Others Section DOI : 10.7860/JCDR/2018/36358.12048
Year : 2018 | Month : Sep | Volume : 12 | Issue : 09 Page : KE01 - KE07

Typhoid Diagnostics: Looking Beneath the Surface

Tarang Sharma1, Shinjini Bhatnagar2, Ashutosh Tiwari3

1 PhD Student, Centre for Bio-design and Diagnostics, Translational Health Science and Technology Institute, Faridabad, Haryana, India.
2 Dean, Paediatric Biology Centre, Translational Health Science and Technology Institute, Faridabad, Haryana, India.
3 Assistant Professor, Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.


NAME, ADDRESS, E-MAIL ID OF THE CORRESPONDING AUTHOR: Dr. Ashutosh Tiwari, Assistant Professor, Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India.
E-mail: ashutosht@hotmail.com
Abstract

Typhoid fever is a systemic life-threatening infection caused by S. typhi, which specifically infects humans. It is a major public health concern in the developing world. The clinical symptoms of typhoid often overlap with other febrile infections, affecting timely and accurate diagnosis. The current gold standard, blood or bone marrow culture is used for definite diagnosis, however its utility is limited due to the requirement of dedicated culture facilities, technical expertise and prior antibiotic use. The existing serological tests demonstrate variable and suboptimal sensitivity and specificity, because of cross-reactivity exhibited by the conserved, somatic antigens of Lipopolysaccharides (LPS) and terminal regions of flagellin. The lack of accurate detection further leads to imprudent antibiotic use, causing complications and morbidity, and even contributes to drug resistance. This emphasises the need for superior tests for efficient typhoid diagnosis. The need has propelled the pursuit of infection-specific markers using high-yield approaches like proteomics, metabolomics, transcriptomics and potential markers have been enumerated. Bio-marker discovery serves as a start point in diagnostic development and the subsequent assessment of the diagnostic tests must be governed by pre-defined metrics to determine their accuracy and field-feasibility. The evaluation of sensitivity and specificity in endemic regions is particularly important. In the present review, we discuss the existing diagnostic modalities; their challenges and the novel approaches being utilised for the discovery of specific biomarkers. We also review the new diagnostic tests under development while also discussing the important metrics to be considered during development of improved tests.

How to cite this article

Tarang Sharma, Shinjini Bhatnagar, Ashutosh Tiwari, .TYPHOID DIAGNOSTICS: LOOKING BENEATH THE SURFACE.Journal of Clinical and Diagnostic Research [serial online]2018 Sep[cited:2019 Aug 21] 09 KE01 - KE07
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Introduction

Typhoid fever is a febrile illness caused by Salmonella enterica serovar Typhi (S. typhi). The global incidence estimates vary between 11.9 million and 26.9 million cases per year, and mortality estimates range between 129000 and 161000 annually [1]. The prevalence of typhoid is high in developing nations with poor hygiene standards [2,3]. It mainly afflicts children, and vaccines based on Vi and Ty21 are recommended for routine immunisation in endemic countries [2,4]. Typhoid manifests 1-2 weeks post-ingestion of an infectious dose of S. typhi, with highly non-specific clinical features, making differential diagnosis difficult [5]. S. typhi causes systemic infection by evading host’s immune system and penetrating deeper tissue [6-8]. Despite its high disease burden, accurate diagnosis of typhoid is still challenging. The gold standard-blood and bone marrow aspirate culture requires adequate infrastructure and trained personnel, unavailable in resource-poor regions. Although, rapid diagnostic tests like TUBEX and Typhidot have shown improved diagnostic accuracy over the routinely used Widal test and can potentially be employed as Point-Of-Care Tests (POCTs), however they lack optimal sensitivity and specificity [9,10], since, most of them employ detection of antibodies to surface antigens that are conserved among Enterobacteriaceae [11], and against which background antibody titre is high in the endemic regions. This diagnostic gap leads to imprecise disease burden estimates, misdiagnosis, and drives imprudent antibiotic use in endemic regions [12], thus, resulting in antimicrobial resistance [13,14]. Therefore, there is an urgent need for superior typhoid diagnostics, and to establish a well-defined metric system for gauging the operational features of new tests for their systemic validation [15]. The major challenge while developing improved diagnostics is either the low bacterial count or the lack of a specific biomarker [16]. Therefore, Rapid Diagnostic Tests (RDTs) employing more specific antigens are needed [17].

Recently, techniques like transcriptomics, metabolomics, and proteomics have been exploited to identify biomarkers unique to S. typhi bacteremia patients. The candidates identified through techniques like proteomics have demonstrated good diagnostic potential in some recent studies [18]. Additionally, some novel methods are also being explored, such as IgA detection based on a new format [19,20].

In the present review, we briefly discuss the current typhoid diagnostics and highlight the approaches for the identification of specific biomarkers. We also outline the diagnostic metrics essential for the development and evaluation of new assays, and the new tests for typhoid diagnosis.

Current Diagnostic Platforms

Pathogen detection:S. typhi detection via blood or marrow culturing has been the mainstay of clinical diagnosis. The turnover period of blood culture is 2-4 days. Fully automated blood culture systems are popular in the developed countries. However, these are not viable for the resource poor regions. Although, highly specific, blood culturing has a low and variable sensitivity (40-60%) due to the transient bacterial presence in blood and previous antibiotic treatment [21,22]. Positive detection requires higher blood volume (5-10 mL). Bone marrow culture with a sensitivity of 80-95% is valuable in cases where there is prior antimicrobial treatment, or in long-standing illness. However, it is invasive and demands expertise and equipment, hindering its regular use.

Nucleic acid-based detection: Amplification of S. typhi-specific genes using Polymerase Chain Reaction (PCR) or real-time PCR approach is currently being explored. While Massi MN et al., amplified fliC gene from all culture positive and negative blood samples with a higher copy number in culture positives vs. culture negatives [23]. A higher copy number contradicts the microbiological results, indicating the presence of more dead than live bacteria in patients. The most frequently assessed molecular targets include; the Hd Flagella gene, fliC-d [24], the Vi capsular gene viaB [25], the tyvelose epimerase gene (tyv), groEL gene [26], of which the fliC-d and the Vi locus are non-specific [27]. Prabagaran SR et al., have demonstrated the nested-multiplex PCR technique to detect fliC and viaB operon as an alternative detection method [28]. The clinical efficacy of this costly approach is uncertain due to its low sensitivity (38-42%) [29].

Surface-antigen detection: Body fluids such as urine show intermittent presence of bacterial antigens and have been utilised for detection. Antigenic cross-reactivity is overcome by using Monoclonal Antibodies (MAbs) specific to surface antigens, and sensitivities up to 95% have been reported [30-32]. In an ELISA based study, Vi antigen showed 73% sensitivity which increased to 97% with sequential urine collection, although specificity remained suboptimal. O9 and Hd antigens were positive in less than 50% of culture-confirmed cases. The sensitivity was found to be highest in the first week [33]. Studies using surface antigens as markers have shown inconsistent results and are not in clinical use [34].

Antibody detection: Widal test involves the detection of agglutinating antibodies against LPS(O) and flagellar (H) antigens in suspected patients and is extensively used in endemic regions [35]. The test requires paired serum taken 10 days apart, and a positive outcome is established by a four-fold increase in antibody titre, which often occurs before the clinical onset of Typhoid. This makes it challenging to demonstrate the rise each time for positive diagnosis [36]. A single agglutination test has inadequate sensitivity and specificity [37]. The RDTs aim at reliable, rapid, uncomplicated and point-of-care diagnosis, and mainly exploit the O and H antigens of S. typhi, in different formats. Inhibition assay format based TUBEX®TF test detects IgM against an immuno-dominant and rare O9-antigen [37]. The ability of serum antibodies to inhibit the binding between the O9-specific mAb-coated coloured latex particles and LPS antigen coated magnetic beads further improves its specificity [38]. Another immuno-dot based method, Typhidot detects specific IgG and IgM against a 50 kDa Outer Membrane Protein (OMP) [39]. Its modification, Typhidot-M prevents masking of IgM by IgG antibodies during re-infection, through inactivation of IgG, thus improving accuracy [40,41]. The operational and technical features of commonly used commercial RDTs are summarised in [Table/Fig-1] [6,9,35,40-56].

Technical and operational features of commercial Rapid Diagnostic Tests for Typhoid diagnosis [6,9,35,40,41,68,69,83-95].

Name of testTyphidotTyphidot-MTubex-TFEnterocheck-WBMulti-Test-Dip-S-TicksTyphiRapid IgM & IgG IgM (Combo)LifeAssay TestitSD BiolineWidalMega SalmonellaPanBio
Manufacturer(Malaysian Biodiagnostic Research, Malaysia)(Malaysian Biodiagnostic Research, Malaysia)(IDL Biotech, Sollentuna, Sweden)(Zephyr Biomedicals, Goa, India)(PanBio Indx, Inc., Baltimore, MD, USA)(CTK Biotech, Inc., San Diego, CA, USA)(Life Assay Diagnostics (Pty), Africa)(Standard Diagnostics Inc., Korea)(Mega Diagnostics, CA, USA)(PanBio Indx, Inc., Baltimore, MD, USA)
TECHNICAL FEATURES
Assay formatDot Blot /ImmunodotInhibition magnetic binding assayDipstickImmuno-chromatographicLateral-flow assay (ICT LFA)Slide or Tube AssayELISA
Target of detection (antibody)IgM & IgGIgM (after IgG removal)IgMIgMIgM & IgGIgMIgMIgG/IgMIgM & IgGIgM & IgGIgM & IgG
Antigen50 kDa OMP of S. TyphiO9 antigen (a-D tyvelose) of S. TyphiLPS50 kDa OMPLPSUndefined antigenFlagellar (H) & LPS (O)Undisclosed antigenLPS
Sensitivitya67-98%47-98%56-100%89%89%89-100%59%69%Variable91%;78%
Specificitya58-100%63-93%58-100%97%53%85-89%98%79%,49%,80%,
OPERATIONAL FEATURES
Turnaround time2.5-3 hours10 minutes15-30 minutes90 minutes15-30 minutes2 hours-overnight (classical);5 minutes (rapid centrifugation)2-3 hours
Sample volume (μL)2.530-40 (one drop)30-40105-10300 (two dilutions)5-10
Infrastructure requirednonenonenonenoneCentrifuge, incubatorIncubator, Microplate ELISA reader
Storage temperature2-8°C4°C to 28°C4°C to 28°C4°C to 28°C2-8°C2-8°C
Costb ($)544104.90.54.9
Remarks1. High IgG concentration may give false negative for IgM2. Difficulty in differentiation between acute and convalescent cases.3. Qualitative assay; Quantitative determination of antibodies levels not possible1. False positives in case of Salmonella Enteritidis infection-common O9 antigen, may lead to improper treatment.2. Subjective interpretation of color reactions.3. Haemolysis may result in difficulty in interpretation1. Specific IgG may compete with the IgM for sites and may result in a false negative.2. High titer Rheumatoid factor may result in a false positive reaction3. Cross reactivity may be observed with S. paratyphi infection1. Qualitative or semi-quantitative results1. Suboptimal specificity-Cross-reactive O&H Ags.2. Lack of standardisation of reagents3. Subjective result interpretation1. Requires laboratory infrastructure & skilled personnel.2. Processing time 3-4 h
References[40,41,83-86][6,9,40,41,83,85,87][87,88][40,68,69,89,90][35,91-94][40,95]

aDenotes a range of sensitivity and specificity values from different studies with a range of sample sizes and methodologies

bThe cost of tests have been determined from the following references: [40,87]


Strategies for Improving Typhoid Diagnostics

Discovery of novel biomarkers: The specificity of the antigens used significantly affects the performance of a test, especially in endemic regions with a high background antibody titre to conserv surface antigens. Therefore, identifying specific, sero-reactive biomarkers would enable more accurate detection [57]. S.typhi expresses many factors to counter host defenses or gaining replicative niche. They may exhibit good diagnostic potential. The next-generation approaches for typhoid bio-marker identification are described below. These are also summarised in [Table/Fig-2] [18,58-73].

Next generation approaches for typhoid biomarker identification [18,58-73].

CategoryTechniqueMethodologyPotential biomarkers/ gene targets/ metabolic markersBiomarker; method; (Current development/ evaluation status)
ProteomicsIn vivo-induced antigen technology (IVIAT)Screening of S. typhi protein library expressed in Escherichia coli with convalescent-phase sera to identify immuno-reactive clonesPagC, TcfB, and STY0860 and STY3683(45)CdtB, yajI, pspB, pilL, ybgF : Detection of antigen specific IgM in plasma from patients with confirmed typhoid (93)HlyE (t1477); Detection of specific IgA, IgG, IgM by ELISA(18)HlyE; Detection of salivary anti-HlyE IgA (71)HlyE: Detection of specific antibodies by Immunoblot and ELISA(72,94)(Clinical Evaluation)
liquid chromatography-mass spectrometry (LC-MS)-based proteomics strategyIdentification of S. typhi-specific virulence factors expressed in conditions that mimic the infective state by comparative proteomics analysis with S. typhimuriumCdtB, HlyE, and gene products of t0142, t1108, t1109, t1476, and t1602(95)
Immuno-affinity Proteomics-based Technology (IPT)Capture of S. typhi proteins using columns charged with antibodies (IgM, IgG & IgA) from infected patients followed by identification of immunogenic proteins by mass spectrometryHlyE, CdtB, PltA, and STY1364(47)
Stable Isotope Labelling with Amino acids in Cell culture (SILAC) technologyQuantitative comparison of the proteomes of S. typhi and S. typhimurium for identification of serovar specific biomarkersrfbE (CDP-tyvelose-2-epimerase), and rfbV (Putative glycosyl transferase)(49)
Antibody in lymphocyte supernatant [ALS] assayIdentification of S. typhi specific antigens by probing S. typhi protein arrays with ALS of patients with confirmed S. typhi bacteraemiaHlyE, CdtB, PhoN, SthD, SthA, BcfA, HpcR, Prc, EutN, and OmpS2(53)
Immune profilingIdentification of S. typhi specific antigens by probing its proteome microarray with sera from infected patientsHemolysin E (hlyE, t1477) & putative toxin-like protein (cdtB, t1111), ssaP(t1285), pilL(t4239), pspB (t1594), yajI (t2449), ybgF(t2126)(48,72)
TranscriptomicscDNA Microarray and Selective capture of transcribed sequences (SCOTS) to identify high quality transcript profiles from intracellular bacteriaSCOTS to identify global genes expression of S. typhi during infection (human macrophages)SCOTS and microarray hybridisation to identify S. typhi transcripts expressed in the blood of patients infected with S. typhi in Bangladeshup regulated genes: STY1482, STY1353, STY1361-STY1367, STY2000-STY2002(60)STY3639, STY4609, STY4543, STY2701, STY2244, STY0417(61)Preliminary reports (Preclinical phase)
mRNA Profiling and microarrayRNA transcriptional profiling of peripheral blood to characterise host response to typhoid infection431 host transcripts abundant in acute typhoid patients (genes encoding intracellular proteins, proteins involved in innate & adaptive immunity etc.) (98)Transporter genes ABCA7, ABCC5 & ABCD4 & ATPase were enriched (99)
MetabolomicsTwo-dimensional gas chromatography with time-of-flight mass spectrometry(GCxGC/TOFMS)GCxGC/TOFMS on plasma from patients with S. typhi and S. paratyphi A infections and asymptomatic controls306 metabolites separated the controls from the S. Typhi infections (2,4dihydroxybutanoic acid, phenylalanine, and pipecolic. Etc.)(57)
GCxGC/TOFMS on plasma from patients with culture confirmed typhoid fever, clinically suspected typhoid, and other febrile diseases (malaria)24 metabolites to identify typhoid fever patients including glycerol-3-phosphate, stearic acid, linoleic acid, pyruvic acid, and creatinine(58)
GenomicsComparative Genomics and PCRGenomic comparison of S. typhi with other enteric pathogens, in silico analysis revealed 6 specific genes; STY0307, STY0201, STY0322, STY0326, STY2020, and STY20215 PCR assays using STY0307, STY0201 STY0322, STY0326, STY2020, and STY2021 were developed and found to be highly specific at single-gene target resolution(59)STY3007, STY0201, STY0207: Fluorescence polarisation based nucleic acid test (96)STY3007, STY0201; Magneto-DNA nanoparticle system (97)Preliminary reports (Preclinical phase)

Immuno-proteomics: In Vivo-Induced-Antigen Technology (IVIAT): Researchers have looked into the immunogenic antigens and S. typhi-specific antibody responses in typhoid patients using immuno-proteomics [19,20]. Harris JB et al., identified 35 immunogenic S. typhi antigens expressed specifically during infection using IVIAT [58]. A protein library of S. typhi was screened to identify immunoreactive clones using convalescent-phase sera, previously adsorbed against in vitro-grown S. typhi and E. coli. PagC, TcfB, STY1648 and STY3683 proteins were reported as S.typhi-specific. In a similar study, YncE was identified as a potential biomarker for carriers [74]. Since, IVIAT does not consider non-protein antigens and the possibility of cross-reactivity of homologous antigens, it warrants additional evaluation to assess the true role of such antigens in diagnostics.

Liquid Chromatography-Mass Spectrometry (LC-MS)-Based Proteomics (LC-MS): By utilising LC-MS based proteomics strategy, a group has characterised the S. typhi proteome under the conditions that mimic infective stage, with the goal to identify putative S. typhi specific virulence factors. Further, the comparison between the proteomes of S. typhi and S. typhimurium revealed that proteins like CdtB, HlyE, and gene products of t0142, t1108, t1109, t1476, and t1602, were unique to S. typhi [59].

Immunoaffinity Proteomics-based Technology (IPT): This involves direct screening of S. typhi lysate against columns charged with IgG, IgM, or IgA fractions, obtained from the patient’s blood [60], using IPT this group identified 57 S.typhi antigens including known immunogenic proteins (PagC, HlyE, OmpA, and GroEL) and several S. typhi and S. paratyphi A specific proteins (HlyE, CdtB, PltA, and STY1364). Isotype characterisation of antibody responses is possible, thereby, determining the actual stage of infection during which antigenic presentation occurs, like in the case of S.typhi, which has both mucosal and systemic phases of infection. Proteins including HlyE, GroEL, and DnaK were identified by the IgM fraction (acute stage), while PagC, HlyE, CdtB, PltA, PhoN, and GroEL, reacted with either acute- or convalescent-phase IgG antibody fraction. Some proteins like the PhoP-regulated protein, PhoN and bacterioferritin reacted only with convalescent-phase IgG, thus indicative of a maturing immune response.

Microarray: Liang L et al., carried out microarray immune-profiling of S. typhi proteome by probing it with sera of acute typhoid patients and controls, and identified potential IgM and IgG candidate antigens, which were differentially reactive in infected patients vs healthy controls [61]. Amongst other identified antigens, two serodiagnostic candidates; haemolysin E (hlyE) and putative toxin-like protein (CdtB) were also identified in a previous study [60].

SILAC: One group identified several serovar-specific proteins by quantitatively comparing the proteomes of S. typhi and S. typhimurium using Stable Isotope Labeling with Amino acids in Cell culture (SILAC) technology [75]. rfbE (CDP-tyvelose-2-epimerase), and rfbV (Putative glycosyl transferase) were a few S. typhi specific antigens amongst the total of 15 antigens, which warrant further assessment.

Antibody in Lymphocyte Supernatant (ALS) assay:S. typhi interacts with the intestinal mucosa and induces mucosal immune response. Activated mucosal lymphocytes migrate from intestinal tissue and circulate within peripheral blood before rehoming to mucosa [76]. This response peaks 1-2 weeks post-infection, and could be measured using Peripheral Blood Mononuclear Cells (PBMCs) in an ASC assay [77] or in PBMC supernatants (ALS assay) [62]. By probing S. typhi protein arrays with ALS of infected patients, researchers identified immuno-reactive antigens that could discriminate between healthy, acute and non-typhoidal cases [78]. Among these antigens, Haemolysin E (HlyE), a pore forming toxin, showed highest immunoreactivity. Measuring ALS response is advantageous over serum for the diagnosis of acute infection, especially in endemic regions [79].

Metabolomics: This approach involves identification of host metabolites in response to infection [63,80]. It offers a holistic snapshot of infection, as it considers the host-pathogen interactions. By utilising two-dimensional gas chromatography with time-of-flight mass spectrometry (GC/MS), on plasma samples from typhoid, paratyphoid patients, and controls, one group has identified serovar-specific metabolic biomarkers [64]. Recently, they also identified reproducible metabolite profiles, significantly discriminating S.typhi-culture-positive individuals from those with alternative febrile diseases [65]. Although, it has limitations in terms of cost-effectiveness and portability, a unique combination of identified markers could be used for developing a sensitive and field deployable point-of-care test, requiring small volumes of blood.

Transcriptomics: cDNA Microarray and Selective Capture of Transcribed Sequences (SCOTS): SCOTS is a PCR hybridisation method of capturing bacterial genes expressed in vivo under specific growth conditions by subtractive hybridisation and PCR amplification with tagged primers. Using DNA microarray and SCOT-cDNA hybridisation approach, a group has obtained high quality global transcript profile of intracellular S. typhi from infected human macrophages [66]. Similarly, one group has identified S. typhi mRNA expressed in the blood of infected patients in order to provide insight into its pathogenesis and also identify novel host-pathogen interactions [67].

mRNA profiling and microarray: A group has investigated host responses to typhoid fever by transcriptional profiling of peripheral blood of naturally infected patients, using microarray analysis, and found that typhoid fever stimulates a distinct and reproducible peripheral blood signature that is changes during treatment, convalescence and returns to normal as found in case of uninfected controls [81]. Using similar technique another group has identified host transcriptional profiles of S. typhi infected young children, to provide a detailed genomic component of immunological response, and propose insights for better diagnosis and treatment [82]. The unique transcriptomic signature has the potential to form the basis of future RDTs [83].

Genomics: Advances in the field of genomics have enabled S. typhi genome sequencing and helped in the improved understanding of the pathogen and identification of specific gene targets. However, commonly used target genes like H antigen flagellar gene (fliC-d) and Vi capsular antigen gene (viaB) etc., are not specific to S. typhi only, thus diagnostic markers that can detect pathogens at single-gene target resolution could lead to a simpler, cost-effective, and more functional DNA-based detection method due to less primers requirement for target detection. A group has identified new DNA markers specific for S.typhi by using the genome database and nucleic acid sequence alignment tools (BLASTn). The diagnostic sensitivities and specificities of the primers designed for amplifying specific gene sequences was validated using a panel of confirmed bacteria S.typhi, non-Typhi Salmonella, and non-Salmonella clinical isolates [84].

Development of new tests: A next-generation serology-based test appears to be the most simple and achievable that which could be modified to a POC format. RDT with a dipstick or immunochromatographic formats score well over conventional formats in rapidity, simplicity, requirement of minimal sample manipulation and must be considered while developing a diagnostic assay [85]. For typhoid diagnosis, TUBEX and Typhidot fare better than conventional microbiological culturing and Widal test due to their desirable operational features but are found to be unreliable in the endemic regions [9,86].

RDTs based on immunochromatographic format; TyphiDot Rapid (TR-02), Enterocheck, and OnSite Typhoid IgG/IgM Combo Rapid Test performed well upon evaluation and exhibited high diagnostic accuracy, along with operational features of rapidity, affordability, user-friendliness and easy interpretation [40,42,43]. All these tests are robust and can be used at the outpatient clinics and primary health centres requiring minimal sample volume. However, they warrant rigorous evaluation in different settings. Moreover, similar platforms can employ more specific antigens to improve their diagnostic accuracy.

Although, minimally invasive, blood can be replaced with non-invasive sample sources such as saliva, stool, and urine [30]. Salivary IgA against S. typhi antigens have been observed during acute typhoid (~19 mg/mL) [68,87], saliva is devoid of the Rh factor; thereby, improving the specificity. LPS and HlyE-specific salivary-IgA antibodies have been detected efficiently in acute infection but large-scale studies are required [69,88,89]. One study has utilised a recently published proteome microarray data [90] to determine the diagnostic utility of haemolysin E (HylE, t1477) antigen, as well as LPS for typhoid fever. The study shows that the HylE and LPS-specific IgA ELISA distinguished well between typhoid patients and healthy controls [18]. Moreover, LPS specific IgA seems to be a useful indicator of acute infection due to its transient appearance during infection. The use of IgA or LPS for diagnosis is not novel, but together they function as a good marker for acute typhoid. Additionally, Typhoid-Paratyphoid Test (TPT), which involves detection of IgA antibodies against S. typhi in patients’ blood, using ALS based assay, has shown promise. It takes advantage of infection-induced systemic migration of activated lymphocytes for the detection of antigen-specific lymphocytes in circulation [91]. The TP Test exhibited significant sensitivity (100%) and specificity (78-97%) when evaluated in Bangladesh and outperformed commonly used RDTs [19,20]. It has been used in both ELISA and immunodot format, suggesting that a lateral flow or dipstick format is possible. Although, long incubation period limits its use as a POC test, studies show that it could be performed in resource-poor regions, requiring just 1 mL of blood with minimal expertise, and lab facilities, showing its immense utility if it is developed further.

Test metrics: The test performance can be influenced by variables like population characteristics at different settings, disease prevalence and genetic variability of the host and pathogen. This also includes characteristics like the physical format, nature of antigen/antibody employed, and the skills needed to perform a test. Therefore, we need a standardised and unbiased selection process to determine the field-feasibility of a diagnostic test in a setting.

Although, there is a lack of well-defined quality standards in development and evaluations of typhoid diagnostics [92], the “ASSURED” criteria proposed by WHO [93], can be extended to typhoid diagnostics. Some key metrics that can be followed while developing superior typhoid diagnostics have been briefly described. Operationally, an ideal diagnostic test must be rapid, to allow early initiation of treatment. It must be field-deployable, cost-effective, non-invasive and require minimal infrastructure and skilled personnel. Other features such as test throughput, heat-stability of reagents, quality control, portability, simplicity of performing and evaluating the test should be assessed consistently. Technical metrics such as high diagnostic accuracy, identified by high sensitivity and specificity (100% or approaching that of the gold standard) and reproducibility are required. Furthermore, a high Negative Predictive Value (NPV) and a Positive Predictive Value (PPV) that make a test reliable and allow for accurate diagnosis are must [85].

Test evaluation: The quality of studies evaluating typhoid RDTs has been suboptimal [94]. A major barrier to the development and evaluation of new tests is the lack of an appropriate gold standard; as errors in determining sensitivity and specificity arise if the comparator is not sensitive and specific enough [95]. The highest grade of reference standard is the bone marrow culture or blood PCR in addition to the blood culture [94]. A Composite Reference Standard (CRS) could help in determining the diagnostic accuracy in the absence of a single reference standard [96]. Moreover, the establishment of bio-banks housing confirmed patient samples and its availability to researchers is essential for evaluation of new tests [97].

Conclusion

S. typhi is an immuno-modulatory pathogen which evades the host immune defenses and delays clinical presentation. This temporal gap between infection and disease acts as a confounder to early typhoid diagnosis. The transient presence of bacteria in the blood makes a positive blood culture challenging. Additionally, several other pathogens can cause symptoms clinically similar to typhoid fever, thus affecting timely and accurate diagnosis and appropriate treatment which may contribute to antibiotic resistance. High-throughout technologies have expanded the understanding of S. typhi pathogenesis. It is crucial to channel these towards diagnostic development through identification of potential biomarkers. Identified biomarkers like HlyE and CdtB have shown good diagnostic potential during preliminary evaluation. An ideal typhoid diagnostic test should be affordable, simple, rapid, non-invasive, and require minimal laboratory infrastructure, and skills. RDTs follow these metrics and could serve as ideal typhoid diagnostic tests provided desired sensitivity and specificity is achieved. Utilisation of S. typhi-specific IgA responses of typhoid patients in TP Test is another promising approach. Furthermore, alternate markers such as infection-specific metabolic signatures could form the foundation of new RDTs. However, clinical validation and rigorous field evaluations of the new tests and identified biomarkers needs to be done under harshest of conditions at the developmental stage for confirming the robustness of the test. This demands an accurate reference standard, thus necessitating the use of a composite reference standard and establishment of well-characterised bio-banks. Further, researchers, policymakers, and other stakeholders must come together to bridge the gap between the development of improved diagnostics, their evaluation, and implementation.

aDenotes a range of sensitivity and specificity values from different studies with a range of sample sizes and methodologiesbThe cost of tests have been determined from the following references: [40,87]

References

[1]Steele AD, Hay Burgess DC, Diaz Z, Carey ME, Zaidi AKM, Challenges and opportunities for typhoid fever control: a call for coordinated action Clin Infect Dis 2016 62(Suppl 1):S4-8.  [Google Scholar]

[2]Bhan MK, Bahl R, Bhatnagar S, Typhoid and paratyphoid fever Lancet 2005 366(9487):749-62.  [Google Scholar]

[3]Crump JA, Luby SP, Mintz ED, The global burden of typhoid fever Bull World Health Organ 2004 82(5):346-53.  [Google Scholar]

[4]Typhoid vaccines: WHO position paper Wkly Epidemiol Rec 2008 83(6):49-59.  [Google Scholar]

[5]Huang DB, DuPont HL, Problem pathogens: extra-intestinal complications of Salmonella enterica serotype Typhi infection Lancet Infect Dis 2005 5(6):341-48.  [Google Scholar]

[6]House D, Bishop A, Parry C, Dougan G, Wain J, Typhoid fever: pathogenesis and disease Current Opinion in Infectious Diseases 2001 14(5):573  [Google Scholar]

[7]Jepson MA, Clark MA, The role of M cells in Salmonella infection Microbes Infect 2001 3(14-15):1183-90.  [Google Scholar]

[8]Vazquez-Torres A, Jones-Carson J, Bäumler AJ, Falkow S, Valdivia R, Brown W, Extraintestinal dissemination of Salmonella by CD18-expressing phagocytes Nature 1999 401(6755):804-08.  [Google Scholar]

[9]Thriemer K, Ley B, Menten J, Jacobs J, Ende J van den, 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(12):e81263  [Google Scholar]

[10]Keddy KH, Sooka A, Letsoalo ME, Hoyland G, Chaignat CL, Morrissey AB, Sensitivity and specificity of typhoid fever rapid antibody tests for laboratory diagnosis at two sub-Saharan African sites Bull World Health Organ 2011 89(9):640-47.  [Google Scholar]

[11]Espersen F, Høiby N, Hertz JB, Cross-reactions between Salmonella typhi and 24 other bacterial species Acta Pathol Microbiol Scand B 1980 88(4):243-48.  [Google Scholar]

[12]Jin C, Gibani MM, Moore M, Juel HB, Jones E, Meiring J, Efficacy and immunogenicity of a Vi-tetanus toxoid conjugate vaccine in the prevention of typhoid fever using a controlled human infection model of Salmonella typhi: a randomised controlled, phase 2b trial Lancet 2017 390(10111):2472-80.  [Google Scholar]

[13]Koirala KD, Thanh DP, Thapa SD, Arjyal A, Karkey A, Dongol S, 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(5):2761-62.  [Google Scholar]

[14]Yan M, Li X, Liao Q, Li F, Zhang J, Kan B, The emergence and outbreak of multidrug-resistant typhoid fever in China Emerg Microbes Infect 2016 5(6):e62  [Google Scholar]

[15]Baker S, Hombach J, Marks F, What have we learned from the typhoid fever surveillance in africa program? Clin Infect Dis 2016 62(suppl_1):S01-03.  [Google Scholar]

[16]Wain J, Diep TS, Ho VA, Walsh AM, Nguyen TT, Parry CM, Quantitation of bacteria in blood of typhoid fever patients and relationship between counts and clinical features, transmissibility, and antibiotic resistance J Clin Microbiol 1998 36(6):1683-87.  [Google Scholar]

[17]Baker S, Favorov M, Dougan G, Searching for the elusive typhoid diagnostic BMC Infect Dis 2010 10:45  [Google Scholar]

[18]Felgner J, Jain A, Nakajima R, Liang L, Jasinskas A, Gotuzzo E, Development of ELISAs for diagnosis of acute typhoid fever in Nigerian children PLoS Neglected Tropical Diseases 2017 11(6):e0005679  [Google Scholar]

[19]Khanam F, Sheikh A, Sayeed MA, Bhuiyan MS, Choudhury FK, Salma U, Evaluation of a typhoid/paratyphoid diagnostic assay (TPTest) detecting anti-salmonella IgA in secretions of peripheral blood lymphocytes in patients in Dhaka, Bangladesh PLoS Neglected Tropical Diseases 2013 7(7):e2316  [Google Scholar]

[20]Islam K, Sayeed MA, Hossen E, Khanam F, Charles RC, Andrews J, Comparison of the performance of the TPTest, tubex, typhidot and widal immunodiagnostic assays and blood cultures in detecting patients with typhoid fever in Bangladesh, including using a bayesian latent class modeling approach PLoS Neglected Tropical Diseases 2016 10(4):e0004558  [Google Scholar]

[21]Biologicals WHOD of V and. Background document: the diagnosis, treatment and prevention of typhoid fever. 2003 [cited 2017 Nov 5]; Available from: http://www.who.int/iris/handle/10665/68122  [Google Scholar]

[22]Wain J, Diep TS, Bay PVB, Walsh AL, Vinh H, Duong NM, Specimens and culture media for the laboratory diagnosis of typhoid fever J Infect Dev Ctries 2008 2(6):469-74.  [Google Scholar]

[23]Massi MN, Shirakawa T, Gotoh A, Bishnu A, Hatta M, Kawabata M, Quantitative detection of Salmonella enterica serovar Typhi from blood of suspected typhoid fever patients by real-time PCR Int J Med Microbiol 2005 295(2):117-20.  [Google Scholar]

[24]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(6):1439-43.  [Google Scholar]

[25]Nizami SQ, Bhutta ZA, Siddiqui AA, Lubbad L, Enhanced detection rate of typhoid fever in children in a periurban slum in Karachi, Pakistan using polymerase chain reaction technology Scand J Clin Lab Invest 2006 66(5):429-36.  [Google Scholar]

[26]Ali A, Haque A, Haque A, Sarwar Y, Mohsin M, Bashir S, Multiplex PCR for differential diagnosis of emerging typhoidal pathogens directly from blood samples Epidemiol Infect 2009 137(1):102-07.  [Google Scholar]

[27]Nair S, Alokam S, Kothapalli S, Porwollik S, Proctor E, Choy C, Salmonella enterica serovar Typhi strains from which SPI7, a 134-kilobase island with genes for Vi exopolysaccharide and other functions, has been deleted J Bacteriol 2004 186(10):3214-23.  [Google Scholar]

[28]Prabagaran SR, Kalaiselvi V, Chandramouleeswaran N, Deepthi KNG, Brahmadathan KN, Mani M, Molecular diagnosis of Salmonella typhi and its virulence in suspected typhoid blood samples through nested multiplex PCR Journal of Microbiological Methods 2017 139:150-54.  [Google Scholar]

[29]Nga TVT, Karkey A, Dongol S, Thuy HN, Dunstan S, Holt K, The sensitivity of real-time PCR amplification targeting invasive Salmonella serovars in biological specimens BMC Infectious Diseases 2010 10:125  [Google Scholar]

[30]Chaicumpa W, Thin-Inta W, Khusmith S, Tapchaisri P, Echeverria P, Kalambaheti T, Detection with monoclonal antibody of Salmonella typhi antigen 9 in specimens from patients J Clin Microbiol 1988 26(9):1824-30.  [Google Scholar]

[31]John TJ, Sivadasan K, Kurien B, Evaluation of passive bacterial agglutination for the diagnosis of typhoid fever J Clin Microbiol 1984 20(4):751-53.  [Google Scholar]

[32]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(9):2513-15.  [Google Scholar]

[33]Fadeel MA, Crump JA, Mahoney FJ, Nakhla IA, Mansour AM, Reyad B, 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(3):323-28.  [Google Scholar]

[34]Mukherjee C, Malik A, Khan HM, Malik A, Rapid diagnosis of typhoid fever by co-agglutination in an Indian hospital J Med Microbiol 1993 39(1):74-77.  [Google Scholar]

[35]Olopoenia LA, King AL, Widal agglutination test-100 years later: still plagued by controversy Postgraduate Medical Journal 2000 76(892):80-84.  [Google Scholar]

[36]House D, Chinh NT, Diep TS, Parry CM, Wain J, Dougan G, Use of paired serum samples for serodiagnosis of typhoid fever J Clin Microbiol 2005 43(9):4889-90.  [Google Scholar]

[37]Dutta S, Sur D, Manna B, Sen B, Deb AK, Deen JL, Evaluation of new-generation serologic tests for the diagnosis of typhoid fever: data from a community-based surveillance in Calcutta, India Diagnostic Microbiology and Infectious Disease 2006 56(4):359-65.  [Google Scholar]

[38]Biologicals WHOD of V and. Background document: the diagnosis, treatment and prevention of typhoid fever. 2003 [cited 2016 Dec 1]; Available from: http://www.who.int/iris/handle/10665/68122  [Google Scholar]

[39]Jackson AA, Ismail A, Ibrahim TA, Kader ZS, Nawi NM, Retrospective review of dot enzyme immunoassay test for typhoid fever in an endemic area Southeast Asian J Trop Med Public Health 1995 26(4):625-30.  [Google Scholar]

[40]Kawano RL, Leano SA, Agdamag DMA, Comparison of Serological test kits for diagnosis of typhoid fever in the Philippines J Clin Microbiol 2007 45(1):246-47.  [Google Scholar]

[41]Naheed A, Ram PK, Brooks WA, Mintz ED, Hossain MA, Parsons MM, Clinical value of TubexTM and Typhidot® rapid diagnostic tests for typhoid fever in an urban community clinic in Bangladesh Diagnostic Microbiology and Infectious Disease 2008 61(4):381-86.  [Google Scholar]

[42]Siba V, Horwood PF, Vanuga K, Wapling J, Sehuko R, Siba PM, Evaluation of serological diagnostic tests for typhoid fever in papua new guinea using a composite reference standard Clin Vaccine Immunol 2012 19(11):1833-37.  [Google Scholar]

[43]Tarupiwa A, Tapera S, Mtapuri-Zinyowera S, Gumbo P, Ruhanya V, Gudza-Mugabe M, Evaluation of TUBEX-TF and OnSite Typhoid IgG/IgM Combo rapid tests to detect Salmonella enterica serovar Typhi infection during a typhoid outbreak in Harare, Zimbabwe BMC Research Notes 2015 8:50  [Google Scholar]

[44]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(4):654-57.  [Google Scholar]

[45]Choo KE, Davis TM, Ismail A, Tuan Ibrahim TA, Ghazali WN, Rapid and reliable serological diagnosis of enteric fever: comparative sensitivity and specificity of Typhidot and Typhidot-M tests in febrile Malaysian children Acta Trop 1999 72(2):175-83.  [Google Scholar]

[46]Dong B, Galindo CM, Shin E, Acosta CJ, Page AL, Wang M, Optimizing typhoid fever case definitions by combining serological tests in a large population study in Hechi City, China Epidemiol Infect 2007 135(6):1014-20.  [Google Scholar]

[47]Olsen SJ, Bleasdale SC, Magnano AR, Landrigan C, Holland BH, Tauxe RV, Outbreaks of typhoid fever in the United States, 1960-99 Epidemiol Infect 2003 130(1):13-21.  [Google Scholar]

[48]Olsen SJ, Pruckler J, Bibb W, Thanh NTM, Trinh TM, Minh NT, Evaluation of rapid diagnostic tests for typhoid fever J Clin Microbiol 2004 42(5):1885-89.  [Google Scholar]

[49]Anusha R, Ganesh R, Lalitha J, Comparison of a rapid commercial test, Enterocheck WB(®), with automated blood culture for diagnosis of typhoid fever Ann Trop Paediatr 2011 31(3):231-34.  [Google Scholar]

[50]Moore CE, Pan-Ngum W, Wijedoru LPM, Sona S, Nga TVT, Duy PT, Evaluation of the diagnostic accuracy of a typhoid IgM flow assay for the diagnosis of typhoid fever in cambodian children using a Bayesian latent class model assuming an imperfect gold standard Am J Trop Med Hyg 2014 90(1):114-20.  [Google Scholar]

[51]Pastoor R, Hatta M, Abdoel TH, Smits HL, Simple, rapid, and affordable point-of-care test for the serodiagnosis of typhoid fever Diagn Microbiol Infect Dis 2008 61(2):129-34.  [Google Scholar]

[52]Levine MM, Grados O, Gilman RH, Woodward WE, Solis-Plaza R, Waldman W, Diagnostic value of the Widal test in areas endemic for typhoid fever Am J Trop Med Hyg 1978 27(4):795-800.  [Google Scholar]

[53]Ley B, Mtove G, Thriemer K, Amos B, von Seidlein L, Hendriksen I, Evaluation of the Widal tube agglutination test for the diagnosis of typhoid fever among children admitted to a rural hospital in Tanzania and a comparison with previous studies BMC Infect Dis 2010 10:180  [Google Scholar]

[54]Lunguya O, Phoba MF, Mundeke SA, Bonebe E, Mukadi P, Muyembe JJ, The diagnosis of typhoid fever in the Democratic Republic of the Congo Trans R Soc Trop Med Hyg 2012 106(6):348-55.  [Google Scholar]

[55]Parry CM, Hoa NTT, Diep TS, Wain J, Chinh NT, Vinh H, Value of a single-tube widal test in diagnosis of typhoid fever in Vietnam J Clin Microbiol 1999 37(9):2882-86.  [Google Scholar]

[56]Gopalakrishnan V, Sekhar WY, Soo EH, Vinsent RA, Devi S, Typhoid fever in Kuala Lumpur and a comparative evaluation of two commercial diagnostic kits for the detection of antibodies to Salmonella typhi Singapore Med J 2002 43(7):354-58.  [Google Scholar]

[57]Liang L, Felgner PL, A systems biology approach for diagnostic and vaccine antigen discovery in tropical infectious diseases Curr Opin Infect Dis 2015 28(5):438-45.  [Google Scholar]

[58]Harris JB, Baresch-Bernal A, Rollins SM, Alam A, LaRocque RC, Bikowski M, Identification of in vivo-induced bacterial protein antigens during human infection with Salmonella enterica serovar Typhi Infect Immun 2006 74(9):5161-68.  [Google Scholar]

[59]Ansong C, Yoon H, Norbeck AD, Gustin JK, McDermott JE, Mottaz HM, Proteomics analysis of the causative agent of typhoid fever J Proteome Res 2008 7(2):546-57.  [Google Scholar]

[60]Charles RC, Sheikh A, Krastins B, Harris JB, Bhuiyan MS, LaRocque RC, Characterization of anti-Salmonella enterica serotype Typhi antibody responses in bacteremic Bangladeshi patients by an immunoaffinity proteomics-based technology Clin Vaccine Immunol 2010 17(8):1188-95.  [Google Scholar]

[61]Liang L, Juarez S, Nga TVT, Dunstan S, Nakajima-Sasaki R, Davies DH, Immune profiling with a Salmonella typhi antigen microarray identifies new diagnostic biomarkers of human typhoid Sci Rep [Internet] 2013 Jan 9 :3Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3540400/  [Google Scholar]

[62]Chang HS, Sack DA, Development of a novel in vitro assay (ALS assay) for evaluation of vaccine-induced antibody secretion from circulating mucosal lymphocytes Clin Diagn Lab Immunol 2001 8(3):482-88.  [Google Scholar]

[63]Preez I du, Loots DT, New sputum metabolite markers implicating adaptations of the host to Mycobacterium tuberculosis, and vice versa Tuberculosis 2013 93(3):330-37.  [Google Scholar]

[64]Näsström E, Thieu NTV, Dongol S, Karkey A, Vinh PV, Thanh TH, Salmonella typhi and Salmonella paratyphi A elaborate distinct systemic metabolite signatures during enteric fever e Life Sciences 2014 3:e03100  [Google Scholar]

[65]Näsström E, Parry CM, Thieu NTV, Maude RR, Jong HK de, Fukushima M, Reproducible diagnostic metabolites in plasma from typhoid fever patients in Asia and Africa e Life Sciences 2017 6:e15651  [Google Scholar]

[66]Faucher SP, Porwollik S, Dozois CM, McClelland M, Daigle F, Transcriptome of Salmonella enterica serovar Typhi within macrophages revealed through the selective capture of transcribed sequences Proc Natl Acad Sci USA 2006 103(6):1906-11.  [Google Scholar]

[67]Sheikh A, Charles RC, Sharmeen N, Rollins SM, Harris JB, Bhuiyan MS, In vivo expression of Salmonella enterica serotype typhi genes in the blood of patients with typhoid fever in Bangladesh PLOS Neglected Tropical Diseases 2011 5(12):e1419  [Google Scholar]

[68]Chau PY, Tsang RS, Lam SK, La Brooy JT, Rowley D, Antibody response to the lipopolysaccharide and protein antigens of Salmonella typhi during typhoid infection. II. Measurement of intestinal antibodies by radioimmunoassay Clin Exp Immunol 1981 46(3):515-20.  [Google Scholar]

[69]Herath HMTU, Early diagnosis of typhoid fever by the detection of salivary IgA J Clin Pathol 2003 56(9):694-98.  [Google Scholar]

[70]Tran Vu Thieu N, Trinh Van T, Tran Tuan A, Klemm EJ, Nguyen Ngoc Minh C, Voong Vinh P, An evaluation of purified Salmonella typhi protein antigens for the serological diagnosis of acute typhoid fever Journal of Infection [Internet] Available from: http://www.sciencedirect.com/science/article/pii/S016344531730141X  [Google Scholar]

[71]Ong EBB, Anthony AA, Ismail A, Ismail A, Lim TS, Cloning, expression, and purification of the hemolysin/cytolysin (HlyE antigen) from Salmonella enterica serovar Typhi: potential application for immunoassay development Diagn Microbiol Infect Dis 2013 77(1):87-89.  [Google Scholar]

[72]Park KS, Charles RC, Ryan ET, Weissleder R, Lee H, Fluorescence polarization-based nucleic acid testing for rapid and cost-effective diagnosis of infectious disease Chemistry 2015 21(46):16359-63.  [Google Scholar]

[73]Park KS, Chung HJ, Khanam F, Lee H, Rashu R, Bhuiyan MT, A magneto-DNA nanoparticle system for the rapid and sensitive diagnosis of enteric fever Sci Rep [Internet] 2016 :6Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5015101/  [Google Scholar]

[74]Charles RC, Sultana T, Alam MM, Yu Y, Wu-Freeman Y, Bufano MK, Identification of Immunogenic salmonella enterica serotype typhi antigens expressed in chronic biliary carriers of S. typhi in Kathmandu, Nepal PLOS Neglected Tropical Diseases 2013 7(8):e2335  [Google Scholar]

[75]Wang Y, Huang KY, Huo Y, Proteomic comparison between Salmonella typhimurium and Salmonella typhi J Microbiol 2014 52(1):71-76.  [Google Scholar]

[76]Kantele JM, Arvilommi H, Kontiainen S, Salmi M, Jalkanen S, Savilahti E, Mucosally activated circulating human B cells in diarrhea express homing receptors directing them back to the gut Gastroenterology 1996 110(4):1061-67.  [Google Scholar]

[77]Kantele A, Peripheral blood antibody-secreting cells in the evaluation of the immune response to an oral vaccine Journal of Biotechnology 1996 44(1):217-24.  [Google Scholar]

[78]Charles RC, Liang L, Khanam F, Sayeed MA, Hung C, Leung DT, Immunoproteomic analysis of antibody in lymphocyte supernatant in patients with typhoid fever in Bangladesh Clin Vaccine Immunol 2014 21(3):280-85.  [Google Scholar]

[79]Carter MJ, Mitchell RM, Meyer Sauteur PM, Kelly DF, Trück J, The antibody-secreting cell response to infection: kinetics and clinical applications Front Immunol [Internet] 2017 :8Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451496/  [Google Scholar]

[80]Langley RJ, Tsalik EL, Velkinburgh JC van, Glickman SW, Rice BJ, Wang C, An integrated clinico-metabolomic model improves prediction of death in sepsis Science Translational Medicine 2013 5(195):195ra95-195ra95.  [Google Scholar]

[81]Thompson LJ, Dunstan SJ, Dolecek C, Perkins T, House D, Dougan G, Transcriptional response in the peripheral blood of patients infected with Salmonella enterica serovar Typhi Proc Natl Acad Sci USA 2009 106(52):22433-38.  [Google Scholar]

[82]Khoo SK, Petillo D, Parida M, Tan AC, Resau JH, Obaro SK, Host response transcriptional profiling reveals extracellular components and ABC (ATP-binding cassette) transporters gene enrichment in typhoid fever-infected Nigerian children BMC Infect Dis 2011 11:241  [Google Scholar]

[83]Herberg JA, Kaforou M, Wright VJ, Shailes H, Eleftherohorinou H, Hoggart CJ, Diagnostic test accuracy of a 2-transcript host RNA signature for discriminating bacterial vs viral infection in febrile children JAMA 2016 316(8):835-45.  [Google Scholar]

[84]Goay YX, Chin KL, Tan CLL, Yeoh CY, Ja’afar JN, Zaidah AR, Identification of five novel Salmonella typhi-specific genes as markers for diagnosis of typhoid fever using single-gene target PCR assays Biomed Res Int [Internet] 2016 2016:8905675Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5126401/  [Google Scholar]

[85]Miller E, Sikes HD, Addressing barriers to the development and adoption of rapid diagnostic tests in global health Nanobiomedicine (Rij) [Internet] 2015 2:6Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4652944/  [Google Scholar]

[86]Keddy KH, Sooka A, Letsoalo ME, Hoyland G, Chaignat CL, Morrissey AB, Sensitivity and specificity of typhoid fever rapid antibody tests for laboratory diagnosis at two sub-Saharan African sites Bull World Health Organ 2011 89(9):640-47.  [Google Scholar]

[87]Jenkins GN, The physiology and biochemistry of the mouth 1978 4th edOxford, PhiladelphiaBlackwell Scientific Publications:viii:599  [Google Scholar]

[88]Zaka-ur-Rab Z, Abqari S, Shahab T, Islam N, Shukla I, Evaluation of salivary anti-Salmonella typhi lipopolysaccharide IgA ELISA for serodiagnosis of typhoid fever in children Archives of Disease in Childhood 2012 97(3):236-38.  [Google Scholar]

[89]Chin KL, Redhuan NEM, Balaram P, Phua KK, Ong EBB, Detection of salivary IgA antibodies against the HlyE antigen as a diagnosis of typhoid fever J Clin Diagn Res 2016 10(6):DM01-03.  [Google Scholar]

[90]Davies DH, Jain A, Nakajima R, Liang L, Jasinskis A, Supnet M, Serodiagnosis of acute typhoid fever in Nigerian pediatric cases by detection of serum IgA and IgG against hemolysin E and lipopolysaccharide Am J Trop Med Hyg 2016 95(2):431-39.  [Google Scholar]

[91]Sheikh A, Bhuiyan MS, Khanam F, Chowdhury F, Saha A, Ahmed D, 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(11):1587-94.  [Google Scholar]

[92]Andrews JR, Ryan ET, Diagnostics for invasive Salmonella infections: current challenges and future directions Vaccine 2015 33(0 3):C8-15.  [Google Scholar]

[93]Mabey D, Peeling RW, Ustianowski A, Perkins MD, Diagnostics for the developing world Nat Rev Microbiol 2004 2(3):231-40.  [Google Scholar]

[94]Wijedoru L, Mallett S, Parry CM, Rapid diagnostic tests for typhoid and paratyphoid (enteric) fever Cochrane Database Syst Rev 2017 5:CD008892  [Google Scholar]

[95]TDR Diagnostics Evaluation Expert PanelBanoo S, Bell D, Bossuyt P, Herring A, Mabey D, Evaluation of diagnostic tests for infectious diseases: general principles Nat Rev Microbiol 2010 8(12 Suppl):S17-29.  [Google Scholar]

[96]Storey HL, Huang Y, Crudder C, Golden A, de los Santos T, Hawkins K, A Meta-Analysis of Typhoid diagnostic accuracy studies: a recommendation to adopt a standardized composite reference PLoS ONE 2015 10(11):e0142364  [Google Scholar]

[97]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(6):711-25.  [Google Scholar]