A method for the diagnosis of a urinary tract infection

Abstract

A method for diagnosing a urinary tract infection in a subject, the method comprising measuring the expression level of one or more of proteins selected from MMP9, IL8, MPO and HNE in a urine sample obtained from the subject; and comparing the expression level of the one or more proteins to a reference level; wherein when the protein expression levels are increased or there is a positive expression level when compared to the reference level, the subject is predicted to have a urinary tract infection.

Description

[0001] Title

[0002] A Method for the Diagnosis of a Urinary Tract Infection

[0003] Field of the Invention

[0004] The invention relates to the diagnosis of Urinary Tract Infections (UTIs) by determining the levels of a biomarker in a urine sample. Specifically, the invention relates to the diagnosis of a complicated or uncomplicated UTI by determining the levels of a combination of biomarkers in a biological sample.

[0005] Background to the Invention

[0006] Urinary tract infection (UTI) represents the leading cause of infection worldwide. UTI results from the presence and multiplication of microorganisms together with tissue invasion in one or more structures of the urinary tract which encompasses the kidneys, ureters, bladder, and urethra. UTIs can be caused by numerous microorganisms, however, the most prevalent microorganism associated with UTI is Escherichia coli, whereby uropathogenic Escherichia coli has been identified as the etiologic agent responsible for more than 80% of all UTI's. Symptomatic patients suffering with lower UTI will present with pain during urination, frequent urination, feeling the need to urinate despite having an empty bladder, and incontinence. Symptomatic patients with upper UTI will present with pain and tenderness in the upper back and sides, chills, fever, nausea and vomiting.

[0007] Incidence of UTI is influenced by age, sex, spinal injuries, catheterization and patients’ immune status. Unnecessary antibiotic usage is significant due to poor clinical decisions, poor rapid urinalysis screening technologies and prolonged turnaround time (TAT) for microbiology culture reports. For symptomatic patient’s, a pure bacterial culture with a colony count of between2and 105cfu / ml is diagnostic depending on the patient demographic. Contaminated urine samples are a regular feature in standard urinalysis, and this often leads to repeat requests which may or may not be followed up, and in many cases the repeat urine sample gives a different picture due to antibiotic intervention. Contamination rates rise to 40% in complicated UTI groups such as those who are elderly catheterised.

[0008] Many laboratories now employ boric acid to improve the stability of the sample. Diagnostic accuracy depends a lot on preanalytical practices, e.g., collection, transport, and storage. Most of the errors in urine analysis involve the pre-analytical phase, and the literature encourages more efforts to be focused here.

[0009] Several guidelines now exist for both management and diagnosis of UTIs. Different guidelines exist for different at-risk groups, e.g., complicated vs uncomplicated UTI, lower UTI (cystitis) v upper UTI (Pyelonephritis), children, over 65’s, catheterized, immunocompromised, and antenatal. The common definition to cover all cases is the presence of symptoms: >104white blood cells / ml and bacterial growth of between 102cfu / ml and 105cfu / ml and strong emphasis on pure growth, a catch all approach.

[0010] The current gold-standard for UTI diagnosis is based on a 1950s urine culture method combined with the presence of a clinical indication (such as dysuria, frequency of urination, etc.) with white blood cell (WBC) microscopy commonly used in combination, or as a negative screen to reflect urine cultures. The current turnaround times from sampling to an initial result is between 24-48 hours. The combination of both culture and microscopy is often termed as urinalysis and is carried out in the laboratory by specially trained technicians. Although adequate for the diagnosis of uncomplicated UTI, the diagnostic performance of current gold standard urinalysis is often poor for complicated UTI due to often non-specific clinical indications and / or the presence of asymptomatic bacteriuria and concomitant pyuria which often causing false positive results.

[0011] Automated microscopy has improved diagnostic precision and increased throughput compared to manual microscopy. However, it has failed to improve turnaround time in most laboratories, even for negative samples, because of issues such as a requirement to manually review results (to improve test sensitivity); the presence of contamination can confound results; and automation is currently unable to differentiate between live and dead bacteria.

[0012] For point of care testing, urine reagent strips more commonly known as urine dipsticks, have previously been applied to assess the presence of UTI. Urinary dipsticks offer quick test results but have notably limited sensitivity (~45-60%), with some calling for this test to be abandoned in the clinical setting. In terms of clinical guidance by regulatory bodies, urinary dipsticks are only recommended for uncomplicated UTIs as a potential negative screen, with their application to complicated UTIs being dismissed due to the lack of sensitivity, specificity, and poor diagnostic accuracy. The ability to accurately diagnose those with complicated UTI is absent from current clinical practice, with a growing body of peer-reviewed literature highlighting the diagnostic challenges associated with complicated UTIs, particularly among elderly, long-term care, and catheterized populations, underscoring an unmet clinical need.

[0013] Some attempts have been made to use biomarkers as a means to diagnose UTIs, for example, as follows: WO 2018 / 158583 describes determining the levels of a selection of biomarkers in a urine sample to diagnose a UTI; US2019064165 describes compositions, methods and test devices for determining the presence of active leucocyte cells such as leukocyte esterase (LE) or HNE in an electrochemical assay to diagnose the presence of a UTI; and US2012115174 describes diagnostic aids for cystitis and, more specifically, to urinary trypsin inhibitors as a differential diagnostic aid for interstitial cystitis. US 2024 / 142466 describes a method of determining levels of one or more biomarkers from a long list of biomarkers to assess if a UTI is present; US 2010 / 166739 describes methods of diagnosing interstitial cystitis by detecting IL8 in a sample; US 2019 / 064165 describes detecting HNE in urine as a biomarker for the presence of a UTI. Gadalla Amal A. H. et al. (Scientific Reports, vol. 9(1) (2019)) describes a list of biomarkers in Tables 1 and 2 for indicating the presence of a UTI and Horvath Jozsef et al. (GMS Infectious Diseases, vol. 8, pp: 1-11 (2020)) describes MMP as a biomarker for a UTI in children.

[0014] It is an object of the claimed invention to overcome at least one of the abovereferenced problems.

[0015] Summary of the Invention

[0016] Complicated UTIs represent the occurrence of UTIs in patient cohorts with the most clinical significance related to the wrongful diagnosis and management. Complicated UTIs cannot be diagnosed accurately using current gold standard methods. In particular, antimicrobial stewardship is complex within these patient groups due to the lack of an effective point of care test leading to UTIs being over-diagnosed. This leads to associated antibiotic treatment being over-prescribed, which is a key contributor the development of antibiotic resistance and an increased risk of urosepsis. The occurrence of UTI in catheterised patients is a complicated UTI with significant clinical implications. Catheter-acquired UTIs represent the leading cause of hospital acquired infections (HAIs) worldwide and are a key driver of both urosepsis and bloodstream infections. The need for an accurate and rapid diagnostic method for use in a laboratory setting or as a point of care kit is essential to improve management and outcomes of patients with UTIs, and in particular patients with complicated UTIs.

[0017] This invention provides a method of diagnosing or detecting the presence of a urinary tract infection (UTI) in a subject by detecting (typically in a urine sample) one or more biomarkers selected from matrix metalloproteinase-8 (MMP8), matrix metalloproteinase-9 (MMP9), human neutrophil elastase (HNE), myeloperoxidase (MPO), interleukin-8 (IL8), interleukin- 6 (IL6), neutrophil gelatinase-associated lipocalin (NGAL), and lactoferrin. Preferably, at least two or three biomarkers from MMP9, HNE, MPO, and IL8 are selected. The invention is particularly relevant in diagnosing or detecting the presence of a complicated UTI in a subject.

[0018] According to the invention, there is provided a method and kit as set out in the appended claims.

[0019] According to the present invention, there is provided a method for diagnosing a (complicated or uncomplicated) urinary tract infection in a subject, the method comprising: measuring the expression level of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, HNE, in a urine sample obtained from the subject; and comparing the expression level of the one or more proteins, or fragments of said protein, to a reference level; wherein when the protein expression levels are increased or there is a positive expression level when compared to the reference level, the subject is predicted to have a (complicated or uncomplicated) urinary tract infection. Preferably, at least two or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least two proteins, or fragments of said proteins, are IL8 and MMP9. Preferably, at least three or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least three proteins, or fragments of said proteins, are IL8, MMP9, and MPO. Preferably, at least four or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least four proteins, or fragments of said proteins, are HNE, MMP9, MPO and IL8. Preferably, at least two or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least two proteins, or fragments of said proteins, are IL8 and MMP9. Preferably, at least three or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least three proteins, or fragments of said proteins, are ILS, MMP9, and MPO. Preferably, at least four proteins, or fragments of said proteins, are MMP9, IL8, MPO, and HNE.

[0020] In one aspect, there is provided a method for monitoring a (complicated or uncomplicated) urinary tract infection in a subject, the method comprising: measuring the expression level of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE in a urine sample obtained from the subject; and comparing the expression level of the one or more proteins to a reference level from either a control sample or from earlier measurements taken from a sample obtained from the same subject; wherein when the protein expression are unchanged, increased or are positive relative to the reference measurements (for example, from the control sample or the earlier measurements taken from the sample obtained by the subject), the subject is predicted to have a persisting (complicated or uncomplicated) urinary tract infection; and wherein when the protein expression levels are decreased or absent relative to the reference measurements or the earlier measurements, the subject is predicted to be recovering from or has recovered from the (complicated or uncomplicated) urinary tract infection. Preferably, at least two or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least two proteins, or fragments of said proteins, are IL8 and MMP9. Preferably, at least three or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least three proteins, or fragments of said proteins, are IL8, MMP9, and MPO. Preferably, at least four or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE and wherein said at least four proteins, or fragments of said proteins, are HNE, MMP9, MPO and IL8. Preferably, at least two or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least two proteins, or fragments of said proteins, are IL8 and MMP9. Preferably, at least three or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least three proteins, or fragments of said proteins, are IL8, MMP9, and MPO. Preferably, at least four proteins, or fragments of said proteins, are MMP9, IL8, MPO, and HNE.

[0021] In one aspect, there is provided a method of treating a (complicated or uncomplicated) urinary tract infection in a subject, the method comprising: measuring the expression level of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE in a urine sample obtained from the subject; and comparing each measurement with a respective reference measurement; wherein when the expression level of the at least one or more proteins is increased or positive relative to the reference measurement, the subject is treated for a (complicated or uncomplicated) urinary tract infection. Preferably, at least two or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least two proteins, or fragments of said proteins, are IL8 and MMP9. Preferably, at least three or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least three proteins, or fragments of said proteins, are IL8, MMP9, and MPO. Preferably, at least four or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least four proteins, or fragments of said proteins, are HNE, MMP9, MPO and IL8. Preferably, at least two or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least two proteins, or fragments of said proteins, are IL8 and MMP9. Preferably, at least three or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least three proteins, or fragments of said proteins, are IL8, MMP9, and MPO. Preferably, at least four proteins, or fragments of said proteins, are MMP9, IL8, MPO, and HNE.

[0022] In one aspect, there is provided a method of monitoring treatment of a (complicated or uncomplicated) urinary tract infection in a subject, the method comprising: measuring the expression level of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE in a urine sample obtained from the subject prior to receiving treatment to define a reference expression level in the subject; measuring the expression level of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE in a urine sample obtained from the subject following treatment; and comparing the expression level of the one or more proteins to a reference level; wherein when the measurements obtained after treatment are unchanged, increased or positive relative to the measurements obtained before treatment, the treatment is predicted to be ineffective against the (complicated or uncomplicated) urinary tract infection; and wherein when the expression level of the one or more proteins obtained after treatment are decreased or absent relative to the measurements obtained before treatment, the treatment is predicted to be effective against the (complicated or uncomplicated) urinary tract infection. Preferably, at least two or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE and wherein said at least two proteins, or fragments of said proteins, are IL8 and MMP9. Preferably, at least three or more proteins, or fragments of said protein, are selected from MMP9, ILS, MPO, and HNE, and wherein said at least three proteins, or fragments of said proteins, are ILS, MMP9, and MPO. Preferably, at least four or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least four proteins, or fragments of said proteins, are HNE, MMP9, MPO and IL8. Preferably, at least two or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least two proteins, or fragments of said proteins, are IL8 and MMP9. Preferably, at least three or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least three proteins, or fragments of said proteins, are IL8, MMP9, and MPO. Preferably, at least four proteins, or fragments of said proteins, are MMP9, IL8, MPO, and HNE.

[0023] In one aspect, there is provided a method for diagnosing a urinary tract infection in a subject, the method comprising measuring the protein expression levels of MMP9, IL8, and MPO in a urine sample obtained from the subject; and comparing the combined protein expression levels of MMP9, IL8, and MPO to a reference level; wherein when the combined protein expression levels of MMP9, IL8, and MPO are increased or there is a positive combined protein expression level of MMP9, IL8, and MPO when compared to the reference level, the subject is predicted to have a urinary tract infection; and wherein the subject is selected from a catheterised patient, a male patient over 65 years of age, and a female patient over 65 years of age.

[0024] In one aspect, there is provided a method for monitoring a urinary tract infection in a subject, the method comprising measuring the protein expression levels of MMP9, IL8, and MPO in a urine sample obtained from the subject; and comparing the combined protein expression level of MMP9, IL8, and MPO to a reference level from either a control sample or from earlier protein expression level measurements taken from a previously obtained sample from the subject; wherein when the combined protein expression levels of MMP9, IL8, and MPO are unchanged, increased or are positive relative to the reference levels, the subject is predicted to have a persisting urinary tract infection; and wherein when the combined protein expression levels of MMP9, IL8, and MPO are decreased or absent relative to the reference level, the subject is predicted to be recovering from or has recovered from the urinary tract infection; and wherein the subject is a catheterised patient, a male patient over 65 years of age, and a female patient over 65 years of age. In one aspect, there is provided a method of monitoring treatment of a urinary tract infection in a subject, the method comprising: measuring the protein expression level of MMP9, IL8, and MPO in a urine sample obtained from the subject prior to receiving treatment to define a reference expression level in the subject; measuring the protein expression levels of MMP9, IL8, and MPO in a urine sample obtained from the subject following treatment; and comparing the combined protein expression levels of MMP9, IL8, and MPO to the reference expression levels obtained before treatment with each measurement obtained after treatment; wherein when the combined protein expression levels of MMP9, IL8, and MPO obtained after treatment are unchanged, increased or positive relative to the measurements obtained before treatment, the treatment is predicted to be ineffective against the urinary tract infection; and wherein when the combined protein expression levels of MMP9, IL8, and MPO obtained after treatment are decreased or absent relative to the measurements obtained before treatment, the treatment is predicted to be effective against the urinary tract infection; and wherein the subject is a catheterised patient, a male patient over 65 years of age, and a female patient over 65 years of age.

[0025] In one aspect, there is provided a method for diagnosing a complicated urinary tract infection in a subject, the method comprising measuring the protein expression level of MMP9, IL8, and MPO in a urine sample obtained from the subject; and comparing the combined protein expression levels of MMP9, IL8, and MPO to a reference level; wherein when the combined protein expression levels of MMP9, IL8, and MPO are increased or there is a positive combined protein expression level of MMP9, IL8, and MPO when compared to the reference level, the subject is predicted to have a complicated urinary tract infection; and wherein the subject is a catheterised patient, a male patient over 65 years of age, and a female patient over 65 years of age.

[0026] In one aspect, there is provided a method for diagnosing a urinary tract infection in a catheterised subject, the method comprising measuring the protein expression levels of MMP9, MPO, and IL8, in a urine sample obtained from the catheterised subject; and comparing the combined protein expression levels of MMP9, MPO, and IL8 to a reference level; wherein when the combined protein expression levels of MMP9, MPO, and IL8 are increased or there is a positive combined protein expression level of MMP9, MPO, and IL8 when compared to the reference level, the catheterised subject is predicted to have a urinary tract infection. In one aspect, there is provided a method for diagnosing a urinary tract infection in a subject, the method comprising measuring the protein expression levels of MMP9, IL8, and MPO in a urine sample obtained from the subject; and comparing the combined protein expression levels of MMP9, IL8, and MPO to a reference level; wherein when the combined protein expression levels of MMP9, IL8, and MPO are increased or there is a positive combined protein expression level of MMP9, IL8, and MPO when compared to the reference level, the subject is predicted to have a urinary tract infection.

[0027] In one aspect, there is provided a method for monitoring a urinary tract infection in a subject, the method comprising measuring the protein expression levels of MMP9, IL8, and MPO in a urine sample obtained from the subject; and comparing the combined protein expression level of MMP9, IL8, and MPO to a reference level from either a control sample or from earlier protein expression level measurements taken from a previously obtained sample from the subject; wherein when the combined protein expression levels of MMP9, IL8, and MPO are unchanged, increased or are positive relative to the reference levels, the subject is predicted to have a persisting urinary tract infection; and wherein when the combined protein expression levels of MMP9, IL8, and MPO are decreased or absent relative to the reference level, the subject is predicted to be recovering from or has recovered from the urinary tract infection.

[0028] In one aspect, there is provided a method of monitoring treatment of a urinary tract infection in a subject, the method comprising measuring the protein expression level of MMP9, IL8, and MPO in a urine sample obtained from the subject prior to receiving treatment to define a reference expression level in the subject; measuring the protein expression levels of MMP9, IL8, and MPO in a urine sample obtained from the subject following treatment; and comparing the combined protein expression levels of MMP9, IL8, and MPO to the reference expression levels obtained before treatment with each measurement obtained after treatment; wherein when the combined protein expression levels of MMP9, IL8, and MPO obtained after treatment are unchanged, increased or positive relative to the measurements obtained before treatment, the treatment is predicted to be ineffective against the urinary tract infection; and wherein when the combined protein expression levels of MMP9, IL8, and MPO obtained after treatment are decreased or absent relative to the measurements obtained before treatment, the treatment is predicted to be effective against the urinary tract infection. In one aspect, there is provided a method for diagnosing a complicated urinary tract infection in a subject, the method comprising measuring the protein expression level of MMP9, IL8, and MPO in a urine sample obtained from the subject; and comparing the combined protein expression levels of MMP9, IL8, and MPO to a reference level; and wherein when the combined protein expression levels of MMP9, IL8, and MPO are increased or there is a positive combined protein expression level of MMP9, IL8, and MPO when compared to the reference level, the subject is predicted to have a complicated urinary tract infection.

[0029] In one aspect, the treatment is an antibiotic selected from trimethoprim, sulfamethoxazole, fosfomycin, nitrofurantoin, cephalexin, ceftriaxone, aminopenicillins (penicillin, amoxicillin carboxypenicillin, p-lactamase inhibitor), clavulanate, fluoroquinolones (such as ciprofloxacin, levofloxacin, moxifloxacin, ofloxacin, gemifloxacin, and delafloxacin), and triazaacenaphthylenes (gepotidacin).

[0030] In one aspect, the urinary tract infection is selected from pyelonephritis, acute pyelonephritis, chronic pyelonephritis, epididymitis, cystitis, vaginitis, prostatitis, and urethritis.

[0031] In one aspect, at least one protein is MMP9. In one aspect, at least one protein is MPO. In one aspect, at least one protein is IL8. In one aspect, at least one protein is HNE.

[0032] In one aspect, at least two or more proteins selected are from MMP9, IL8, MPO, and HNE, and are MMP9 and IL8, MMP9 and MPO, or IL8 and MPO.

[0033] In one aspect, at least three or more proteins are selected from MMP9, IL8, MPO, and HNE, and are MMP9, IL8 and MPO, MMP9, MPO and HNE, and MMP9, IL8 and HNE.

[0034] In one aspect, only MMP9, IL8, and MPO are selected. In one aspect, all of MMP9, IL8, MPO, and HNE are selected.

[0035] In one aspect, the complicated urinary tract infection is selected from chronic pyelonephritis, epididymitis, and acute pyelonephritis, cystitis, and urethritis.

[0036] In one aspect, the complicated urinary tract infections of cystitis and urethritis are typically in catheterised subjects, paediatric subjects (<12 years old), geriatric subjects (women and men >65 years old), male of any age, subjects with diabetes, antenatal subjects, subjects with a recurring UTI or subjects with anatomical abnormalities related to the urinary tract such as an enlarged bladder.

[0037] In one aspect the uncomplicated urinary tract infection is cystitis and / or urethritis in non-pregnant females between 12-65 years old.

[0038] In one aspect, the subject is selected from a cohort defined as a whole population, females only, males only, females older than 65 years of age, males older than 65 years age, females younger than 12 years of age, females between 12 and 65 years of age males younger than 12 years old, males between 12 and 65 years of age, antenatal subjects, diabetic subjects, catheterised subjects, and non-catheterised subjects. Typically, subjects selected from females older than 65 years of age, males older than 65 years age, females younger than 12 years of age, males younger than 12 years old, males between 12 and 65 years of age, diabetic subjects, and catheterised subjects would only have a complicated UTI. Typically, non-pregnant females between 12-65 years of age would only have an uncomplicated UTI.

[0039] In one aspect, when the cohort is a catheterised subject, the one or more proteins are selected from MMP9, HNE, MPO, and IL8.

[0040] In one aspect, when the cohort is a non-catheterised subject, the one or more proteins are selected from MMP9, HNE, MPO, and IL8.

[0041] In one aspect, when the cohort is females only, the one or more proteins are selected from MMP9, HNE, MPO, and IL8.

[0042] In one aspect, when the cohort is males only, the one or more proteins are selected from MMP9, HNE, MPO, and IL8.

[0043] In one aspect, when the cohort is females over the age of 65, the one or more proteins are selected from MMP9, HNE, MPO, and IL8.

[0044] In one aspect, when the cohort is males over the age of 65, the one or more proteins are selected from MMP9, HNE, MPO, and IL8.

[0045] In one aspect, when the cohort is a female under the age of 12, the one or more proteins are selected from MMP9, HNE, MPO, and IL8. In one aspect, when the cohort is a male under the age of 12, the one or more proteins are selected from MMP9, HNE, MPO, and ILS.

[0046] In one aspect, when the cohort is a female between the ages of 12 and 65, the one or more proteins are selected from MMP9, HNE, MPO, and IL8.

[0047] In one aspect, when the cohort is a male between the ages of 12 and 65, the one or more proteins are selected from MMP9, HNE, MPO, and IL8.

[0048] In one aspect, when the cohort is an antenatal female, the one or more proteins are selected from MMP9, HNE, MPO, and IL8.

[0049] In one aspect, when the cohort is a catheterised subject, the proteins are MMP9, MPO, and IL8.

[0050] In one aspect, when the cohort is a non-catheterised subject, the proteins are MMP9, MPO, and IL8.

[0051] In one aspect, when the cohort is females only, the proteins are MMP9, MPO, and IL8.

[0052] In one aspect, when the cohort is males only, the proteins are MMP9, MPO, and IL8.

[0053] In one aspect, when the cohort is females over the age of 65, the proteins are MMP9, MPO, and IL8.

[0054] In one aspect, when the cohort is males over the age of 65, the proteins are MMP9, MPO, and IL8.

[0055] In one aspect, when the cohort is a female under the age of 12, the proteins are MMP9, MPO, and IL8.

[0056] In one aspect, when the cohort is a male under the age of 12, the proteins MMP9, MPO, and IL8.

[0057] In one aspect, when the cohort is a female between the ages of 12 and 65, the proteins are MMP9, MPO, and IL8.

[0058] In one aspect, when the cohort is a male between the ages of 12 and 65, the proteins are MMP9, MPO, and IL8. In one aspect, when the cohort is an antenatal female, the proteins are MMP9, MPO, and ILS.

[0059] In one aspect, there is provided a method for diagnosing a complicated urinary tract infection in a subject, the method comprising: measuring the expression level of one or more of proteins selected from MMP9, IL8, MPO and HNE in a biological sample obtained from the subject; and comparing the expression level of the one or more proteins to a reference level; wherein when the protein expression levels are increased or there is a positive expression level when compared to the reference level, the subject is predicted to have a complicated urinary tract infection.

[0060] In one aspect, there is provided a method for monitoring a complicated urinary tract infection in a subject, the method comprising: measuring the expression level of one or more of proteins selected from MMP9, IL8, MPO and HNE in a biological sample obtained from the subject; and comparing the expression level of the one or more proteins to a reference level from either a control sample or from earlier expression level measurements taken from the sample obtained by the subject; wherein when the protein expression levels are unchanged, increased or are positive relative to the reference levels, the subject is predicted to have a persisting complicated urinary tract infection; and wherein when the protein expression are decreased or absent relative to the reference level, the subject is predicted to be recovering from or has recovered from the complicated urinary tract infection.

[0061] In one aspect, there is provided a method of treating a complicated urinary tract infection in a subject, the method comprising: measuring the expression level of one or more of proteins selected from MMP9, IL8, MPO and HNE in a biological sample obtained from the subject; and comparing the expression level of the one or more proteins to a reference level; wherein when the expression level of the at least one or more proteins is increased or positive relative to the reference level, the subject is treated for a complicated urinary tract infection.

[0062] In one aspect, there is provided a method of monitoring treatment of a complicated urinary tract infection in a subject, the method comprising: measuring the expression level of one or more of proteins selected from MMP9, IL8, MPO and HNE in a biological sample obtained from the subject prior to receiving treatment to define a reference expression level in the subject; measuring the expression level of one or more of proteins selected from MMP9, ILS, MPO and HNE, in a urine sample obtained from the subject following treatment; and comparing the expression level of the one or more proteins to a reference level obtained before treatment with each measurement obtained after treatment; wherein when the protein expression levels obtained after treatment are unchanged, increased or positive relative to the measurements obtained before treatment, the treatment is predicted to be ineffective against the complicated urinary tract infection; and wherein when the protein expression levels obtained after treatment are decreased or absent relative to the measurements obtained before treatment, the treatment is predicted to be effective against the complicated urinary tract infection.

[0063] In one aspect, there is provided a method for diagnosing a complicated urinary tract infection in cohort of subjects selected from a whole population, females only, males only, females older than 65 years of age, males older than 65 years age, females younger than 12 years of age, males younger than 12 years old, males between 12 and 65 years of age, antenatal subjects, diabetic subjects, catheterised subjects and non-catheterised subjects, the method comprising: measuring the expression level of one or more proteins selected from MMP9, HNE, MPO, and IL8, in a biological sample obtained from the subject; and comparing the expression level of the one or more proteins to a reference level; wherein when the protein expression levels are increased or there is a positive expression level when compared to the reference level, the subject in the cohort is predicted to have a complicated urinary tract infection.

[0064] In one aspect, there is provided a method for diagnosing a complicated urinary tract infection in, for example, a catheterised subject, the method comprising: measuring the expression level of one or more proteins selected from MMP9, HNE, MPO, and IL8, in a biological sample obtained from the subject; and comparing the expression level of the one or more proteins to a reference level; wherein when the protein expression levels are increased or there is a positive expression level when compared to the reference level, the catheterised subject is predicted to have a complicated urinary tract infection.

[0065] In one aspect, the biological sample is selected from blood, blood derivatives, urine, urine derivatives, saliva, sweat, cerebrospinal fluid, semen, and lymph. Preferably, the biological sample is urine or urine derivatives. As described herein, expression levels of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE can be increased and / or present in a urine sample obtained from a subject with a (complicated or uncomplicated) urinary tract infection when compared to a urine sample obtained from a subject who does not have a urinary tract infection. Accordingly, in one aspect of any of the embodiments, described herein is a method of treating a (complicated or uncomplicated) urinary tract infection in a subject in need thereof, the method comprising administering trimethoprim, fosfomycin, nitrofurantoin, cephalexin, ceftriaxone, and / or fluoroquinolones to a subject determined to have an expression level of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE that is increased relative to a reference sample. In one aspect of any of the embodiments, described herein is a method of treating a (complicated or uncomplicated) urinary tract infection in a subject in need thereof, the method comprising: a) determining the expression level of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE in a sample obtained from a subject; and b) administering an antibiotic to the subject if the expression level of the one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE is increased or positive relative to a reference sample. Preferably, the expression levels of MMP9, MPO, and IL8, or fragments of said proteins, are determined and combined.

[0066] In some embodiments of any of the aspects, the method comprises administering an antibiotic to a subject previously determined to have an increased or positive expression level of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE relative to a reference sample. In some embodiments of any of the aspects, described herein is a method of treating a (complicated or uncomplicated) urinary tract infection in a subject in need thereof, the method comprising: a) first determining the expression level of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE in a urine sample obtained from the subject, and b) then administering an antibiotic to the subject if the expression level of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO and HNE is increased or positive relative to a reference sample. Preferably, at least two or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least two proteins, or fragments of said proteins, are IL8 and MMP9. Preferably, at least three or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least three proteins, or fragments of said proteins, are IL8, MMP9, and MPO. Preferably, at least four proteins, or fragments of said proteins, are MMP9, IL8, MPO, and HNE. Preferably, the expression levels of MMP9, MPO, and IL8, or fragments of said proteins, are determined and combined.

[0067] In one aspect of any of the embodiments, described herein is a method of treating a (complicated or uncomplicated) urinary tract infection in a subject in need thereof, the method comprising: a) determining if the subject has an increased expression level of or a positive expression of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE in a urine sample obtained from the subject; and b) administering an antibiotic to the subject if the expression level of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE is increased or positive relative to a reference sample. In some embodiments of any of the aspects, the step of determining if the subject has an increased expression level or a positive expression of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE can comprise i) obtaining or having obtained a sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine the presence or absence of, and to measure the expression level of, one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE, relative to a reference sample. In some embodiments of any of the aspects, the step of determining if the subject has an increased expression level or a positive expression of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE can comprise performing or having performed an assay on a sample obtained from the subject to determine the presence or absence of, and to measure the expression level of, one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE, in the sample. In some embodiments of any of the aspects, the step of determining if the subject has an increased expression level of, or a positive expression of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE, can comprise ordering or requesting an assay on a sample obtained from the subject to determine the expression level or positive expression of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an increased expression level or positive expression of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE, can comprise receiving the results of an assay on a sample obtained from the subject to determine the expression levels or positive expression of one or more of proteins, or fragments of said protein, selected from MMP9, ILS, MPO, and HNE. In some embodiments of any of the aspects, the step of determining if the subject has an increased expression level or a positive expression of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE can comprise receiving a report, results, or other means of identifying the subject as a subject with an increased expression level of, or a positive expression of, one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE. Preferably, at least two or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least two proteins, or fragments of said proteins, are IL8 and MMP9. Preferably, at least three or more proteins, or fragments of said protein, are selected from MMP9, IL8, MPO, and HNE, and wherein said at least three proteins, or fragments of said proteins, are IL8, MMP9, and MPO. Preferably, at least four proteins, or fragments of said proteins, are MMP9, IL8, MPO, and HNE.

[0068] In one aspect of any of the embodiments, described herein is a method of treating a (complicated or uncomplicated) urinary tract infection in a subject in need thereof, the method comprising: a) determining if the subject has an increased expression level or a positive expression of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE; and b) instructing or directing that the subject be administered an antibiotic if the expression level of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE is increased or positive relative to a reference sample. In some embodiments of any of the aspects, the step of determining if the subject has an increased expression level or a positive expression of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE relative to a reference, can comprise i) obtaining or having obtained a sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine the expression level or the positive expression of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE. In some embodiments of any of the aspects, the step of determining if the subject has an increased expression level or positive expression of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE relative to a reference sample, can comprise performing or having performed an assay on a sample obtained from the subject to determine the expression levels or positive expression of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE. In some embodiments of any of the aspects, the step of determining if the subject has an increased expression levels or positive expression of one or more of proteins, or fragments of said protein, selected from MMP9, ILS, MPO and HNE relative to a reference sample, can comprise ordering or requesting an assay on a sample obtained from the subject to determine the expression levels or positive expression of one or more of proteins, or fragments of said protein, selected from MMP9, IL8, MPO, and HNE. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results and / or treatment recommendations in view of the assay results. Preferably, the expression levels of MMP9, MPO, and IL8, or fragments of said proteins, are determined and combined.

[0069] In one embodiment, the levels of a polypeptide or a protein in a sample can be detected by a lateral flow immunoassay test (LFIA), also known as the immunochromatographic assay, or strip test. LFIAs are a simple device intended to detect the presence (or absence) of antigen, e.g., a polypeptide, in a fluid sample. There are currently many LFIA tests used for medical diagnostics, either for home testing, point of care testing, or laboratory use. LFIA tests are a form of immunoassay in which the test sample flows along a solid substrate via capillary action. After the sample is applied to the test strip it encounters a colored reagent (generally comprising antibody specific for the test target antigen) bound to microparticles which mixes with the sample and transits the substrate encountering lines or zones which have been pretreated with another antibody or antigen. Depending upon the level of target polypeptides present in the sample the colored reagent can be captured and become bound at the test line or zone. LFIAs are essentially immunoassays adapted to operate along a single axis to suit the test strip format or a dipstick format. Strip tests are extremely versatile and can be easily modified by one skilled in the art for detecting an enormous range of antigens from fluid samples such as urine, blood, water, and / or homogenized tissue samples etc. Strip tests are also known as dip stick tests, the name bearing from the literal action of "dipping" the test strip into a fluid sample to be tested. LFIA strip tests are easy to use, require minimum training and can easily be included as components of point-of-care test (POCT) diagnostics to be use on site in the field. LFIA tests can be operated as either competitive or sandwich assays. Sandwich LFIAs are similar to a sandwich enzyme-linked immunosorbent assay (ELISA). The sample first encounters colored particles which are labeled with antibodies raised to the target antigen. The test line will also contain antibodies to the same target, although it may bind to a different epitope on the antigen. The test line will show as a colored band in positive samples. In some embodiments of any of the aspects, the lateral flow immunoassay can be a double antibody sandwich assay, a competitive assay, a quantitative assay or variations thereof. Competitive LFIAs are similar to competitive ELISA. The sample first encounters colored particles which are labeled with the target antigen or an analogue. The test line contains antibodies to the target / its analogue. Unlabeled antigen in the sample will block the binding sites on the antibodies preventing uptake of the colored particles. The test line will show as a colored band in negative samples. There are a number of variations on lateral flow technology. It is also possible to apply multiple capture zones to create a multiplex test.

[0070] The use of "dip sticks" (such as Multistix®) or LFIA test strips and other solid supports have been described in the art in the context of an immunoassay for a number of antigen biomarkers. U.S. Pat. Nos. 4,943,522; 6,485,982; 6,187,598; 5,770,460; 5,622,871 ; 6,565,808, U. S. patent applications Ser. No. 10 / 278,676; U.S. Ser. No. 09 / 579,673 and U.S. Ser. No. 10 / 717,082, which are incorporated herein by reference in their entirety, are non-limiting examples of such lateral flow test devices. Examples of patents that describe the use of "dip stick" technology to detect soluble antigens via immunochemical assays include, but are not limited to US Patent Nos. 4,444,880; 4,305,924; and 4,135,884; which are incorporated by reference herein in their entireties. The apparatuses and methods of these three patents broadly describe a first component fixed to a solid surface on a "dip stick" which is exposed to a solution containing a soluble antigen that binds to the component fixed upon the "dip stick," prior to detection of the component-antigen complex upon the stick. It is within the skill of one in the art to modify the teachings of this "dip stick" technology for the detection of polypeptides using antibody reagents as described herein.

[0071] In some embodiments of any of the aspects, detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means. The detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies). The detectable label can be linked by covalent or non-covalent means to the reagent. Alternatively, a detectable label can be linked such as by directly labeling a molecule that achieves binding to the reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules. Detectable labels can include, but are not limited to, colloidal gold nanoparticles, colored latex beads, carbon nanoparticles, quantum dots, magnetic reporter particles, radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds (such as Europium fluorescent labels), metal chelates, and enzymes.

[0072] In other embodiments, the detection reagent is labelled with a fluorescent compound. When the fluorescently labeled reagent is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. In some embodiments of any of the aspects, a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine, Cy3™, Cy5™, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, fluorescent CPNs™ tandem conjugates such as phycoerythrin-Cy5™, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™, 6-carboxyfhiorescein (commonly known by the abbreviations FAM and F), 6-carboxy-2',4',7',4,7-hexachlorofiuorescein (HEX), 6- carboxy-4',5'-dichloro-2',7'-dimethoxyfiuorescein (JOE or J), N,N,N',N'-tetramethyl- 6carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5- carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc.’ BODIPY dyes and quinoline dyes. In some embodiments of any of the aspects, a detectable label can be a radiolabel including, but not limited to3H,125l,35S,14C,32P, and33P. In some embodiments of any of the aspects, a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase. An enzymatic label can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes contemplated for use to detectably label an antibody reagent include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In some embodiments of any of the aspects, a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. In some embodiments of any of the aspects, a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.

[0073] In some embodiments of any of the aspects, detection reagents can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, gold, or biotin. Other detection systems can also be used, for example, a biotin-streptavidin system. In this system, the antibodies immunoreactive ( / .e., specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromagenic substrate. Such streptavidin peroxidase detection kits are commercially available, e. g. from DAKO; Carpinteria, CA. A reagent can also be detectably labeled using fluorescence emitting metals such as152Eu, or others of the lanthanide series. These metals can be attached to the reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0074] A level which is less than a reference level can be a level which is less by at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 80%, at least about 90%, or less relative to the reference level. In some embodiments of any of the aspects, a level which is less than a reference level can be a level which is statistically significantly less than the reference level.

[0075] A level which is more than a reference level can be a level which is greater by at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 500% or more than the reference level. In some embodiments of any of the aspects, a level which is more than a reference level can be a level which is statistically significantly greater than the reference level.

[0076] In some embodiments of any of the aspects, the methods, assays, and systems described herein can further comprise a step of obtaining or having obtained a test sample from a subject. In some embodiments of any of the aspects, the subject can be a mammal. In some embodiments of any of the aspects, the mammal can be selected from a human subject, primates, non-human primates, farm animals, veterinary mammals, and mammals kept in captivity, and other mammals and higher mammals. In some embodiments of any of the aspects, the subject can be a subject in need of treatment for (e.g., having or diagnosed as having a urinary tract infection) or a subject at risk of or at increased risk of developing a urinary tract infection as described elsewhere herein.

[0077] Definitions

[0078] In the specification, the term “urinary tract infection (UTI)” should be understood to mean an infection in any part of the mammalian urinary system, that is, the kidneys, ureters, bladder, and urethra. Most infections involve the lower urinary tract (the bladder and the urethra). Examples of UTIs include acute pyelonephritis (kidney), cystitis (bladder), and urethritis (urethra). UTIs can be categorized into two distinct disease states: complicated and uncomplicated UTIs.

[0079] In the specification, the term “complicated UTI” should be understood to mean UTIs in patients with underlying medical conditions, co-morbidities, and / or other factors which complicate and effect the efficacy of current diagnostics methods and treatment of UTIs. Such underlying conditions, co-morbidities, and other factors include the presence of fever, catheterization (all catheter-related UTIs are a complicated UTI), sterile pyuria, asymptomatic bacteriuria (ASB), recurrent UTI, diabetes, anatomical abnormalities related to the urinary tract (such as enlarged bladder, bladder neck obstruction), impaired renal function, delirium, geriatric populations, the immunocompromised, pregnant women, post-menopausal women, male subjects (of any age), the elderly (anyone over 65 years of age), those in long term care and paediatric patients <12 years old. Complicated UTIs are particularly difficult to diagnose in catheterized patients and the elderly, especially those within long term care. This is fundamentally due to the presence of ASB and concomitant pyuria in the urine due to reasons other than infection. For long term care patients, the incidence of delirium and cognitive decline restricts the ability for patients to confer symptoms clearly and reliably to the clinician, augmenting UTI miss-management. All upper urinary tract infections are deemed complicated UTI. Complicated UTIs are associated with a significantly higher risk of morbidity and mortality, requiring generally longer and different treatment regimens and patient management to uncomplicated UTI. An uncomplicated UTI is a UTI in a nonpregnant female between the ages of 12 to 65. For all other patient cohorts mentioned, the presence of a UTI would be deemed as a complicated UTI.

[0080] In the specification, the term “uncomplicated UTI” should be understood to mean a urinary tract infection that occurs in the lower urinary tract (bladder and urethra) of otherwise healthy, nonpregnant females between 12-65 years old. Subjects with uncomplicated UTI are mostly treated on an outpatient basis and often resolve spontaneously without treatment. Current diagnostic procedures, including urine culture and microscopy are deemed adequate for the diagnosis of uncomplicated cases with urinary dipsticks being sometimes recommended as a negative screen.

[0081] In the specification, the term “subject”, “individual” or “patient” should be understood to mean all mammals, for example, a human, primates, non-human primates, farm animals (such as pigs, horses, goats, sheep, cows (including bulls, bullocks, heifers etc.), donkey, reindeer, etc.), veterinary mammals (such as dogs, cats, rabbits, hamsters, guinea pigs, mice, rats, ferrets, etc.), and mammals kept in captivity (such as lions, tigers, elephants, zebras, giraffes, pandas, rhino, hippopotamus, etc.), and other mammals and higher mammals for which the use of the invention is practicable.

[0082] In the specification, the term “antenatal” should be understood to mean during pregnancy.

[0083] As used herein, the terms “lateral flow device (LFD)” or a “test cartridge” primarily refer to the sandwich lateral flow assays of the type that generally comprise a strip of capillary flow materials having a longitudinal dimension and a capillary flow path along the longitudinal dimension. The strip generally comprises discrete overlapping sections including a sample pad, a conjugate release pad, a detection membrane (i.e., cellulose or a cellulose derivative such as nitrocellulose), and an adsorbent pad. The sample pad is generally formed from a wicking material, the details of which will be known to those skilled in the art, although it may also be provided by a proximal part of the detection membrane. The conjugate release pad generally comprises a conjugate dispersed within a dissolvable matrix (often salt or sugar) that releases the conjugate upon dissolution by the sample fluid. The conjugate comprises an analyte-specific antibody conjugated to a detectable label. The conjugate release pad may also be provided by a proximal part of the detection membrane. Further description of the components of the sections of the lateral flow assay are not provided herein but will be known to a person skilled in the art and are described in EP0349215, incorporated herein in its entirety.

[0084] As used herein, the term “biomarker” primarily refers to any or all of the biomarkers listed above. The biological fluids of interest to the present invention are primarily blood and blood derivatives (serum, plasma, etc.), and urine, but may also include other biological fluids such as saliva, sweat, cerebrospinal fluid, semen, and lymph. Non- biological synthetic fluids may also be of interest to the invention such as artificial urine, synthetic blood, synthetic cerebrospinal fluid, synthetic interstitial fluid, buffer solutions and synthetic calibration standards. The non-biological synthetic fluids may be used for assessment of, for example, an immune response in pre-clinical studies for antibiotic efficacy. In one embodiment, the biological sample employed in the methods of the invention is undiluted (especially when the sample is urine from a person who is dehydrated), which provides an advantage insofar as the user is not required to dilute the sample and makes the test suitable as a home-use test, or the biological sample is diluted. In one embodiment, the sample is diluted no more than 2, 3, 4, 5, 6, 7, 8, or 9- fold.

[0085] As used herein, the term “conjugate” refers to a biomarker-specific antibody, or biomarker-specific antibody fragment, conjugated to a detectable label. Generally, the antibody is a monoclonal antibody, but may also be a polyclonal antibody. When the biomarker is a human biomarker, the antibody is generally a non-human antibody, for example a mouse or goat antibody that is specific for the biomarker in question. The purpose of the conjugate is to bind to any biomarker in the sample, and travel along the flow path in the strip to the detection membrane where the conjugate binds to the test line (via the biomarker part of the conjugate binding to the immobilised antibody in the test zone).

[0086] As used herein, the term “detectable label” refers to a label that can be detected (for example, some emit a signal that is detectable), for example an optical, fluorescent, luminescent, magnetic, or electrical signal. Examples of detectable labels useful for the present invention include gold nanoparticles, coloured latex beads, magnetic particles, carbon nanoparticles, selenium nanoparticles, silver nanoparticles, quantum dots, up converting phosphors, organic fluorophores, textile dyes, enzymes, liposomes and others. As used herein, the term “test zone” refers to an area of the detection membrane containing a binding ligand for the biomarker. Generally, the binding ligand for the biomarker is provided along a line ( / .e., test line) that is generally perpendicular to the longitudinal axis of the test strip. Generally, the binding ligand for the biomarker is a biomarker-specific antibody ( / .e., capture antibody). When the biomarker is MMP, for example, the binding ligand is generally an MMP-specific antibody. The detection membrane may contain one or more test zones, and each may contain different concentrations of biomarker-binding ligand.

[0087] As used herein, the term “control zone” refers to an area of the detection membrane, generally distal of the test zone, containing a binding ligand for the biomarker-binding ligand. Generally, the binding ligand is provided along a line ( / .e., control line) that is generally perpendicular to the longitudinal axis of the test strip. Generally, the binding ligand for the capture (biomarker-specific) antibody is an antibody, for example an anti- IgG antibody or antibody-binding protein.

[0088] As used herein, the term “competitive analyte zone” refers to an area of the detection membrane containing biomarker. Generally, the biomarker is provided along a line ( / .e., a test line) that is generally perpendicular to the longitudinal axis of the test strip, or provided as a series or pattern of dots or lines. Generally, the binding ligand for the biomarker is a biomarker-specific antibody. The biomarker generally binds directly to the membrane, for example the nitrocellulose, and is typically not immobilised to the membrane via a binding partner.

[0089] As used herein, the term “detectable label fingerprint” refers to the combination of the intensities of the detectable label at the test zone, control zone and competitive biomarker zone. Generally, the detectable label is an optical label that can be read by a reader, for example an optical label scanner, and then converted into a set of detectable label intensity values. However, the intensities may be non-optical intensities, for example magnetic or electrical intensities, or the like. The computational model may be a linear regression model, that may employ Principal Component Analysis (PCA).

[0090] In the specification, the terms “reference level” or “reference” or "control sample", each of which can be used interchangeably, should be understood to be a reference level obtained from either a sample obtained from a subject who does not have a UTI (complicated or uncomplicated), or is from a sample obtained from a subject who already has been diagnosed with a UTI and has been already provided with a treatment. The samples can be pooled together in their correct cohorts to provide a library of known values that can be used to create threshold expression levels for the biomarkers, where if the value is above the threshold value, the subject has a UTI or a persisting UTI, and where if the value is below the threshold value, the subject does not have a UTI and / or the UTI is shown to be responding to or has responded to treatment. The method can distinguish a complicated UTI in a patient cohort whereby the diagnosis of UTI is compromised or difficult due to the presence of other complex symptoms and co-morbidities, such as those who are catheterised (where current testing is sub-optimal). In addition, the method can also distinguish UTI in an uncomplicated group, for example, the method can distinguish a UTI in nonpregnant women between the ages of 12-65 where symptoms similar to an uncomplicated UTI may present which is not a UTI.

[0091] Brief Description of the Drawings

[0092] The invention will be more clearly understood from the following description of an aspect thereof, given by way of example only, with reference to the accompanying drawings, in which:

[0093] Figure 1 illustrates an AUC-ROC for MMP9 (concentrations - ng / ml) in differentiating patients with a probable UTI from those with no UTI, in the whole population.

[0094] Figure 2 illustrates an AUC-ROC for MPO (concentrations - ng / ml) in differentiating patients with a probable UTI from those with no UTI, in the whole population.

[0095] Figure 3 illustrates an AUC-ROC for MMP8 (concentrations - ng / ml) in differentiating patients with a probable UTI from those with no UTI, in the whole population.

[0096] Figure 4 illustrates an AUC-ROC for HNE (concentrations - ng / ml) in differentiating patients with a probable UTI from those with no UTI, in the whole population.

[0097] Figure 5 illustrates an AUC-ROC for IL8 (concentrations - ng / ml) in differentiating patients with a probable UTI from those with no UTI, in the whole population. Figure 6 illustrates an AUC-ROC for NGAL (concentrations - ng / ml) in differentiating patients with a probable UTI from those with no UTI, in the whole population.

[0098] Figure 7 illustrates an AUC-ROC for Lactoferrin (concentrations - ng / ml) in differentiating patients with a probable UTI from those with no UTI, in the whole population.

[0099] Figure 8 illustrates an AUC-ROC for IL6 (concentrations - ng / ml) in differentiating patients with a probable UTI from those with no UTI, in the whole population.

[0100] Figure 9 illustrates an AUC-ROC for MMP9 and HNE in differentiating patients with a probable UTI from those with no UTI, in the whole population.

[0101] Figure 10 illustrates an AUC-ROC for MMP9 and MPO in differentiating patients with a probable UTI from those with no UTI, in the whole population.

[0102] Figure 11 illustrates an AUC-ROC for MMP9 and IL8 in differentiating patients with a probable UTI from those with no UTI, in the whole population.

[0103] Figure 12 illustrates an AUC-ROC for MMP9 and MMP8 in differentiating patients with a probable UTI from those with no UTI, in the whole population.

[0104] Figure 13 illustrates an AUC-ROC for the MMP9, HNE and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in the whole population.

[0105] Figure 14 illustrates an AUC-ROC for the MMP9, MPO and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in the whole population.

[0106] Figure 15 illustrates an AUC-ROC for the MMP9, HNE and MPO logistic regression model in differentiating patients with a probable UTI from those with no UTI, in the whole population.

[0107] Figure 16 illustrates an AUC-ROC for the MMP9, HNE and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in females > 65 years old.

[0108] Figure 17 illustrates an AUC-ROC for the MMP9, HNE and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in males > 65 years old.

[0109] Figure 18 illustrates an AUC-ROC for the MMP9, HNE and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in females <12 years old. Figure 19 illustrates an AUC-ROC for the MMP9, HNE and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in males <12 years old.

[0110] Figure 20 illustrates an AUC-ROC for the MMP9, HNE and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in females aged 12 - 65 years old.

[0111] Figure 21 illustrates an AUC-ROC for the MMP9, HNE and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in males aged 12 - 65 years old.

[0112] Figure 22 illustrates an AUC-ROC for the MMP9, HNE and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in females.

[0113] Figure 23 illustrates an AUC-ROC for the MMP9, HNE and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in males.

[0114] Figure 24 illustrates an AUC-ROC for the MMP9, HNE and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in antenatal subjects.

[0115] Figure 25 illustrates an AUC-ROC for the MMP9, HNE and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in catheterised subjects.

[0116] Figure 26 illustrates an AUC-ROC for the MMP9, HNE and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in non-catheterised subjects.

[0117] Figure 27 illustrates an AUC-ROC for the MMP9, MPO and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in females > 65 years old.

[0118] Figure 28 illustrates an AUC-ROC for the MMP9, MPO and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in males > 65 years old.

[0119] Figure 29 illustrates an AUC-ROC for the MMP9, MPO and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in females < 12 years old.

[0120] Figure 30 illustrates an AUC-ROC for the MMP9, MPO and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in males < 12 years old. Figure 31 illustrates an AUC-ROC for the MMP9, MPO and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in females between 12 -65 years old.

[0121] Figure 32 illustrates an AUC-ROC for the MMP9, MPO and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in males between 12 -65 years old.

[0122] Figure 33 illustrates an AUC-ROC for the MMP9, MPO and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in females.

[0123] Figure 34 illustrates an AUC-ROC for the MMP9, MPO and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in males.

[0124] Figure 35 illustrates an AUC-ROC for the MMP9, MPO and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in antenatal subjects.

[0125] Figure 36 illustrates an AUC-ROC for the MMP9, MPO and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in catheterised subjects.

[0126] Figure 37 illustrates an AUC-ROC for the MMP9, MPO and IL8 logistic regression model in differentiating patients with a probable UTI from those with no UTI, in non-catheterised subjects.

[0127] Figure 38 illustrates an AUC-ROC for the MMP9, HNE and MPO logistic regression model in differentiating patients with a probable UTI from those with no UTI, in females > 65 years old.

[0128] Figure 39 illustrates an AUC-ROC for the MMP9, HNE and MPO logistic regression model in differentiating patients with a probable UTI from those with no UTI, in males > 65 years old.

[0129] Figure 40 illustrates an AUC-ROC for the MMP9, HNE and MPO logistic regression model in differentiating patients with a probable UTI from those with no UTI, in females < 12 years old.

[0130] Figure 41 illustrates an AUC-ROC for the MMP9, HNE and MPO logistic regression model in differentiating patients with a probable UTI from those with no UTI, in males < 12 years old.

[0131] Figure 42 illustrates an AUC-ROC for the MMP9, HNE and MPO logistic regression model in differentiating patients with a probable UTI from those with no UTI, in females between 12 -65 years old. Figure 43 illustrates an AUC-ROC for the MMP9, HNE and MPO logistic regression model in differentiating patients with a probable UTI from those with no UTI, in males between 12 -65 years old.

[0132] Figure 44 illustrates an AUC-ROC for the MMP9, HNE and MPO logistic regression model in differentiating patients with a probable UTI from those with no UTI, in females.

[0133] Figure 45 illustrates an AUC-ROC for the MMP9, HNE and MPO logistic regression model in differentiating patients with a probable UTI from those with no UTI, in males.

[0134] Figure 46 illustrates an AUC-ROC for the MMP9, HNE and MPO logistic regression model in differentiating patients with a probable UTI from those with no UTI, in antenatal subjects.

[0135] Figure 47 illustrates an AUC-ROC for the MMP9, HNE and MPO logistic regression model in differentiating patients with a probable UTI from those with no UTI, in catheterised subjects.

[0136] Figure 48 illustrates an AUC-ROC for the MMP9, HNE and MPO logistic regression model in differentiating patients with a probable UTI from those with no UTI, in non-catheterised subjects.

[0137] Detailed Description of the Drawings

[0138] There is an unmet need for accurate detection of UTIs in subjects, and particularly in complicated UTI patient cohorts. Urinary dipsticks and standard diagnostic procedures such as standard urine culture and microscopy, often yield false positives due the underlying presence of asymptomatic bacteriuria, with concomitant pyuria, in complicated UTI patient cohorts. The Applicants performed a study across 554 patients with suspected UTI to assess the efficacy of a panel of biomarkers to diagnose the presence of a UTI or having no UTI. The biomarkers were selected from the group comprising: matrix metalloproteinase-8 (MMP8), matrix metalloproteinase-9 (MMP9), human neutrophil elastase (HNE), myeloperoxidase (MPO), interleukin-8 (IL8), interleukin-6 (IL6), neutrophil gelatinase-associated lipocalin (NGAL), and lactoferrin, or combinations thereof, for use in the detection of (complicated or uncomplicated) UTIs, as a first line screening tool for the laboratory diagnosis of (complicated or uncomplicated) UTI. These combinations were compared to a reference method of light microscopy and the accepted gold standard of microbial culture performed as per Public Health England UK Standards for Microbiology Investigations Investigation (SMI) of Urine B41. First line urine culture was performed using Chromagar with discordant samples reflexed to test for antimicrobial substances and expanded culture for fastidious organisms. The use of several of these biomarkers for the diagnosis of UTI has been previously described, for example, in WO 2018 / 158583, which is incorporated herein by reference in its entirety.

[0139] Methods:

[0140] Patients and sample collection

[0141] Urine specimens were collected in sterile universal containers and were included in the study. All specimens represented excess remnants of urine specimens submitted for routine standard of care testing which otherwise would have been discarded and were enrolled in accordance with local ethics guidelines. A total of 554 urine specimens were eligible for inclusion in this study.

[0142] Participants of all ages both male and female exhibiting at least one clinical sign or symptoms of a UTI were eligible for inclusion as per the UK Standards for Microbiology Investigations (SMI) B41 guidelines for urine investigations. These clinical symptoms included dysuria, frequency, nocturia, urgency, difficulty urinating, low abdominal pain, pelvic pain pressure, low back pain, or haematuria. Urine samples were enrolled both from inpatients and those in the community and were tested within 24 hours of collection. Specimens were excluded if they did not meet the inclusion criteria or if the specimen was not set up on all methods (a combination of microscopy and culture) on the same day.

[0143] Light microscopy

[0144] Light microscopy was performed on all specimens. A KOVA counting chamber (Langan Bach Services, Ireland) was loaded with fresh urine using a capillary tube (Vitrex, Denmark). Each specimen was examined under light microscopy (X20 or X40 objective lens) using an Olympus BX51 light microscope (Olympus, UK). Samples were considered screen positive if they had a white blood cell count (WBC) of > 5x104WBC / ml. Discordant samples were reflexed to determine counts of > 1x104WBC / ml as per PHE UK standard for Microbiology Investigations (SMI) - Investigation of Urine B41 Issue 8.7.

[0145] Standard urine culture

[0146] Standard first line urine culture was performed on each urine specimen by pipetting 100pl (Eppendorf, UK) of urine onto UTI clarity agar (Fannin L.I.P, Ireland) and inoculating the entire surface of the agar using a disposable ‘hockey stick’ (Sarstedt, Germany) in order to obtain quantitative colony counts. Plates were incubated at 37°C for 24 hours under aerobic conditions. Following incubation plates were inspected for growth, if colonies were present, they were counted by eye and identified using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI- TOF) on the MicroFlex mass spectrometer (Bruker Daltonics, USA). The number of CFU / ml was calculated using the formula stated in the UK SMI B41 guidelines (PHE, 2019).

[0147] Test for antimicrobial substances and expanded urine culture

[0148] In cases where the biomarker results were discordant to the reference method, specimens were tested for antimicrobial substances and then for fastidious organisms. Detection of antimicrobial substances was performed as per UK Standards for Microbiology Investigations B41. A 0.5 McFarland of Bacillus subtilis control strain (NTCC 10400) was lawned onto Muller Hinton agar (Fannin L.I.P, Ireland) and allowed to dry before the plate was inoculated with 10 pl of urine. Plates were incubated at 37°C for 24 hours under aerobic conditions. The presence of antimicrobial substances is represented by the presence of a clear zone in the Bacillus growth indicating antimicrobial activity in the urine sample that may inhibit culture of target microorganisms. Where the test for antimicrobial substances was negative, expanded quantitative urine culture (EQUC) was performed in accordance with UK SMI B41 guidelines. Using a pipette (Eppendorf, UK) 100 pl of urine was inoculated onto Chocolate Agar + Bacitracin agar (Fannin L.I.P, Ireland) and Fastidious Anaerobic Agar (FAA) with 7% Horse Blood + Neomycin (Fannin, L.I.P, Ireland). The inoculum was spread over the entire surface of the plate with a sterile ‘hockey stick’ (Sarstedt, Germany). A Metronidazole disc (Thermo Scientific, USA) was placed on FFA plates to help identify the presence of anaerobes. Following 48 hours incubation at 37°C in anaerobic conditions the plates were inspected for growth. Colonies counted by eye and the number of CFU / ml calculated using the formula stated in the UK SMI B41 (PHE, 2019). All bacterial growth was also identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) on the MicroFlex mass spectrometer (Bruker Daltonics, USA). Each colony was spotted on a target plate, allowed to air dry before 1 pl of matrix solution was added to each spot (Bruker Daltonics, USA). Once dry the target plate was inserted into the MicroFlex mass spectrometer and the results analysed using the MALDI Biotyper 3.0 software (Burker Daltonics, USA). Stratifications and Patient Groups

[0149] The following patient groups were sampled:

[0150] • Females > 65 yo

[0151] • Males > 65 yo

[0152] • Females < 12 yo

[0153] • Males < 12 yo

[0154] • Males > 12 yo & <65 yo

[0155] • Females >12 yo & <65 yo

[0156] • Antenatal

[0157] • Catheterized patients (also referred to as catheter specimens of urine (CSU))

[0158] • Males

[0159] • Females (non-pregnant)

[0160] • CSU V Non-CSU

[0161] Biomarker selection: A panel of biomarkers including MMP9, IL8, MPO, MMP8, IL6, HNE, NGAL and lactoferrin were selected for their potential to diagnose complicated or uncomplicated UTI. Firstly, each biomarker is assessed individually in its ability to differentiate UTI from no UTI from a high confidence diagnostic group including eligible patients from all patient cohorts listed above. Following logistical regression analysis of individual biomarker performances, two marker and three marker combinations were proposed and applied to the same samples to assess the ability of sequential addition of different biomarkers in different combinations to improve diagnostic accuracy. The best combination predicted from the logistical regression was applied to specific patient cohorts stratified to assess the performance of the assay across different patient cohorts, including cohorts whereby a UTI would be deemed a complicated UTI (CSU, Females >65, Males >65, Females 12, Males <12, Males >12 & <65) and cohorts whereby a UTI would be deemed an uncomplicated UTI (nonpregnant females of any age). Poor performing biomarkers were excluded from further analysis following individual biomarker performance analysis.

[0162] Biomarker measurement & quantification’. The quantitative detection of the biomarkers under investigation during the clinical study was carried out using an optimised antigen / antibody capture methodology in lateral flow strip format. Firstly, analytical studies were carried out using spiked urine samples with a range of defined concentrations of each biomarker to develop calibration curves. Lateral flow strips were plotted with seven antibody pairs on a dipstick format. A lateral flow reading instrument (Chembio® Cube Reader), based on reflectance of light intensity, was used to analyse the resulting line intensities across the range of defined concentrations for each biomarker, producing measurable units called cube units. Each markers' cube units were converted into concentrations (ng / ml) using four-parameter logistic (4PL) regression - a mathematical model particularly suited for dose-response curves in bioassays. Firstly, known concentrations of each biomarker were measured, using their respective assays, generating a set of datapoints that paired these concentrations with corresponding cube units obtained from the lateral flow test. Then, 4PL regression was applied to these datapoints to create standard curves, for each assay, that best fit the sigmoidal nature of the relationship between the concentrations and corresponding cube units, serving as a mathematical representation of how the lateral flow test responds to different concentrations of biomarkers. Once the 4PL regression models were established for each assay, concentrations were extrapolated from new cube units that were not part of the original dataset used to create the standard curves. Clinical specimens, meeting exclusion / inclusion criteria (see patient sample and collection eligibility above), were then selected. For each clinical specimen, 1.6ml of urine from each was added to a cartridge containing 5 lateral flow strips plotted with each of the candidate antibody pairs, with associated line intensities taken by the reader and cube units produced. The resulting cube units were converted into concentrations for each utilizing the 4PL method defined. Concentrations were subject to logistical regression for development of the algorithm for classification as No or Probable UTI.

[0163] Logistic Regression modelling

[0164] Logistic regression was used to produce the diagnostic models. The concept underlying logistic regression, in this context, is the identification of a statistical relationship (correlation) between the concentrations of certain biomarkers in an individual’s urine, and the presence or absence of a UTI. The model combines these biomarkers in an equation, creating a unified predictive model that considers the joint effect of the biomarkers and their combined influence on the probability of having a UTI, with the outcome being risk scores for each patient (values between 0 and 1 , representing their probability of having a UTI). Optimal risk scores were developed using methods standard to the art and applied to the dataset to provide diagnostic accuracy data including area under curve (AUC), sensitivity and specificity. Assessment of diagnostic accuracy

[0165] ROC (receiver operating characteristic) curves were constructed for the logistic regression models, as well as the individual biomarkers, to evaluate their ability in distinguishing between patients with probable (complicated or uncomplicated) UTIs and those without any UTI, as indicated by AUC (area under the ROC curve) plots. These curves depict the True Positive Rate (TPR) plotted against the False Positive Rate (FPR), of the individual biomarkers at various concentration thresholds, and of the logistic regression models at various risk score thresholds, providing insights into their overall effectiveness in differentiating the two groups.

[0166] Sensitivity and Specificity - Individual biomarkers and logistic regression models

[0167] Sensitivity and specificity were measured at various concentration thresholds for each biomarker, and, considering the logistic regression models’ output, which consists of risk scores ranging from 0 to 1 for each patient, different combinations of sensitivity and specificity were also generated for the models. These combinations were dependent on the various possible decision thresholds (risk scores above which a patient is classified as having a UTI by the outcome of the combination score), with higher thresholds prioritising specificity, and lower thresholds prioritising sensitivity. Optimum risk scores were selected based on adherence to a specificity target of >95%. The analysis involved evaluating and comparing each model's maximum sensitivity achieved at risk score thresholds meeting this target. Final diagnostic accuracy results were based on optimal risk scores utilized for each dataset.

[0168] Three biomarker combination vs individual biomarkers and a Multistix® dipstick

[0169] The aim of this analysis was to assess whether the three-biomarker combination of MMP9, MPO, and IL8 outperformed the biomarkers when taken individually and versus a dipstick (such as a Multistix® dipstick). Specifically, the aim was to determine whether the three-biomarker combination’s sensitivity was significantly higher when each biomarker was constrained to match the specificity achieved by the three- biomarker combination. This analysis was only performed on patient cohorts whereby sample size was large enough to provide statistical significance.

[0170] Method

[0171] For each of the following patient groups: Whole Population, All Females, All Males, >65's and CSU samples, the three-biomarker combination specificity was determined using pre-define risk score thresholds: High Likelihood of Infection : Risk Score > 0.46

[0172] Equivocal : Risk Scores between 0.11 and 0.46

[0173] Low Likelihood of Infection : Risk Score < 0.11

[0174] To allow for a fair comparison, concentration thresholds (ng / mL) were selected for each biomarker that achieved a specificity as close as possible to that of the three-biomarker combination within each subgroup, while maximizing sensitivity (this accounts for the differences in sensitivity and specificity reported where individual biomarker specificity was not aligned with the three-biomarker combination model). This ensured that false positive rates were held constant and allowed for a meaningful comparison of sensitivity. For Multistix® comparisons, the existing binary result (a positive result = the presence of either leukocyte esterase or nitrites, and a negative result = the absence of both) was used. To reflect the intended clinical application, patients with an equivocal result from the three-biomarker combination were excluded when calculating its sensitivity and specificity (this accounts for the differences in sensitivity and specificity reported for the three-biomarker combination model where the equivocal results were not removed). In contrast, all patients were included in the analyses of individual biomarkers (MMP9, MPO, IL8) and the Multistix® dipstick, as these tests do not incorporate an indeterminate range. This ensured that comparisons between the three- biomarker combination and other methods were clinically relevant and aligned with each tool’s intended use.

[0175] To assess whether the three biomarker combinations’ sensitivity was statistically higher than that of each individual biomarker and Multistix® dipstick, a two-proportion z-test was used, which is a statistical test that is used to compare two proportions to see if their difference is statistically significant. Sensitivity and specificity were reported with 95% confidence intervals, calculated using the Wilson score method.

[0176] Results

[0177] Population Statistics

[0178] A total of 554 patient samples were measured, with 19% (N=104) being diagnosed as having a probable UTI, 32% (N=177) with no UTI, 10% (N=54) with a possible UTI, 3% (N=15) with an indeterminate diagnosis, 7% (N=41) with a probable colonization / contamination / faulty collection / transport diagnosis and 29% (N=163) with an uncertain diagnosis (Table 1). Table 1 : Patient diagnosis categories - sample sizes and proportions

[0179] The high confidence diagnosis group, made up of those with either a probable or no UTI, consisted of 281 patients, with 53% (N=149) being female, 41% (1X1=114) being over the age of 65, 11% (N=32) being under the age of 12 and 23% (N=66) being catheterized (Table 2).

[0180] Table 2: Cohort stratifications - sample sizes and proportions (probable UTI and No UTI groups only).

[0181] Individual biomarker performance:

[0182] Upon assessment of performance at various concentration thresholds, it was observed that only MMP9, on its own, could achieve a sensitivity exceeding 90% at the target specificity of >95% across the whole population (see Figure 1) with MPO exhibiting a sensitivity of 79.8% (see Figure 2), MMP8 82.7% (see Figure 3), HNE 80.8% (see

[0183] Figure 4), and IL8 72.1% (see Figure 5). The performance of IL-6, NGAL and Lactoferrin was significantly poor (see Figures 6-8) and were excluded from any further evaluation. Two-biomarker combinations

[0184] Logistical regression identified three two-biomarker combinations which showed promise in raising assay performance:

[0185] • MMP9+HNE

[0186] • MMP9+MPO

[0187] • MMP9+IL8

[0188] The MMP9+HNE (see Figure 9), MMP9+MPO (see Figure 10) and MMP9+IL8 (see Figure 11) combinations demonstrated comparable performance, achieving AUCs of 98.3% , 98.2% and 97.9%, respectively. All combinations achieved a maximum sensitivity of 90.4% (94 / 104) at specificities adhering to the >95.0% target (95.5% (169 / 177), 95.5% (169 / 177) and 96.0% (170 / 177) respectively), at optimum risk score thresholds.

[0189] Three-biomarker combinations

[0190] Logistical regression identified three three-biomarker combinations which showed promise in raising assay performance:

[0191] • MMP9+HNE+IL8

[0192] • MMP9+MPO+IL8

[0193] • MMP9+HNE+MPO

[0194] The MMP9+HNE+IL8 combination achieved an AUC of 98.5% and a sensitivity of 94.2% (98 / 104) achieved at a specificity of 95.4% (169 / 177) (see Figure 13).

[0195] The MMP9+MPO+IL8 combination had an AUC of 98.4%, with a sensitivity of 92.3% (96 / 104) achieved at a specificity of 96.6% (171 / 177) (see Figure 14).

[0196] The MMP9+HNE+MPO combination achieved an a AUCs of 98.3%. sensitivity of 90.4% (94 / 104) and with specificities of 96.1% (170 / 177) (see Figure 15).

[0197] MMP9+HNE+IL8, MMP9+MPO+IL8, and MMP9+HNE+MPO combinations were selected for further investigation for diagnosis of complicated UTI cohorts. Diagnostic performance of three-biomarker combinations by patient cohort

[0198] Diagnostic performance in geriatric patients(>65 years old):

[0199] In females > 65 years old the MMP9+HNE+IL8 and MMP9+MPO+IL8 combinations both achieved 100% AUC (Figures 16 and 27, respectively), with sensitivities of 100% (27 / 27) each, and specificities (24 / 24) each, at their respective optimal risk score thresholds. The MMP9+HNE+MPO combination achieved an AUC of 99.8% in this group (Figure 38), with a sensitivity of 96.3% (26 / 27) at a specificity of 95.8% (23 / 24) at a risk score threshold. For men > 65 years old, all combinations achieved >99% AUC, with sensitivities and specificities >95% for each combination (see Figures 17, 28, 39).

[0200] Diagnostic performance in the under 12 years old age group:

[0201] The combinations also performed well patients under the age of 12, despite this group having a small sample size, with 100% AUC across all combinations, and sensitivities and specificities of 100% (2 / 2) and 100% (15 / 15) respectively for each combination in females < 12 (see Figures 18, 29 and 40).. Sensitivities and specificities of 100% (3 / 3). 100% (12 / 12) were achieved in males < 12 (see Figures 19 and 41) in the MMP9+HNE+IL8 and MMP9+HNE+MPO combinations, respectively. The MMP9+MPO+IL8 combination, however, achieved an AUC of 100% but a sensitivity of 66.7% (2 / 3) and a specificity of 100% (12 / 12) at its optimal risk score threshold in males < 12 (see Figure 30).

[0202] Diagnostic performance for those between 12-65 years old:

[0203] For females, AUCs, specificity, and sensitivity for the MMP9+MPO+IL8 combination was 95.7%, 91.1% and 91.6% respectively for those aged 12-65 (see Figure 31). AUC, specificity, and sensitivity stood at 95.2%, 88.89% and 91.67% respectively for the MMP9+HNE+IL8 combination (see Figure 20). AUC, specificity, and sensitivity stood at 95.1%, 91.11 % and 86.11% respectively for the MMP9+HNE+MPO combination (see Figure 42). For males 12-65, with all three combinations achieving 100% (7 / 7) sensitivity and 95.7% (45 / 47) specificity at their relative optimal risk score thresholds. AUCs achieved for MMP9+HNE+IL8, MMP9+HNE+MPO and MMP9+IL8+MPO were 99.4%, 100% and 98.9% respectively (see Figures 21 , 43, and 32, respectively).

[0204] Antenatal group

[0205] High sensitivity across all three combinations at their respective risk score thresholds in the antenatal group (100% (3 / 3) sensitivity for each combination), specificity for the MMP9+MPO+IL8 and MMP9+MPO+HNE combinations (see Figures 35 and 46, respectively) achieved 62.5% (5 / 8) specificity and the MMP9+HNE+IL8 combination (see Figure 24) achieving a specificity of 75.0% (6 / 8) at their respective optimal risk score thresholds.

[0206] Catheterized vs non-catheterized patients

[0207] The performance seen in the CSU cohort was comparable to that of the non-CSU cohort in the MMP9+HNE+IL8 and MMP9+MPO+IL8 combinations. Both combinations achieved ACUs sensitivities and specificities of >97%, 93.1% (27 / 29) and 100% (37 / 37), respectively, in the CSU cohort (see Figures 25 and 36), compared to AUCs, sensitivities and specificities of >99% (+ / - 0.1%),, 92.0% (69 / 75) and 95.7% (134 / 140), respectively, in the non-CSU cohort (see Figures 26 and 37) at their optimal risk score threshold. The MMP9+MPO+HNE combination achieved a AUC, sensitivity and specificity of 96.9%, 86.2% (25 / 29) and 100% (37 / 37), respectively, in the CSU cohort (see Figure 47), and an AUC, sensitivity and specificity of 99%, 92.0% (69 / 75) and 95.0% (133 / 140), respectively, in the non-CSU cohort (see Figure 48) at its optimal risk score threshold. Specifically, the AUCs in the catheterised patients were 97.2%, 97.1% and 96.9% for MMP9+HNE+IL8, MMP9+MPO+IL8 and MMP9+MPO+HNE combinations respectively. ACU's in non-catheterised patients were 99%, 99.1% and 98.7% for MMP9+HNE+MPO, MMP9+HNE+IL8 and MMP9+IL8+MPO respectively.

[0208] Diagnostic performance of three biomarker combination vs individual biomarkers and the Multistix® dipstick

[0209] Across all groups, the three-biomarker combination of MMP9, MPO, and IL8 consistently showed higher sensitivity than any single marker or Multistix® result at matched specificity levels. In all comparisons, the increase in sensitivity was statistically significant (p < 0.05). Statistical comparisons showed that the three- biomarker combination sensitivity was significantly higher than all individual biomarkers and Multistix® in the Whole Population, All Females, All Males, the CSU group and >65s (p< 0.05 for all comparisons). Table 3: Whole population analysis

[0210] For the whole population, biomarker thresholds were selected to match the three- biomarker combination specificity of 98.2% while maximising sensitivity.

[0211] Table 4: Female population analysis

[0212] In the female subgroup, biomarker thresholds were selected to match the three- biomarker combination specificity of 97.4%, while maximising sensitivity.

[0213] Table 5: Male population analysis

[0214] For males, biomarker thresholds were chosen to match the three-biomarker combination specificity of 98.9%, while maximising sensitivity. Table 6: Catheterised population analysis M

[0215] In the catheterised (catheter specimen of urine (CSU)) sample group, thresholds were selected to match the three-biomarker combination for 100% specificity, while maximising sensitivity.

[0216] Table 7: Over 65 years of age population analysis

[0217] In the >65s group, thresholds were selected to match the three-biomarker combination for 100% specificity, while maximising sensitivity.

[0218] Discussion

[0219] The claimed invention provides for the diagnosis of (complicated or uncomplicated) UTI through detection of one or more biomarkers. The biomarkers could be detected, for example, on a lateral flow strip where the colour change on the test line of the strip is read and quantified automatically by a reader.

[0220] This study, conducted on a diverse population of 554 patients, yielded valuable insights into the diagnosis of complicated and uncomplicated UTIs, using biomarkers in urine. The study focused on eight biomarkers: MMP8, MMP9, HNE, NGAL, MPO, IL6, IL8, and lactoferrin, with the goal of developing a multiplex lateral flow test to support clinical decision-making. The analysis covered population statistics, assessment of individual biomarker performance, and the evaluation of the diagnostic accuracy of various logistic regression models in differentiating individuals with a probable UTI, from those with no UTI.

[0221] Population statistics revealed a nuanced distribution of diagnoses, with 19% diagnosed with a probable UTI, 32% with no UTI, 10% with a possible UTI, and 29% with an uncertain diagnosis. The high-confidence diagnosis group, comprising of probable (N=104) and no UTI (N=177) cases, provided a robust sample size for further analysis. Individual biomarker performance, as assessed by AUC-ROC, highlighted MMP9 as the most accurate in distinguishing probable UTI from no UTI present, with an AUC of 97.6%, followed by MPO, MMP8, HNE, IL8, NGAL, Lactoferrin, and finally, IL6, with an AUC of 60.0%. Sensitivity and specificity analysis highlighted the challenges of achieving both metrics simultaneously, with only MMP9 being able to achieve a sensitivity exceeding 90% at the predetermined target specificity of >95%.

[0222] Logistic regression models, including two-marker and three-marker combinations using four candidate biomarkers, were thoroughly evaluated, and the transition from individual markers to combination combinations marked a significant enhancement in diagnostic performance. The two-marker combinations achieved sensitivities remaining just above 90% at specificities above the 95% target. Three-marker combinations achieved greater sensitivity and specificity. The MMP9+HNE+IL8, MMP9+MPO+IL8, and MMP9+HNE+MPO combinations showed the best performance, all achieving an AUC of >98%.

[0223] Due to their superior performance during biomarker evaluation, the MMP9+HNE+IL8, MMP9+MPO+IL8, and MMP9+HNE+MPO combinations were selected for further investigation to evaluate performance in diagnosing complicated UTI. Cohort-specific analyses was conducted to assess the robustness of these combinations to diagnose complicated UTI. Combination performance excelled in females aged over 65 years old, in which all three-biomarker combinations achieved an AUC of 100%, and sensitivities of 100% at specificities exceeding 95.0%. The combinations also performed well in pediatrics (children under 12 years old), with the MMP9+HNE+IL8 and MMP9+HNE+MPO combinations achieving 100% sensitivity and specificity. The combinations performed well in catheterized patients. Performance across catheterized and non-catheterized cohorts remained comparable in the MMP9+HNE+IL8 and MMP9+MPO+IL8 combinations, both achieving >90% sensitivity at specificities exceeding the target of 95%, emphasizing the robustness and consistency of the combinations in different clinical contexts.

[0224] Men of any age with a UTI are classified as having a complicated UTI. The combinations performed well in men of any age, with sensitivity and specificity remaining high in males aged 12-65, with all three combinations achieving 100% sensitivity and 95.7% specificity at their respective optimal thresholds. The ability to have diagnostic performance of over 90% sensitivity at 95% specificity for the diagnosis of complicated UTI in these patient cohorts is novel, providing a promising solution to current diagnostic stewardship compromises.

[0225] In the female 12-65 yrs cohort the MMP9+MPO+IL8 combination was the only combination with both sensitivity and specificity >90%. UTIs in females between 12-65 yrs with no other co-morbidities are generally classified as uncomplicated UTI. These results highlight the utility of the MMP9+MPO+IL8 combination for both complicated UTI and uncomplicated UTI and provides for a unified combination across all patient cohorts. This three-marker combination therefore provides a promising solution for point of care diagnosis of both complicated and uncomplicated UTI using the same test. The ability to utilize a single test for all suspected UTI cases would be favorable in practice to enable standardized diagnostic stewardship at point of care for all patients with suspected UTI. Such a versatile test is absent from clinical diagnosis of UTI.

[0226] The three best performing biomarker combinations all consisted of MMP9 in combination with either IL8 +MPO, IL8+HNE or HNE+MPO. Both IL8 and MMP9 have distinct functions to both MPO and HNE in the UTI immune response. When combined with IL8 (chemotactic recruitment) and MMP9 (neutrophil migration), MPO completes a coherent immune response signature sequence of recruitment, migration and microbial clearance without the functional redundancy created by pairing MPO and HNE in a combination. This combined with the performance of the MMP9+MPO+IL8 combination in all cohorts including the female 12-65 yrs cohort provides for the MMP9+MPO+IL8 combination as the optimal and preferred combination.

[0227] Analysis of MMP9+MPO+IL8 performance vs individual biomarker and Multistix® in All Females, All Males, CSU (catheterised) and >65s (female and male) patient cohorts demonstrated consistently higher sensitivity for MMP9+MPO+IL8 than any single marker or Multistix® result at matched specificity levels. The increased sensitivity was statistically significant (p < 0.05) in the Whole Population, All Females, All Males, and notably in the complicated CSU (catheterised) cohort and >65s elderly (female and male) cohort.

[0228] Conclusion:

[0229] The present invention relates to a specific combination of host response biomarkers for the detection of urinary tract infections (UTIs). Each three-marker combination showed AUC's above 95% for the diagnosis of UTIs across the various patient cohorts studied here, including women aged between 12-65 years old, opening the door for a point of care test with comparable performance in the diagnosis of both uncomplicated and complicated UTI, potentially allowing standardization of diagnostic stewardship of all those with a UTI. This is a promising discovery as the use of dipsticks, a common point of care diagnostic tool, is not recommended for the diagnosis of complicated UTI. While each of the individual biomarkers included in the combination(s) is known in the art, it has unexpectedly been found that their combined use synergistically and significantly enhances diagnostic accuracy. Clinical data demonstrate that the combination yields statistically superior performance relative to any of the individual biomarkers used alone (p < 0.05). Notably, this improvement is particularly evident in subpopulations of patient that are typically difficult to diagnose, including elderly individuals and patients with indwelling urinary catheters. In these cohorts, conventional diagnostics frequently yield suboptimal performance due to non-specific symptoms, asymptomatic bacteriuria, concomitant pyruia and baseline physiological variation. The combined biomarkers described, however, enables a more accurate and reliable determination of infection status in such cases. The enhanced diagnostic performance in clinically relevant subgroups was not predictable from the known properties of the individual biomarkers in the literature.

[0230] In the specification the terms "comprise, comprises, comprised and comprising" or any variation thereof and the terms “include, includes, included and including" or any variation thereof are considered to be totally interchangeable and they should all be afforded the widest possible interpretation and vice versa.

[0231] The invention is not limited to the aspects hereinbefore described but may be varied in both construction and detail.

Claims

Claims1. A method for diagnosing a urinary tract infection in a subject, the method comprising: measuring the protein expression levels of MMP9, IL8, and MPO in a urine sample obtained from the subject; and comparing the combined protein expression levels of MMP9, IL8, and MPO to a reference level; wherein when the combined protein expression levels of MMP9, IL8, and MPO are increased or there is a positive combined protein expression level of MMP9, IL8, and MPO when compared to the reference level, the subject is predicted to have a urinary tract infection; and wherein the subject is selected from a catheterised patient, a male patient over 65 years of age, and a female patient over 65 years of age.

2. A method for monitoring a urinary tract infection in a subject, the method comprising: measuring the protein expression levels of MMP9, IL8, and MPO in a urine sample obtained from the subject; and comparing the combined protein expression level of MMP9, IL8, and MPO to a reference level from either a control sample or from earlier protein expression level measurements taken from a previously obtained sample from the subject; wherein when the combined protein expression levels of MMP9, IL8, and MPO are unchanged, increased or are positive relative to the reference levels, the subject is predicted to have a persisting urinary tract infection; and wherein when the combined protein expression levels of MMP9, IL8, and MPO are decreased or absent relative to the reference level, the subject is predicted to be recovering from or has recovered from the urinary tract infection; and wherein the subject is a catheterised patient, a male patient over 65 years of age, and a female patient over 65 years of age.

3. A method of monitoring treatment of a urinary tract infection in a subject, the method comprising: measuring the protein expression level of MMP9, IL8, and MPO in a urine sample obtained from the subject prior to receiving treatment to define a reference expression level in the subject;measuring the protein expression levels of MMP9, ILS, and MPO in a urine sample obtained from the subject following treatment; and comparing the combined protein expression levels of MMP9, IL8, and MPO to the reference expression levels obtained before treatment with each measurement obtained after treatment; wherein when the combined protein expression levels of MMP9, IL8, and MPO obtained after treatment are unchanged, increased or positive relative to the measurements obtained before treatment, the treatment is predicted to be ineffective against the urinary tract infection; and wherein when the combined protein expression levels of MMP9, IL8, and MPO obtained after treatment are decreased or absent relative to the measurements obtained before treatment, the treatment is predicted to be effective against the urinary tract infection; and wherein the subject is a catheterised patient, a male patient over 65 years of age, and a female patient over 65 years of age.

4. The method of Claim 3, wherein the treatment is an antibiotic selected from trimethoprim, fosfomycin, nitrofurantoin, cephalexin, ceftriaxone, aminopenicllins, fluoroquinolones and gepotidacin.

5. The method of any one of the preceding claims, wherein the urinary tract infection is a complicated urinary tract infection or an uncomplicated urinary tract infection.

6. The method of Claim 5, wherein the complicated urinary tract infection is selected from pyelonephritis, chronic pyelonephritis, acute pyelonephritis, epididymitis, prostatitis, cystitis, vaginitis, and urethritis.

7. A method for diagnosing a complicated urinary tract infection in a subject, the method comprising: measuring the protein expression level of MMP9, IL8, and MPO in a urine sample obtained from the subject; and comparing the combined protein expression levels of MMP9, IL8, and MPO to a reference level; wherein when the combined protein expression levels of MMP9, IL8, and MPO are increased or there is a positive combined protein expression level of MMP9, IL8, and MPO when compared to the reference level, the subject is predicted to have a complicated urinary tract infection; andwherein the subject is a catheterised patient, a male patient over 65 years of age, and a female patient over 65 years of age.

8. A method for diagnosing a urinary tract infection in a catheterised subject, the method comprising: measuring the protein expression levels of MMP9, MPO, and IL8, in a urine sample obtained from the catheterised subject; and comparing the combined protein expression levels of MMP9, MPO, and IL8 to a reference level; wherein when the combined protein expression levels of MMP9, MPO, and IL8 are increased or there is a positive combined protein expression level of MMP9, MPO, and IL8 when compared to the reference level, the catheterised subject is predicted to have a urinary tract infection.

9. A method for diagnosing a urinary tract infection in a subject, the method comprising: measuring the protein expression levels of MMP9, IL8, and MPO in a urine sample obtained from the subject; and comparing the combined protein expression levels of MMP9, IL8, and MPO to a reference level; wherein when the combined protein expression levels of MMP9, IL8, and MPO are increased or there is a positive combined protein expression level of MMP9, IL8, and MPO when compared to the reference level, the subject is predicted to have a urinary tract infection.

10. A method for monitoring a urinary tract infection in a subject, the method comprising: measuring the protein expression levels of MMP9, IL8, and MPO in a urine sample obtained from the subject; and comparing the combined protein expression level of MMP9, IL8, and MPO to a reference level from either a control sample or from earlier protein expression level measurements taken from a previously obtained sample from the subject; wherein when the combined protein expression levels of MMP9, IL8, and MPO are unchanged, increased or are positive relative to the reference levels, the subject is predicted to have a persisting urinary tract infection; andwherein when the combined protein expression levels of MMP9, IL8, and MPO are decreased or absent relative to the reference level, the subject is predicted to be recovering from or has recovered from the urinary tract infection.11 . A method of monitoring treatment of a urinary tract infection in a subject, the method comprising: measuring the protein expression level of MMP9, IL8, and MPO in a urine sample obtained from the subject prior to receiving treatment to define a reference expression level in the subject; measuring the protein expression levels of MMP9, IL8, and MPO in a urine sample obtained from the subject following treatment; and comparing the combined protein expression levels of MMP9, IL8, and MPO to the reference expression levels obtained before treatment with each measurement obtained after treatment; wherein when the combined protein expression levels of MMP9, IL8, and MPO obtained after treatment are unchanged, increased or positive relative to the measurements obtained before treatment, the treatment is predicted to be ineffective against the urinary tract infection; and wherein when the combined protein expression levels of MMP9, IL8, and MPO obtained after treatment are decreased or absent relative to the measurements obtained before treatment, the treatment is predicted to be effective against the urinary tract infection.

12. The method of Claim 11 , wherein the treatment is an antibiotic selected from trimethoprim, fosfomycin, nitrofurantoin, cephalexin, ceftriaxone, aminopenicllins, fluoroquinolones and gepotidacin.

13. The method of any one of Claims 9 to 12, wherein the urinary tract infection is a complicated urinary tract infection or an uncomplicated urinary tract infection.

14. The method of Claim 13, wherein the uncomplicated urinary tract infection is urethritis and / or cystitis in non-pregnant females between 12 and 65 years old.

15. The method of Claim 13, wherein the complicated urinary tract infection is selected from pyelonephritis, chronic pyelonephritis, acute pyelonephritis, epididymitis, prostatitis, cystitis, vaginitis, and urethritis.

16. A method for diagnosing a complicated urinary tract infection in a subject, the method comprising: measuring the protein expression level of MMP9, IL8, and MPO in a urine sample obtained from the subject; and comparing the combined protein expression levels of MMP9, IL8, and MPO to a reference level; and wherein when the combined protein expression levels of MMP9, IL8, and MPO are increased or there is a positive combined protein expression level of MMP9, IL8, and MPO when compared to the reference level, the subject is predicted to have a complicated urinary tract infection.

17. The method of Claim 15 or Claim 16, wherein the complicated urinary tract infection of cystitis and urethritis is typically in men of any age, subjects with an indwelling catheter, a subject with diabetes, a subject with anatomical abnormalities related to the urinary tract, a subject with a recurring UTI, antenatal subjects, and in paediatric patients under 12 years old.

18. The method of any one of Claims 9 to 16, wherein the subject is selected from a cohort defined as a whole population, females only, males only, females younger than 12 years of age, males younger than 12 years old, males between 12 and 65 years of age, females between 12 and 65 years of age, antenatal subjects, diabetic subjects, and non-catheterised subjects.

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