Vaccine 33 (2015) C8–C15

Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Review

Diagnostics for invasive Salmonella infections: Current challenges and

future directions
Jason R. Andrews a,∗ , Edward T. Ryan b
a
Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, United States
b
Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, Boston, MA 02114, United States

a r t i c l e i n f o a b s t r a c t

Article history: Invasive Salmonellosis caused by Salmonella enterica serotype Typhi or Paratyphi A, B, C, or invasive
Available online 30 April 2015 non-typhoidal Salmonella serotypes, is an immensely important disease cluster for which reliable, rapid
diagnostic tests are not available. Blood culture remains the gold standard but is insensitive, slow, and
Keywords: resource-intensive. Existing molecular diagnostics have poor sensitivity due to the low organism burden
Salmonella in bodily fluids. Commercially available serologic tests for typhoidal Salmonella have had limited sensitiv-
Invasive
ity and specificity. In high burden, resource-limited settings, reliance on clinical diagnosis or inaccurate
Enteric fever
tests often results in frequent, unnecessary treatment, which contributes selective pressure for the emer-
Typhoid
Paratyphoid
gence of antimicrobial resistance. This practice also results in inadequate therapy for other etiologies of
Non-typhoidal acute febrile illnesses, including leptospirosis and rickettsial infections. A number of novel serologic,
Diagnostics molecular, transcriptomic and metabolomic approaches to diagnostics are under development. Target
product profiles that outline specific needs may focus development and investment, and establish bench-
marks for accuracy, cost, speed, and portability of new diagnostics. Of note, a critical barrier to diagnostic
assay rollout will be the low cost and low perceived harm of empiric therapy on behalf of providers and
patients, which leaves few perceived incentives to utilize diagnostics. Approaches that align incentives
with societal goals of limiting inappropriate antimicrobial use, such as subsidizing diagnostics, may be
essential for stimulating development and uptake of such assays in resource-limited settings. New diag-
nostics for invasive Salmonellosis should be developed and deployed alongside diagnostics for alternative
etiologies of acute febrile illnesses to improve targeted use of antibiotics.
© 2015 Published by Elsevier Ltd.

1. Introduction of disability from an infectious disease in the developing world

Invasive Salmonellosis is caused by Salmonella enterica serotype
Typhi (Salmonella. Typhi) or Paratyphi A and B (Salmonella. Paraty-
phi), the causes of enteric fever, Salmonella Paratyphi C, which
causes septicemia and metastatic purulent infections, or invasive
non-typhoidal Salmonella (iNTS) serotypes, including Salmonella.
Enteriditis and Salmonella. Typhimurium. Invasive non-typhoidal
Salmonellosis has its highest burden among immunocompromised
or malnourished individuals, especially children infected with
HIV in resource-limited areas, among whom case fatality is high
[1]. Similarly, enteric fever remains among the leading causes
∗ Corresponding author at: Division of Infectious Diseases and Geographic
Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Lane
Building, L141, Stanford, CA 94305-5107, United States. Tel.: +1 650 497 2679;
fax: +1 650 723 3474.
E-mail addresses: jandr@stanford.edu (J.R. Andrews), etryan@mgh.harvard.edu
(E.T. Ryan).
[2]. Recent estimates of typhoidal Salmonella incidence have
varied substantially [3–6], and iNTS estimates are sparse [7–9], in
large part due to poor access to reliable diagnostics, particularly
in low-resource outpatient settings where patients with these
illnesses typically present for medical care. Measured by its burden
and influence on antibiotic use, invasive Salmonellosis is perhaps
the most important infectious disease cluster for which rapid and
reliable (>90% sensitivity and specificity) diagnostics do not exist.
This diagnostic gap leads to under-diagnosis as well as inaccu-
rate, over-diagnosis of enteric fever especially, the latter of which
may lead to inappropriate and excessive antibiotic use. This results
in selective pressure for the emergence of resistant bacteria, at a
time in which highly resistant Gram-negative infections, including
Salmonella [10–13], threaten to undermine reductions in case fatal-
ity rates for bacterial infections [14]. Additionally, inappropriate
targeting of antibiotics for Salmonellosis results in inadequate ther-
apy for other treatable infections, such as leptospirosis, rickettsia,
and brucellosis. It also poses a challenge to the targeted rollout

http://dx.doi.org/10.1016/j.vaccine.2015.02.030
0264-410X/© 2015 Published by Elsevier Ltd.
 J.R. Andrews, E.T. Ryan / Vaccine 33 (2015) C8–C15
Table 1
Characteristics of currently available diagnostics for invasive Salmonellosis.
Diagnostic class Sensitivity
Blood culture Low
Bone marrow cultures High
Rapid serology (Widal, Tubex, Typhidot, RTI) Low–moderate
Antigen Moderate
Polymerase chain reaction (PCR) Variable
and evaluation of more effective, conjugated enteric fever vaccines,
which are on the horizon [15,16]. A recent review (2011) of diag-
nostics for enteric fever provided a detailed summary of the state
of existing diagnostics, with an emphasis on serologic assays and
nucleic acid amplification-based tests [17]. Here, we briefly review
the literature on currently available diagnostic approaches for both
enteric fever and iNTS, and then provide an overview of diag-
nostic strategies under development, desirable test characteristics
according to their utilization goal, and the development and imple-
mentation challenges for scale-up of new Salmonella diagnostics.
2. Available diagnostic approaches for enteric fever
Essentially all enteric fever diagnosis begins with evaluation of
clinical signs and symptoms. For perhaps the majority of patients
with suspected enteric fever worldwide, who live in settings where
diagnostic microbiology is unavailable [18], this is also the end
of the diagnostic algorithm, and a decision concerning empiric
treatment is made at this juncture. Unfortunately, clinical diag-
nosis of typhoid is not reliable, as it is difficult to distinguish
typhoid from other co-endemic acute febrile illnesses including
influenza, dengue, leptospirosis, malaria, brucellosis, rickettsial
infections, and other systemic infections. Fever and headache occur
in the majority of patients, and a myriad of non-specific symp-
toms include abdominal pain, myalgias, chills, cough, sore throat,
anorexia and nausea [19–25]. Diarrhea and constipation are both
regularly reported in case series. Hepatomegaly, splenomegaly, and
cervical lymphadenopathy are present in a minority of patients.
Faget’s sign (relative bradycardia in the presence of fever) occurs
in less than half of patients and is not specific for enteric fever.
Rose spots—a salmon-colored maculopapular eruption typically on
the trunk—are seen in less than 30% of cases in most series [21],
and are similarly not pathognomonic [26]. Laboratory abnormal-
ities are also non-specific. Most patients have normal leukocyte
counts, though leukopenia is present in a minority. Mild increases
in hepatic transaminases, creatine kinase and lactic acid dehydro-
genase have been reported but are also common to other infections
in the differential diagnosis [19,21]. While Salmonella Paratyphi A
has been considered to cause a more mild illness than typhoid, a
recent large study from a co-endemic setting found these infections
to be clinically indistinguishable [27]. Studies aiming to develop
and validate prediction rules for enteric fever from clinical features
and laboratory results have had very limited success [28–31].
After clinical diagnosis, the two most common diagnostic proce-
dures for typhoid in use today were developed in the 19th century:
bacterial culture and the Widal test. Since Eberth’s discovery of the
etiological agent of enteric fever in 1880 [32], culture has been
the gold standard of enteric fever diagnosis. Whereas early cul-
ture techniques had low yield, Coleman and Buxton developed
an improved approach by using larger quantities of blood (10 ml)
and broth, and adding Ox-bile which lyses blood cells and inhibits
antibacterial activity [33]. This approach, while effective, prevents
isolation of many other important bacteria. Tryptone soy broths are
among the most commonly used blood culture media today, with
automated systems used in settings with sufficient resources. The
majority of positive cultures are evident within 48 h, and nearly all
C9
Specificity Time to result Laboratory requirements Comments
Excellent 1–5 days Moderate Provides antibiotic susceptibility
Excellent 1–3 days Moderate Invasive; requires trained personnel
Moderate <1 hour Low Only point-of-care tests available
Variable 1–3 hours Moderate Limited evidence on accuracy
Excellent 1–3 hours High Variable sens. among culture-negative
are positive by five days (Table 1). Subculture, biochemical testing,
and agglutination with specific antisera are typically performed to
identify Salmonella serovars.
The sensitivity of culture varies substantially according to the
specific fluid and volume assayed, age of the affected individual,
prior antimicrobial use, and stage of the illness. Bone marrow
cultures have the highest sensitivity (>80%) and are relatively unaf-
fected by antibiotics [34,35]; however, this diagnostic procedure
is not commonly performed in clinical settings where typhoid is
endemic due to its invasive nature and the need for training and
specialized, sterile equipment. The sensitivity of blood culture has
been variably reported at 40–80%, with higher sensitivity in the
first week of illness, when the bacterial concentration in blood is
an order of magnitude higher than in subsequent weeks [34–36].
Unlike with bone marrow cultures, antibiotics substantially dimin-
ish the yield of blood cultures. Stool cultures and rectal swabs have
lower sensitivity (<40%), though they can be enhanced by culturing
three specimens or performing multiple cultures from a single stool
specimen [37]. Duodenal string sampling and culture may provide
a higher yield than stool or rectal swabs [38,39], but with all gas-
trointestinal site sampling, it should be recognized that positivity
may reflect chronic carriage rather than invasive illness. Urine cul-
tures have a low yield and are unlikely to provide an incremental
benefit to diagnostic yield over blood culture. When present, rose
spots can be cultured and provide some enhancement to diagnostic
yield, particularly later in the course of illness [34].
The Widal agglutination test, in which killed Salmonella Typhi
and Paratyphi A antigen is reacted with serum to measure aggluti-
nating antibodies to the flagellar (H) and lipopolysaccharide (O)
antigens, was developed in the 1890s [40], modified and stan-
dardized in the 1950s [41], and today remains in widespread use
throughout typhoid-endemic settings. The simplicity and rapidity
of the test enables its use in settings with minimal laboratory infra-
structure, but misuse and misinterpretation of the results remains
a critical problem. A single agglutination test has limited sensitiv-
ity and specificity, particularly early in the course of illness and in
endemic settings [42,43]; comparison of acute and convalescent
titers improves test accuracy but has limited utility in guiding clin-
ical practice [44,45]. A number of ELISAs have been evaluated for
typhoid diagnosis by targeting antibodies to the O, H and virulence
polysaccharide (Vi) antigens; however, for diagnosis in the acute
phase, these tests suffer from the same limitations as the Widal
test [44,46,47].
Several serologic tests have been developed for point-of-
care diagnosis of enteric fever. The two that have been most
widely studied are TUBEX TF (IDL Biotech, Sweden) and Typhidot
(Malaysian Biodiagnostic Research, Malaysia). TUBEX TF assays for
antibodies to Salmonella Typhi LPS (O9) by quantifying inhibition of
binding between O9 monoclonal antibodies and LPS-coupled mag-
netic particles [48]. Typhidot is a miniaturized dot-blot ELISA that
detects IgM and IgG antibodies to a 50 kDa Salmonella Typhi outer
membrane protein (OMP). Typhidot-M uses the same approach to
detect IgM to OMP after removal of total serum IgG, to improve
specificity for recent infection [49]. A recent systematic review
and meta-analysis of TUBEX TF and Typhidot found sensitivity of
56–95% and 56–84%, respectively, with specificity of 72–95% and
31–97% [50]. Several assays based on antigen detection (O9, Vi,
C10 J.R. Andrews, E.T. Ryan / Vaccine 33 (2015) C8–C15
Table 2
Novel diagnostic approaches for enteric fever and invasive non-typhoidal Salmonellosis.
Class Examples
Simplified culture PortaTherm
PCR enhancement Enrichment, selective human DNA removal
Isothermal nucleic acid LAMP
amplification
DNA capture probes Microwave-accelerated metal-enhanced
fluorescence (MAMEF)
IgA assays TPTest
Transcriptomic cDNA microarrays
Metabolomic Gas chromatography with time-of-flight mass
spectrometry
and Hd) in urine or plasma have been evaluated and have had
moderate sensitivity and specificity [51–56]; despite adaptation
into dot blot and immunochromatographic assays [57,58], none
appear to have gained widespread use. While point-of-care sero-
logic tests have the advantage of rapid time-to-result and minimal
laboratory infrastructure requirements, their modest accuracy and
moderate costs have limited their use in routine clinical settings
in high-burden countries.
A number of studies have assessed the yield of nucleic acid
amplification tests for enteric fever diagnosis; flagellin genes (fliC-
d for Salmonella Typhi, fliC-a for Salmonella Paratyphi A) have been
most commonly targeted, but an array of alternative PCR gene tar-
gets have been assessed [59–63]. Parry et al. reviewed published
literature on PCR assays for enteric fever performed on blood, and
found excellent specificity (100%) but variable sensitivity [17]. In
general, the sensitivity is very good (most studies >90%) among
blood culture-positive patients but lower in blood culture-negative
patients (3–13% in the two largest studies) [62,63]. PCR assays tar-
geting fliC have also been applied to urine, showing promising
results, though further validation of accuracy is needed [60,64].
Thus, in comparison with culture and conventional identification
methods, the primary gain of PCR is rapidity of result rather than
improved sensitivity. The downsides of PCR are that it requires
fairly sophisticated laboratory infrastructure, which is limited in
settings where typhoid is endemic, and current configurations do
not provide information on antimicrobial resistance, for which a
diversity of genes are responsible.
3. Available diagnostic approaches for iNTS
As with enteric fever, most treatment for iNTS is based on clini-
cal presentation alone given the dearth of diagnostic infrastructure
in settings where iNTS is common. However, iNTS causes a similarly
non-specific febrile syndrome. Splenomegaly and hepatomegaly
may be suggestive of iNTS in children [65,66], but are also com-
monly seen with malaria and other infections that are co-endemic
in Africa, where most iNTS cases arise. Respiratory symptoms and
signs (cough, tachypnea) are not uncommon, and pneumonia is fre-
quently present [67,68]. Diarrhea is present in a minority of iNTS
infections among children [65,69]. In a large prospective study
in Tanzania, Nadjm and colleagues found that application of the
WHO criteria to target antimicrobial therapy failed to identify one-
third of invasive bacterial infections, among which non-typhoidal
Salmonella were the leading cause [68].
For culture-based and molecular diagnostics, similar chal-
lenges present with typhoidal Salmonella—low bacterial burden
(1 CFU/ml) in blood—may confound sensitive diagnosis of iNTS
[70]. PCR assays have been clinically validated for diagnosis of
enterocolitis [71], or for use on cultured isolates [72], but not for
direct diagnosis of invasive infections.
Potential advantages Refs
Minimal laboratory requirements; ability to [75]
confirm at central laboratory
Rapidity, specificity [76,77]
Rapidity, minimal laboratory requirements [81,82]
Rapidity, sensitivity, specificity [83]
Sensitivity, specificity, minimal laboratory [85]
requirements
Broad spectrum pathogen diagnosis, potential [91]
prognostic information
Broad spectrum pathogen diagnosis, potential [93]
prognostic information
In contrast with typhoidal Salmonella, there is more limited
experience with serologic assays for iNTS. Despite similarity
between the O antigens of Salmonella Typhi and Salmonella Enter-
itidis, the Widal agglutination test performs poorly [73]. Kuhn et
al. recently reviewed ELISAs for non-typhoidal Salmonella infec-
tions, which were based on Salmonella Enteritidis and Salmonella
Typhimurium LPS, and found most sensitivities in the 70–95%
range, with specificities >90%, though most studies were small and
many did not report specificity [74]. The higher specificity, in com-
parison with ELISAs for enteric fever, may reflect their evaluation in
low endemic settings. At present, there is a lack of standardization
of ELISA assays for non-typhoidal salmonelloses. A challenge with
developing serologic tests for iNTS is the diversity of non-typhoidal
serovars globally, which will require empirically informed, and per-
haps locally targeted, selection of LPS antigens to achieve adequate
sensitivity. Additionally, it will be difficult for serologic diagnostics
using current technologies to distinguish between the two syn-
dromes iNTS can cause: self-limiting gastroenteritis and invasive
systemic infection.
4. Novel diagnostic approaches for enteric fever and iNTS
All of the currently available diagnostic approaches for invasive
Salmonellosis have limitations in terms of speed, sensitivity, infra-
structure requirements, and suitability to point-of-care application.
To date, a rapid, accurate, point-of-care diagnostic has remained
elusive. However, a number of promising approaches are under
development, which may address many of the limitations of the
existing diagnostics. Here, we briefly review some of these tech-
nologies (Table 2).
Microbiologic culture has excellent specificity; however, draw-
backs include lower sensitivity, 24–72 hour incubation need,
and—critical for resource-limited settings—requirement for lab-
oratory capacity. To mitigate at least the need for laboratory
infrastructure at the point-of-care, novel approaches have been
explored, including the development of an electricity-free incu-
bation system and colorimetric readout that permits sensitive
culturing of blood in areas with extremely limited resources [75].
Similarly, much of the non-culture based work under devel-
opment has focused on addressing the limitations of molecular
diagnostics in terms of sensitivity, speed, and instrumentation
requirements. Zhou and Pollard demonstrated increases to the
sensitivity of PCR using a three-hour enrichment step in culture
media [76], and separately using selective removal of human DNA
from blood using ox-bile for cell lysis and enzymatic digestion [77].
Several isothermal nucleic acid amplification procedures have
been developed and commercialized in recent years, eliminating
the need for thermocycling instruments [78–80]. Among these,
loop-mediated isothermal amplification (LAMP) has received the
most attention. LAMP assays involve amplification at 60 ◦ C with
 J.R. Andrews, E.T. Ryan / Vaccine 33 (2015) C8–C15
interpretation through a visual or fluorometric readout, and have
the advantage over PCR of less inhibition by biological fluids, which
reduces the sample preparation requirements. LAMP assays for
Salmonella Typhi have been developed and studied using simulated
specimens and a human challenge model [81,82]. However, there
remains a need for minimally instrumented, low-cost approaches
to DNA extraction and concentration in order for molecular
diagnostics to be widely adoptable in resource-limited settings.
Novel amplification-free molecular methods may provide even
more rapid diagnosis of Salmonella. Tennant and colleagues have
demonstrated detection of 1 CFU/ml of non-typhoidal Salmonella in
under 30 s using Microwave-Accelerated Metal-Enhanced Fluores-
cence (“MAMEF”) [83]. Evaluation of this technology using clinical
specimens, and toward typhoidal Salmonella, is underway.
Newer generation serologic approaches may yield more accu-
rate immunodiagnosis. One approach involves detection of IgA
antibodies produced by activated lymphocytes that migrate in the
peripheral blood after infection [84]. This assay, referred to as
TPTest (typhoid and paratyphoid fever test), had 100% sensitiv-
ity and 78–97% specificity in an endemic setting, and has been
adapted into an immunodot format for use in settings without
ELISA capability [85]. The procedure does require a centrifuge and
incubator, and the chief limitation is the 24–48 h incubation period,
which precludes use as a rapid point-of-care diagnostic. Separately,
immunoscreening techniques have led to the identification of a
novel protein (YncE), the IgG response to which appears to be spe-
cific for asymptomatic carriage, and could lead to the development
of improved screening for chronic carriers of Salmonella Typhi [86].
One area of growing interest in diagnostic development has
been transcriptional profiling of host mRNA in peripheral blood
cells, which has been applied in diagnosis of influenza [87], tuber-
culosis [88], pertussis [89], and other infectious diseases [90].
Thompson and colleagues identified distinctive gene expression
signatures in patients during acute disease, convalescence and
recovery from typhoid [91]. While their study did not specifi-
cally evaluate these signatures from a diagnostic perspective, this
transcriptomic approach could potentially be adapted for diag-
nostic purposes. Metabolomic approaches are among the newest
class of diagnostic approaches in infectious diseases, and utilize a
pathogen non-specific screening of metabolite signals in biologi-
cal samples to identify infectious agents. Exploratory studies have
examined specific metabolites to differentiate bacterial infections,
including Staphylococcus aureus and Escherichia coli [92]. Näsström
and colleagues used gas chromatography with time-of-flight mass
spectrometry on plasma samples and found that a combination
of six metabolites could accurately distinguish Salmonella Typhi,
Salmonella Paratyphi A and control patients [93]. While the cost,
equipment, and analytic requirements of these approaches are cur-
rently far too high for their use in routine diagnosis, particularly in
resource-limited settings, it is conceivable that insights generated
from this work, combined with decreasing costs of analytic tools,
may lead to novel classes of tests for enteric fever that convey both
diagnostic and prognostic information.
5. Target product profiles for enteric fever and iNTS
diagnostics
There is an increasing recognition that diagnostic develop-
ment should be guided by specific needs for their utilization—for
example, sensitive, rapid screening tests for use in clinics versus
comprehensive resistance assays to be implemented in reference
laboratories. Such detailed “target product profiles” (TPPs) have
been developed for a number of tropical infectious diseases,
including malaria [94], tuberculosis [95], and helminthiases [96].
TPPs are typically constructed by convening experts from the
C11
scientific community, technical agencies, industry, implementa-
tion agencies, clinicians, and national programs to identify and
then prioritize specific diagnostic gaps. For enteric fever and iNTS,
a set of TPPs is not currently available to our knowledge. While
we do not attempt to propose a comprehensive list of needs nor
would we prioritize those needs in the absence of input of relevant
stakeholders, we here highlight what we perceive as several key
focus areas for enteric fever diagnostics.
Perhaps the most obvious acute need is for a rapid diagnostic
test with improved accuracy over existing rapid tests. A test with
greater sensitivity would give clinicians confidence in excluding
Salmonellosis as a diagnosis, and since Salmonella is likely present
in a minority of acute febrile illnesses, this would likely have the
greatest impact on treatment, as long as specificity remains rea-
sonably good. Such a test could also be used to focus limited
resources for blood culture, which is more costly, in settings where
Salmonella is the most common recoverable organism in blood
cultures. Because most treatment is provided in outpatient sett-
ings, including medical shops, the ideal rapid diagnostic would
not require a formal laboratory, sophisticated ancillary instru-
mentation, extensive manual procedures, or advanced laboratory
training. The result readout should be easily interpretable by
non-laboratory personnel. Containing costs will be essential to
maximize use in high-burden settings.
For inpatient settings, where patients with more severe febrile
illnesses are likely to receive empiric therapy to cover an array of
potential bacterial pathogens, speed and cost may be less important
than accuracy and compatibility with drug susceptibility testing.
Patients who are hospitalized with suspected typhoid are more
likely to have already received empiric oral therapy as outpatients,
and assessing for drug resistance is therefore more important in
guiding therapy. The current standard against which this diagnostic
would be measured is blood culture, which has limited sensitiv-
ity, particularly for patients with recent antibiotic exposure, and
is not available in many hospitals in high burden settings due to
laboratory infrastructure and personnel requirements.
Screening for chronic carriage is not commonly performed in
endemic settings, but may be valuable in reducing transmission,
particularly in transitional countries that are moving toward elim-
ination of endemic enteric fever [97]. Because this is likely to
be pursued in more advanced economies where sanitation has
reduced transmission, and the urgency of providing antibiotics in
carriage is less, cost and rapidity of diagnostics for carriage may be
less critical compared with diagnostics for acute infection.
Finally, there have been few examples of sustained surveillance
for Salmonella infections in resource-limited settings, particularly
in clinics or medical shops where most patients initially present
with fever. As conjugate vaccines become available, reliable surveil-
lance methods will become increasingly critical for guiding vaccine
deployment and ongoing assessment of vaccine impact. Diagnostic
speed may be sacrificed but accuracy will be critical. Blood culture,
which has excellent specificity, is currently filling this niche, but is
unavailable in settings without microbiology laboratories, limiting
the breadth of surveillance.
Convening experts and relevant stakeholders to refine and pri-
oritize enteric disease diagnostic needs will be critical to focusing
limited resources on those tests that will have the appropriate com-
binations of accuracy, speed, cost, and infrastructure requirements
to enable them to be adopted broadly for patient care or disease
surveillance.
6. Challenges for development and rollout of enteric fever
and iNTS diagnostics
Among the central challenges to establishing a robust diag-
nostics pipeline for Salmonellosis is stimulating development
C12 J.R. Andrews, E.T. Ryan / Vaccine 33 (2015) C8–C15
and commercialization in the absence of a clear, profitable
market for diagnostics. The overwhelming majority of enteric
fever and iNTS infections occur among the poor in low- and
middle-income countries, whose limited purchasing power is typ-
ically directed toward empiric antibiotics. Purely market-based
approaches driven by private industry have failed to generate
new diagnostics for many tropical infectious diseases. Nonprofit
product development partnerships (PDPs), which collaborate with
academic, industry, and governmental partners to accelerate diag-
nostic development and scale up, have emerged as a successful
alternative model. For diseases of poverty, the Foundation for Inno-
vative New Diagnostics (FIND) is the largest and most successful
PDP, having launched important diagnostic tests for tuberculosis,
malaria, and human African trypanosomiasis. FIND has identi-
fied non-malaria acute febrile illnesses as an important focus for
diagnostic development and has initiated early-phase activities
including disease mapping and target product prioritization, but
has not reported specific diagnostics under development for enteric
fever.
Upon development of candidate diagnostics, there are several
challenges in conducting high-quality, clinical validation studies.
First is the absence of an accurate, non-invasive reference standard.
Blood culture is the most commonly used reference standard in
Salmonella studies given its excellent specificity, but as previ-
ously noted has limited and variable sensitivity. Many studies
report sensitivity among blood culture-positive cases and speci-
ficity in healthy controls or individuals with confirmed alternative
diagnoses; this approach, while a reasonable compromise, is sub-
optimal when the goal is to know the overall sensitivity (including
culture-negative cases) and unbiased specificity. Paired serologic
tests from the acute and convalescent period may improve diagnos-
tic accuracy [44], but it can be challenging to obtain convalescent
samples consistently in resource-limited settings. Use of multiple
reference diagnostics, along with reliable diagnostics for alterna-
tive infections, combined with latent class analysis (which accounts
for imperfect reference standards), can enable more accurate esti-
mation of sensitivity and specificity and should be encouraged for
studies of novel diagnostics [98].
Once successful next generation diagnostics for enteric fever
and iNTS are validated and brought to market, there will be critical
questions about how to spur their rollout in high-burden, resource-
limited settings. Chief among these is how to incentivize the use of
a diagnostic when the alternative—empiric treatment—is low cost
and has limited “apparent” harms (e.g. adverse effects, toxicities)
at the individual level. For example, point-of-care serologic tests
such as Tubex and Typhidot are estimated to cost $2–4 in kit costs
alone, not accounting for labor costs and other laboratory consu-
mables [43,99], while a 7-day course of the most commonly used
treatments (Ciprofloxacin/Ofloxacin, Cefixime, Azithromycin) fre-
quently retails for $1.00–$3.00 in South Asia. Similar challenges
have been found with other disease-specific diagnostics, such as
leptospirosis, for which empirical therapy has been found to be
more cost-effective than diagnostic-guided treatment across a
wide range of assumptions [100].
The patient-level harm of empiric treatment for Salmonella
infections that receives less consideration is the failure to consider
and treat the alternative diagnosis. In Nepal, for example, more
than 500,000 cases of typhoid are reported in the public sector
each year, nearly all empiric diagnoses [101]. This contrasts to 6
cases of leptospirosis and 1 case of rickettsial infection for the most
recent year available [101]. Surveillance studies, however, suggest
that these alternative diagnoses—which require different antibiotic
therapy—are combined at least as common as enteric fever [102].
Untreated morbidity and mortality from these other infections may
be considerable [103,104]. Additionally, the societal costs of indis-
criminate antibiotic use may be substantial, as bacterial resistance
rises and microbiota change amid selective pressure of antimicro-
bials [105].
The fundamental challenge is one of aligning incentives for the
use of diagnostics by providers and patients with those of soci-
etal goals of improving health by appropriate therapy for infectious
diseases and limiting unnecessary antibiotic use to contain drug
resistance. An analogous, informative situation has been the chal-
lenge of scaling up rapid diagnostic tests (RDTs) for malaria, which
is similarly over-treated and for which increasing resistance has
also been problematic. Subsidizing RDTs for malaria at private drug
shops led to increased uptake, improved targeting of treatment and
less unnecessary, empiric therapy [106,107]. Similar strategies are
being examined for tuberculosis diagnostics in India [108], though
attention will need to be given to ensure that wealth disparities in
access to diagnostics do not arise, as may occur with market-based
subsidies.
Finally, it is important that the invasive Salmonella diagnostics
development and rollout proceed in parallel with (1) enhanced
surveillance for Salmonella and other febrile illnesses; and (2)
improved diagnostics for other acute febrile illnesses. There has
been a dearth of sustained public health surveillance of enteric fever
or iNTS, particularly in rural areas [5,7]. If the prevalence of invasive
Salmonellosis is low among acute febrile illnesses, even a diagnostic
with a specificity of 95% may generate more false positives than true
positives. Informing the pre-test probability of disease is critical to
appropriate use of and geographic rollout of imperfect diagnostics,
particularly given the non-specific clinical presentation of invasive
Salmonella infections.
In the near term, a case could be made that a critical component
of a best diagnostic for Salmonellosis, in terms of limiting unnec-
essary treatment, may therefore be the corollary development and
use of accurate diagnostics for other common, alternative infections
such as malaria, influenza or rickettsia. Diagnostics for invasive
Salmonellosis and acute febrile illnesses will need to be comple-
mentary, and should be deployed as part of integrated approaches
that are informed by local epidemiology, and aimed at reducing
inappropriate antibiotic usage and enhanced targeting of therapy.
Conflicts of interest
None.
Funding
This work was supported in part by awards from the Bur-
roughs Wellcome Fund/American Society of Tropical Medicine and
Hygiene (JRA), and the U.S. National Institutes of Health (AI100023;
ETR). The funders had no role in decision to publish or preparation
of the manuscript.
References
[1] Feasey NA, Dougan G, Kingsley RA, Heyderman RS, Gordon MA.
Invasive non-typhoidal salmonella disease: an emerging and
neglected tropical disease in Africa. Lancet 2012;379:2489–99,
http://dx.doi.org/10.1016/S0140-6736(11)61752-2.
[2] Murray CJL, Vos T, Lozano R, Naghavi M, Flaxman AD, Michaud C,
et al. Disability-adjusted life years (DALYs) for 291 diseases and
injuries in 21 regions, 1990–2010: a systematic analysis for the
Global Burden of Disease Study 2010. Lancet 2012;380:2197–223,
http://dx.doi.org/10.1016/S0140-6736(12)61689-4.
[3] Crump JA, Luby SP, Mintz ED. The global burden of typhoid fever. Bull World
Health Organ 2004;82:346–53.
[4] Buckle GC, Walker CLF, Black RE. Typhoid fever and paratyphoid fever: Sys-
tematic review to estimate global morbidity and mortality for 2010. J Glob
Health 2012;2:010401, http://dx.doi.org/10.7189/jogh.02.010401.
[5] Mogasale V, Maskery B, Ochiai RL, Lee JS, Mogasale VV, Ramani E, et al. Burden
of typhoid fever in low-income and middle-income countries: a system-
atic, literature-based update with risk-factor adjustment. Lancet Glob Health
2014;2:e570–80, http://dx.doi.org/10.1016/S2214-109X(14)70301-8.
 J.R. Andrews, E.T. Ryan / Vaccine 33 (2015) C8–C15
[6] Arndt MB, Mosites EM, Tian M, Forouzanfar MH, Mokhdad AH, Meller M, et al.
Estimating the burden of paratyphoid a in Asia and Africa. PLoS Negl Trop Dis
2014;8:e2925, http://dx.doi.org/10.1371/journal.pntd.0002925.
[7] Reddy EA, Shaw AV, Crump JA. Community-acquired bloodstream infec-
tions in Africa: a systematic review and meta-analysis. Lancet Infect Dis
2010;10:417–32, http://dx.doi.org/10.1016/S1473-3099(10)70072-4.
[8] Gordon MA, Graham SM, Walsh AL, Wilson L, Phiri A, Molyneux E, et al.
Epidemics of invasive Salmonella enterica serovar enteritidis and S. enterica
Serovar typhimurium infection associated with multidrug resistance among
adults and children in Malawi. Clin Infect Dis Off Publ Infect Dis Soc Am
2008;46:963–9, http://dx.doi.org/10.1086/529146.
[9] Sigaúque B, Roca A, Mandomando I, Morais L, Quintó L, Sacarlal J,
et al. Community-acquired bacteremia among children admitted to a
rural hospital in Mozambique. Pediatr Infect Dis J 2009;28:108–13,
http://dx.doi.org/10.1097/INF.0b013e318187a87d.
[10] Holt KE, Phan MD, Baker S, Duy PT, Nga TVT, Nair S, et al. Emer-
gence of a globally dominant IncHI1 plasmid type associated with
multiple drug resistant typhoid. PLoS Negl Trop Dis 2011;5:e1245,
http://dx.doi.org/10.1371/journal.pntd.0001245.
[11] Koirala KD, Thanh DP, Thapa SD, Arjyal A, Karkey A, Dongol S, et al. Highly
resistant Salmonella enterica Serovar Typhi with a novel gyrA mutation
raises questions about the long-term efficacy of older fluoroquinolones
for treating typhoid fever. Antimicrob Agents Chemother 2012;56:2761–2,
http://dx.doi.org/10.1128/AAC.06414-11.
[12] Kingsley RA, Msefula CL, Thomson NR, Kariuki S, Holt KE, Gordon MA,
et al. Epidemic multiple drug resistant Salmonella Typhimurium causing inva-
sive disease in sub-Saharan Africa have a distinct genotype. Genome Res
2009;19:2279–87, http://dx.doi.org/10.1101/gr.091017.109.
[13] Savard P, Gopinath R, Zhu W, Kitchel B, Rasheed JK, Tekle T,
et al. First NDM-positive Salmonella sp. strain identified in the
United States. Antimicrob Agents Chemother 2011;55:5957–8,
http://dx.doi.org/10.1128/AAC.05719-11.
[14] Bhutta ZA, Naqvi SH, Razzaq RA, Farooqui BJ. Multidrug-resistant typhoid in
children: presentation and clinical features. Rev Infect Dis 1991;13:832–6.
[15] Szu SC. Development of Vi conjugate – a new generation
of typhoid vaccine. Expert Rev Vaccines 2013;12:1273–86,
http://dx.doi.org/10.1586/14760584.2013.845529.
[16] World Health Organization. Expert consultation to review evidence in sup-
port of the use of typhoid conjugate vaccines. Geneva, Switzerland: WHO;
2014, http://www.who.int/immunization/research/meetings workshops/
Execsum Expert consulation typhoid ViCV July2014.pdf?ua=1.
[17] Parry CM, Wijedoru L, Arjyal A, Baker S. The utility of diagnostic tests for
enteric fever in endemic locations. Expert Rev Anti Infect Ther 2011;9:711–25,
http://dx.doi.org/10.1586/eri.11.47.
[18] Archibald LK, Reller LB. Clinical microbiology in developing countries. Emerg
Infect Dis 2001;7:302–5.
[19] Stuart BM, Pullen RL. Typhoid; clinical analysis of 360 cases. Arch Intern Med
Chic Ill 1908 1946;78:629–61.
[20] Wicks AC, Holmes GS, Davidson L. Endemic typhoid fever. A diagnostic pitfall.
Q J Med 1971;40:341–54.
[21] Hoffman TA, Ruiz CJ, Counts GW, Sachs JM, Nitzkin JL. Waterborne typhoid
fever in Dade County, Florida. Clinical and therapeutic evaluation of 105 bac-
teremic patients. Am J Med 1975;59:481–7.
[22] Briedis DJ, Robson HG. Epidemiologic and clinical features of sporadic
Salmonella enteric fever. Can Med Assoc J 1978;119:1183–7.
[23] Klotz SA, Jorgensen JH, Buckwold FJ, Craven PC. Typhoid fever. An epi-
demic with remarkably few clinical signs and symptoms. Arch Intern Med
1984;144:533–7.
[24] Gupta SP, Gupta MS, Bhardwaj S, Chugh TD. Current clinical patterns of
typhoid fever: a prospective study. J Trop Med Hyg 1985;88:377–81.
[25] Patel TA, Armstrong M, Morris-Jones SD, Wright SG, Doherty T.
Imported enteric fever: case series from the hospital for tropical dis-
eases, London, United Kingdom. Am J Trop Med Hyg 2010;82:1121–6,
http://dx.doi.org/10.4269/ajtmh.2010.10-0007.
[26] Kennedy H. Are the rose spots of typhoid fever always diagnostic of that
affection? The Lancet 1863;82:725–6.
[27] Maskey AP, Day JN, Phung QT, Thwaites GE, Campbell JI, Zimmerman M, et al.
Salmonella enterica serovar Paratyphi A and S. enterica serovar Typhi cause
indistinguishable clinical syndromes in Kathmandu, Nepal. Clin Infect Dis Off
Publ Infect Dis Soc Am 2006;42:1247–53, http://dx.doi.org/10.1086/503033.
[28] Ross IN, Abraham T. Predicting enteric fever without bacteriological culture
results. Trans R Soc Trop Med Hyg 1987;81:374–7.
[29] Vollaard AM, Ali S, Widjaja S, Asten HAGH van, Visser LG, Surjadi C, et al.
Identification of typhoid fever and paratyphoid fever cases at presenta-
tion in outpatient clinics in Jakarta. Indonesia Trans R Soc Trop Med Hyg
2005;99:440–50, http://dx.doi.org/10.1016/j.trstmh.2004.09.012.
[30] Hosoglu S, Geyik MF, Akalin S, Ayaz C, Kokoglu OF, Loeb M. A simple validated
prediction rule to diagnose typhoid fever in Turkey. Trans R Soc Trop Med Hyg
2006;100:1068–74, http://dx.doi.org/10.1016/j.trstmh.2005.12.007.
[31] Kuvandik C, Karaoglan I, Namiduru M, Baydar I. Predictive value of clinical
and laboratory findings in the diagnosis of the enteric fever. New Microbiol
2009;32:25–30.
[32] The bacillus of typhoid fever. Br Med J 1881;2:877–8.
C13
[33] Coleman W, Buxton B. The bacteriology of the blood in typhoid fever. Am J
Med Sci 1907;133:896–903.
[34] Gilman RH, Terminel M, Levine MM, Hernandez-Mendoza P, Hornick RB. Rela-
tive efficacy of blood, urine, rectal swab, bone-marrow, and rose-spot cultures
for recovery of Salmonella typhi in typhoid fever. Lancet 1975;1:1211–3.
[35] Wain J, Bay PVB, Vinh H, Duong NM, Diep TS, Walsh AL, et al. Quantitation
of bacteria in bone marrow from patients with typhoid fever: relation-
ship between counts and clinical features. J Clin Microbiol 2001;39:1571–6,
http://dx.doi.org/10.1128/JCM.39.4.1571-1576.2001.
[36] Wain J, Diep TS, Ho VA, Walsh AM, Nguyen TT, Parry CM, et al. Quantita-
tion of bacteria in blood of typhoid fever patients and relationship between
counts and clinical features, transmissibility, and antibiotic resistance. J Clin
Microbiol 1998;36:1683–7.
[37] Wain J, Diep TS, Bay PVB, Walsh AL, Vinh H, Duong NM, et al. Specimens and
culture media for the laboratory diagnosis of typhoid fever. J Infect Dev Ctries
2008;2:469–74.
[38] Gilman RH, Hornick RB. Duodenal isolation of Salmonella typhi by string cap-
sule in acute typhoid fever. J Clin Microbiol 1976;3:456–7.
[39] Gilman RH, Islam S, Rabbani H, Ghosh H. Identification of gallbladder typhoid
carriers by a string device. Lancet 1979;1:795–6.
[40] Widal F. Serodiagnostic de la fiévre typhoide a-propos d’uve modification par
M.M.C. Nicolle et A. Halipre. Bull Soc Med Hop Paris 1896;13:561–6.
[41] Felix A, Bensted HJ. Proposed standard agglutinating sera for typhoid and
paratyphoid A and B fevers. Bull World Health Organ 1954;10:919–26.
[42] Parry CM, Hoa NT, Diep TS, Wain J, Chinh NT, Vinh H, et al. Value of a single-
tube widal test in diagnosis of typhoid fever in Vietnam. J Clin Microbiol
1999;37:2882–6.
[43] Dutta S, Sur D, Manna B, Sen B, Deb AK, Deen JL, et al. Evaluation of new-
generation serologic tests for the diagnosis of typhoid fever: data from a
community-based surveillance in Calcutta, India. Diagn Microbiol Infect Dis
2006;56:359–65, http://dx.doi.org/10.1016/j.diagmicrobio.2006.06.024.
[44] House D, Chinh NT, Diep TS, Parry CM, Wain J, Dougan G, et al. Use of
paired serum samples for serodiagnosis of typhoid fever. J Clin Microbiol
2005;43:4889–90, http://dx.doi.org/10.1128/JCM.43.9.4889-4890.2005.
[45] Willke A, Ergonul O, Bayar B. Widal test in diagnosis of typhoid fever in Turkey.
Clin Diagn Lab Immunol 2002;9:938–41.
[46] Sippel J, Bukhtiari N, Awan MB, Krieg R, Duncan JF, Karamat KA, et al.
Indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosor-
bent assays (ELISAs) and IgM capture ELISA for detection of antibodies to
lipopolysaccharide in adult typhoid fever patients in Pakistan. J Clin Microbiol
1989;27:1298–302.
[47] Quiroga T, Goycoolea M, Tagle R, Gonzalez F, Rodriguez L, Villarroel
L. Diagnosis of typhoid fever by two serologic methods. Enzyme-linked
immunosorbent assay of antilipopolysaccharide of Salmonella typhi antibod-
ies and Widal test. Diagn Microbiol Infect Dis 1992;15:651–6.
[48] Lim PL, Tam FC, Cheong YM, Jegathesan M. One-step 2-minute test to detect
typhoid-specific antibodies based on particle separation in tubes. J Clin Micro-
biol 1998;36:2271–8.
[49] Bhutta ZA, Mansurali N. Rapid serologic diagnosis of pediatric typhoid fever
in an endemic area: a prospective comparative evaluation of two dot-enzyme
immunoassays and the Widal test. Am J Trop Med Hyg 1999;61:654–7.
[50] Thriemer K, Ley B, Menten J, Jacobs J, van den Ende J. A systematic review
and meta-analysis of the performance of two point of care typhoid fever
tests, Tubex TF and Typhidot, in endemic countries. PloS One 2013;8:e81263,
http://dx.doi.org/10.1371/journal.pone.0081263.
[51] Taylor DN, Harris JR, Barrett TJ, Hargrett NT, Prentzel I, Valdivieso C, et al.
Detection of urinary Vi antigen as a diagnostic test for typhoid fever. J Clin
Microbiol 1983;18:872–6.
[52] Banchuin N, Appassakij H, Sarasombath S, Manatsathit S, Rungpitarangsi B,
Komolpit P, et al. Detection of Salmonella typhi protein antigen in serum and
urine: a value for diagnosis of typhoid fever in an endemic area. Asian Pac J
Allergy Immunol Launched Allergy Immunol Soc Thail 1987;5:155–9.
[53] Chaicumpa W, Thin-Inta W, Khusmith S, Tapchaisri P, Echeverria P, Kalamba-
heti T, et al. Detection with monoclonal antibody of Salmonella typhi antigen
9 in specimens from patients. J Clin Microbiol 1988;26:1824–30.
[54] West B, Richens JE, Howard PF. Evaluation in Papua New Guinea of a urine
coagglutination test and a Widal slide agglutination test for rapid diagnosis
of typhoid fever. Trans R Soc Trop Med Hyg 1989;83:715–7.
[55] Chaicumpa W, Ruangkunaporn Y, Burr D, Chongsa-Nguan M, Echeverria P.
Diagnosis of typhoid fever by detection of Salmonella typhi antigen in urine. J
Clin Microbiol 1992;30:2513–5.
[56] Fadeel MA, Crump JA, Mahoney FJ, Nakhla IA, Mansour AM, Reyad B, et al.
Rapid diagnosis of typhoid fever by enzyme-linked immunosorbent assay
detection of Salmonella serotype typhi antigens in urine. Am J Trop Med Hyg
2004;70:323–8.
[57] Nguyen NQ, Tapchaisri P, Chongsa-nguan M, Cao VV, Doan TT, Sakolvaree
Y, et al. Diagnosis of enteric fever caused by Salmonella spp. in Vietnam by
a monoclonal antibody-based dot-blot ELISA. Asian Pac J Allergy Immunol
Launched Allergy Immunol Soc Thail 1997;15:205–12.
[58] Preechakasedkit P, Pinwattana K, Dungchai W, Siangproh W, Chaicumpa
W, Tongtawe P, et al. Development of a one-step immunochromato-
graphic strip test using gold nanoparticles for the rapid detection of
Salmonella typhi in human serum. Biosens Bioelectron 2012;31:562–6,
http://dx.doi.org/10.1016/j.bios.2011.10.031.
C14 J.R. Andrews, E.T. Ryan / Vaccine 33 (2015) C8–C15
[59] Song JH, Cho H, Park MY, Na DS, Moon HB, Pai CH. Detection of Salmonella typhi
in the blood of patients with typhoid fever by polymerase chain reaction. J
Clin Microbiol 1993;31:1439–43.
[60] Hatta M, Smits HL. Detection of Salmonella typhi by nested polymerase
chain reaction in blood, urine, and stool samples. Am J Trop Med Hyg
2007;76:139–43.
[61] Sánchez-Jiménez MM, Cardona-Castro N. Validation of a PCR for diagno-
sis of typhoid fever and salmonellosis by amplification of the hilA gene
in clinical samples from Colombian patients. J Med Microbiol 2004;53:
875–8.
[62] Nandagopal B, Sankar S, Lingesan K, Appu KC, Padmini B, Sridharan G,
et al. Prevalence of Salmonella typhi among patients with febrile illness
in rural and peri-urban populations of Vellore district, as determined by
nested PCR targeting the flagellin gene. Mol Diagn Ther 2010;14:107–12,
http://dx.doi.org/10.2165/11534350-000000000-00000.
[63] Chaudhry R, Chandel DS, Verma N, Singh N, Singh P, Dey AB. Rapid diagnosis of
typhoid fever by an in-house flagellin PCR. J Med Microbiol 2010;59:1391–3,
http://dx.doi.org/10.1099/jmm.0.020982-0.
[64] Kumar G, Pratap CB, Mishra OP, Kumar K, Nath G. Use of urine with nested
PCR targeting the flagellin gene (fliC) for diagnosis of typhoid fever. J Clin
Microbiol 2012;50:1964–7, http://dx.doi.org/10.1128/JCM.00031-12.
[65] Mandomando I, Macete E, Sigaúque B, Morais L, Quintó L, Sacarlal J, et al. Inva-
sive non-typhoidal Salmonella in Mozambican children. Trop Med Int Health
2009;14:1467–74, http://dx.doi.org/10.1111/j.1365-3156.2009.02399.x.
[66] Wilkens J, Newman MJ, Commey JO, Seifert H. Salmonella bloodstream infec-
tion in Ghanaian children. Clin Microbiol Infect Off Publ Eur Soc Clin Microbiol
Infect Dis 1997;3:616–20.
[67] Schwarz NG, Sarpong N, Hünger F, Marks F, Acquah SE, Agyekum A, et al.
Systemic bacteraemia in children presenting with clinical pneumonia and
the impact of non-typhoid salmonella (NTS). BMC Infect Dis 2010;10:1–5,
http://dx.doi.org/10.1186/1471-2334-10-319.
[68] Nadjm B, Amos B, Mtove G, Ostermann J, Chonya S, Wangai H, et al. WHO
guidelines for antimicrobial treatment in children admitted to hospital in an
area of intense Plasmodium falciparum transmission: prospective study. BMJ
2010;340:c1350.
[69] Kariuki S, Revathi G, Kariuki N, Kiiru J, Mwituria J, Hart CA. Characterisa-
tion of community acquired non-typhoidal Salmonella from bacteraemia and
diarrhoeal infections in children admitted to hospital in Nairobi, Kenya. BMC
Microbiol 2006;6:101, http://dx.doi.org/10.1186/1471-2180-6-101.
[70] Gordon MA, Kankwatira AMK, Mwafulirwa G, Walsh AL, Hopkins MJ,
Parry CM, et al. Invasive non-typhoid salmonellae establish systemic
intracellular infection in HIV-infected adults: an emerging disease patho-
genesis. Clin Infect Dis Off Publ Infect Dis Soc Am 2010;50:953–62,
http://dx.doi.org/10.1086/651080.
[71] Lin L-H, Tsai C-Y, Hung M-H, Fang Y-T, Ling Q-D. Rectal swab
sampling followed by an enrichment culture-based real-time PCR
assay to detect Salmonella enterocolitis in children. Clin Microbiol
Infect Off Publ Eur Soc Clin Microbiol Infect Dis 2011;17:1421–5,
http://dx.doi.org/10.1111/j.1469-0691.2010.03450.x.
[72] Tennant SM, Diallo S, Levy H, Livio S, Sow SO, Tapia M, et al. Identification
by PCR of non-typhoidal Salmonella enterica serovars associated with inva-
sive infections among febrile patients in Mali. PLoS Negl Trop Dis 2010:4,
http://dx.doi.org/10.1371/journal.pntd.0000621.
[73] Strid MA, Dalby T, Mølbak K, Krogfelt KA. Kinetics of the human antibody
response against Salmonella enterica Serovars Enteritidis and Typhimurium
determined by lipopolysaccharide enzyme-linked immunosorbent assay. Clin
Vaccine Immunol 2007;14:741–7, http://dx.doi.org/10.1128/CVI.00192-06.
[74] Kuhn KG, Falkenhorst G, Ceper TH, Dalby T, Ethelberg S, Mølbak K, et al.
Detecting non-typhoid Salmonella in humans by ELISAs: a literature review.
J Med Microbiol 2012;61:1–7, http://dx.doi.org/10.1099/jmm.0.034447-0.
[75] Andrews JR, Prajapati K, Eypper E, Shrestha P, Shakya M, Pathak K, et al. Eval-
uation of an electricity-free, culture-based approach for detecting typhoidal
Salmonella bacteremia during enteric fever in a high burden, resource-limited
setting. PLoS Negl Trop Dis 2013;7:e2292.
[76] Zhou L, Pollard AJ. A fast and highly sensitive blood culture PCR method for
clinical detection of Salmonella enterica serovar Typhi. Ann Clin Microbiol
Antimicrob 2010;9:14, http://dx.doi.org/10.1186/1476-0711-9-14.
[77] Zhou L, Pollard AJ. A novel method of selective removal of human DNA
improves PCR sensitivity for detection of Salmonella Typhi in blood samples.
BMC Infect Dis 2012;12:164, http://dx.doi.org/10.1186/1471-2334-12-164.
[78] Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N,
et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res
2000;28:E63.
[79] Piepenburg O, Williams CH, Stemple DL, Armes NA. DNA detec-
tion using recombination proteins. PLoS Biol 2006;4:e204,
http://dx.doi.org/10.1371/journal.pbio.0040204.
[80] Vincent M, Xu Y, Kong H. Helicase-dependent isothermal DNA amplification.
EMBO Rep 2004;5:795–800, http://dx.doi.org/10.1038/sj.embor.7400200.
[81] Francois P, Tangomo M, Hibbs J, Bonetti E-J, Boehme CC, Notomi T,
et al. Robustness of a loop-mediated isothermal amplification reaction
for diagnostic applications. FEMS Immunol Med Microbiol 2011;62:41–8,
http://dx.doi.org/10.1111/j.1574-695X.2011.00785.x.
[82] Darton T, Waddington C, Blohmke C, Jones C, Zhou L, Pollard A. Detecting
typhoid using LAMP in a S. Typhi controlled human infection model. Philadel-
phia (PA): IDWeek 2012; 2012.
[83] Tennant SM, Zhang Y, Galen JE, Geddes CD, Levine MM. Ultra-fast and sen-
sitive detection of non-typhoidal Salmonella using microwave-accelerated
metal-enhanced fluorescence (MAMEF). PLoS ONE 2011;6:e18700,
http://dx.doi.org/10.1371/journal.pone.0018700.
[84] Sheikh A, Bhuiyan MS, Khanam F, Chowdhury F, Saha A, Ahmed D,
et al. Salmonella enterica serovar Typhi-specific immunoglobulin A antibody
responses in plasma and antibody in lymphocyte supernatant specimens in
Bangladeshi patients with suspected typhoid fever. Clin Vaccine Immunol
2009;16:1587–94, http://dx.doi.org/10.1128/CVI.00311-09.
[85] Khanam F, Sheikh A, Sayeed MA, Bhuiyan MS, Choudhury FK, Salma
U, et al. Evaluation of a typhoid/paratyphoid diagnostic assay (TPTest)
detecting anti-Salmonella IgA in secretions of peripheral blood lympho-
cytes in patients in Dhaka, Bangladesh. PLoS Negl Trop Dis 2013;7:e2316,
http://dx.doi.org/10.1371/journal.pntd.0002316.
[86] Charles RC, Sultana T, Alam MM, Yu Y, Wu-Freeman Y, Bufano MK, et al.
Identification of immunogenic Salmonella enterica serotype Typhi antigens
expressed in chronic biliary carriers of S. Typhi in Kathmandu. Nepal PLoS Negl
Trop Dis 2013;7:e2335, http://dx.doi.org/10.1371/journal.pntd.0002335.
[87] Zaas AK, Chen M, Varkey J, Veldman T, Hero III AO, Lucas J, et al.
Gene expression signatures diagnose influenza and other symptomatic
respiratory viral infections in humans. Cell Host Microbe 2009;6:207–17,
http://dx.doi.org/10.1016/j.chom.2009.07.006.
[88] Kaforou M, Wright VJ, Oni T, French N, Anderson ST, Bangani N, et al. Detec-
tion of tuberculosis in HIV-infected and -uninfected African adults using
whole blood RNA expression signatures: a case–control study. PLoS Med
2013;10:e1001538, http://dx.doi.org/10.1371/journal.pmed.1001538.
[89] Ge Y, Zhao K, Qi Y, Min X, Shi Z, Qi X, et al. Serum microRNA expression profile
as a biomarker for the diagnosis of pertussis. Mol Biol Rep 2013;40:1325–32,
http://dx.doi.org/10.1007/s11033-012-2176-9.
[90] Ramilo O, Allman W, Chung W, Mejias A, Ardura M, Glaser C,
et al. Gene expression patterns in blood leukocytes discrimi-
nate patients with acute infections. Blood 2007;109:2066–77,
http://dx.doi.org/10.1182/blood-2006-02-002477.
[91] Thompson LJ, Dunstan SJ, Dolecek C, Perkins T, House D, Dougan G, et al.
Transcriptional response in the peripheral blood of patients infected with
Salmonella enterica serovar Typhi. Proc Natl Acad Sci U S A 2009;106:22433–8,
http://dx.doi.org/10.1073/pnas.0912386106.
[92] Antti H, Fahlgren A, Näsström E, Kouremenos K, Sundén-Cullberg
J, Guo Y, et al. Metabolic profiling for detection of Staphylococcus
aureus infection and antibiotic resistance. PLoS ONE 2013;8:e56971,
http://dx.doi.org/10.1371/journal.pone.0056971.
[93] Näsström E, Thieu NTV, Dongol S, Karkey A, Vinh PV, Thanh TH, et al.
Salmonella Typhi and Salmonella Paratyphi A elaborate distinct sys-
temic metabolite signatures during enteric fever. eLife 2014;3:e03100,
http://dx.doi.org/10.7554/eLife.03100.
[94] The malERA Consultative Group on Diagnoses and Diagnostics. A research
agenda for malaria eradication: diagnoses and diagnostics. PLoS Med
2011;8:e1000396, http://dx.doi.org/10.1371/journal.pmed.1000396.
[95] Kik SV, Denkinger CM, Casenghi M, Vadnais C, Pai M. Tuberculosis diag-
nostics: which target product profiles should be prioritised? Eur Respir J
2014;44:537–40, http://dx.doi.org/10.1183/09031936.00027714.
[96] Solomon AW, Engels D, Bailey RL, Blake IM, Brooker S, Chen J-X, et al. A diag-
nostics platform for the integrated mapping, monitoring, and surveillance of
neglected tropical diseases: rationale and target product profiles. PLoS Negl
Trop Dis 2012;6:e1746, http://dx.doi.org/10.1371/journal.pntd.0001746.
[97] Gunn JS, Marshall JM, Baker S, Dongol S, Charles RC, Ryan
ET. Salmonella chronic carriage: epidemiology, diagnosis, and
gallbladder persistence. Trends Microbiol 2014;22:648–55,
http://dx.doi.org/10.1016/j.tim.2014.06.007.
[98] Walter SD, Irwig LM. Estimation of test error rates, disease prevalence and rel-
ative risk from misclassified data: a review. J Clin Epidemiol 1988;41:923–37.
[99] Olsen SJ, Pruckler J, Bibb W, Thanh NTM, Trinh TM, Minh NT, et al. Evaluation
of rapid diagnostic tests for typhoid fever. J Clin Microbiol 2004;42:1885–9,
http://dx.doi.org/10.1128/JCM.42.5.1885-1889.2004.
[100] Suputtamongkol Y, Pongtavornpinyo W, Lubell Y, Suttinont C, Hoontrakul
S, Phimda K, et al. Strategies for diagnosis and treatment of suspected
leptospirosis: a cost–benefit analysis. PLoS Negl Trop Dis 2010;4:e610,
http://dx.doi.org/10.1371/journal.pntd.0000610.
[101] Department of Health Services. Annual Report: 2067/68 (2010/2011). Kath-
mandu, Nepal: Government of Nepal, Ministry of Health and Population;
2011.
[102] Murdoch DR, Woods CW, Zimmerman MD, Dull PM, Belbase RH, Keenan AJ,
et al. The etiology of febrile illness in adults presenting to Patan hospital in
Kathmandu, Nepal. Am J Trop Med Hyg 2004;70:670–5.
[103] Ko AI, Galvão Reis M, Ribeiro Dourado CM, Johnson WD, Riley LW. Urban
epidemic of severe leptospirosis in Brazil. Salvador Leptospirosis Study Group.
Lancet 1999;354:820–5.
[104] Kumar V, Kumar V, Yadav AK, Iyengar S, Bhalla A, Sharma N, et al.
Scrub typhus is an under-recognized cause of acute febrile illness
 J.R. Andrews, E.T. Ryan / Vaccine 33 (2015) C8–C15 C15

with acute kidney injury in India. PLoS Negl Trop Dis 2014;8:e2605,
http://dx.doi.org/10.1371/journal.pntd.0002605.
[105] Goossens H, Ferech M, Vander Stichele R, Elseviers M. Outpa-
tient antibiotic use in Europe and association with resistance:
a cross-national database study. The Lancet 2005;365:579–87,
http://dx.doi.org/10.1016/S0140-6736(05)17907-0.
[106] Cohen JL, Dickens WT. Adoption of over-the-counter malaria diagnostics
in Africa: the role of subsidies, beliefs, externalities and competition. In:
Laxminarayan R, Macauley MK, editors. Value Inf. Netherlands: Springer;
2012. p. 173–91.
[107] Cohen J, Fink G, Berg K, Aber F, Jordan M, Maloney K, et al. Feasibility
of distributing rapid diagnostic tests for malaria in the retail sector: evi-
dence from an implementation study in Uganda. PLoS ONE 2012;7:e48296,
http://dx.doi.org/10.1371/journal.pone.0048296.
[108] Anand G. Plan to fight deadly TB strain gains in India. Wall Str J 2013. Available
at: http://www.mcgill.ca/tb/files/tb/wsj march17 2013.pdf