Detection of lipoarabinomannan and host biomarkers to diagnose tuberculosis

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

Description

[0001] DETECTION OF LIPOARABINOMANNAN AND HOST BIOMARKERS TO DIAGNOSE TUBERCULOSIS

[0002] BACKGROUND OF THE INVENTION

[0003] There is an urgent need for disruptive and accurate tests for tuberculosis (TB), which is historically the biggest killer of mankind, and now the commonest cause of death in South Africa. It is estimated that TB impacts the country’s GDP by 2 to 3% per annum (about 9 billion USD per year) (KPMG Report (2017)). This exemplar reflects what is happening in many African and Asian low and middle income countries. On a global level, TB is now again the most common infectious diseases killer worldwide (surpassing COVID-19), resulting in ~11 million newly ill patients every year, and TB is also the most common cause of chronic pulmonary disability worldwide. A startling statistic is that 2 out of every 5 newly ill persons with TB continue to remain undetected and live in a community-based setting. Thus, better diagnostic tools are required.

[0004] However, a significant unmet need facing the development of better TB diagnostics are the lack of clinically useful biomarkers, and lack of a simple and affordable sputum-independent detection platform for TB both at the bedside and in the community. There are 2 key issues.

[0005] Firstly, and most importantly, as outlined above, almost 40% of the world’s TB cases go undiagnosed or unreported. Globally this amounts to over 4 million newly ill TB patients per annum (WHO Report (2021 )). Most of these persons reside in the periurban informal settlements of large cities in Africa and Asia. Finding such cases would be greatly facilitated by the availability of a low cost scalable and easily to use screening (triage) test (i.e. test with high sensitivity but modest specificity that would be able to earmark persons for more specific and intensive testing in health care facilities). There is no such approved test in clinical use and it represents a major unmet need. The WHO have provided target product profile metrics for such a screening test [> 90% sensitivity and > 70% specificity] (WHO Report (2014), Denkinger etal. (2015)). This would allow almost all the cases of TB to be detected cheaply and at scale using a simple test, and referred onward so that the diagnosis can be confirmed with a more specific test (hence high sensitivity and modest specificity requirement). Second, there is a need for an effective sputum-independent confirmatory (rulein) test for TB (for example using urine, unlike sputum, that is almost always accessible and available). The problem is particularly acute for the -50% of patients co-infected with HIV who fail to produce sputum or whose sputum contains very few bacteria (< 50 bacilli / ml). The current frontline PCR-based TB diagnostic test used in many low and middle income countries (Gene Xpert Ultra; Cepheid) fails to address this issue (because one third of TB patients cannot produce sputum or have extra-pulmonary TB), and the existing urine lipoarabinomannan (LAM) assay (Alere Determine™ TB LAM Ag) is insensitive (detects less than 50% of TB-HIV co-infected patients and < 15% of HIV uninfected persons) and is unsuitable for use in sputum samples (Peter et al. (2012a), Peter etal. (2016), Peter etal. (2012b)). A more sensitive assay [SILVAMP TB-LAM (FujiLAM) lateral flow assay] is under development, but only detects < 50% of TB in HIV uninfected persons (Broger et al. (2020)). Thus, LAM alone as a urine biomarker will not solve the problem. One or more additional biomarkers are required.

[0006] The field of urine, saliva, blood and serum diagnostics has thus far has restricted itself to using either M.tb-derived biomarkers (e.g. LAM or TB DNA etc.) or host biomarkers (e.g. IFN-gamma, IP-10 etc.) alone. Little research has been conducted on the utility of combining mycobacterial and host biomarkers in a unified method for the diagnosis of TB. Thus this specific hypothesis, i.e. that host and antigenspecific glycolipid-based biomarkers can serve as a screening test for TB, remains, until now, unclarified.

[0007] To address these unmet needs the present inventors have carried out a mass spectrometry-based discovery proteomics analysis of urine samples from a cohort of patients with suspected TB (both HIV positive and HIV negative) and have identified a set of 36 previously unreported mycobacterial proteins in urine that correlate with disease status, and which distinguish active TB from latent TB infection and from non- TB (those without active TB). A subset of these novel biomarkers has been disclosed in an UK patent application number 1314873.9. A follow-up targeted study revealed a biosignature of twelve mycobacterial and host-derived urinary proteins, allowing for multidimensional and combinatorial biomarker detection to diagnose TB. These urinary proteins were disclosed in international patent publication WO 2021 / 064592A1 .

[0008] There are currently no screening tests or confirmatory tests which combine host biomarkers and TB antigen-specific biomarkers to make a preliminary diagnosis of TB infection. Further, there are no urine-based screening tests for diagnosing TB infection in a subject.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention relates to screening and confirmatory diagnostic tests and kits for diagnosing tuberculosis in a subject. The tests rely on the identification of a combination of host specific biomarkers and TB antigen-specific biomarkers to diagnose the presence of tuberculosis infection in a subject. The invention further relates to the identification of the host specific biomarkers and TB antigen-specific biomarkers from a sample of a subject, preferably a urine sample.

[0011] According to a first aspect of the invention there is provided for a method for diagnosing a Mycobacterium tuberculosis (M. tb) infection in a subject, the method consisting of or comprising of (i) providing a sample obtained from the subject, wherein the sample is a urine sample, (ii) detecting the presence of at least one host biomarker in the sample, and (iii) detecting the presence of at least one M. tb biomarker in the sample. Wherein, the presence of the at least one host biomarker together with the presence of the at least one M. tb biomarker indicates the presence of M. tb infection in the subject.

[0012] In a first embodiment of the first aspect of the invention the sample is selected from the group consisting of or comprising of blood, cerebrospinal fluid, pericardial fluid, peritoneal fluid, faeces, lymph, mucous, plasma, saliva, semen, serum, sputum, synovial fluid, urine, and vaginal fluid. In a preferable embodiment the sample is a urine sample.

[0013] In a second embodiment of the first aspect of the invention the at least one host biomarker is selected from the group consisting of or comprising of C-reactive protein (CRP), alpha- 1 -antichymotrypsin (SERPINA3) or resistin (RETN).

[0014] In a third embodiment of the first aspect of the invention the at least one M. tb biomarker is lipoarabinomannan (LAM).

[0015] In a fourth embodiment of the first aspect of the invention the method consists of or comprises detecting the presence of at least one of the following combinations of biomarkers in the sample (i) CRP and LAM, or (ii) RETN and LAM, or (iii) SERPINA3 and LAM, or (iv) RETN, CRP and LAM, or (v) RETN, SERPINA3 and LAM, or (vi) CRP, SERPINA3 and LAM. In a fifth embodiment of the first aspect of the invention the steps of detecting the presence of the at least one M. tb biomarker and the at least one host biomarker are by means of immunoassay. Preferably, the immunoassay is a lateral flow assay.

[0016] In a fifth embodiment of the first aspect of the invention the M. tb infection is an active infection, or a latent infection.

[0017] In a sixth embodiment of the first aspect of the invention the subject is a human.

[0018] In a second aspect of the invention there is provided for a kit for detecting at least one host biomarker and at least one M. tb biomarker in a urine sample from a subject, the kit consisting of or comprising of (i) a host biomarker capture molecule which binds to at least one host biomarker in the sample, (ii) a host biomarker detection molecule which detects the binding of the host biomarker capture molecule to the at least one host biomarker in the sample, (iii) a M. tb biomarker capture molecule which binds to at least one M. tb biomarker in the sample, and (iv) a M. tb biomarker detection molecule which detects the binding of the M. tb capture molecule to the at least one M. tb biomarker.

[0019] In a first embodiment of the second aspect of the invention the kit further consists of or comprises of a means for obtaining the sample from the subject. In a preferred embodiment the sample is selected from the group consisting of or comprising of blood, cerebrospinal fluid, pericardial fluid, peritoneal fluid, faeces, lymph, mucous, plasma, saliva, semen, serum, sputum, synovial fluid, urine, and vaginal fluid. Most preferably the sample is a urine sample.

[0020] In a second embodiment of the second aspect of the invention the at least one host biomarker is selected from the group consisting of or comprising of C-reactive protein (CRP), alpha- 1 -antichymotrypsin (SERPINA3) or resistin (RETN).

[0021] In a third embodiment of the second aspect of the invention the at least one M. tb biomarker is lipoarabinomannan (LAM).

[0022] In a fourth embodiment of the second aspect of the invention the kit is for detecting the presence of at least one of the following combinations of biomarkers in the sample (i) CRP and LAM, or (ii) RETN and LAM, or (iii) SERPINA3 and LAM, or (iv) RETN, CRP and LAM, or (v) RETN, SERPINA3 and LAM; or (vi) CRP, SERPINA3 and LAM.

[0023] In a fifth embodiment of the second aspect of the invention the host biomarker capture molecule and the M. tb biomarker capture molecule are independently selected from the group consisting of or comprising of a probe, an antibody, an antibody fragment, an aptamer or a ligand.

[0024] In a sixth embodiment of the second aspect of the invention the host biomarker detection molecule and the M. tb biomarker detection molecule are independently selected from the group consisting of or comprising of a probe, an antibody, an antibody fragment, an aptamer or a ligand.

[0025] In a seventh embodiment of the second aspect of the invention the host biomarker detection molecule and the M. tb biomarker detection molecule are independently conjugated, either covalently or non-covalently to a detection label. Preferably, the detection label is selected from the group consisting of or comprising of colourimetric labels, fluorescent labels, chemiluminescent labels, biotin, phosphorbased labels, thermal-based labels, enzymatic labels, gold nanoparticles, silver nanoparticles and magnetic beads.

[0026] BRIEF DESCRIPTION OF THE FIGURES

[0027] Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

[0028] Figure 1 : Significant differences in biomarker peptide levels. Mass spectrometry readings showing significant differences at peptide level for CRP (A), RETN (B) and Serpina3 (C) in TB and non-TB groups (N = 118).

[0029] Figure 2: Significant biomarker differences at protein level in the urine. Significant differences in protein concentration (using commercially procured ELISAs) for CRP (A), RETN (B) and Serpina3 (C) in TB and non-TB samples (N = 40).

[0030] DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

[0032] The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. As used throughout this specification and in the claims which follow, the singular forms “a”, “an” and “the” include the plural form, unless the context clearly indicates otherwise.

[0033] The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms “comprising”, “containing”, “having” and “including” and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0034] The following abbreviations are used in the specification and have the corresponding meanings provided:

[0035] CAP - Community-Acquired Pneumonia

[0036] CRP - C-Reactive Protein

[0037] ELISA - Enzyme-Linked Immunosorbent Assay

[0038] EPTB - Extrapulmonary TB

[0039] HIV - Human Immunodeficiency Virus

[0040] LAM - Lipoarabinomannan

[0041] LFA - Lateral Flow Assay

[0042] LFI - Lateral Flow Immunoassays

[0043] LTBI - Latent Tuberculosis Infection

[0044] M. tb - Mycobacterium tuberculosis

[0045] POC - Point Of Care

[0046] PTB - Pulmonary Tuberculosis

[0047] RETN - Resistin

[0048] SERPINA3 - Alpha-1 -antichymotrypsin

[0049] TB - Tuberculosis

[0050] WHO - World Health Organization

[0051] As used herein, the term “biomarker” refers to a parameter that can be objectively measured as an indicator of normal or pathogenic biological processes or as an indicator of pharmacological responses to therapeutic interventions. Biomarkers are used for screening and monitoring (repeated at timed intervals) and are determined and applied on an individual basis or may be related to a population group. Population groups at risk for a disease may be identified by deviations from normal mean values and individual variations will be reflected in statistical terms. When the inter-individual variation of the biomarker is large in comparison to intra-individual variation, analysis of paired samples (before, during and after the exposure) may greatly enhance the power of the biomarker to detect exposure. In infectious diseases, biomarkers can be either host or pathogen derived. An alternative to pathogen detection is quantification of host derived biomarkers, such as serum proteins. Host serum protein biomarkers that indicate a high likelihood for active TB disease represent attractive targets for integration into screening tests. Serum proteins are generally of higher abundance than pathogen products, are amenable to existing POC technologies such as lateral flow immunoassay (LFA) and have been shown to discriminate between different infections when combined as biosignatures. Various inflammatory parameters can be combined into a multiple biomarker panel to significantly improve the prediction accuracy of the prognosis and the response to antibiotic chemotherapy.

[0052] Biomarkers can be applied in a direct and rapid diagnostic method using non- invasive or minimally invasive specimens (e.g., breath, urine, finger stick whole blood). To be used at POC, biomarkers need to perform in settings with limited laboratory facilities; be affordable and easy to use.

[0053] As used herein, “latent TB infection” or “latent TB” refers to a ‘carrier state’ of M. tuberculosis infection where an individual carries the TB bacteria, but the infection is well contained by the host’s immune system. Hence, unlike “active TB” or “active TB disease”, individuals with LTBI are asymptomatic, and not contagious to others. However, this condition may progress or reactivate to active disease at any time point. The development of active TB disease depends on a variety of risks and medical conditions. Individuals with LTBI are commonly offered preventative therapy to avoid active TB from occurring. Preventative treatment is an important strategy to reduce TB morbidity and mortality rates in many countries. It is estimated that up to 10% of people infected with M. tuberculosis will develop active TB in their lifetime. With an estimated two billion people (i.e. , one third of the world’s population) infected, the large global reservoir of LTBI represents a huge pool of contagious disease. Diagnosing LTBI, and preventative treatment thereof, can significantly reduce the risk of disease, and prevent outbreaks from recent transmission. On a global level, achieving a significant reduction in the burden of TB cases cannot be achieved without also including the detection and treatment of LTBI. About 5-15% of LTBI progress to active TB with pulmonary and / or extra pulmonary involvement. “Active TB” or “active TB disease” refers to a disease state of uncontrolled M. tuberculosis growth which occurs when TB bacteria are able to overcome a person’s immune system. Active TB can affect any organ of the body but is most commonly a disease of the lung. A person with active TB will often have symptoms which are not specific for tuberculosis (e.g., a cough, night sweats and weight loss). Direct detection of M. tuberculosis bacilli in sputum microscopy or TB culture is the hallmark of disease and is considered the gold standard of TB diagnosis.

[0054] In tuberculosis endemic areas, TB is strongly associated with the presence of chronic respiratory disease in adults. Efforts to improve long-term lung health should be part of tuberculosis care. “Chronic respiratory diseases” or “other respiratory diseases” are a group of disorders that primarily affect the lungs and airways. It is associated with significant morbidity and mortality. The World Health Organization estimates that 4.6 million people die prematurely each year as a result of chronic respiratory disease, accounting for more than 5% of global deaths; almost 90% occur in low and middle-income countries.

[0055] The twin epidemics of HIV and TB place enormous strain on the limited healthcare resources in high burden areas. A common clinical problem is the differentiation of bacterial infection from TB in HIV-seropositive patients. Community- acquired pneumonia (CAP) is a common cause of disease and hospital admission among HIV-seropositive patients. Streptococcus pneumoniae and Mycobacterium tuberculosis are two of the most frequent bacterial causes of CAP in this population and may have similar clinical presentations. In addition, HIV co-infection often alters the radiographic presentation of pulmonary tuberculosis (PTB), such that there is a higher prevalence of lobar and lower zone consolidation, making it even more difficult to distinguish these infections from other common bacterial causes of pneumonia. Thus, patients admitted to hospital who are frequently treated empirically for multiple organisms, whilst awaiting results of investigations, may have a delayed treatment response which can be detrimental to patient recovery outcomes. Prompt diagnosis and initiation of appropriate treatment is important to ensure optimisation of patient recovery within a short period of time and reduce mortality in all inpatients especially those who are severely immunocompromised. This approach is costly, exposes patients to potentially harmful chemotherapy and may lead to inappropriate therapy that negatively influences morbidity and mortality.

[0056] People living with HIV are more likely than others to become infected with TB Worldwide, and TB is known to be one of the leading causes of death among people living with HIV. Without treatment, as with other opportunistic infections, HIV and TB can shorten lifespan. People infected with HIV, and who also have either latent TB or TB disease can be effectively treated. The first step is to ensure that people living with HIV are tested for TB; If identified to have TB infection, further tests are needed to rule out TB disease. The next step is to start treatment for latent TB or TB disease based on test results. Untreated latent TB can quickly progress to TB disease in people living with HIV since the immune system is already weakened, therefore, without treatment, TB disease can progress to mortality.

[0057] HIV associated TB further complicates diagnosis and treatment of patients. Infected patients also have a lower rate of positivity using the TB skin test or Interferon Gamma Release Assays due to their inherent immunocompromised status. Thus, a diagnostic method that can efficiently detect both HIV positive and HIV negative TB disease, and / or TB infection is needed.

[0058] Among people living with HIV, TB remains the main cause of death accounting for 1 in 3 HIV-related deaths. A major barrier to implementing systematic screening of high-risk groups is the lack of an adequate TB screening test. Novel approaches to TB screening are therefore critical in achieving global targets for TB elimination. Studies have shown that treating patients with LTBI reduces the risk of active TB in people living with HIV especially when diagnostic tests are used to identify those infected with TB. Therefore, as indicated in the WHO End TB Strategy, the diagnosis and treatment of LTBI are important elements in the control and elimination of TB disease worldwide.

[0059] Lateral Flow Immunoassays (LFA’s) are a diagnostic test used to confirm the presence or absence of a target analyte, such as pathogens or biomarkers in humans or animals, or contaminants in water supplies, foodstuffs, or animal feeds. The most known type of lateral flow rapid test strip is the pregnancy test. LFAs typically contain a control line to confirm the test is working properly, along with one or more target or test lines. They are designed to incorporate intuitive user protocols and require minimal training to operate. They can be qualitative and read visually or provide data when combined with reader technology. Lateral flow tests are widely used across many industries for point of care testing. They can be performed by professionals, trained lay users or a patient, and in a range of settings including the laboratory, clinic, or home.

[0060] Different types of paper-based immunoassays are known in the art, including colorimetric-based, fluorescence-based, and electrochemical based immunoassays. The advantages of colorimetric-based immunoassay include low cost, equipment-free, rapid, naked-eye readable, and suitable for high-throughput screening on-site; however, the improvements in accuracy, sensitivity, and quantification are still challenges. Recent developments have proposed the use of advanced labels based on naked-eye detection, fluorescent or chemiluminescent reading, and surface- enhanced Raman spectroscopy.

[0061] Immunoassays can be performed either in competitive or sandwich-type configurations. The competitive-type assay is based on the competition between the analyte in the sample which is unlabelled and a labelled analyte for a limited number of captured Abs. The enzyme immunoassay or enzyme-linked immunosorbent assay (ELISA) is the most commonly used technique employed as a diagnostic tool for clinical purposes. Two approaches can be employed: the first is based on immobilized antibodies that react with free antigens in competition with labelled antigens; the second consists in immobilized antigens and labelled antibodies. The working principle of ELISA consists in the immobilization of an unknown amount of antigen onto a solid surface, and then a specific antibody is added over the surface so that it can bind to the antigen. This antibody is usually labelled with an enzyme; the substrate of the enzyme is then added, which will be converted into a compound that can be quantified by means of optical methods.

[0062] A biosensor is a device that measures specific molecules, including biomarkers, in order to provide information about health and disease. Designed for the purpose, biosensors are generally highly selective due to the possibility to tailor the specific interaction of compounds by immobilizing biological recognition elements on the sensor substrate that have a specific binding affinity to the desired molecule. The most well-known biosensor is the glucose sensor which measures the concentration of glucose in the blood or in the skin. With a POC biosensor, patients and doctors immediately get the information they need in order to optimise treatment, reduce unnecessary hospital visits and enable self-management of health and disease. The recent developments made in the immunosensor field, regarding the incorporation of nanomaterials for increased sensitivity, multiplexing or microfluidic-based devices, have potential for promising use in industry and clinical analysis.

[0063] A biosensor uses an enzyme linked recognition element to recognise the target and catalyse the specific reaction to generate a signal. The recognition element captures the target analyte, enabling the isolation of the analyte and thus facilitating quantitative analysis. Suitable recognition elements may include aptamers, enzymes, antibodies, nucleic acids, such as miRNA, whole cells, and receptors. A frequently used format is the amperometric immunosensor, where proteins are labelled with enzymes producing an electroactive product from an added substrate. The most commonly used labels are enzymes such as peroxidase, glucose oxidase, alkaline phosphatase, catalase or luciferase. Some other labels such as electroactive compounds (ferrocene or In2+ salts), fluorescent reagents (rhodamine, fluorescein, Cy5, ruthenium diamine complexes, phosphorescent porphyrin dyes, etc.), and metallic nanoparticle (gold or silver produced in situ electrochemically) are also used.

[0064] In electrochemical biosensors an electrode provides a solid support for the immobilization of the capture binder (e.g., antibody), as well as a sensing means for the electrons produced from the biological reaction.

[0065] Recognition molecules in the biosensor bind to the biomarkers which then leads to a signal. Preferably, the biosensor is a disposable sensor device.

[0066] Biosensors may further include a reader instrument that measures the signal coupled to software that translates the signal into meaningful information.

[0067] As used herein, the term “sample” refers to a sample obtained from a biological source, or a “biological sample”. Typically, a suitable biological sample may include a saliva, sputum, blood, plasma, cerebrospinal fluid, pericardial fluid, peritoneal fluid, serum, lymph, tissue or urine sample. In a preferred embodiment, the sample is a urine sample.

[0068] Typically, detection comprises contacting the sample with selective reagents such as probes, primers, antibodies, aptamers or ligands, and thereby detecting the presence, or measuring the level or amount, of biomarker protein present in the sample. The contacting may be made under any condition suitable for a detectable complex, for example nucleic acid hybridisation or antibody-antigen complexation, formed between the reagent and the nucleic acids or proteins of the sample.

[0069] Specifically, detecting the expression level of the biomarker(s) of the present invention in the sample may also be performed by directly measuring the level of biomarker protein present in the sample. As used herein, the “level of biomarker protein” means the quantity or concentration of the biomarker protein. Methods for measuring the level of biomarker protein comprise contacting a sample with a binding partner, for example an antibody, capable of selectively interacting with the biomarker protein present in the sample. The presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. For example, such assays include, but are not limited to, lateral flow assays, Western blots, agglutination tests, enzyme-labelled and mediated immunoassays, such as ELISAs, biotin / avidin type assays, radioimmunoassays, immunoelectrophoresis, immunoprecipitation, etc. The reactions of the aforementioned assays generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody. Such methods generally involve separation of unbound protein and transferring the protein to a solid support to which antigen-antibody complexes are bound. Solid supports suitable for use in these methods include substrates such as nitrocellulose (e.g., membrane or microtiter well), polyvinylchloride (e.g., sheets or microtiter wells), polystyrene latex (e.g., beads or microtiter plates), polyvinylidine fluoride, diazotized paper, nylon membranes, activated beads, magnetically responsive beads, etc. Preferably, a lateral flow assay can be used.

[0070] The methods of the invention may further comprise a step of comparing biomarker levels with a control reference sample or with a reference value.

[0071] As used herein, the term “control” relates to a healthy subject, i.e., a subject who does not have TB, or to a subject with ORD. The control may also be a subject that has LTB but does not have active TB. Thus, the biomarkers of the present invention may be used to distinguish between a subject that has active TB from a subject with a latent TB infection.

[0072] Typically, a “reference value” is determined experimentally, empirically, or theoretically. For example, the reference value may the mean expression level of the biomarker(s) of the invention in a healthy individual or a population of healthy individuals, wherein the term “healthy individual” denotes a subject which is known to be healthy, i.e., which does not suffer, and never has suffered, from TB. Alternatively, the reference value may be in relation to the quantity or level of the biomarkers in a known disease state and is the mean expression level or quantity of the biomarker(s) of the invention in a diseased individual or a population of diseased individuals, wherein the term “diseased individual” denotes a subject which is known to have TB or ORD. In some embodiments of the present invention the biomarkers may be used to distinguish between a subject that has active TB based on a reference value for a subject or a population of subjects with a latent TB infection.

[0073] In some embodiments, the reagents for detecting the biomarker(s) and / or measuring the expression level or quantity of biomarker of the present invention may be provided in a kit, together with instructions for use. The following example is offered by way of illustration and not by way of limitation.

[0074] EXAMPLE

[0075] Study recruitment and classification

[0076] Patients suspected of TB were recruited in the Western Cape, South Africa. All patients provided written informed consent and the studies from which the samples were collected were approved University of Cape Town, Faculty of Health Sciences Human Research Ethics Committee. Patients underwent a series of microbiological tests (smear and / or culture and / or GeneXpert MTB / RIF and a TB-Determine Alere LAM lateral flow assay) and a HIV lateral flow assay. Confirmed active TB was classified as those with a microbiological confirmed test for TB. Confirmed non-TB was classified as those who have no microbiological evidence of TB (all tests negative) and were not initiated onto TB treatment and showed improvement on follow-up. Patients were classified according to the results into four clinical groups: confirmed active TB and HIV infected (N = 28), confirmed active TB and HIV uninfected (N = 30), confirmed non-TB and HIV infected (N = 26) and confirmed non-TB and HIV uninfected (N = 34). Mid-stream urine samples were collected and stored at -20 °C until processing. The samples were processed in randomised batches of 12.

[0077] Sample processing: protein precipitation, in-solution tryptic digest and desalting Urine samples were allowed to thaw at room temperature. Following vortexing, aliquots of 4 ml were transferred into glass vials for protein precipitation with methanol and chloroform at a ratio of 1 : 0.75. The precipitate was separated into phases by centrifugation at 4000RCF for 2 minutes, and the top phase was removed. The remaining protein precipitate was washed with 1 volume of methanol and pelleted by centrifugation at 4000RCF for 2 minutes. The supernatant was discarded, and the pellet was dried in a fume hood for 1 hour. The pellet was resuspended in 200 pl denaturation buffer (6 M urea, 2 M thiourea in 10 mM tris pH 8.0), vortexed and the sample was transferred into an Eppendorf tube for storage at -20 °C.

[0078] Protein samples (processed above) were quantified using a modified Bradford assay (Bio-Rad) with the addition of 90 pl of 0.1 M HCL A 100 pg aliquot of each sample was transferred into a fresh Eppendorf tube. Samples were reduced with 1 mM DTT (dithiothreitol) for 1 hour at room temperature, alkylated with 5.5 mM iodoacetamide for 20 minutes at room temperature, and then diluted 5x with 50 mM ammonium bicarbonate. Trypsin (New England Biolabs) was added to each sample at a ratio of 50:1 , and the samples were incubated at room temperature overnight for 16 hours. The addition of 0.1% formic acid (FA) halted the tryptic digest.

[0079] Tryptic peptides were desalted on C18 stage tips. The C18 plugs were first activated with solvent B [60% acetonitrile (ACN), 0.1 % FA], equilibrated with solvent A (2% ACN, 0.1 % FA). For each sample, 10 pg of peptides was bound to the C18 by slow centrifugation, then washed 3x with solvent A. A glass insert was placed into the Eppendorf tube and the desalted peptides were eluted 3x with 40 pl of solvent C (80% ACN, 0.1% FA) into the glass insert. The peptides were dried in a vacuum centrifuge at 35°C for 1 hour and stored at -20°C until use.

[0080] Sample application to liquid chromatography mass spectrometry (LCMS)

[0081] Peptide samples were reconstituted in solvent A to a final concentration of 200 ng / pl. Samples were loaded into the Dionex Ultimate 3000 autosampler. A total of 400 ng of each sample were loaded onto an in-house packed 4 cm Luna C18 5 pm trap column at a flow rate of 5 pl / min. The valve was then switched to bring the trap column in line with an in-house packed 40 cm Aeris peptide C18 3.6 pm analytical column (ID 75 pm). The mobile phase gradient went from 6-35% B (ACN with 0.1% FA) over 190 minutes at 0.4 pl / min and 40°C. Analytes eluted directly into a Thermo QExactive hybrid quadrupole Orbitrap instrument, which acquired MS and MS / MS scans in top 10 mode. MS scans were acquired at a resolution of 70000 with an AGC target of 3e6 or an injection time of 250 ms. MS / MS scans were acquired at a resolution of 17500 with an AGC target of 5e4 or an injection time of 80 ms. The dynamic exclusion window was 30 seconds, and the normalised collision energy was 28.

[0082] Investigation at peptide level

[0083] Data were analysed using MaxQuant version 1.5.3.12 with match between runs, intensity-based absolute quantification (iBAQ), and label-free quantification (LFQ) on. The database used was downloaded from Uniprot in August 2016 and was a combined Homo sapiens and Mycobacterium tuberculosis H37Rv database. The MaxQuant output folder ‘.txt’ was analysed using an in-house QC pipeline in R to assess the quality of the runs. Contaminant and reverse hits were removed from the data. The stratified LFQ data (peptide level data) was filtered to evaluate the top differential protein hits. This included the biomarkers outlined in this application CRP, RETN and SerpinA3 (Table 1 ). The LFQ per biomarker was stratified into 2 groups, (i) confirmed TB and (ii) confirmed non-TB. CRP, SerpinA3 and RETN peptide levels were all significantly higher in the confirmed active TB group in comparison to the confirmed non-TB group (p-value < 0.0001 for all biomarkers) (Figure 1 ).

[0084] Table 1 : The combinatorial and multidimensional biosignature encompasses LAM (Mycobacterium tuberculosis biomarker) along with below mentioned gene identifiers, proteins and / or peptides and their downstream functions in humans.

[0085] Investigation at protein level and lycolipid level

[0086] To validate the peptide level data, 40 bio-banked urine samples were selected (N = 21 confirmed TB and N = 19 confirmed non-TB).

[0087] Once thawed at room temperature, these samples were applied to commercial immunoassays kits i.e. ELISA kits as per the manufacturer’s instructions [Human RETN (Resistin) ELISA Kit (Catalogue no: E-EL-H1213), Elabscience; Human Serpin A3 / Alpha-1 -Antichymotrypsin ELISA Kit (EH411 RB), Invitrogen and Human CRP SimpleStep ELISA® Kit (C-Reactive Protein) (ab260058)], and the Abbott Determine LAM® assay (LAM) (7D2741 ). CRP, SerpinA3 and RETN peptide (Figure 1 ) and protein levels (Figure 2), were all significantly higher in the confirmed active TB group in comparison to the confirmed non-TB group (p-value = 0.001 1 , 0.0082, 0.0035, respectively) (Figure 2. Thus, the LFQ peptide data and ELISA protein data, per biomarker, were compared across 2 groups, confirmed TB and confirmed non-TB and underwent an unpaired t-test with a Mann-Whitney rank sum test (Figure 1 and 2).

[0088] Diagnostic accuracy, including 95% confidence intervals (95% Cis), was assessed using sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the receiver operator characteristic curve (AUROC) was confirmed in active TB and non-TB groups at various cut points per biomarker (alone or in combination) with the TB-Determine immmunoassay. Cut points in combination with TB-Determine LAM lateral flow assay and / or other host-derived biomarkers that were closest to the WHO targeted product profiles for a triage (-95% sensitivity & -70% specificity) (Table 2), and as a rule-in test for TB (-65% sensitivity & >95% specificity) (Table 3) were selected. This was to expressly test the hypothesis of whether TB antigen-specific biomarkers (e.g. LAM glycolipid) together with human host biomarkers could be useful as a screening or non-screening diagnostic test for TB. There were two meaningful outcomes of the evaluation.

[0089] Firstly, for the rule-out / triage / screening test the best performer was CRP (at a cut point of 155 pg / ml) in combination with LAM with a sensitivity, specificity, PPV and NPV of 85.8%, 79%, 81.9% and 83.4%, respectively (Table 2). Such a test would be useful for detecting undiagnosed patients in the community using a low cost user- friendly assay, and such persons could then be targeted for testing at clinic or hospital levels with a more specific and confirmatory test for TB. Thus, the novel finding that we have demonstrated is that combining a mycobacterial-specific glycolipid antigen (like LAM) can be combined with host-specific biomarkers (like CRP) to screen for TB.

[0090] Table 2: Biomarker performance meets triage test target product profiles (-95% sensitivity & -70% specificity). In the 2 biomarker performance panel ie: LAM and CRP, Resistin or SerpinA3, a positive test was defined as 1 out of the 2 biomarkers being present [CRP (cutpoint: 155 pg / ml), RETN (2000 pg / ml) and Serpina3 (1 10 ng / ml)]. Best performer 2 biomarker panel is CRP and LAM (bold). Statistical analyses were performed using GraphPad Prism (version 6.0), Medcalc, version 18.6, and Microsoft Excel. A p-value of < 0.05 was considered statistically significant.

[0091] Second, for the rule-in test 2 x 3 biomarker panels showed similar performance with a sensitivity, specificity, PPV and NPV of 52.4%, 89.5%, 84% and 63%, respectively (Table 3). These were (i) Resistin (cut point = 2000 pg / ml), SerpinA3 (cut point = 185 ng / ml) and LAM and (ii) Serpin A3 (cut point 185 ng / ml), CRP (155 pg / ml) and LAM. Such an optimised test, if developed using these biomarkers, could be useful to rule-in the diagnosis of TB in problematic sub-groups of participants e.g. children, adults unable to expectorate sputum, immunosuppressed persons where conventional tests perform poorly. Again we have demonstrated that combining a mycobacterial- specific glycolipid antigen (like LAM) can be combined with host-specific biomarkers to rule-in a diagnosis of TB. Table 3: Biomarker performance close to meeting non-sputum-based test target product profile (-65% sensitivity & >95% specificity). In the 3 biomarker performance panel, a positive test was defined as 2 out of the 3 biomarkers being present [CRP (cut-point: 155 pg / ml), RETN (2000 pg / ml) and Serpina3 (185 ng / ml)].

[0092] * and **: in these instances, by chance, although the numerators (*) are the same they are made up of different individuals making up the total of those having TB) but with a positive test (n= 1 1 )* or without TB but with a negative test**.

[0093] REFERENCES

[0094] Broger T, Nicol MP, Sigal GB et al. (2020) Diagnostic accuracy of 3 urine lipoarabinomannan tuberculosis assays in HIV-negative outpatients. J Clin Invest. 130 (11 ) : 5756-5764.

[0095] Denkinger CM, Kik SV, Cirillo DM et al. (2015) Defining the Needs for Next Generation Assays for Tuberculosis. JID. 21 1 (Suppl 2) : S29-S38.

[0096] KPMG LLP. (2017) Global Economic Impact of Tuberculosis: A reporter for Results UK.

[0097] Peter JG, Theron G, Dheda K. (2012a) Urine antigen test for diagnosis of HIV- associated tuberculosis. Lancet Infect Dis. 12(1 1 ):825;826-7.

[0098] Peter JG, Zijenah LS, Chanda D. (2016) Effect on mortality of point-of-care, urine-based lipoarabinomannan testing to guide tuberculosis treatment initiation in HIV-positive hospital inpatients: a pragmatic, parallel-group, multicountry, open-label, randomised controlled trial. Lancet. 387 (10024) : 1187-97.

[0099] Peter JG, Cashmore TJ, Meldau R, Theron G, van Zyl-Smit R, Dheda K. (2012b) Diagnostic accuracy of induced sputum LAM ELISA for tuberculosis diagnosis in sputum-scarce patients. Int J T uberc Lung Dis. 16 (8) : 1 108-12.

[0100] World Health Organization (2021 ). Global Tuberculosis Report.

[0101] World Health Organization (2014). High-priority target product profiles for new tuberculosis diagnostics: report of a consensus meeting. Geneva: World Health Organization.

Claims

CLAIMS1 . A method for diagnosing a Mycobacterium tuberculosis (M. tb) infection in a subject, the method comprising:(i) providing a urine sample obtained from the subject,(ii) detecting the presence of at least one host biomarker in the sample, and(iii) detecting the presence of at least one M. tb biomarker in the sample, wherein the presence of the at least one host biomarker together with the presence of the at least one M. tb biomarker indicates the presence of M. tb infection in the subject.

2. The method of claim 1 , wherein the at least one host biomarker is selected from the group consisting of C-reactive protein (CRP), alpha-1 - antichymotrypsin (SERPINA3) or resistin (RETN).

3. The method of claim 1 or 2, wherein the at least one M. tb biomarker is lipoarabinomannan (LAM).

4. The method of any one of claims 1 to 3, comprising detecting the presence of the following combinations of biomarkers in the sample:(i) CRP and LAM;(ii) RETN and LAM;(iii) SERPINA3 and LAM;(iv) RETN, CRP and LAM;(v) RETN, SERPINA3 and LAM; or(vi) CRP, SERPINA3 and LAM.

5. The method of any one of claims 1 to 4, wherein the steps of detecting the presence of the at least one M. tb biomarker and the at least one host biomarker are by means of immunoassay.

6. The method of claim 5, wherein the immunoassay is a lateral flow assay.

7. The method of any one of claims 1 to 6, wherein the M. tb infection is an active infection, or a latent infection.

8. The method of any one of claims 1 to 7, wherein the subject is a human.

9. A kit for detecting at least one host biomarker and at least one M. tb biomarker in a urine sample from a subject, the kit comprising:(i) a host biomarker capture molecule which binds to at least one host biomarker in the sample,(ii) a host biomarker detection molecule which detects the binding of the host biomarker capture molecule to the at least one host biomarker in the sample,(iii) a M. tb biomarker capture molecule which binds to at least one M. tb biomarker in the sample, and(iv) a M. tb biomarker detection molecule which detects the binding of the M. tb capture molecule to the at least one M. tb biomarker.

10. The kit of claim 9, wherein the kit further comprises a means for obtaining the sample from the subject.

11. The kit of claim 9 or 10, wherein the at least one host biomarker is selected from the group consisting of C-reactive protein (CRP), alpha-1 - antichymotrypsin (SERPINA3) or resistin (RETN).

12. The kit of any one of claims 9 to 11 , wherein the at least one M. tb biomarker is lipoarabinomannan (LAM).

13. The kit of any one of claims 9 to 12, wherein the kit is for detecting the presence of the following combinations of biomarkers in the sample:(i) CRP and LAM;(ii) RETN and LAM;(iii) SERPINA3 and LAM;(iv) RETN, CRP and LAM;(v) RETN, SERPINA3 and LAM; or(vi) CRP, SERPINA3 and LAM.

14. The kit of any one of claims 9 to 13, wherein the host biomarker capture molecule and the M. tb biomarker capture molecule are independently selected from the group consisting of a probe, an antibody, an antibody fragment, an aptamer or a ligand.

15. The kit of any one of claims 9 to 14, wherein the host biomarker detection molecule and the M. tb biomarker detection molecule are independently selected from the group consisting of a probe, an antibody, an antibody fragment, an aptamer or a ligand.

16. The kit of any one of claims 9 to 15, wherein the host biomarker detection molecule and the M. tb biomarker detection molecule are independently conjugated covalently or non-covalently to a detection label.

17. The kit of claim 16, wherein the detection label is selected from the group consisting of colourimetric labels, fluorescent labels, chemiluminescent labels, biotin, phosphor-based labels, thermal-based labels, enzymatic labels, gold nanoparticles, silver nanoparticles and magnetic beads.