antigens, was performed in two ways:
(i) as a semiquantitative slide agglutination test with visual examination as per the package
insert; (ii) as a Widal test18 performed with a single tube, as described by Parry et
al.19 The presence or absence of visible agglutination indicates the presence or absence of
the corresponding antibody to the O and H antigens ofSalmonella Typhi. We defined the
positivity cut-off point for the slide and tube agglutination reactions for both O and H
antigens as antibody titres 1:80.
IDL TUBEX TF (IDL Biotech AB, Bromma, Sweden)
This semiquantitative colorimetric test detects anti-O:9 antibody titres in patient
specimens on visual examination.20 A positive TUBEX result was defined as a reading
of 4, as per manufacturers instructions. The manufacturers warn that the test may have
to be repeated after 48 hours if indeterminate results are obtained.

Typhidot (Malaysian Biodiagnostic Research, Bangi, Malaysia)

This qualitative antibody detects the presence of IgM and IgG antibodies to a 50kDa outer
membrane protein.21 A positive Typhidot result (IgG and IgM) was defined as a visible
reaction of an intensity equal to or greater than that of the control reaction on the
commercially prepared filter paper. The manufacturers warn that if indeterminate results
are obtained, the test may have to be repeated after 48 hours.
Statistical methods
Data were captured into Excel 2003 (Microsoft Corporation, Redmond, United States of
America) and converted to STATA version 11 (StataCorp. LP, College Station, USA), in
which analysis was performed by Stat/Transfer version 10 (Circle Systems, Seattle, USA).
Statas diagt command was used to determine each tests sensitivity, specificity and
positive and negative predictive values (PPV and NPV, respectively),22 which are
presented with the understanding that exposure to antimicrobials could have affected the
final results. Analysis was performed at a two-sided significance level of 5%. Pretest
probabilities of background typhoid fever rates were also calculated at 5% and 50% to
ensure that the results were applicable even in conditions of few typhoid fever outbreaks
since incidence would be higher during outbreaks and of lower endemicity, given that
study patients were selected on the probability of having typhoid fever.
Research ethics
The NICD has blanket ethics clearance in relationship to its surveillance duties (M0604
49), but further approval was obtained from the Committee for Research on Human
Subjects (CRHS) at the University of the Witwatersrand to update CRHS on this aspect of
typhoid fever surveillance.
The part of the study conducted in the United Republic of Tanzania was approved by the
Kilimanjaro Christian Medical Centre (KCMC) Research Ethics Committee, the United
Republic of Tanzania National Institutes for Medical Research National Research Ethics
Coordinating Committee, and an institutional review board of Duke University Medical
Center.
Results
Participants
Ninety-two patients were enrolled: 53 (58%) in South Africa (between 25 May 2007 and
10 November 2009) and 39 (42%) in the United Republic of Tanzania (between 17
September 2007 and 25 August 2008). Participants had a median age of 24 years (range:
< 1 to 96). Twenty-five (27%) patients (23 South African and 2 Tanzanian) were under the
age of 15 years; the ages of two participants (2.2%) were unknown. Forty-two (46%)
patients were female; the sex of two (2%) was not available. Thirty-six (39%) blood
cultures grew a pathogen; 28 (78%) of these cultures grewSalmonella Typhi. Other
pathogens isolated included Salmonella Typhimurium,Streptococcus pneumoniae,
Staphylococcus aureus and Mycobacterium tuberculosis (one culture each)
and Cryptococcus neoformans (four cultures). Of the 92 blood cultures, 52 (57%) were
negative and 4 (4%) grew organisms considered to be contaminants.
In compliance with eligibility criteria, no South African patient was taking antimicrobials
at the time of the blood culture. Of the 20 Tanzanian patients whose blood cultures were
negative for Salmonella Typhi, 2 (10%) had received trimethroprimsulfamethoxazole and
8 (40%) had received quinine or sulfadoxine-pyrimethamine or had an unknown history of
antimicrobial exposure. Of 19 Tanzanian patients with blood cultures positive
for Salmonella Typhi, 17 (89%) had received antibacterials or antimalarials or had an
unknown history of antimicrobial exposure.
Test results

Blood cultures were done as soon as patients were admitted to hospital. Serological tests
were performed within the subsequent 6 months at the South African site and within
2 years at the Tanzanian site. Table 1 shows the characteristics of the three assays, the
equipment required to perform each test and the results of the technologists assessments
regarding ease of use and perceived laboratory costs. None of the sera gave indeterminate
results.
Estimates

Sensitivity, specificity and predictive values are shown in Table 2. Of 28 patients with a
blood culture positive for Salmonella Typhi, 1 (4%) was positive on the
Cromotest semiquantitative slide O test; 14 (50%) were positive on the
Cromotestsemiquantitative slide H test; 2 (7%) were positive on the Cromotest Widal O
agglutination test; 4 (14%) were positive on the Cromotest Widal H agglutination test;
and 19 (68%) were positive on the TUBEX test. Of 27 patients with a blood culture
positive for Salmonella Typhi with sufficient serum available for testing, 19 (70%) were
positive on the Typhidot IgG test and 17 (63%) on the Typhidot IgM test. The positive
and negative predictive values for each of the pretest probability calculations are presented
in Table 3.
Table 2. Sensitivity, specificity and predictive values of four rapid diagnostic tests
for typhoid fever as determined by comparison with blood culture results
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Table 3. Predictive values of four rapid diagnostic tests for typhoid fever as
determined by comparison with blood culture results under assumed pretest probabilities
of 5% and 50%
html, 5kb
Discussion
All four typhoid rapid antibody tests performed poorly compared with blood culture.
Some tests performed better than others, but none stood out in all respects. In sub-Saharan
Africa, cost and ease of use are important considerations in addition to diagnostic
accuracy.
The single-tube Widal and Typhidot tests were found to require the most non-kit
laboratory supplies, consumables and equipment, and this increased the overall cost of the
test. The semiquantitative slide agglutination and TUBEX tests had shorter turnaround
times than the Widal tube and Typhidot tests. However, the results of all the tests were
available the same day the specimen was received in the laboratory.
Of the four tests evaluated, the semiquantitative slide agglutination test performed the
worst. It had very poor specificity and low PPV and NPV, even though it was performed
under optimal conditions in a national reference laboratory. This poor performance was
further compounded by substantial inter-test variability, which suggests that in a field
situation results would not be comparable between sites.23Hence, the slide agglutination
test should not be used as a diagnostic tool. Although the sensitivity and specificity of the
H slide agglutination test appeared to be greater, this was offset by the inconsistent results
obtained with the O slide agglutination. Others have noted this disparity between the
sensitivity and specificity of the Widal test containing O and H antigens.19
The single-tube Widal agglutination test also performed poorly. The original Widal
agglutination test was described using paired sera obtained 10 days to 2 weeks apart and
examined for a twofold or greater change in titre.18 It is possible that the Widal test would
have performed better in our study had we used paired sera, but we chose to apply the test
under the conditions normally found in clinical practice. In our experience, patients rarely
return for outpatient follow-up once treated, so that obtaining paired sera in a routine

clinical setting is unlikely. Recently, the use of paired sera has been re-examined and has
been shown to improve both the sensitivity and specificity of serological tests for typhoid
fever.24
Both the TUBEX and Typhidot tests had lower sensitivity than the semiquantitative
slide agglutination and the Widal tests, but they had considerably greater specificity. In our
setting, TUBEX had marginally less sensitivity but more specificity than the
Typhidot IgM test and it had a slightly better PPV. TyphidotIgG was comparable to
TUBEX with respect to sensitivity, specificity and PPV, but none of these tests performed
as well as the blood culture comparator assay.
This study had several limitations. Typhoid fever was confirmed by blood culture in
almost one third of the study participants, a much larger proportion than expected under
field conditions in sub-Saharan Africa, where typhoid fever is relatively
uncommon.12,13 Lowering the pretest probability for typhoid fever to 5% further
degraded the performance characteristics of the typhoid rapid antibody tests (Table 3),
which suggests that these tests would not be useful in routine diagnostic situations. At a
pretest probability of 50%, higher than the actual fraction of blood-culture-positive cases
used in this evaluation, the performance of the new rapid antibody tests improved. Hence,
these tests can perhaps be judiciously used during outbreaks.
The time elapsed between the onset of symptoms and serum collection can affect the
performance of antibody-based tests.25 We did not analyse this aspect to reflect how the
tests would be used under routine health-care conditions in sub-Saharan Africa. Similarly,
human immunodeficiency virus (HIV) infection is highly prevalent in sub-Saharan
Africa26 and we enrolled participants without reference to their HIV serostatus to reflect
field conditions, although many of our patients could have been HIV-infected. HIV
infection rates among 1504 adult outpatients tested in Nelspruit (RFH) were reportedly as
high as 45% in 2010 (G Hoyland, personal communication). The prevalence of HIV
infection among participants in the study on febrile illness at KCMC and Mawenzi
Regional Hospital was 39% for adolescents and adults and 12% for infants and
children.15,16 Although recent studies at KCMC have shown that HIV appears to protect
against typhoid fever, disease may still occur in HIV-infected individuals.15 It is possible
that HIV-associated immune dysregulation affects the production of antibodies specific
to Salmonella Typhi outer membrane proteins, present in both the Typhidot and the older
Widal tests. This has been observed in patients infected with invasive nontyphoidal Salmonella (NTS).27 This theoretical effect can also impair antibody binding in
the TUBEX test, which is based on the O9 antigen. The production of antibodies
against Salmonella lipopolysaccharide (LPS) is increased in patients with invasive NTS
infection who are also HIV-infected. If antibody production were also higher in HIV+
typhoid fever patients, TUBEXwould have performed better than the other typhoid rapid
antibody tests, but it did not.
The sensitivity of blood culture is known to be less than 100%, even in the absence of
antimicrobial exposure, and is further reduced by patient antimicrobial use. Although two
Tanzanian patients had been exposed to antibacterials, they represented only 3.8% of the
52 patients whose blood cultures were negative forSalmonella Typhi. These patients
probably affected our results very little. Furthermore, blood culture sensitivity was
optimized in our study because we used modern blood culture techniques.28
Our findings on the Widal test and the newer typhoid rapid antibody tests are similar to
those from studies conducted in Asia and Egypt;810,25,29,30 none of the rapid tests
performed nearly as well as blood culture for the diagnosis of typhoid fever. Some reports
suggest that the Typhidot test may be more useful in Asia.31,32However, the true
incidence of typhoid fever in the catchment population differed in these studies and in

ours. The pretest probability of typhoid fever was artificially elevated in our evaluation
because we included South African patients suspected of having typhoid fever and
specifically analysed a subset of antisera from the United Republic of Tanzania in which
half of the cases were known to have typhoid fever. The earlier studies also focused on
paediatric populations and allowed for inclusion of microbiologically unconfirmed
typhoid fever.31,32
NTS bacteraemia, which is predominantly caused by Salmonella serotypes Typhimurium
and Enteritidis, is much more common in sub-Saharan Africa than typhoid fever.12,13 An
important limitation of our study is the absence of cases of NTS in the control group; one
patient in our study had Salmonella Typhimurium bacteraemia and none
had Salmonella Enteritidis bacteraemia. It has been observed in previous studies that
bacteraemia due to Salmonella Enteritidis may result in false-positive results with
TUBEX because they have an O9 antigen in common.33Although the patient
with Salmonella Typhimurium had negative typhoid rapid antibody tests, we could not
examine the rate of false positives for the TUBEX test in patients
with Salmonella Enteritidis bacteraemia.
In conclusion, typhoid rapid antibody tests appear to correlate poorly with blood culture
results in sub-Saharan Africa, even in a study with inflated pretest probability. While such
tests may be useful for rapidly diagnosing typhoid fever in emergencies e.g. during
outbreaks, when pretest probability would be high, and following blood culture
confirmation of initial cases their performance is unlikely to justify deployment in
routine care settings in sub-Saharan Africa. TUBEX and Typhidotappeared to have
comparable performance and were more specific although less sensitive than the
semiquantitative slide agglutination test and the unpaired Widal test. Unpaired Widal and
semiquantitative slide agglutination are unreliable, with poor specificity and PPV. It is
important to remember that antimicrobial susceptibility testing and molecular
epidemiological linkage cannot be elicited on serological diagnosis. Blood culture before
initiating antimicrobial therapy remains the diagnostic method of choice.
Acknowledgements
We thank EDRU laboratory staff members: Mimmy Ngomane, Florah Mnyameni,
Innocent Mtambo and Mzikazi Dickmolo for their assistance. KHK has a dual
appointment with Faculty of Health Sciences, University of the Witwatersrand,
Johannesburg, South Africa. JAC has dual appointments with Duke Global Health
Institute, Duke University, Durham, United States of America; Kilimanjaro Christian
Medical Centre, Moshi, United Republic of Tanzania and Kilimanjaro Christian Medical
College, Tumaini University, Moshi, United Republic of Tanzania.
Funding:
This work was supported in part by IDL Biotech AB, Bromma, Sweden (TUBEX) and by
Malaysian Biodiagnostic Research, Bangi, Malaysia (Typhidot), who supplied kits for
evaluation and training in the use of these kits. Additional work was funded by WHO,
Geneva, Switzerland. Research done in the United Republic of Tanzania was supported by
an International Studies on AIDS Associated Co-infections (ISAAC) award, a United
States National Institutes of Health (NIH) funded program (U01 AI062563). Authors
received support from NIH awards ISAAC (ABM, JAC); AIDS International Training and
Research Program D43 PA-03018 (JAC); the Duke Clinical Trials Unit and Clinical
Research Sites U01 AI069484 (JAC), and the Center for HIV/AIDS Vaccine Immunology
U01 AI067854 (JAC).
Competing interests:

None declared.
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