The present invention relates to novel biomarker combinations for use in the diagnosis of typhoid fever. In particular, to the measurement of the relative levels or concentrations of antibodies to specific targets in a sample obtained from a subject.

Enteric (typhoid) fever is a systemic infection caused by Salmonella enterica serovars Typhi (S. Typhi) and Paratyphi A (S . Paratyphi A). There are an estimated 20 million cases of typhoid fever (caused by S . Typhi only) worldwide annually leading to approximately 200,000 deaths. The organisms are transmitted via the faecal-oral route and the disease remains common in low/middle income countries in South/Southeast Asia and sub-Saharan Africa, where sanitary conditions facilitate transmission. Despite S . Paratyphi A being an emergent cause of typhoid fever in parts of South and Southeast Asia, S . Typhi (typhoid fever) remains the most commonly reported etiological agent of enteric fever in Asia and Africa.

Typhoid fever occurs only in humans, making it a disease that can technically be eradicated. Indeed, typhoid fever has all but been eliminated from several countries in Southeast Asia where it was the most common cause of hospitalised febrile disease 20-30 years ago. Elimination in these areas is generally attributed to extensive improvements in sanitation rather than en masse immunization programmes. Limited data regarding the long-term impact of mass immunization for typhoid fever and the performance of licensed vaccines has hindered immunization as a sustainable typhoid control and elimination strategy. The future consideration of rational control measures for typhoid fever (including the introduction of new conjugate vaccines) will rely on accurately assessing disease burden, which requires a reliable diagnostic approach.

All commonly used typhoid diagnostics perform poorly and are a roadblock for disease control efforts. Currently, the only reliable method for the identification of febrile individuals with typhoid fever is the culture of a causative organism from a sterile biological specimen. Therefore, blood culture is the universal gold standard for typhoid fever diagnostics. However, this procedure is restricted to laboratories with adequate equipment and microbiology training, and the method has a limited sensitivity as a consequence of a low concentration of organisms in peripheral circulation (< l CFU/mL). This bacterial load issue has a similar impact on other methods that detect physical evidence of the infecting organisms (such as antigen or nucleic acid) . These methods are often reported to be highly sensitive, but have a non- physiological performance; pretreatment with antimicrobials is likely to compound this issue further. New typhoid diagnostics are a necessity and various approaches have been evaluated, including innate immune responses, antibody in lymphocyte supernatants (ALS), and the identification of metabolomic signatures. However, these approaches are still restricted to research laboratories and are not yet ready to be developed into simple, rapid diagnostic tests (RDT).

A further approach for typhoid diagnostics is the use of serological assays, i.e. the detection of antibody against the infecting pathogen. A range of serological RDTs to identify patients with typhoid fever are available, these tests generally detect antibody against Salmonella flagellin (H-antigen) or Lipopolysaccharide (LPS; O-antigen). However, these antigens are highly cross-reactive with other members of the Enterobacteriaceae, giving less than desirable operational levels of sensitivity and specificity for typhoid fever diagnosis. These data suggest that the main obstacles for developing new serological diagnostic tests for typhoid fever is a lack of understanding regarding the short-term antibody responses during infection and the identification of organism specific antigens.

An aim of the present invention is to provide alternative and more specific markers for use in the detection of typhoid fever. According to a first aspect the present invention provides a method of determining the typhoid status of a subject comprising:

a) obtaining a sample provided by a subject;

b) screening the sample for the presence of antibodies to the expression product of one or more genes selected from the group consisting of STY0452 (SEQ ID NO: 1), STY0796 (SEQ ID NO: 2). STY 1086 (SEQ ID NO: 3), STY 1372 (SEQ ID NO: 4), STY 1612 (SEQ ID NO: 5), STY4539 (SEQ ID NO: 6), STY 1522 (SEQ ID NO: 7), STY 1703 (SEQ ID NO: 8), STY 1767 (SEQ ID NO: 9), STY 1886 (SEQ ID NO : 10), STY3208 (SEQ ID NO : 1 1) and STY4910 (SEQ ID NO: 12);

c) screening the sample for the presence of antibodies to the Vi polysaccharide; and d) determining if the levels of antibody determined in (b) and (c) are diagnostic of typhoid fever.

The Vi polysaccharide of Salmonella Typhi is a linear homopolymer of poly- alpha( l→4)GalNAcp variably O acetylated at the C-3 position.

Preferably the method of the invention is for determining whether a subject has typhoid fever. Preferably the method of the invention is for determining whether a subject has an active S . Typhi infection. In a preferred embodiment, in step (b) the presence of antibodies to the expression product of at least one of STY4539 (PilL), STY1886 (CdtB) and STY 1703 are determined. In another embodiment the presence of antibodies to the expression product of STY4539 (PilL) and STY1886 (CdtB) are determined in (b). In another embodiment the presence of antibodies to the expression product of STY 1703 and STY 1886 (CdtB) are determined in (b).

In steps (b) and (c) the antibody titre to a specific antigen may be determined, rather than just the absence or presence of antibodies directed to a specific antigen. In step (d) the levels observed in (b) and (c) may be compared to a control. The control may be the levels of antibodies observed in the local population who do not have typhoid disease.

The method may be used to detect IgM antibodies.

The level of antibody may be determined by any appropriate method, including immunoassay, spectrometry, western blot, ELISA, immunoprecipitation, isoelectric focusing, SDS-PAGE, radioimmunoassay (RIA), fluoroimmunoassay, surface enhanced Raman spectroscopy (SERS), or combinations thereof.

The presence, and optionally the level, of the antibody biomarkers in a sample may be determined by using epitopes from the expression products of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and the Vi polysaccharide or an antigenic part therof. The expression products of the genes are preferably proteins.

The sample may be a sample of blood, serum, plasma, sputum or saliva obtained from a subject. Preferably the method of the invention does not include the step of obtaining the sample .

The sample may be obtained from a mammal, the mammal may be human. Preferably the method of the invention is carried out in vitro.

By using the novel biomarker combination of the invention a sensitivity of at least 80%, preferably at least 85%, preferably at least 90%, for the detection of typhoid fever can be achieved. Sensitivity measures the proportion of positives that are correctly identified as such (e.g., the percentage of sick people who are correctly identified as having typhoid fever), specificity measures the proportion of negatives that are correctly identified as such (e.g., the percentage of healthy people who are correctly identified as not having typhoid fever). One advantage of the method of the invention is that it is much more accurate than current methods to detect typhoid fever. This means that patients can be given the optimal treatment and that the unnecessary use of antimicrobials can be reduced.

The method of the invention also has the advantage that areas where typhoid is occurring can be identified and targeted immunisation programmes can be introduced. The method also allow the burden of the disease to more accurately assessed.

The method of the invention may be used to triage patients so that only those with typhoid fever are treated for typhoid fever. This will reduce the unnecessary administration of antibiotics to patients who do not have typhoid fever.

The method of the invention may also be used in combination with markers for other diseases, for example, markers for dengue fever and/or malaria and/or for any other diseases that present in a manner similar to typhoid fever. According to a further aspect the invention provides a biomarker panel for determining the typhoid status of a subject, wherein the panel comprises antibodies to the expression product of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and antibodies to the Vi polysaccharide.

According to a yet further aspect the invention provides a method for detecting typhoid in an in vitro sample obtained from a subject, wherein the sample is contacted with a solid-state device onto which has been immobilised probes derived from the expression products of one or more of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and the Vi polysaccharide. The probes may be proteins or peptides derived from the expression product of STY4539 (PilL) and/or STY1886 (CdtB) and/or STY 1703, preferably from STY4539 (PilL) and/or STY 1886 (CdtB).

The solid-state device may comprise a substrate having a probe or multiple different probes immobilised upon it that bind specifically to the biomarkers being screened for. The probe may be a protein or a polypeptide, or polysaccharide.

The device may also comprise probes specific for other diseases, for example, dengue fever and/or malaria.

The substrate may be any surface able to support one or more probes. It may be polymer based, metallic, ceramic or any other suitable material.

According to a further aspect, the invention provides a method of detecting antibodies to the expression product of one or more genes selected from the group consisting of

STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703,

STY 1767, STY 1886, STY3208 and STY4910; and antibodies to the Vi polysaccharide, said method comprising:

a) obtaining a sample provided by a subject;

b) detecting whether antibodies to the expression product of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and antibodies to the Vi polysaccharide are present in the sample;

wherein the antibodies are detected by contacting the sample with the expression product or peptides derived from the expression of product of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and Vi polysaccharide or an antigenic fragment of the Vi polysaccharide; and

detecting binding between the expression product or peptide derived therefrom of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and the Vi polysaccharide or a fragment thereof; and antibodies in the sample.

According to a yet further aspect, the invention provides a method of diagnosing typhoid fever in a patient, said method comprising

a) obtaining a sample from a subject;

b) detecting whether antibodies to the expression product of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372,

STY 1612, STY4539, STY1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and antibodies to the Vi polysaccharide or a fragment thereof are present in the sample;

wherein the antibodies are detected by contacting the sample with the expression product or peptides derived from the expression of product of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and Vi polysaccharide or an antigenic fragment of the Vi polysaccharide; and

detecting binding between the expression product or peptide derived therefrom of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and the Vi polysaccharide or a fragment thereof; and antibodies in the sample; and

c) diagnosing the patient has typhoid fever when levels of antibodies to the expression product or peptide derived therefrom of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and the level of antiobodies to the Vi polysaccharide or an antigenic fragment of the Vi polysaccharide are above that in a control.

According to a another aspect, the invention provides a method of diagnosing and treating typhoid fever in a subjecr, said method comprising

a) obtaining a sample from a subject;

b) detecting whether antibodies to the expression product of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and antibodies to the Vi polysaccharide or a fragment thereof are present in the sample;

wherein the antibodies are detected by contacting the sample with the expression product or peptides derived from the expression of product of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and Vi polysaccharide or an antigenic fragment of the Vi polysaccharide; and

c) diagnosing the patient has typhoid fever by comparing the levels of antibodies to the expression product or peptide derived therefrom of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and the level of antibodies to the Vi polysaccharide or an antigenic fragment of the Vi polysaccharides; observed in the sample with a control; and

c) diagnosing the patient has typhoid fever when levels of antibodies to the expression product or peptide derived therefrom of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and the level of antiobodies to the Vi polysaccharide or an antigenic fragment of the Vi polysaccharide are above that in a control; and

d) administering an effective amount of an antibiotic to the subject. Preferably if a subject is found to have typhoid fever they will be treated with an antibiotic, preferably fluoroquinolone and/or azithromycin.

In another aspect of the invention the biomarker concentration values, or antibody titres, determined in the method of the invention are inputted into a statistical methodology to produce an output value that correlates with the chances that the patient has typhoid fever. Preferably the statistical method considers the expected levels of the biomarkers that would be observed in a subject without typhoid fever. This may be based on levels seen in a control local population of subjects that do not have typhoid fever as determined by other means.

Preferably, the statistical methodology used is logistic regression, decision trees, support vector machines, neural networks, random forest or another machine-learning algorithm. The performance of the results of the applied statistical methods used in accordance with the present invention can be best described by their receiver operating characteristics (ROC). The ROC curve addresses both the sensitivity (the number of true positives) and the specificity (the number of true negatives) of the test. Therefore, sensitivity and specificity values for a given combination of biomarkers are an indication of the performance of the test. For example, if a biomarker combination has a sensitivity and specificity value of 85 %, this means that out of 100 patients, 85 will be correctly identified/diagnosed from the determination of the presence of the particular combination of biomarkers as positive for the disease, while out of 100 patients who do not have the disease 85 will accurately test negative for the disease . A suitable statistical classification model, such as logistic regression, can be derived for a combination of biomarkers. Moreover, the logistic regression equation can be extended to include other (clinical) variables such as age and gender of the patient. In the same manner as described before, the ROC curve can be used to access the performance of the discrimination between patients and controls by the logistic regression model. Therefore, the logistic regression equation can be used apart or combined with other clinical characteristics to aid clinical decision making. Although a logistic regression equation is a common statistical procedure used in such cases and may be used in the context of the current invention, other mathematical/statistical, decision trees or machine learning procedures can also be used. One convenient goal to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single number. The most common global measure is the area under the curve (AUC) of the ROC plot. The area under the ROC curve is a measure of the probability that the perceived measurement will allow correct identification of a condition. Values typically range between 1.0 (perfect separation of the test values of the two groups) and 0.5 (no apparent distributional difference between the two groups of test values), any result below 0.5 is considered to be worse than a random guess. The area does not depend only on a particular portion of the plot such as the point closest to the diagonal or the sensitivity at 90% specificity, but on the entire plot. This is a quantitative, descriptive expression of how close the ROC plot is to the perfect one (area = 1 .0) . In the context of the present invention, the two different conditions can be whether a patient has or does not have typhoid fever.

In a preferred embodiment, the step of comparing the antibody levels in a sample with a control can comprise querying a database of reference levels of the test biomarkers from a plurality of reference samples, wherein the database of known antibody levels comprises the antibody levels of at least the test biomarkers for samples of bodily fluids taken from subjects diagnosed with typhoid disease and/or with samples from subjects who do not have typhoid. Preferably the database comprises samples from at least 10 subjects with typhoid and 10 without. Preferably the data base is queried by undertaking statistical analysis, such as a multivariate statistical analysis, preferably a least squares fit analysis such as a partial least squares regression analysis and more preferably a partial least squares discriminant analysis, of the known reference antibody levels against the antibody levels in the sample from the subject being tested/studied.

In further complimentary embodiments, at least one reference group antibody profile may be obtained by: analyzing reference samples obtained from patients with a diagnosis of typhoid or patients who do not have typhoid, measuring the reference samples for the reference antibody levels of at least the test biomarkers; undertaking statistical analysis of the reference antibody levels of each reference sample relative to the reference antibody levels of the ensemble of reference samples to determine a relative antibody profile for each reference sample; and generating a reference group antibody profile by mapping the relative antibody profile of each reference sample to the known typhoid status of each reference sample. Additionally, the step of calculating a statistical score may further comprise : undertaking statistical analysis of the antibody levels of the test biomarkers in the test sample relative to the antibody levels of the ensemble of reference samples to determine a relative test antibody profile for the test sample; and determining the statistical fit between the relative test antibody profile and the relative antibody profile and assigning a statistical score based on the statistical fit.

By referencing the obtained antibody levels with a database of reference samples of known reference antibody levels and known diagnoses, statistically relevant patterns may be drawn between the obtained antibody levels and the reference antibody levels. In essence, each newly obtained series of antibody levels of the test biomarkers is compared to the reference antibody levels for the test biomarkers and mapped onto the statistical distribution obtained between the reference antibody levels and profiles and the diagnosis associated with each reference antibody level and profile . This allows a statistical score to be attributed to the obtained test sample relative to the reference samples or diagnoses used to generate the reference antibody levels and profiles. For example, a higher score may represent an increased likelihood that the test sample is from a subject suffering from typhoid. The score may be a value that indicates how closely the test sample conforms to the antibody profile of the determined diagnosis.

The statistical analysis may use partial least squares fit techniques and optionally or preferably may use partial least squares discriminant analysis. The statistical analysis may be a multivariate statistical analysis. The statistical analysis may interrogate the internal correlation structure and relative antibody levels between the at least three biomarkers However, other statistical techniques may be used relevant to the regression analysis performed. It can be appreciated that different statistical techniques may provide differing confidence intervals. The choice of statistical technique is typically dependent upon the suitability of the data.

According to a further aspect of the present invention, there is provided a system for calculating the probability that a subject has typhoid, the system comprising: a test sample of bodily fluid obtained from a subject; a panel of test biomarkers; a processor for undertaking statistical analysis on the antibody levels of the biomarkers from the panel; a database containing one or more reference group expression profiles; and an output device for signalling the results of the statistical analysis, wherein the processor determines a statistical score based on a comparison between the antibody levels of the panel of test biomarkers in the test sample and the reference group antibody profiles representing the probability that the antibody levels of the test biomarkers in the test sample diagnose typhoid.

The system may be used to assist in the diagnosis, prediction or monitoring of typhoid and may also form part of a test available to medical practitioners to assist diagnosis and monitor disease progression.

According to yet further aspect the method of the invention may be used to monitor the progression of typhoid fever in a subject or to monitor the response of a subject to treatment for typhoid fever. According to a further aspect the invention may provide a kit for determining the typhoid status of subject, wherein the kit comprises the expression product or peptides derived from the expression of product of one or more genes selected from the group consisting of STY0452, STY0796. STY 1086, STY 1372, STY 1612, STY4539, STY 1522, STY 1703, STY 1767, STY 1886, STY3208 and STY4910; and the Vi polysaccharide or an antigenic fragment of the Vi polysaccharide. The peptides and polysaccharide or fragments thereof may be on a chip for high throughput screening. The kit could comprise a multi-well plate or microfluidic card or multi-plex chip prepared with reagents to capture and quantify the markers constituting the biomarker panel or fingerprint, as well as a database containing disease reference profiles and a computer module facilitating comparison of the test results with the reference panel using appropriate statistics. Equipment needed to read the plate or microfluidic card or chip would be standard high throughput laboratory equipment such as Luminex or Mesoscale Discovery or quantitative PCR or microarray platforms. The kit may comprise instructions for suitable operational parameters in the form of a label or separate insert. The instructions may inform a consumer about how to collect the sample . The skilled person will understand that optional features of one embodiment or aspect of the invention may be applicable, where appropriate, to other embodiments or aspects of the invention. Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying figures.

Figure 1 - depicts Table 1 which includes details of the plasmid constructs of Salmonella Typhi antigens generated and used in this invention.

Figure 2 - depicts Table 2 which includes details of the Salmonella Typhi antigens expressed in this study for serological testing.

Figure 3 - depicts Table 3 which includes details of the sensitivity and specificity of multiple antigens for typhoid fever diagnosis using blood culture positive patients only as positive reference group (n= 19) .

Figure 4 - depicts Table 4 which includes details of the sensitivity and specificity of multiple antigens for typhoid fever diagnosis using blood culture and PCR positive patients as positive reference group (n=32).

Figure 5 - illustrates the correlation of IgM measurements between Salmonella Typhi antigens. The data presented is a representative selection of the data obtained which shows a correlation in IgM measurements in human plasma for the antigens encoded by STY4539, STY 1703, STY 1886 and the Vi antigen.

The histograms, on the diagonal, show the distribution of IgM levels by optical density to the highlighted S. Typhi antigen. The scatterplot, above the histogram, plots IgM measurements between the two antigens on a right angle to the scatterplot and describes the correlation between antibody responses to two selected antigens. The numerals below the histogram depict the Spearman correlation coefficient (rho) values of the mirrored scatterplot.

Figure 6 - illustrates IgM responses against Salmonella Typhi antigens in a Bangladeshi cohort of febrile patients and controls. The boxplots show IgM measurements (optical density) in plasma in afebrile controls (light grey), febrile patients with an infection other than typhoid fever (medium grey) and typhoid patients (dark grey). Dark horizontal lines represent the mean, with the box representing the 25th and 75th percentiles, whiskers represent the 5th and 95th percentiles, and outliers are represented by dots. Figure 6 A shows boxplots of antibody responses against (in order) STY0452, STY0769,

STY 1086, STY 1372, STY 1612, STY 1522, STY 1703, STY 1767, STY3208 and STY4190. All mean antibody measurements are statistically significant between the healthy controls and typhoid infections and between other infections and typhoid infections, with the exception of STY 1522 (/?<0.05 Wilcoxon Rank sum). Figure 6B shows boxplots of antibody responses against

(in order) STY4539, STY 1886, Vi. All mean antibody measurements are statistically significant between the healthy controls and typhoid infections and between other infections and typhoid infections (p<0.05 Wilcoxon Rank sum).

Figure 7 - shows the results of experiments assessing the sensitivity and specificity of IgM against Salmonella Typhi antigens for the diagnosis of typhoid fever. Receiver operating characteristic (ROC) curves summarise the antibody responses against antigen combinations for the diagnosis of typhoid fever. The x-axis displays the false positivity rate (Specificity) and the y-axis displays true positive rate (Sensitivity). The performance of two, three and four antigens are shown by the dotted, light grey and dark grey lines, respectively. A) shows a ROC curve produced when the positive references are typhoid cases confirmed by blood culture and PCR amplification (n=32). B) shows a ROC curve produced when the positive references are typhoid cases confirmed by blood culture only (n= 19).

Figure 8 - demonstrates that febrile patients with an IgM profile indicative of typhoid fever can be detected using the invention. The hyperlane plots predict the number of undiagnosed febrile patients that have an IgM measurement indicative of typhoid fever. The black circles represent the negative controls, which includes healthy controls and patients other with infections. The white squares represent typhoid cases confirmed by blood culture or PCR. The white triangles are febrile patients with an IgM profile indicative of not having typhoid fever, whilst the grey triangles are febrile patients defined as having a typhoid infection using pre-defined hyperlane IgM profile . Figure 8A shows a plot where hyperlane was defined using the positive reference as the typhoid cases confirmed by blood culture or PCR (n=32); the selected combination of antigens was STY 1703 and Vi. Figure 8B shows a plot where hyperlane was defined using the positive reference as the typhoid cases confirmed by blood culture only (n= 19); the selected combination of antigens was STY 1886 and

Vi.

Figure 9 - shows the correlation of IgM measurements between Salmonella Typhi antigens. The plots show a correlation in IgM measurements in human plasma for the antigens encoded by STY0452, STY0769, STY 1086, STY 1372,

STY 1612, STY 1522, STY 1703, STY 1767, STY3208, STY4190, STY4539, STY 1886 and the Vi antigen. The histograms, below the plots, show the distribution of IgM levels by optical density to the highlighted S . Typhi antigen. The scatterplots, above the histograms, plot the IgM measurements of the two antigens on a right angle to the scatterplot and describe the correlation between antibody responses to two selected antigens. The numerals below the histogram depict the Spearman correlation coefficient (rho) values of the mirrored scatterplot. Figure 10 - details the sequence listing for SEQ ID NOS 1 to 12.

Materials and Methods

Ethical approval

The study was conducted according to the principles expressed in the Declaration of Helsinki. The Bangladesh National Research Ethical Committee (BMRC/NREC/2010- 2013/1543), the Chittagong Medical College Hospital Ethical Committee, the Oxford Tropical Research Ethics Committee (OXTREC 53-09) and the Research Ethics committee of the Liverpool School of Tropical Medicine gave ethical approval for the study. Informed written or thumbprint consent was taken from the subject, their parent or caretaker for all enrollees.

Study site, population and study design

The site and recruitment for this study has been descried previously Maude et al. (2015) Trop Med Int Health 20: 1376-84. Briefly, Chittagong Medical College Hospital (CMCH) is a 1 ,000-bed hospital serving Chittagong and the surrounding province . Adults and children (>6 months) consecutively admitted from January to June 2012 to the adult and pediatric wards at CMCH with an axillary temperature of >38 °C up to 48 hours after admission and history of fever for <2 weeks were eligible for the study.

The study was originally designed to compare blood culture alongside PCR amplification, rapid serological diagnostics tests and other approaches for the diagnosis of acute typhoid fever. Blood was collected from patients and controls at the time of admission to the study in EDTA tubes and separated into cells and plasma on the day of collection before storage at -20°C on site at CMCH. Here the gold standards for typhoid fever diagnosis are blood culture and PCR amplification from blood using a previously described method - Mga et al (2010) BMC Infect Dis 10: 125.

Blood (5- 12 mL for adults and 1- 12 mL) was cultured using Bact/Alert-FA and PF blood culture bottles, bottles were incubated in the Bact/Alert automated system (Biomerieux, Marcy l'Etoile, France) for five days. The patient demographics and diagnostic testing results for this study are reported elsewhere Maude et al. (2015) Trop Med Int Health 20: 1376-84. For the purposes of this investigation plasma samples from 40 healthy adult control subjects, 32 cases of confirmed typhoid fever ( 16 cases confirmed by blood culture, 13 cases confirmed by PCR and three cases confirmed by both blood culture and PCR), 17 cases from patients with confirmed febrile diseases other than typhoid fever and 243 febrile patients with undiagnosed conditions (332 patient samples in total) were subjected to serological assays using the S. Typhi antigens.

PCR amplification, gene expression and protein purification

\2 S. Typhi antigens were selected that gave a differential serodiagnostic signal using protein microarray screening for further expression and purification (Table 1 in Figure 1). The coding sequences of the selected genes, excluding trans-membrane domains and other proteomic features likely to hinder protein solubility and stop codons, were cloned into the 5 ^' Ncol and 3 ^' Noil restriction sites of pET28b vector (Novagen, United Kingdom) for further His-Tag purification. E. coli DH5a were transformed with the plasmid constructs for stable storage and E. coli BL21 -DE3 PLYSS (Promega, WI, USA) were used for expression and purification. The predicted protein sizes ranged from 5.7 KDal to 52.6 KDal in size (Table li n Figure 1). For protein expression, the E. coli BL21 -DE3 PLYSS strains harboring unique plasmid constructs (pEK90-pEK109) containing the genes of interest were inoculated into Luria-Bertani (LB) broth containing l OO mg/L kanamycin (Sigma, MO, USA), and incubated at 37°C overnight with gentle circular agitation (40-50rpm). Overnight cultures were diluted ( 1 : 100) into LB broth and incubated at 37°C with agitation until an optical density (OD ₆₀ o) of 0.5 was reached. Expression of the exogenous proteins was induced by the addition of isopropyl- -D-thiogalactoside (IPTG) (Sigma-Aldrich, United Kingdom), to a final concentration of 0. 1 mM. Bacterial cells were harvested after three hours of incubation at 24°C with gentle agitation.

For soluble proteins bacterial pellets were resuspended in 50 mM phosphate buffer (pH 8) containing 300 mM NaCl and 10 mM imidazole . The suspension was gently agitated at ambient temperature for 30 minutes prior to sonication (3 cycles of 10 seconds for 2 minutes with 10 seconds in between cycles). Cell debris and the membrane fragment were pelleted by centrifugation at 16,000 x g at 4°C for 30 minutes and discarded. Supernatants were filtered through a 0.45 μιη membrane before being rocked at 4°C with nickel coated agarose beads (Ni-NTA, Invitrogen) for two hours. Protein bound Ni-NTA beads were loaded into gravity flow columns (Qiagen, Hilden, Germany) and washed with 20mM imidazole in phosphate buffer. Proteins were eluted with 250 mM imidazole in phosphate buffer. For insoluble proteins a denaturing protocol was performed by firstly incubating the bacterial cells in an 8M urea (pH 7.8) solution containing 20mM sodium phosphate and 500mM NaCl. The denatured cellular matter was incubated as before with Ni-NTA beads and non- specific bound proteins were removed with two washes in 4M Urea (first wash pH6; second wash pH5) solution containing 20mM Sodium Phosphate and 500 mM NaCl. Proteins were eluted with 4M Urea (pH3) in a solution containing 20mM Sodium Phosphate buffer and 500mM NaCl. Proteins were renatured after purification in 50mM Sodium Phosphate solution and 500mM NaCl. The purity and size of the expressed and purified proteins were assessed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations were quantified using Bradford reagent (Bio-Rad).

Enzyme-linked immunosorbent assays (ELISAs) using S. Typhi protein antigens ELISAs to detect antigen specific IgM in human plasma samples were performed as described by Karkey et al (2013) PLos Negl Trop Dis 7:e2391 with 12 purified protein antigens (Seq ID Nos: 1 to 12) and S. Typhi Vi polysaccharide antigen (provided by Sclavo Berhing Vaccines Institute for Global Health, Siena, Italy). Briefly, 96 well flat-bottom ELISA plates (Nunc 2404, Thermo Scientific) were coated overnight with Ι ΟΟμΙ per well of the various antigens (final concentrations; 7μ1/ιη1 of protein antigens and ^g/ml for the Vi polysaccharide antigen in 50mM Carbonate Bicarbonate buffer). Coated plates were washed and blocked with 5% fat free milk solution in phosphate buffered saline. After 2 hours of blocking, plates were washed and incubated with Ι ΟΟμΙ (per well) of a 1 :200 dilution of plasma at ambient temperature for 2 hours. Plates were washed again and incubated with Ι ΟΟμΙ per well of alkaline phosphatase- conjugated anti-human IgM at ambient temperature for one hour. The final ELISA Plates were developed using p-Nitrophenyl phosphate (SigmaFAST N 1891 , Sigma- Aldrich, United Kingdom) substrate for 30 minutes at ambient temperature and the final absorbance were read at dual wavelengths (405 nm and 490 nm) using an automated microplate reader (Biorad). End point positive absorbance results were defined as optical densities (OD) greater than the absorbance obtained for the blank control wells plus four times the standard deviation.

Statistical analysis

A geometric mean optical density was calculated to summarize the IgM response to the S. Typhi antigens in each arm of validation group, including the negative reference population samples (healthy controls and other confirmed febrile infections) and the positive reference population (febrile patients confirmed to be infected with S. Typhi). The Wilcoxon signed-rank test was used to test the null hypothesis; no difference in optical densities between the patient groups. Spearman's rho was used to investigate potential correlations between IgM antibody responses against the various antigens. Receiver operating characteristic (ROC) curves were used to determine the optimal cut-off and the specificity and sensitivity of the various antigens. A performance estimation of more than one antigen combination was evaluated using Support Vector Machine (SVM). A SVM is a supervised learning model that analyses data for classification and regression analysis using a training and test data set. In this study this was used to investigate the performance of combining multiple antigens for serological testing. All analyses were performed with R software (version 3.3. 1 ; R Foundation for Statistical Computing). All confidence intervals (CIs) are reported 2- sided at the 95% intervals; all other significant testing were performed 2-sided with a significance level of p<0.05.

Results

Protein purification

12 protein antigens of SEQ ID NOS : l to 12 were expressed and purified see Table 2 (Figure 2 and Figure 10). Six of these antigens gave a serodiagnostic signal on an immunoarray with IgG (CT18 naming convention Parkhill et al (2001) Nature 413 : 848-52; STY 1552, STY 1703, STY 1767, STY 1886, STY3208 and STY4190) and six were serodiagnostic with IgM (STY452, STY796, STY 1086, STY 1372, STY 1612 and STY4539) Liang et al (2013) Sci Rep 3 : 1043). Examination of these twelve His- tagged fusion proteins demonstrated they were highly pure and of the desired size, ranging from 9.2 KDal to 52.6 KDal. Acute IgM antibody responses against Salmonella Typhi antigens

IgM is the first antibody isotype to increase in the early stages of infection/exposure; therefore the concentration of IgM was assessed against the various purified S. Typhi antigens in early time point plasma samples taken from patients with febrile disease in Bangladesh. These blood samples were collected from patients on the first day of admission to CMCH and represent the type of sampling that would be required for an acute diagnostic test for typhoid fever. Indirect ELISAs were performed independently employing the purified protein antigens and the Vi polysaccharide to detect IgM in the plasma of 40 healthy control subjects, 17 febrile individuals with a confirmed infection other that enteric fever, and 32 individuals with either blood culture or PCR (or both) confirmed typhoid infections (n= 89 samples). The resulting IgM concentrations in these samples were measured by calculating the endpoint optical density from the ELISAs.

Detectable IgM was measured against all twelve of the purified S. Typhi proteins and the Vi polysaccharide in all of the 89 samples subjected to ELISAs. The IgM titers were compared from each of the antigens individually to assess the performance of the antigens and to identify potential correlations between potential serological targets. It was found that early IgM responses against the majority of the novel S. Typhi protein antigens, with the exception of STY 1522 (rho<0.7), were highly correlated with one other (rho>0.8). Notably, the IgM response to the protein antigens correlated only weakly to those directed against the Vi polysaccharide (rho<0.6). A summary of these data for the antigens encoded by STY452, STY 1086, STY3208, STY 1886 and the Vi antigen are shown in Figure 5. The diagnostic potentiation of IgM against Salmonella Typhi antigens

The diagnostic potential of IgM directed at the purified antigens for identifying patients with typhoid fever was considered. Encouragingly, IgM against all twelve of the protein antigens and the Vi polysaccharide was significantly elevated in the plasma of the typhoid patients in comparison to the healthy controls (p<0.05) (Figure 6a and 6b). Furthermore, there was a significant differentiation in the plasma IgM titers between the typhoid patients and those with febrile disease with an alternative confirmed etiology with all antigens with the exception of STY 1522 (/?<0.05)

By assessing antibody titer it was surmised that three of the better performing antigens, with respect to differentiating between the patient groups and afebrile controls, were STY4539, STY 1886 and Vi (Figure 6b). The mean IgM responses (OD values) in the afebrile controls, the other confirmed infections and the typhoid infections were 0.29, 0.27 and 0.42 (against STY4539), 0.17, 0.18 and 0.25 (against STY 1886), and 0.22, 0.21 , 0.35 (against Vi), respectively. This segregation between the patient groups was highly significant, resulting in /^-values of 0.0001 , 0.003 for IgM against STY4539; <0.0001 , 0.004 for IgM against STY 1886; and 0.0001 , 0.0001 for IgM against Vi, between healthy controls and between other febrile diseases and typhoid infections, respectively (Wilcoxon signed-rank test used for all comparisons). Sensitivity and specificity of the serodiagnostic antigens

To further assess the IgM responses against the various S. Typhi antigens for the purposes of diagnostic testing, specificity and sensitivity was calculated using a validation group incorporating a negative reference group and two positive reference groups. The negative reference group (n=57) was the combination of data from the afebrile controls (n=40) and from those with a confirmed diagnosis other than typhoid fever (n=17 cases). The assay results were validated independently with two sets of positive reference data, these were blood culture confirmed S. Typhi patients (n= 19) (Table 3, Figure 3) and a combination of blood culture confirmed S. Typhi along with those with a positive PCR amplification result for S. Typhi from blood (n=32) (Table 4, Figure 4). The IgM responses against each of the antigens generated a set of continuous data that was used to generate receiver operating characteristic (ROC) curves to optimize the index cut off value . The defined cut-off values of the thirteen antigens corresponded with a range of specificities between 0.58 and 0.84 and sensitivities ranging from 0.50 to 0.84; areas under the ROC curve (AUC) ranged from 0.7 to 0.85. However, when used alone none of the antigens demonstrated specificities or sensitivities >0.8. As predicted earlier, Vi, STY4359 and STY 1886 were the three antigens with the greatest serodiagnostic capacity in discriminating typhoid cases from afebrile controls and other infections. The specificities and sensitivities for identifying typhoid patients by IgM titers against Vi, STY4539 and STY 1886 were 0.8, 0.68; 0.82, 0.62 and 0.82, 0.62, respectively. Correspondingly, the AUCs were 0.835 (95%CI: 0.71 1 , 0.958), 0.767 (95%CI: 0.612, 0.921) and 0.767 (95%CI: 0.62, 0.913). To enhance the performance of serology testing in defining typhoid cases, a support vector machine (SVM) was employed to identify for combinations of two or more antigens across all 13 antigens that may increase the overall sensitivity and specificity for detecting typhoid patients. A cross-validation was incorporated in a leave-one-out- cross scheme for the SVM. Using confirmed S. Typhi infection by blood culture or PCR as the positive reference (n=32), 1 1 combinations of two to four antigens were found that gave specificities from 0.81 to 0.86, sensitivities from 0.81 to 0.84 (Table 3 and Table 4) and AUCs from 0.865 to 0.867 (Figure 3a). 17 combinations of two to four antigens were identified when using the positive reference as culture confirmed S. Typhi only (n= 19), obtaining specificities from 0.88 to 0.94, sensitivities from 0.84 to 0.89 (Table 2, Figure 2) and AUCs from 0.859 to 0.912 (Figure 7b).

For the positive reference sets, IgM against Vi contributed to all of the combinations, while STY 1703, STY 1886 and STY4539 were present in more than half of the SVM combinations. The remaining nine antigens contributed to at least one combination that gave specificities and sensitivities >0.8. These results demonstrated that, in the majority of examples, a combination of up to four antigens was directly associated with an increased performance of the IgM serology. However, the best performing antigens for the identification of typhoid patients by IgM were Vi in combination with either STY 1703 or STY 1886 (Table 3 and Table 4, Figures 3 and 4). Identifying typhoid cases in patients with undiagnosed febrile disease

One of the greatest challenges in diagnosing patients with typhoid fever is estimating the proportion of patients that may have typhoid fever but are blood culture negative. In this Bangladeshi cohort there were 243 patients with a febrile disease of unknown etiology. In order to estimate the proportion of patients in this population that may have typhoid fever the SVM cutoffs were applied and combined with the IgM titers against two S. Typhi antigens. Two independent analyses were performed; the first combined IgM titres against STY 1703 and Vi and used a combination of culture confirmed S. Typhi and a positive PCR amplification result for S. Typhi from blood as the positive reference group (Figure 8a). Using these conservative criteria it was found that 83/243 (34%) of the undiagnosed febrile patient group had IgM titers indicative of typhoid fever. Using the less stringent criteria by using only the blood culture confirmed patients as the positive reference and a combination of IgM against Vi and STY 1886 we found that 145/243 (58%) febrile cases had a profile indicative of typhoid fever (Figure 8b). These results demonstrate the insensitivity of the blood culture method.

Discussion

The diagnosis of typhoid fever is complicated by indistinguishable clinical symptoms of many febrile diseases and the lack of a reliable gold standard test. In the data presented here, the IgM response against all 12 antigens studied was significantly higher in patients with typhoid fever than both afebrile controls and patients with febrile diseases other than typhoid fever. Furthermore, through inference from the AUC under the ROC curve the best three performing antigens were identified, which were encoded by STY4539 (PilL) and STY 1886 (CdtB) in combination with the Vi polysaccharide. PilL is a putative exported protein and a component of the type IV pili encoded adjacent to the genes encoding Vi on SPI-7. The PilL protein has been shown to be induced in human derived macrophages, and the type IV pili to which it is associated facilitates entry into host intestine epithelial cells. CdtB, encoded by STY 1886, is one of the two A sub-units of typhoid toxin, an AB type toxin. Typhoid toxin is a virulence-associated factor of S. Typhi, which is currently thought to be associated with the early symptoms of typhoid fever. This data confirms that this component of typhoid toxin is immunogenic and may be important biomarker of acute typhoid.