Introduction

Overview of typhoid fever and its global burden

Typhoid fever is a life-threatening infection and a significant global health problem. It is a febrile
illness caused by Gram-negative bacterium Salmonella enterica serovar Typhi (S. Typhi) and
usually spread through contaminated food or water and impacts the lives of people living in areas
without access to clean water and sanitation (Jessie et al., 2023; WHO, 2023; Basnyat et al.,
2021). In 2019 alone, typhoid caused an estimated 110 000 deaths and 8.1 million disability-
adjusted life-years (GBD, 2019). The burden of Salmonella typhi has historically been
challenging to quantify due to the reliance on blood culture for diagnosis and limited reliable
laboratory tests (Crump et al., 2004). However, recent investments in regional surveillance have
improved our understanding, revealing significant incidence and mortality rates, especially in
many African and South Asian countries (Jessie at al., 2023). Kim et al. (2019) found that 42 out
of 57 African countries reported at least one instance of culture-confirmed typhoid fever between
1900 and 2018. It was also recorded that reported cases has risen over time across Africa, with
significant variation observed both between countries and over different periods. Additionally,
outbreaks of typhoid fever were documented in 15 countries, with both their frequency and
magnitude increasing over time. A study by Antillón et al. (2017) estimated that 17.8 million
cases of typhoid fever occur each year in low- and middle-income countries LMICs (95%
credible interval: 6.9-48.4 million) with Central Africa experiencing the highest incidence of
typhoid, followed by select countries in Central, South, and Southeast Asia. This incidence is
typically peaked in the 2-4 year old age group.

Figure 1.0 Global incidence of typhoid fever (Meiring et al., 2023)

From March 1, 2010, to January 31, 2014, blood cultures from 13,431 febrile patients yielded
135 isolates of Salmonella enterica serotype Typhi (S Typhi) and 94 isolates of invasive non-
typhoidal Salmonella (iNTS). At nine sites, Salmonella spp accounted for 33% or more of all
bacterial pathogens. The adjusted incidence rate (AIR) of S Typhi per 100,000 person-years of
observation varied from 0 (95% CI 0-0) in Sudan to 383 (95% CI 274-535) at one site in Burkina
Faso (Marks et al., 2017)
Importance of accurate diagnosis of typhoid fever
Accurate and rapid diagnosis of typhoid fever could significantly improve management of cases
with reliable antibiotics to which S. Typhi or S. Paratyphi are sensitive (Andrews and Ryan,
2015). The diagnosis of typhoid in most low-resource settings is made based on clinical criteria,
which is challenging due to the nonspecificity of typhoid symptoms. Because typhoid symptoms
are common to many other illnesses such as malaria and dengue, patients are frequently
misdiagnosed (Crump, 2012). Difficulties with accurate typhoid diagnostics and appropriate
treatment can lead to more serious complications and can contribute to drug resistance (Coalition
Against Typhoid, 2018). Findings by Marchello et al. (2020) identify considerable typhoid-
associated illness and death that could be averted by accurate diagnosis and prevention measures
including typhoid conjugate vaccine introduction. Ohanu et al., (2019) reported that Widal test
performed poorly as a diagnostic test in Nigeria and has led to emerging multi-drug resistance
due to inaccurate diagnosing of S. typhi. The data also indicated that periodic surveillance of
antibiotic susceptibility is critical for optimal typhoid therapy. High incidences of MDR S. Typhi
are calculated in locations with a high burden of typhoid, specifically in children aged <15 years
(Park et al., 2018). Poor diagnosis continues to hinder effective control of concurrent typhoid
fever due to non-specific clinical presentation of the disease, lack of resources, insufficient
access to health facilities, and lack of trained health care providers (Uneke, 2008).
The challenges associated with diagnosing typhoid fever in Salmonella endemic
communities.
Challenge of diagnosing typhoid fever in Salmonella endemic communities lies in the lack of
reliable rapid diagnostic tests, leading to over-reliance on inaccurate tests and consequent
unnecessary antibiotic treatment leading to antimicrobial resistance and inadequate therapy for
other febrile illnesses (Andrews and Ryan, 2015). Culture-based approaches for isolating S.
Typhi in endemic countries face several challenges, including prolonged time for obtaining
results (typically 5–7 days), low sensitivity, insufficient infrastructure, and a shortage of trained
personnel. Additionally, the requirement for antisera to confirm biochemical results indicating S.
Typhi poses a resource burden on diagnostic laboratories in these settings (Ajibola et al., 2018).
In areas of endemicity, there are low background level of antibodies in the normal population,
this makes it difficult to determine the appropriate cut-off point for a positive result due to
differences between areas and between times (Crump et al., 2015).
Pathogenesis of S. typhi
S. Typhi is typically ingested through contaminated food or water. It passes through the stomach
to invade the gut epithelium, mostly in the distal ileum. The bacterium actively attaches to host
cells by unidentified adhesion molecules, potentially involving fimbriae, though specific
interactions remain unclear (Townsend et al., 2001). Salmonella spp. invade epithelial cells
through bacterial-mediated endocytosis, involving cytoskeletal rearrangement and disruption of
the epithelial cell brush border which is facilitated by an adherent and invasive phenotype
activated in conditions resembling the human small intestine. This invasive phenotype is partly
mediated by the salmonella pathogenicity island (SPI)-1, which encodes a type III secretion
system delivering bacterial proteins into host cells and enhancing adherence and invasion
efficiency (Sukhan, 2000; Kingsley and Baumler, 2000). After entering the small intestine, S.
Typhi migrates via M cells into the mesenteric lymph nodes, leading to transient primary
bacteremia. Some bacteria escape APCs and reach Peyer's patches, where dendritic cells present
bacterial antigens and activates T and B lymphocytes. This dissemination results in subsequent
infection of various organs, with re-entry into the bloodstream marking clinical disease onset
(Everest et al., 2001; Andrade and Andrade Júnior, 2003)

DIAGNOSIS OF TYPHOID FEVER

Typhoid fever is difficult to diagnose due to the lack of sensitivity of the exiting methods. Blood
culture is a standard diagnostic method used to detect the presence of Salmonella typhi in the
bloodstream mainly due to the fact that about 80% or more of patients with typhoid fever have
the causative organism in their blood (WHO, 2003). Blood culture method is about 40% to 60%
sensitive (Parry et al., 2011). 2-15ml of blood is collected from the patient suspected of having
typhoid fever depending on the age (WHO, 2003). The collected blood sample is then inoculated
into blood culture bottles containing tryptic soy broth or brain heart infusion broth. The
inoculated blood culture bottles are then placed in a suitable incubator set at the optimal
temperature (usually around 37°C or 98.6°F) for the growth of Salmonella typhi. The bottles are
left in the incubator for a predetermined period, typically up to 7 days. The volume of blood used
and the amount of time involved to yield a result is a major setback for this method ( Getahun
Strobel et al., 2021). Throughout the incubation period, the bottles are periodically checked for
signs of bacterial growth. If bacterial growth is observed, a small amount of the broth is
subcultured onto agar plates usually blood or McConkey. After incubation, the colonies grown
on agar plates are examined morphologically (size, shape, color, etc.) and subjected to
biochemical tests to confirm the presence of Salmonella typhi. Biochemical tests may include
various reactions to specific substrates that are characteristic of Salmonella species. Motility,
indole, citrate and triple sugar iron (TSI) tests are some biochemical tests performed. TSI test is
used to differentiate enteric bacteria based on their ability to ferment sugars, produce gas, and
reduce sulfur. A TSI agar slant containing three sugars (glucose, lactose, and sucrose) and
ferrous sulfate is inoculated with the bacterial isolate. The slant is then incubated at 37°C for 18-
24 hours. The result is interpreted as follows;
Observation Inference
Red slant/yellow butt Glucose fermentation
Yellow slant/yellow butt Glucose and lactose/sucrose fermentation
Red slant/red butt No sugar fermented
Presence of bubbles or cracks in the agar Gas production

Blackening of the agar due to the reaction of H2S production

hydrogen sulfide with ferrous sulfate.

In some cases, serological tests may be performed on the isolated colonies to further confirm the
identity of Salmonella typhi. Serological tests involve detecting specific antigens or antibodies
associated with the bacterium. The Widal test was developed in the 1890s and modified in the
1950s. Today it is widely used in typhoid endemic regions (Andrews and Ryan 2015). The
Widal test measures agglutinating antibodies in the sera of people suspected of having enteric
fever against lipopolysaccharide (O) and flagellar (H) antigens of Salmonella serovar Typhi.
Regarding the diagnostic standards to be used in interpreting the test, there is disagreement.
(Levine et al.,1978). Interpretation of the Widal test may be aided by knowledge of the
background levels of antibodies in the community, and performance is best among patients with
a high prior probability of enteric fever(Levine et al.,1978). The test is simple and cheap to
perform but its use is discouraged due to its inaccuracy.
Wam et al., in 2019 evaluated the performance of Widal test to stool culture in the diagnosis of
typhoid fever and concluded that Widal test is not reliable for diagnosis of typhoid fever. Hence
health care professionals should develop a rapid, highly sensitive and affordable diagnostic
method that is also capable of differentiating Salmonella infection from other infections. Another
study by Mawazo et al., (2019) to determine the diagnostic performance of the Widal test and
stool culture in typhoid-suspected patients using blood culture as a golden standard concluded
that Widal test is not reliable for diagnosis of typhoid fever because it gives false positive and
negative results frequently. Also, widal test showed poor agreement with the blood culture while
the stool culture showed high agreement. Shahapur et al., 2021 also agreed to the fact that the
widal test gives low positive predictive values reiterating the point that it is not reliable for the
diagnosis of typhoid. Widal test has low sensitivity, specificity and positive predictive value
(Andualem et al.,2014).
BIOMARKERS FOR IDENTIFICATION OF SALMONELLA TYPHI
Existing serological diagnostic tools for enteric fever are based on the detection of antibodies
against Salmonella (lipopolysaccharide) LPS or flagellum, resulting in a high false-positive rate
therefore there is the need for improved diagnostics and therapeutic tools. The development of
sensitive and specific diagnostic tools requires the identification of novel markers capable of
detecting the pathogen. Liang et al.,(2013) used a protein microarray with 2,724 Samonella
serotype Typhi antigen covering over 63% of its proteome to identify antibodies against these
antigens. Antibodies were identified against 16 IgG and 77 IgM antigens that showed differential
reactivity between acute typhoid patients and healthy controls. The study indicated that the
reactive antigens had enrichment properties related to membrane association, secretion, and
protein expression. Using a Naïve Bayes classifier, approximately 72% of the serodiagnostic
antigens were found in the top 25% of the ranked antigen list. These data offer a valuable
resource for better treatment, diagnostics and vaccine development against a significant human
infection.
Herath, (2003) developed an ELISA to detect IgA anti-Salmonella typhi LPS in patients and
concluded that it is only suitable for diagnosis of acute infection. Chin et al., (2016) also agreed
that salivary anti-HlyE IgA antibody can detect typhoid fever in acute infections. Redhuan et al.,
(2017) also evaluated how best salivary immunoglobulin could be used to detect Salmonella
typhi and concluded that salivary IgA anti-50kDa antibody is suitable for routine screening of
typhoid fever. In a study by Goay et al.,(2016), six S. Typhi genes namely,STY0201, STY0307,
STY0322, STY0326, STY2020, and STY2021 were discovered to be specific in silico after a
genomic comparison of S. Typhi with other enteric pathogens was conducted. In vitro, the
specificity of these genes was assessed using six PCR assays, each of which targeted a distinct
gene. Each assay's diagnostic sensitivities and specificities were assessed using 39 clinical
isolates of S. Typhi, 62 Salmonella strains other than Typhi, and 10 non-Salmonella strains. Five
of the genes, STY0307, STY0322, STY0326, STY2020, and STY2021, showed 100% sensitivity
(39/39) and 100% specificity (0/72) in the results. The five PCR assays had a detection limit of
1.28 pg for STY0307, 6.4 pg for STY0326, STY2020, and STY2021, and 32 pg for STY0322. 5
PCR assays using STY0307, STY0322, STY0326, STY2020, and STY2021 were developed and
found to be extremely specific at single-gene target resolution for diagnosis of typhoid fever.
Another study by Franklin et al.,(2020) identified specific biomarkers in the blood for the
detection of typhoid. Two antigens HlyE(for acute typhoid) and YncE( for asymptomatic
carriers) were tested against host antibodies IgG, IgA and IgM for both acute and asymptomatic
carries. The HlyE and YncE were synthesized in the lab and tested against 422 sera samples
from acute typhoid patients, other febrile, food handlers, and healthy individuals. The findings
revealed that when testing for antibodies against HlyE collectively, there was an 83% accuracy
in identifying cases of typhoid while YncE-IgG and IgA detected 16 individuals who potentially
carry the typhoid bacteria based their antibody profile.
Näsström et al.,(2014), performed two dimensional gas chromatography with time-of-flight
mass spectrometry (GCxGC/TOFMS) on plasma from patients with S. Typhi and S. Paratyphi A
infections and asymptomatic controls, in other to understand metabolite signals associated with
typhoid fever concluded that reproducible and serovar specific systemic biomarkers can be
detected during enteric fever diagnostics.
Rapid Diagnostic Tests
The ELISA principle is applied in the Rapid Diagnostic Test or Rapid IgM/IgG
immunochromatographic test (ICT) (Shahapur et al., 2021). RDTs are used to compliment the
results of blood culture and the Widal test. The majority of RDTs have been designed to identify
antibodies against Salmonella antigens and are intended for use with blood, such as venous
whole-blood, serum, or capillary samples. IgM is typically the antibody class seen, indicating a
recent or ongoing infection (REF). They are easily obtained on the market and do not require any
technical expertise to use or analyze the result. Because of their low sensitivity, specificity and
positive predictive values, RDTs usually perform poorly for individual patient diagnosis
(Getahun et al., 2021 and Shahapur et al., 2021). For the diagnosis of typhoid in endemic areas
Getahun et al., 2021 showed that Typhidot Rapid offered the best value in terms of sensitivity,
specificity, affordability, positive and negative predictive values, and ease of use. Typhidot test
is a multistep dot enzyme immunoassay that uses a 50kDa antigen to identify specific S. typhi
IgG and IgM antibodies. Serum of patients and control are incubated and washed. After an
additional washing, the strips are incubated with a color reagent and the intensity of the sample
dots is compared with those of the positive control strips. The detection of IgM reveals acute
typhoid in the initial stage of infection, while the detection of both IgG and IgM suggests acute
typhoid in the middle stage of infection (WHO, 2003). This test is easy to perform but not ideal
for typhoid diagnosis (Ousenu et al.,2021). The TUBEX test detects antibodies against the S.
Typhi lipopolysaccharide (LPS) antigen by hindering the binding between O9 monoclonal
antibodies and LPS-coupled magnetic particles. A positive result is indicated by visible
decolorization of patient serum in the test reagent solution post-magnetic particle separation.
Samples are graded from 0 to 10 based on the reaction mixture's color, with grades exceeding 2
considered positive (Wijedoru et al., 2017). The test is highly selective for Salmonella typhi IgM
antibodies (Kawamo et al., 2007, Tam et al., 2008). The TUBEX Test is a simple, easy and
quick. It saves time, money and labor (Nugraha et al., 2012). It uses the separation of colored
particles to increase its sensitivity and resolution. The TUBEX method is 60% sensitive and 58%
specific (Khanam et al., 2022).
NUCLEIC ACID
This involves the identification of Salmonella typhi by PCR which amplifies Salmonella specific
serovar DNA for diagnosis. The ttr gene which encodes tetrethionate reductase is used as a target
gene to detect Salmonella because it is present in all Salmonella species except that it cannot be
used to differentiate typhoidal salmonella from nontyphoidal salmonella species

DIAGNOSTIC CHALLENGES IN ENDEMIC REGIONS

Typhoid fever is usually endemic in low income communities where there are insufficient
resources for proper diagnosis of the disease. (Ministry of Health and Sanitation. (2015). As such
laboratories rely on tests with low diagnostic sensitivity like the Widal to help doctors make
clinical decisions regarding typhoid fever. Lack of rapid diagnostic test kits that are cheap,
highly sensitive and specific has also impeded the proper diagnosis of the disease.
The availability and inappropriate use of antimicrobials without isolation of S. typhi has in
endemic communities
The availability of antimicrobials in endemic communities a
Antimicrobials are readily available and easily accessible contributing to disease burden.
Antimicrobials lessen the severity of typhoid fever symptoms, but they also prevent people from
seeking official medical attention and obstruct confirmed diagnosis.
Various approaches have been employed to improve the sensitivity of diagnosis by the culture
method. Adding ox bile or bile salt (sodium taurocholate) to the culture media has increased the
frequency of bacterial isolation in a shorter amount of time. More precisely, the contents of bile
lyse blood cells to release bacteria and inhibit antibacterial activity. It also prevents the isolation
of other important bacteria.(REF)

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