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Publication numberWO2016161413 A1
Publication typeApplication
Application numberPCT/US2016/025824
Publication date6 Oct 2016
Filing date4 Apr 2016
Priority date2 Apr 2015
Publication numberPCT/2016/25824, PCT/US/16/025824, PCT/US/16/25824, PCT/US/2016/025824, PCT/US/2016/25824, PCT/US16/025824, PCT/US16/25824, PCT/US16025824, PCT/US1625824, PCT/US2016/025824, PCT/US2016/25824, PCT/US2016025824, PCT/US201625824, WO 2016/161413 A1, WO 2016161413 A1, WO 2016161413A1, WO-A1-2016161413, WO2016/161413A1, WO2016161413 A1, WO2016161413A1
InventorsAndrew SCHWADERER, David Hains, John David Spencer, Joshua WATSON
ApplicantResearch Institute At Nationwide Children's Hospital
Export CitationBiBTeX, EndNote, RefMan
External Links: Patentscope, Espacenet
Urinary tract infection biomarkers
WO 2016161413 A1
Abstract
A method of diagnosing a urinary tract infection in a subject is described. The method includes obtaining a urine sample from the subject; determining the level of human α-defensin 5 (HD5) and/or human neutrophil peptides (HNP)1-3 in the urine sample and comparing it to a corresponding control value; and diagnosing the subject as having a urinary tract infection if the level of HD5 and/or HNP1-3 is greater than the control value. Kits for diagnosing a urinary tract infection in a subject using antibodies specific for HD5 and HNP1-3 are also described.
Claims  (OCR text may contain errors)
CLAIMS What is claimed is:
1. A method of diagnosing a urinary tract infection in a human subject, comprising
obtaining a urine sample from the subject;
determining the level of human oc-defensin 5 (HD5) and/or human neutrophil peptides (HNP)l-3 in the urine sample and comparing it to a corresponding control value; and
diagnosing the human subject as having a urinary tract infection if the level of HD5 and/or HNPl-3 is greater than the control value.
2. The method of claim 1, wherein the levels of both HD5 and HNPl-3 are determined.
3. The method of claim 2, further comprising determining the level of creatinine in the urine sample and using the creatinine level as a control value.
4. The method of claim 3, wherein the human subject is diagnosed as having a urinary tract infection if the level of HD5 is equal to or greater than 150 pg HD5/mg creatinine, and the level of HNPl-3 is equal to or greater than 300 pg HNPl-3/mg creatinine.
5. The method of claim 1, further comprising determining the level of leukocyte esterase (LE) in the urine sample and comparing it to a corresponding control value.
6. The method of claim 5, wherein the level of HD5 is determined in the urine sample.
7. The method of claim 1, wherein the level of HD5 and/or HNPl-3 are determined using an immunoassay.
8. The method of claim 7, wherein the immunoassay is a lateral flow immunoassay.
9. The method of claim 1, wherein the urinary tract infection comprises infection by Escherichia coli.
10. The method of claim 1, wherein the urinary tract infection is a lower urinary tract infection.
11. The method of claim 1, wherein the subject does not have any symptoms of a urinary tract infection.
12. The method of claim 1, wherein the human subject is a human child.
13. The method of claim 1, further comprising providing treatment for urinary tract infection for the human subject identified as having a urinary tract infection.
14. A kit for diagnosing urinary tract infection in a human subject, comprising a first antibody or binding fragment thereof specific for HD5 and/or a second antibody or binding fragment thereof specific for HNPl-3, reagents for conducting the diagnosis, and a package for holding the antibodies and the reagents.
15. The kit of claim 14, wherein the kit further comprises a third antibody or binding fragment thereof specific for leukocyte esterase.
16. The kit of claim 15, wherein the kit includes a first antibody or binding fragment thereof specific for HD5.
17. The kit of claim 14, wherein the kit further comprises a lateral flow strip.
18. The kit of claim 14, wherein the kit further comprises instructions for using the kit to determine the levels of HD5 and/or HNPl-3 in a urine sample.
Description  (OCR text may contain errors)

URINARY TRACT INFECTION BIOMARKERS

GOVERNMENT FUNDING

[0001] This invention was made with Government support under grant number K08DK094970-03 awarded by the National Institutes of Health, and Grant No. UL1TR001070 awarded by the National Center for Advancing Translational Sciences. The Government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application Serial No. 62/142,107, filed on April 2, 2015, which is hereby incorporated by reference in its entirety.

BACKGROUND

[0003] Urinary tract infection (UTI) is a common and potentially severe infection that represents a major burden of healthcare utilization/expenditure and antibiotic exposure in children. UTIs afflict up to 17% of girls, account for 5% of febrile conditions treated in emergency departments and 2% of pediatric hospitalizations, culminating in over $8 billion in medical expenditures each year. UTI is one of the most common reasons for short duration antibiotic exposure for acute treatment and long-term prophylactic antibiotic therapies to prevent recurrent UTI during childhood

[0004] The diagnosis of UTI relies on suggestive symptoms, pyuria, and isolation of a uropathogen in culture. However, UTI symptoms and pyuria in children often are nonspecific, and culture results are not available at the time of initial evaluation. Consequently, providers frequently rely on rapid results of the urine dipstick to decide whether to initiate empiric antibiotic therapy for a presumed UTI while awaiting culture results. This approach may lead to either delayed diagnosis or unnecessary antibiotic therapy because of the limitations of leukocyte esterase (LE) and urinary nitrite on dipstick urinalysis. Specifically, large pediatric studies and meta-analyses of the utility of the urine dipstick for predicting positive urine culture have consistently shown suboptimal sensitivity and/or specificity of individual and combination tests, even though results of individual studies vary widely. Kazi et al, Am J Emerg Med 31:1405-7 (2013); Williams et al, Lancet Infect Dis. 10:240-50 (2010).

SUMMARY OF THE INVENTION

[0005] In one aspect, the present invention provides a method of diagnosing a urinary tract infection in a subject that includes obtaining a urine sample from the subject; determining the level of human oc-defensin 5 (HD5) and/or human neutrophil peptides (HNP) 1-3 in the urine sample and comparing it to a corresponding control value; and diagnosing the subject as having a urinary tract infection if the level of HD5 and/or HNP1-3 is greater than the control value. In some embodiments, the method further includes determining the level of leukocyte esterase (LE) in the urine sample and comparing it to a corresponding control value. A preferred method for determining biomarker levels is an immunoassay.

[0006] In another aspect, the present invention provides a kit for diagnosing urinary tract infection in a human subject. The kit includes a first antibody or binding fragment thereof specific for HD5 and/or a second antibody or binding fragment thereof specific for HNP1-3, reagents for conducting the diagnosis, and a package for holding the antibodies and the reagents. In some embodiments, the kit also includes a third antibody or binding fragment thereof specific for leukocyte esterase.

[0007] Clinicians frequently use urinalysis to diagnose a urinary tract infection (UTI). However, suboptimal test characteristics of leukocyte esterase (LE) and urinary nitrite limit their diagnostic utility. In example 1 described herein, the inventors evaluated two urinary antimicrobial peptides (AMPs) as novel biomarkers to predict positive urine culture in children.

[0008] Pediatric Emergency Department patients were prospectively enrolled if urinalysis and urine culture were performed. Urine concentrations of human a-defensin 5 (HD5) and human neutrophil peptides (HNP) 1-3 were measured by enzyme-linked immunosorbent assay and normalized to urine creatinine (Cr) concentration. Urine culture was the reference standard. Receiver operating characteristic (ROC) curves were constructed for each AMP. Sensitivities and specificities of LE, HD5, HNP1-3, and test combinations were compared. [0009] Of 199 enrolled patients, urine was collected by catheterization in 99 (50%) and clean-catch method in 100 (50%). The urine culture was positive in 29 (15%) patients. The areas under the ROC curves for HD5 and HNPl-3 were 0.86 (95% CI, 0.81-0.92) and 0.88 (95% CI, 0.82-0.93), respectively. Compared to LE alone, the combination test "LE and HD5" increased specificity by 6.5% (95% CI, 2.8-12.2) without affecting sensitivity. In the clean-catch subgroup, combination tests "LE and HD5", "HD5 and HNPl-3", and "LE and HD5 and HNPl-3" all increased specificity by > 10% compared to LE alone without affecting sensitivity. When added to LE, HD5 significantly improved specificity for positive urine culture. Urine AMP profiles are a promising novel strategy as an adjunct to urinalysis to aid UTI diagnosis.

BRIEF DESCRIPTION OF THE FIGURES

[0010] The present invention may be more readily understood by reference to the following figures, wherein:

[0011] Figures 1A-1D provide graphs showing HD5 and HNPl-3 concentrations in culture- negative and culture-positive urine samples (A/B), and ROC curves (C/D). A/B, Horizontal bars represent median values and interquartile ranges. Gray circles and squares indicate individual data points. C/D, The diagonal line represents a test with no diagnostic value.

[0012] Figure 2A provides a graph comparing the sensitivity and specificity of LE, HD5, and HNPl-3, alone and in combination. The graph displays the change in specificity of each individual or combination test compared to LE > trace. Horizontal bars represent 95% confidence intervals.

[0013] Figure 2B provides a graph comparing the sensitivity and specificity of LE, HD5, and HNPl-3, alone and in combination, for the subgroup of patients whose urine was collected by catheterization.

[0014] Figure 2C provides a graph comparing the sensitivity and specificity of LE, HD5, and HNPl-3, alone and in combination, for the subgroup of patients whose urine was collected by clean-catch method. [0015] Figure 3 provides graphs showing ROC curves for urinary antimicrobial peptide detection of positive urine cultures in ED adults ages >18 years. ROC = receiver operating characteristic.

[0016] Figure 4 provides graphs showing ROC curves for urinary antimicrobial peptide detection of positive urine cultures in older ED adults >65 years of age. ROC = receiver operating characteristic.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention provides a method of diagnosing a urinary tract infection in a subject. The method includes obtaining a urine sample from the subject; determining the level of one or more of the biomarkers human oc-defensin 5 (HD5), human neutrophil peptides (HNP)l-3, and leukocyte esterase (LE) in the urine sample and comparing it to a corresponding control value; and diagnosing the subject as having a urinary tract infection if the level of the one or more biomarkers is greater than the corresponding control value(s). Kits for diagnosing a urinary tract infection in a subject using antibodies specific for one or more of HD5, HNP1-3, and LE are also described.

Definitions

[0018] As used herein, the term "diagnosis" can encompass determining the likelihood that a subject will develop a disease, or the existence or nature of disease in a subject. The term diagnosis, as used herein also encompasses determining the severity and probable outcome of disease or episode of disease or prospect of recovery, which is generally referred to as prognosis).

[0019] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic or physiologic effect. The effect may be therapeutic in terms of a partial or complete cure for a disease or an adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and can include inhibiting the disease or condition, i.e. , arresting its development; and relieving the disease, i.e. , causing regression of the disease. [0020] The term therapy, as used herein, encompasses activity carried out to treat a disease. The specific activity carried out to conduct therapy can include use of antibiotics, analgesics, or probiotics.

[0021] The terms "therapeutically effective" and "pharmacologically effective" are intended to qualify the amount of an agent which will achieve the goal of improvement in disease severity and the frequency of incidence over treatment of each agent by itself, while avoiding adverse side effects typically associated with alternative therapies. The effectiveness of treatment may be measured by evaluating a reduction in symptoms.

[0022] The term "biomarkers" generally encompasses biological compounds which are useful for the diagnosis, prediction, prognosis and/or monitoring of urinary tract infection as disclosed herein.

[0023] The terms "subject" and "patient" can be used interchangeably herein, and generally refer to a mammal, including, but not limited to, primates, including simians and humans, equines (e.g. , horses), canines (e.g. , dogs), felines, various domesticated livestock (e.g. , ungulates, such as swine, pigs, goats, sheep, and the like), as well as domesticated pets and animals maintained in zoos. Treatment and evaluation of human subjects is of particular interest. Human subjects can be various ages, such as a child (under 18 years), adult (18 to 59 years) or elderly (60 years or older) human subject.

[0024] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0025] As used herein, the term "about" refers to +/- 10% deviation from the basic value. [0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0027] As used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a sample" also includes a plurality of such samples and reference to "a biomarker" includes reference to one or more biomarkers, and so forth.

Methods of Diagnosing Urinary Tract Infection

[0028] One aspect of the invention provides a method of diagnosing a urinary tract infection in a human subject. The method includes the steps of obtaining a urine sample from the subject; determining the level of human oc-defensin 5 (HD5) and/or human neutrophil peptides (HNP)l-3 in the urine sample and comparing it to a corresponding control value; and diagnosing the human subject as having a urinary tract infection if the level of HD5 and/or HNPl-3 is greater than the control value.

[0029] HD5 and HNPl-3 are both antimicrobial peptides, which are peptides known to play an important role in innate immunity. See Martin et al., Front Immunol. 6:404 (2015). In some embodiments, the levels of only HD5 or HNPl-3 is determined, while in other embodiments the levels of both HD5 and HNPl-3 are determined and used to provide a diagnosis. Human oc-defensin 5 (HD5) is an epithelial-derived antimicrobial peptide produced by intestinal Paneth cells, the female genital tract, and the uroepithelium. Spencer et al., PLoS One 7, e31712 (2012). The amino acid sequence and structure of HD5 have been determined and are known to those skilled in the art. Rajabi et al., J Biol Chem., 287(26):21615-27 (2012).

[0030] Human neutrophil peptides (HNP)l-3 represent a group of three very closely related peptides, namely HNP-1, HNP-2, and HNP-3. Because of their close similarity, HNPl-3 are often analyzed collectively, without trying to distinguish the three peptides. HNP-1, HNP-2, and HNP-3 are encoded by oc-defensin genes DEFAl and DEFA3. It has been noted that rather than regarding DEFAl and DEFA3 as distinct loci, it is more realistic to view them as variant repeats in gene array haplotypes. Aldred et al., Hum Mol Genet. 14(14):2045-52 (2005). Nonetheless, while there is significant overlap between the structures of HNP-1, HNP-2, and HNP-3, their structures are well known, and levels of HNPl-3 can be readily identified by one skilled in the art.

[0031] In further embodiments, the levels of additional biomarkers or control compounds can be determined. In some embodiments, the method of diagnosis also includes the step of determining the level of leukocyte esterase (LE) in the urine sample and comparing it to a corresponding control value. Thus, the invention can include determining HD5 levels and LE levels, determining HNPl-3 and LE levels, or determining the levels of HD5, HNPl-3, and LE. The inventors have shown that use of a plurality of biomarkers can give synergistic diagnostic results, in which the accuracy to the diagnosis is significantly improved compared to the accuracy of the diagnosis when only one biomarker is used.

[0032] The method diagnoses a subject as having a urinary tract infection if the level of HD5 and/or HNPl-3 is greater than the control value. If levels of LE are also determined, the method provides a diagnosis of urinary tract infection if the levels of HD5 and/or HNPl-3 and LE are all greater than the corresponding control values. When the control values are the corresponding levels of the biomarker in a healthy subject, a greater level is simply one in which the amount exceeds than found in a healthy subject. However, more specific numbers may be used, particularly when an internal control such as creatinine is used. Accordingly, in some embodiments, a human subject is diagnosed as having a urinary tract infection if the level of HD5 is equal to or greater than 150 pg HD5/mg creatinine, and the level of HNPl-3 is equal to or greater than 300 pg HNPl-3/mg creatinine, while in a further embodiment the human subject is diagnosed as having a urinary tract infection if the level of HD5 is equal to or greater than 170 pg HD5/mg creatinine, and the level of HNPl-3 is equal to or greater than 350 pg HNPl-3/mg creatinine.

Urinary Tract Infection

[0033] A urinary tract infection (UTI), as defined herein, is an infection of any part of the urinary tract. The urinary tract includes the kidneys, the bladder, the urethra, and the ureter. Infection of the urinary tract typically results in a variety of symptoms, depending on the specific site of infection. The urinary tract includes both the upper and lower urinary tract. The kidneys and most of the upper part of the ureters comprise the upper urinary tract, while the distal parts of the ureters, urinary bladder and urethra make up the lower urinary tract. In some embodiments, the urinary tract infection is in both the upper and lower urinary tract, while in other embodiments, the urinary tract infection is a lower urinary tract infection or an upper urinary tract infection. A urinary tract infection affecting the lower urinary tract it is also referred to as a bladder infection (cystitis), while a UTI affecting the upper urinary tract it is also referred to as a kidney infection (pyelonephritis). There are some differences in the symptoms observed for upper and lower urinary tract infections.

[0034] Infection of the kidneys (e.g., acute pyelonepthritis) can result in upper back and side pain, high fever, shaking and chills, nausea, and vomiting. Infection of the bladder (e.g., cystitis) can result in pelvic pressure, lower abdomen discomfort, frequent and painful urination, and blood in the urine. Infection of the urethra (e.g. , urethritis) typically can be diagnosed based on a burning sensation associated with urination. One or more of these conditions can indicate a urinary tract infection, though it is preferable to confirm the presence of infection since there are other conditions such as irritation of the urethra, vaginitis, interstitial cystitis, or sexually transmitted diseases that can replicate some of these symptoms. For febrile UTI, a fever will be present, and possibly other associated symptoms such as shaking and chills as well.

[0035] Urinary tract infections can be acute or chronic. An acute UTI is typically short term (i.e. , less than one month) and of high intensity, whereas a chronic infection is a longer-term infection (i.e. , lasting at least one month, and up to a number of years). In a chronic infection and/or colonization, the patient typically has bacteria growing in their bladder but they do not have symptoms typically associated with a urinary tract infection. An acute infection is present when the patient has symptoms such as painful urination or fever. A fever, as defined herein, is a body temperature above 100 F. If an acute infection is present simultaneously with a chronic infection, the effects of the acute infection will dominate those of the chronic infection in terms of overall characterization of the infection, for at least the reason that a chronic infection typically shows few effects.

[0036] In some embodiments, the subject is a subject who has an increased risk of having a urinary tract infection. An increased risk refers to a higher likelihood or percent possibility of having a urinary tract infection in comparison with a subject who is not at an increased risk. For example, urinary tract infections occur most frequently in boys and girls during the first year of life. The likelihood of a urinary tract infection drops sharply after the first year, but then gradually increases with age. Gender is also a factor, with women having a high rate of UTIs due to physiological differences. The risk of having a UTI increases even further after menopause in women, and in pregnant women. Andriole, V.T., Patterson, T.F., Med. Clin. North. Am. 75, 359-373 (1991). Other risk factors for a urinary tract infection include taking antibiotics, having a urinary catheter inserted or having surgery on the urinary tract, a high level of sexual activity (Scholes et al., J. Infect Dis. 182, 1177-1182 (2000)), and various diseases or disorders such as urinary tract anatomical defects, vesicoureteral reflux, diabetes, weakened immune system, kidney stones, an enlarged prostate, body paralysis, a history of kidney transplant, HIV status, sickle cell anemia, and nervous system disorders affecting bladder emptying.

[0037] In some embodiments, the subject does not have any symptoms of a urinary tract infection. Urinary tract infections can be asymptomatic. Asymptomatic bacteriuria is a colonization of a portion of the urinary tract by bacteria that does not display the symptoms typically seen for a urinary tract infection. The urine samples obtained from a subject with asymptomatic bacteriuria may look infected (as evaluated by dipstick, for example) and will result in bacterial growth if cultured. However, it is difficult to determine if this represents an early infection that can be treated briefly to avoid complications, or just bladder colonization with non-pathogenic bacteria that does not represent a problem and will likely not be cleared by treatment with antibiotics. Not all asymptomatic infections represent chronic infections. Some types of subjects will be asymptomatic as a result of a lack of inflammatory response due to immunosuppression (e.g., transplant patients) or lack of sensation of symptoms as a result of, for example, having spinal cord injuries or congenital spinal/neural tube defects.

[0038] A urinary tract infection is typically a bacterial infection of the urinary tract. The bacteria can be gram-negative bacteria, or the bacterial can be gram-positive bacteria. For example, the bacteria can be one or more of Escherichia coli, Pseudomonas, Enterococcus, Enterobacter, Klebsiella, or Proteus mirabilis. The majority (80-85%) of bacterial urinary tract infections are caused by E. coli. However, a urinary tract infection can also occur as a result of infection by pathogens other than bacteria. For example, urinary tract infections can also be caused by viruses and fungus. Examples of urinary viral infections include those by BK virus, cytomegalovirus (CMV) and Epstein-Barr virus (EBV). Fungal infection is commonly caused by infection by fungi of the genus Candida. Urine Samples

[0039] A variety of methods are known to those skilled in the art for obtaining a urine or fecal sample. Urine collected in a normal individual by suprapubic aspiration of the bladder is sterile and does not contain leukocytes. This method represents the ideal method for obtaining a urine sample. However, it is not performed routinely in clinical practice in which urine samples are generally obtained after natural micturition; in this setting, some degree of artifactual contamination with normal urethral organisms occurs.

[0040] A standard method for obtaining a urine sample can be referred to as the clean-catch sample method. To obtain an untainted urine sample, doctors usually request a so-called midstream, or clean-catch, urine sample. To provide this, the subject washes the area from which urine will issue, urinate a small amount into the toilet for a few seconds and then stop, position the container to catch the middle portion of the stream, urinate until the collection cup is halfway full (about 2 ounces), and then remove the cup. The collection cup should then be sealed with a cap and given to the doctor or sent to the laboratory for analysis. An advantage of the present invention is its ability to increase UTI diagnostic accuracy in a bag urine sample, which currently is not an accurate method of urine collection for UTI evaluations.

[0041] Alternately, urine can be collection with a catheter. Some patients (for example, small children, elderly patients, or hospitalized patients) cannot provide a urine sample. In such cases, a catheter may be inserted into the bladder to collect urine. This is the best method for providing a contaminant-free sample, but has the disadvantage of possibly introducing or spreading infection.

[0042] The urine sample may be pretreated as necessary by dilution in an appropriate buffer solution and concentrated or fractionated by any number of methods including but not limited to ultracentrifugation, fractionation by fast performance liquid chromatography (FPLC), or precipitation. Any of a number of standard aqueous buffer solutions at physiological pH, such as phosphate, Tris, or the like, can be used.

[0043] A urine sample may be fresh or stored. Urine samples may be or have been stored or banked under suitable tissue storage conditions. The urine sample may have been expressly obtained for the assays of this invention or a urine sample obtained for another purpose which can be subsampled for the assays of this invention. Preferably, urine samples are either chilled or frozen shortly after collection if they are being stored to prevent deterioration of the sample.

Detection Methods

[0044] Once a sample has been obtained, an analytic device is used to measure biomarker levels. The analytic device can be either a portable or a stationary device. In addition to including equipment used for detecting biomarkers, the analytic device can also include additional equipment to provide physical separation of analytes prior to analysis. For example, if the analyte detector is an immunoassay, it may also include an ion exchanger column chromatography to purify the proteins from urine before detection of the biomarkers of interest by immunoassay.

[0045] Levels of biomarkers such as the antimicrobial peptides HD5 and HNP1-3, or LE, can be determined using a variety of different methods. In some embodiments, the levels of biomarkers are determined using an immunoassay. In other embodiments, the levels of the biomarker are determined using a method other than an immunoassay, such as mass spectrometry. For example, biomarkers such as HD5, HNP1-3, or LE can be detected using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF).

[0046] Prior to analysis for the biomarker, it may be preferable to purify the sample. Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the biomarker may be further purified and/or quantified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of pure proteins are immunohistochemistry, ion- exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.

[0047] In certain embodiments, antibodies are provided that are specific for HD5, HNP1-3, or LE. Antibodies provided herein include polyclonal and monoclonal antibodies, as well as antibody fragments that contain the relevant antigen binding domain of the antibodies. The term "antibody" as used herein refers to immunoglobulin molecules or other molecules which comprise at least one antigen-binding domain. The term "antibody" as used herein is intended to include whole antibodies, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, primatized antibodies, multi-specific antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, and totally synthetic and recombinant antibodies. The antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

[0048] Monoclonal antibodies may be produced in animals such as mice and rats by immunization. B cells can be isolated from the immunized animal, for example from the spleen. The isolated B cells can be fused, for example with a myeloma cell line, to produce hybridomas that can be maintained indefinitely in in vitro cultures. These hybridomas can be isolated by dilution (single cell cloning) and grown into colonies. Individual colonies can be screened for the production of antibodies of uniform affinity and specificity. Hybridoma cells may be grown in tissue culture and antibodies may be isolated from the culture medium. Hybridoma cells may also be injected into an animal, such as a mouse, to form tumors in vivo (such as peritoneal tumors) that produce antibodies that can be harvested as intraperitoneal fluid (ascites). The lytic complement activity of serum may be optionally inactivated, for example by heating.

[0049] Protocols for generating antibodies, including preparing immunogens, immunization of animals, and collection of antiserum may be found in Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, N.Y., 1988) pp. 55-120 and A. M. Campbell, Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984).

[0050] The term "antibody fragment" or "binding fragment" as used herein is intended to include any appropriate antibody fragment which comprises an antigen-binding domain that displays antigen binding function. Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab1 fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, T and Abs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. Antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CH3 domains.

[0051] An immunoassay can be used to detect and analyze biomarkers in a sample. An immunoassay is an assay that uses an antibody to specifically bind an antigen (e.g., a biomarker). An immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to a biomarker from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically reactive with that biomarker and not with other proteins, except for polymorphic variants and alleles of the biomarker. This selection may be achieved by subtracting out antibodies that cross-react with the biomarker molecules from other species.

[0052] Generally, a sample obtained from a subject can be contacted with the antibody that specifically binds the biomarker. Optionally, the antibody can be fixed to a solid support to facilitate washing and subsequent isolation of the complex, prior to contacting the antibody with a sample. Examples of solid supports include glass or plastic in the form of, e.g., a micro titer plate, a stick, a bead, or a microbead. Antibodies can also be attached to a probe substrate or a protein chip.

[0053] After incubating the sample with antibodies, the mixture is washed and the antibody- marker complex formed can be detected. This can be accomplished by incubating the washed mixture with a detection reagent. This detection reagent may be, e.g., a second antibody which is labeled with a detectable label. Exemplary detectable labels include magnetic beads, fluorescent dyes, radiolabels, enzymes (e.g., horse radish peroxide, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads. Alternatively, the biomarker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound biomarker- specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the biomarker is incubated simultaneously with the mixture.

[0054] Methods for measuring the amount or presence of an antibody-marker complex include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a gating coupler waveguide method or interferometry). Optical methods include microscopy (both confocal and non- confocal), imaging methods and non-imaging methods. Electrochemical methods include voltammetry and amperometry methods. Radio frequency methods include multipolar resonance spectroscopy. Useful assays are well known in the art, including, for example, an enzyme immune assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western blot assay, or a slot blot assay.

[0055] In some embodiments, the immunoassay is carried out using a lateral flow device. Lateral flow devices, also known as lateral flow immunochromatographic assays, are simple devices intended to detect a target analyte in a sample without the need for specialized and costly equipment. The lateral flow device includes a series of capillary beds, such as pieces of porous paper or sintered polymer, that have the capacity to transport fluid (e.g., urine) spontaneously. The first element (the sample pad) acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid migrates to the second element (conjugate pad) which includes the "conjugate," a dried format of bio-active particles in a salt-sugar matrix that facilitates the chemical reaction between an antigen on the biomarker with an antibody or antibody fragment that specifically binds to the antigen and that has been immobilized on the particle's surface. While the sample fluid dissolves the salt-sugar matrix, it also dissolves the particles and in one combined transport action the sample and conjugate mix while flowing through the porous structure. In this way, the analyte binds to the particles while migrating further through the third capillary bed. This material has one or more areas (often called stripes) where a third molecule has been immobilized. By the time the sample-conjugate mix reaches these strips, analyte has been bound on the particle and the third 'capture' molecule binds the complex. After a while, when more and more fluid has passed the stripes, particles accumulate and the stripe-area changes color. Lateral flow tests can operate as either competitive or sandwich immunoassays. In some embodiments, lateral flow tests can include nanoparticles or paramagnetic particles. Magnetic particles can be used to provide a lateral flow test that quantifies results non-optically.

[0056] Once the levels of the biomarkers have been determined, they can be displayed in a variety of ways. For example, the levels of antimicrobial peptides can be displayed graphically on a display as numeric values or proportional bars (i.e., a bar graph) or any other display method known to those skilled in the art. The graphic display can provide a visual representation of the amounts of the various biomarkers in the samples being evaluated. In addition, in some embodiments, the analytic device can also be configured to display a comparison of the levels of biomarkers in the subject's urine to a control value based on levels of biomarkers in a comparable urine sample, urine samples from a reference cohort, or a standard numerical reference.

Comparison of Biomarker levels to corresponding Control Values

[0057] A method of diagnosing urinary tract infection in a human subject is described. The method includes comparing the levels of one or more biomarkers in a urine sample obtained from a subject to corresponding control values to determine if the subject has an increased risk of having a urinary tract infection. The biomarkers include the antimicrobial peptides HD5 and HNP1-3, as well as LE. Subtypes of HNP (i.e., HNP1, HNP2, or HNP3) may also be used as biomarkers. Corresponding control values are the appropriate control value for the particular biomarker being evaluated. For example, in some embodiments, the corresponding control values are the values of the biomarkers in healthy subjects, while in other embodiments the corresponding control values are another compound (e.g., creatinine) whose level is the same in subjects having and not having a urinary tract infection.

[0058] Control values can be based upon the level of the biomarker (e.g., HD5 and/or HNP1- 3) in comparable samples obtained from a reference cohort. In certain embodiments, the reference cohort is the general population. For example, the reference cohort can be a select population of human subjects. Control values for particular biomarkers may in some cases already be known to those skilled in the art. Alternately, or in addition, control values can be an internal standard whose level does not vary significantly from patients having a UTI and healthy patients. For example, creatinine can be used as an internal standard control.

[0059] In some embodiments, the corresponding control is creatinine and the method includes determining the level of creatinine in the urine sample and using the creatinine level as a control value. Typical urine samples will have a creatinine value between 20-350 mg/dL (milligrams per deciliter), which varies with the gender and muscle mass of the subject. Methods of determining creatinine levels in urine are well known to those skilled in the art, and include the urine albumin test and the urine protein test. One common method to determine creatinine levels is to react creatinine with picric acid to form a red Janovski complex. The color intensity of the complex is directly proportional to the creatinine concentration and can be measured spectrophotometrically at 505 nm.

[0060] The control value can take a variety of forms. The control value can be a single cutoff value, such as a median or mean. Control values of risk predictors in biological samples obtained, such as for example, mean levels, median levels, or "cut-off" levels, are established by assaying a large sample of individuals in the general population or the select population and using a statistical model such as the predictive value method for selecting a positivity criterion or receiver operator characteristic curve that defines optimum specificity (highest true negative rate) and sensitivity (highest true positive rate) as described in Knapp, R. G., and Miller, M. C. (1992). Clinical Epidemiology and Biostatistics. William and Wilkins, Harual Publishing Co. Malvern, Pa., which is specifically incorporated herein by reference. A "cutoff" value can be determined for each biomarker that is assayed.

Methods for treating Urinary Tract Infection

[0061] In some embodiments, the method also includes providing treatment for the human subject identified as having a urinary tract infection. A variety of methods are known for treating a urinary tract infection. Typically, this is done with a therapeutic agent. For example, in some embodiments, the therapeutic agent is an antibiotic. Examples of suitable antibiotics include trimethoprim-sulfamethoxazole, cephalosporins, nitrofurantoin, amoxicillin, Augmentin™, doxycycline, and fluoroquinolones. Pyelonephritis is treated more aggressively than a simple bladder infection using either a longer course of oral antibiotics or intravenous antibiotics. For a description of the various treatment methods for various types of urinary tract infection, see Orenstein et al., Am. Fam. Physician., 59(5): 1225-1234 (1999), the disclosure of which is incorporated by reference herein.

[0062] Urinary tract infections can also be treated with analgesics to relieve the burning pain and urgent need to urinate. For example, the local analgesic phenazopyridine hydrochloride (Pyridium) can be used together with an antibiotic for treatment of a urinary tract infection.

[0063] In some embodiments, a urinary tract infection can be treated by administration of a probiotic to the subject. Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit to the subject. Preferred probiotics for the present invention are non-pathogenic, and/or non-fever- inducing bacteria, such as Lactobacillus bacteria. The presence of benign bacterial flora is important for body function and prevention of infection by pathogenic bacteria. Probiotics can be administered orally, or can be administered directly to the urinary tract. Methods of treating urinary tract infection by administration of probiotics are known to those skilled in the art. Borchert et al., Indian J. Urol. 24, 139-144 (2008).

Kits

[0064] An additional aspect of the invention provides a kit for diagnosing urinary tract infection in a human subject. The kit includes a first antibody or binding fragment thereof specific for HD5, a second antibody or binding fragment thereof specific for HNP1-3, reagents for conducting the diagnosis, and a package for holding the antibodies and the reagents. In some embodiments, the kit also includes a third antibody or binding fragment thereof specific for leukocyte esterase. In further embodiments, the kit also includes reagents useful for determining creatinine levels. Creatinine levels can be determined, for example, using sodium hydroxide and picric acid to carry out a Jaffe reaction.

[0065] A kit generally includes a package with one or more containers holding the reagents, as one or more separate compositions or, optionally, as an admixture where the compatibility of the reagents will allow. The kit can also include other material(s), which may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and/or any other material useful in sample processing, washing, or conducting any other step of the assay. In some embodiments, the kit includes a lateral flow strip which can be used to conduct an immunoassay using the antibodies included in the kit. In some embodiments, such kits may also include control reagents, e.g., known amounts of one or more antimicrobial peptides. Kits can also include a tool for obtaining a urine sample from a subject, such as a urine receptacle or syringe.

[0066] In some embodiments, the reagents include antibodies, or binding fragments thereof, capable of specifically binding to the compound they are capable of detecting. For example, the kit can include an antibodies specific for HD5, HNP1-3, or LE. In some embodiments, multiple concentrations of each antibody can be included in the kit to facilitate the generation of a standard curve to which the signal detected in the test sample can be compared. Alternatively, a standard curve can be generated by preparing dilutions of a single antibody solution provided in the kit.

[0067] As used herein, the terms "specific binding" or "specifically binding", refer to the interaction of an antibody, a protein, or a peptide with a second chemical species, wherein the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally.

[0068] The kits may also include a solid phase, to which the antibodies functioning as capture antibodies and/or detection antibodies in a sandwich immunoassay format are bound. The solid phase may be a material such as a magnetic particle, a bead, a test tube, a microtiter plate, a cuvette, a membrane, a scaffolding molecule, a quartz crystal, a film, a filter paper, a disc or a chip. In some embodiments, the solid phase comprises a portion of a lateral flow test strip. The kit may also include a detectable label that can be or is conjugated to an antibody, such as an antibody functioning as a detection antibody. The detectable label can for example be a direct label, which may be an enzyme, nanoparticle chemiluminophore, fluorophore, fluorescence quencher, chemiluminescence quencher, or biotin. Test kits may optionally include any additional reagents needed for detecting the label.

[0069] The kit can also include instructions for using the kit to diagnose urinary tract infection. Diagnosing urinary tract infection includes using the kit to determine the levels of HD5, HNP1-3, and/or LE in a urine sample. Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" can include the address of an internet site that provides the instructions.

[0070] Examples have been included to more clearly describe particular embodiments of the invention. However, there are a wide variety of other embodiments within the scope of the present invention, which should not be limited to the particular example provided herein.

EXAMPLES

Example 1: Novel Urinary Tract Infection Biomarkers in Children

[0071] Two peptides in the a-defensin family of antimicrobial peptides (AMPs) were evaluated as novel biomarkers for UTI in children. AMPs are small, cationic peptides that participate in the innate immune defense of the kidneys, urinary tract, and other organ systems. Spencer et al., Pediatr Nephrol 29: 1139-49 (2014). AMPs are produced by white blood cells and/or epithelial cells and may be constitutively expressed or induced when pathogens enter the urinary tract. Human a-defensin 5 (HD5) is an epithelial-derived AMP produced by intestinal Paneth cells, the female genital tract, and the uroepithelium. Quayle et al., Am J Pathol 152: 1247-58 (1998). In a prior study of a small number of children, the inventors' research group demonstrated via Western immunoblot and enzyme-linked immunosorbent assay that HD5 was detected in culture-positive but not culture-negative urine samples. Spencer et al., PLoS One 7:e31712 (2012). Human neutrophil peptides (HNP) 1-3 are three closely related a-defensins (collectively known as HNP1-3 and typically measured in aggregate) produced in promyelocytes and stored in the primary granules of neutrophils. Two prior studies of adults demonstrated increased urine concentrations of HNP1-3 during acute UTI (Ihi et al, Clin Infect Dis, 25: 1134-40 (1997)) and HNP1 during chronic pyelonephritis. Tikhonov I, Rebenok A, Chyzh A. A study of interleukin-8 and defensins in urine and plasma of patients with pyelonephritis and glomerulonephritis. Nephrol Dial Transplant 12:2557-61 (1997). Given this prior data, the inventors hypothesized that HD5 and HNP1-3, alone or as adjuncts to dipstick urinalysis, would improve diagnostic accuracy for UTI. Therefore, the objective of this work was to evaluate the performance of urine HD5 and HNP1-3 concentrations as biomarkers for positive urine culture in children. Results

[0072] Study population. During the study period, urine samples were collected from 268 Emergency Department (ED) or Urgent Care (UC) patients who met inclusion criteria. Of those, 34 had received antibiotics within 7 days before presentation and were excluded. Of the remaining 234 patients, 199 provided adequate excess urine sample volume for analysis of both HD5 and HNP1-3 concentrations. Table 1 describes the demographic characteristics and documented clinical signs of symptoms of the study population.

[0073] Table 1: Demographic Characteristics and Documented Clinical Signs and Symptoms of Study Populationa

CVA, costovertebral angle

a Unless otherwise specified, numerical values indicate the number (percent) with the stated demographic characteristic or clinical sign or symptom.

b Subgroup of patients whose urine was collected by catheterization

c Subgroup of patients whose urine was collected by a clean-catch method

d Fisher's exact test (categorical variables) or Mann- Whitney rank-sum test (age)

[0074] Clinical laboratory results. Of the 199 patients, 29 (15%) had urine cultures that yielded > 50 000 CFU/mL of a single uropathogen. The positive urine cultures included 10 (10%) of 99 in the subgroup of patients whose urine was collected by catheterization and 19 (19%) of 100 in the subgroup whose urine was collected by a clean-catch method. Escherichia coli was the most common bacterial isolate, accounting for 25 (86%) positive urine cultures, followed by Staphylococcus saprophyticus (n = 2), Klebsiella pneumoniae (n = 1), and Serratia marcescens (n= 1). Of the 29 patients with a positive urine culture, 23 (79%) had signs or symptoms of upper urinary tract infection, including fever, back/flank pain, costovertebral angle tenderness, or vomiting. Table 2 lists the sensitivities and specificities of LE and nitrite, alone and in combination, for all patients and for subgroups by urine collection method.

[0075] Table 2: Sensitivities and Specificities of Leukocyte Esterase and Nitrite, Alone and in Combination

LE, leukocyte esterase; Se, sensitivity; Sp, specificity

a Subgroup of patients whose urine was collected by catheterization

b Subgroup of patients whose urine was collected by a clean-catch method

[0076] Antimicrobial peptide analyses. Urine HD5 and HNP1-3 concentrations were significantly higher in culture-positive urine samples compared to culture-negative urine samples (Figure 1, Panels A and B). The median HD5 concentration was 590.6 pg per mg creatinine (pg/mgCr) (IQR, 373.1-908.5) in culture-positive urine samples versus 89.6 pg/mgCr (IQR, 12.8-253.5) in culture-negative urine samples (P < .001, Mann- Whitney). The median HNP1-3 concentration was 3801.4 pg/mgCr (IQR, 1776.6-11 861.3) in culture- positive urine samples versus 148.7 pg/mgCr (IQR, 25.7-934.2) in culture-negative urine samples (P < .001, Mann- Whitney). The areas under the ROC curves for HD5 and HNP1-3 were 0.86 (95% CI, 0.81-0.92) and 0.88 (95% CI, 0.82-0.93), respectively (Figure 1, Panels C and D).

[0077] Test comparisons. The sensitivity of the test "LE >trace" was 97%. HD5 and HNP1- 3 thresholds of 174 pg/mgCr and 384 pg/mgCr, respectively, provided maximal specificity of each individual test with sensitivity at least equal to that of "LE > trace". Comparisons of the sensitivities and specificities of LE, HD5, and HNPl-3, alone and in combination, for all patients are shown in Figure 2A. Nitrite was not included in these test comparisons due to its low sensitivity. The specificities of both HD5 and HNPl-3 were lower than the specificity of LE. However, the combination tests "LE and HD5" and "LE and HD5 and HNPl-3" increased specificity by 6% (95% CI, 3%-10%) and 7% (95% CI, 3%-l l%), respectively, compared to LE alone without decreasing sensitivity.

[0078] Analyses by urine collection method. Comparisons of LE, HD5, and HNPl-3 in the catheterization subgroup showed no increased specificity of combination tests versus LE alone (Figure 2B). However, in the clean-catch subgroup, specificity increased 13% (95% CI, 6%-21%) when "LE and HD5" was compared to LE alone (Figure 2C). The combination tests "HD5 and HNPl-3" and "LE and HD5 and HNPl-3" also increased specificity but differed only slightly from "LE and HD5".

Discussion

[0079] In this example, two AMPs were evaluated as novel biomarkers for pediatric urinary tract infection. The inventors demonstrated that both HD5 and HNPl-3 performed well as diagnostic tests to predict positive urine culture in children, with areas under the ROC curves of 0.86 and 0.88 for HD5 and HNPl-3, respectively. For test comparisons, they determined whether combinations of highly sensitive tests would increase specificity without decreasing sensitivity. When compared to LE alone, the combination test "LE and HD5" increased specificity by 6% in all patients and 13% in the clean-catch subgroup without affecting sensitivity. The addition of HNPl-3 to LE did not improve specificity, but the combination test "HD5 and HNPl-3" did increase specificity by 12% in the clean-catch subgroup. The combination test "LE and HD5 and HNPl-3" also increased specificity in all patients and the clean-catch subgroup, but the results were only slightly better than "LE and HD5" without HNPl-3. These results suggest that urine AMPs have potential to improve standard methods for diagnosing UTI in children.

[0080] The rationale for identifying novel UTI biomarkers stems from the limited utility of currently available point-of-care tests for the diagnosis of UTI in children. The high specificity (99%) but low sensitivity (45%) of urinary nitrite in our cohort is comparable to results of prior studies. Kazi et al., Am J Emerg Med 31 : 1405-7 (2013); Williams et al., Lancet Infect Dis. 10:240-50 (2010). Thus, clinicians can expect a positive urine culture when nitrite is detected, but a negative nitrite test result does not rule out a UTI. Regarding LE, three pediatric meta-analyses and one recent, large study reported sensitivities of 72-83% and specificities of 78-87% (5-8). In this study, LE sensitivity (97%) was greater than results of prior studies, while specificity (75%) was comparable. A high false-positive rate of LE may lead to over-diagnoses and unnecessary antibiotic exposure. False-positive LE tests occur because pyuria is associated with a number of other conditions including acute febrile illnesses, urinary calculi, sexually transmitted infections, intrinsic renal diseases, and other disorders. Dieter RS, Compr Ther 26: 150-2 (2000). Congruent results of LE and nitrite (both positive or both negative) help to rule in or rule out a UTI, but the common scenario in which LE is positive but nitrite is negative creates diagnostic uncertainty. Mori et al., Acta Paediatrica 99:581-4 (2010); Whiting et al, BMC Pediatrics 5:4 (2005). Improved rapid tests are needed to aid the accurate diagnosis of UTI.

[0081] In this example, both HD5 and HNP1-3 demonstrated potential to improve UTI diagnostic accuracy. The results show that HD5 and HNP1-3 are induced during UTI. The higher concentrations of HD5 and HNP1-3 in culture-positive than culture-negative urine samples in this study is consistent with prior investigations performed in small numbers of children and adults. Spencer et al., PLoS One 7:e31712 (2012); Ihi et al., Clin Infect Dis 25: 1134-40 (1997). The areas under the ROC curves for HD5 and HNP1-3 were between 0.75 and 0.9 and thus generally indicate "good" overall diagnostic value of the biomarkers. Ray et al, Anesthesiology 112: 1023-40 (2010). However, HNP1-3 did not improve specificity when combined with LE, perhaps because LE and HNP1-3 are both neutrophil markers and therefore indicate pyuria. In contrast, HD5 is expressed throughout the urothelium of the lower urinary tract and in the nephron and collecting tubules of the kidney. As an epithelial-derived AMP, HD5 likely performed well as a UTI biomarker independent of pyuria. Indeed, it was found that the addition of HD5 to LE improved specificity. Still, there were some false-positive HD5 test results. Reasons for false-positive HD5 tests may include urethritis, viral cystitis, UTI due to pathogens that do not grow well in culture, noninfectious inflammatory conditions of the kidneys and urinary tract, and possibly other currently unrecognized stimuli for HD5 expression in the urine. Additionally, contamination of urine specimens with vaginal secretions could potentially result in false-positive HD5 test results because HD5 is produced in the female genital tract. Quayle et al., Am J Pathol 152: 1247-58 (1998). [0082] Novel UTI biomarkers may provide more benefit in certain clinical scenarios than others. In this example, the specificity of LE was much lower in the clean-catch subgroup. The data does not differentiate whether the difference in specificity was, in full or in part, due to the method of collection. Age, toilet-training status, or combinations of factors may have accounted for the difference. Previous studies have not directly compared LE specificity in samples collected by catheterization versus a clean-catch method. A previous study did compare characteristics of the urine dipstick by age group and found lower specificity of the combination test "LE or nitrite" in children aged > 2 years (of whom only 10% were catheterized) than children aged < 2 years (of whom 88% were catheterized) (19). Whatever the reasons for the difference, in our study the greatest improvement in specificity was observed in the clean-catch subgroup, in whom combination tests "LE and HD5", "HD5 and HNP1-3", and "LE and HD5 and HNP1-3" all increased the specificity by > 10% compared to LE alone. Clinically, fewer false-positive tests may translate to decreased unnecessary empiric antibiotic therapy for patients evaluated for a UTI.

[0083] In summary, the inventors found that urine HD5 and HNP1-3 concentrations performed well as biomarkers for predicting positive urine culture in children. When combined with LE, HD5 provided the greatest increase in specificity without decreasing sensitivity, particularly when a clean-catch method of urine collection was used. Urine AMP profiles are a promising novel strategy as an adjunct to dipstick urinalysis to aid diagnosis and guide empiric management when UTI is suspected. Future research is needed to evaluate additional AMPs that may provide further improvements in specificity and/or sensitivity. Also, determination of AMP performance in subgroups of patients, such as those with neurogenic bladder and complicated urinary tracts, warrants investigation.

Methods

[0084] Study enrollment. Children were prospectively enrolled in the ED and main campus UC at Nationwide Children's Hospital, Columbus, OH, from 11/20/2013 to 7/2/2014. Patients aged < 18 years who met the following inclusion criteria were enrolled during times when research staff was available: 1) both dipstick urinalysis and urine culture were performed for any clinical indication, and 2) excess urine sample was available. Patients were excluded if 1) they had received antibiotics in the 7 days before presentation to the ED or UC, or 2) the urine sample collected had insufficient volume for performance of all investigational assays. The institutional review board at Nationwide Children's Hospital approved the study and granted a waiver of informed consent (IRB 13-00090).

[0085] Clinical data collection and analysis. Electronic medical records were reviewed for pertinent demographic, clinical, and laboratory data, including urine dipstick and urine culture results. Fever was considered present when reported by the patient or caregiver or when a temperature > 38C was documented in the ED/UC. Dipstick urinalyses were performed either in the ED or UC laboratory using the CLINITEK Status+ Analyzer (Siemens, Tarrytown, NY, USA) or in the hospital chemistry laboratory using the iChem Velocity™ instrument (Iris Diagnostics, Chatsworth, CA, USA). Urine was plated by clinical microbiology laboratory staff using calibrated loops and cultured on 5% sheep blood and MacConkey agar biplates and incubated at 35C in ambient atmosphere. A positive urine culture result consisted of > 50 000 colony forming units (CFU) per mL of a single uropathogen (21,23). Urine cultures were considered negative if they yielded < 50 000 CFU/mL, mixed bacteria, or likely contaminants such as Lactobacillus species, coagulase- negative staphylococci, or Corynebacterium species.

[0086] Sample collection. After ensuring sufficient urine volume was available for clinical diagnostic tests, excess urine was immediately collected in AssayAssure™ urine collection tubes (Thermo Scientific™, Waltham, MA, USA) containing a bacteriostatic preservative that suppresses nuclease and protease activity and preserves urine specimens at room temperature for up to 26 days according to the manufacturer.

[0087] AssayAssure™ validation. The stability of AMPs was independently verified for up to 14 days in AssayAssure™ preservative by measuring serial urine concentrations of ribonuclease 7 (RNase 7). The inventors chose to measure RNase 7 because it is an AMP that is constitutively expressed in the urine of healthy individuals. On day 0, urine samples from 2 healthy individuals were collected and stored in AssayAssure™ urine collection tubes. On days 1, 2, 7, and 14, an aliquot of each sample was removed and stored at -80C until analyzed. After the 14 day period, all frozen samples were analyzed simultaneously in a single enzyme-linked immunosorbent assay to determine the concentrations of RNase 7, as previously described. Spencer et al., Kidney Int 80: 174-80 (2011). Concentrations of RNase 7 remained stable in AssayAssure™ during the 14 day validation period. [0088] Sample processing and analysis. Within 7 days of collection, study samples were centrifuged at 3000 rpm for 5 minutes. The supernatant was saved in 300 to 500 xL aliquots and stored at -80C until analyzed. To evaluate subgroups by urine collection method, nearly equal numbers of study samples obtained by catheterization and clean-catch method were analyzed. Urine concentrations of HD5 and HNPl-3 were measured in duplicate by enzyme- linked immunosorbent assay using commercial kits according to the manufacturers' instructions (HD5: Uscn Life Science Inc., Wuhan, Hubei, China; HNPl-3: Hycult Biotech Inc., Plymouth Meeting, PA, USA). Study samples with concentrations less than the lower limit of quantification for HD5 or HNPl-3 were assigned a value of one-half the lower limit of quantification as calculated on a log1Q curve. To account for urine dilution, concentrations of HD5 and HNPl-3 were normalized to urine creatinine concentration, which was measured by a colorimetric assay (Oxford Biomedical Research, Oxford, MI, USA).

[0089] Sample size determination. To design a study with 80% power, 176 total patients was adequate to ascertain a 10% difference in specificity between two diagnostic tests with a Type I error of 0.05.

[0090] Statistical analysis. Urine culture was the reference standard for evaluating test characteristics of the urine dipstick and AMPs. First, urine concentrations of each AMP were compared in culture-negative versus culture-positive urine samples using the Mann- Whitney rank-sum test (non-normally distributed, continuous variables). Next, a receiver operating characteristic curve was generated for each AMP, and the area under the curve was calculated using the scientific software GraphPad Prism 6 (GraphPad Software Inc, La Jolla, CA, USA). Optimal threshold values for positive HD5 and HNPl-3 test results were modeled to maximize specificity while ensuring sensitivity no less than that of LE. Last, the sensitivities and specificities of LE, HD5, HNPl-3, and combinations of the aforementioned tests were compared. Test combinations using the conjunction "and" were considered positive only if all components were positive. Test combinations using the conjunction "or" were considered positive if any individual component was positive. Comparisons between subgroups utilized Fisher's exact test (categorical variables) or Mann-Whitney rank-sum test (non-normally distributed, continuous variables). McNemar's test was used to evaluate differences in specificity between two diagnostic tests. All P values were two-sided. Example 2: Use of Urinary Antimicrobial Peptides in Diagnosing Emergency

Department Patients With Positive Urine Cultures

[0091] One potential strategy to improve diagnostic accuracy makes use of the innate immune response. Antimicrobial peptides (AMPs) are key effectors of innate immunity in the urinary tract that have antimicrobial activity through several mechanisms, including inhibition of bacterial binding, cell lysis, and induction of other immune components. Preliminary studies have documented increased urinary levels of several AMPs in response to infection. Zasloff M., J Am Soc Nephrol 18:2810-6 (2007). Human neutrophil peptides 1-3 (HNP1-3) are bactericidal oc-defensins expressed in neutrophils and the kidney that increase in the setting of pyelonephritis. Spencer et al., Pediatr Nephrol 29: 1139-49 (2014). Human oc-defensin 5 (HD5) is produced in urinary tract epithelial cells and is increased in UTI in children. Human beta defensin 2 (hBD-2) is produced in distal nephron epithelial cells and becomes detectable in UTI.5 Cathelicidin (LL-37) displays vitamin D dependent expression in neutrophils and epithelial cells and is induced with UTI. Chromek et al. Nature Med 12:636^41 (2006). The goal of this work was to determine if urinary levels of AMPs increase with positive urine cultures in ED adults. The inventors hypothesized that AMP levels would be greater in those with positive culture, including the older adult subgroup.

METHODS

[0092] Study Design. This was a prospective observational study. Institutional review board approval was obtained from the Ohio State University (which includes Nationwide Children's Hospital through a cooperative agreement) and the Massachusetts General Hospital. Informed consent was obtained from all subjects.

[0093] Study Setting and Population. The study included a convenience sample of adult patients with suspected UTI presenting to our urban, academic ED (OSU Wexner Medical Center) from January 1, 2014, through March 30, 2014. Inclusion criteria were age older than 17 years and completion of a urine culture. Exclusion criteria included incarceration, suicidal or homicidal ideation, prior enrollment, and presenting as a trauma alert.

[0094] Study Protocol. Study staff identified patients in real time on weekdays from 8 AM to 9 PM. Patients completed an initial survey covering demographics, symptoms, and medical history and provided urine and blood samples. Electronic medical record chart review (EPIC, EPIC Systems Corp., Verona, WI) was used to record vital signs, diagnostic study results, and disposition.

[0095] Urine was collected using standard clinical procedures (clean-catch or catheter). Both urine and serum were centrifuged and stored at -80C. Enzyme-linked immunosorbent assays (ELISAs) were performed using HNP1-3 human ELISA kit (Hycult Biotech, Plymouth Meeting, PA), Defensin-5 ELISA kit (Lifeome Biolabs, Oceanside, CA), human beta defensin 2 ELISA kit (Lifeome Biolabs), and LL-37 human ELISA kit (Hycult Biotech). For all but HNP 1-3, ELISA results were divided by urine creatinine (measured using a creatinine microplate assay, Oxford Biomedical Research, Rochester Hills, MI) to standardize for urine concentration. Samples were run in duplicate. Serum 25- hydroxyvitamin D(25(OH)D) levels were measured by liquid chromatography-tandem mass spectrometry.

[0096] Measures. Because the traditional cutoff of 105 CFU/mL may be overly restrictive in symptomatic patients, a positive urinary culture was defined as >104 CFU/mL of one to two organisms from a female clean-catch specimen or >103 CFU/mL from a catheterized specimen, a male specimen, or a specimen from a chronic indwelling catheter. Hooton et al. Clin Infect Dis, 50:625-63 (2010). Urinary tract symptoms included fever, urgency, frequency, suprapubic pain, dysuria, flank pain, hematuria, or new incontinence. In patients with chronic catheterization, they also included fever or altered mental status without other source.

[0097] Data Analysis. Analyses were performed using STATA v.12. Descriptive statistics are reported as proportions, means with standard deviation (SD) or medians with interquartile range (IQR). Continuous variables were tested for normality using the Shapiro-Wilk test. Comparisons between groups were performed using Wilcoxon rank sum tests. We constructed receiver operating characteristic curves and calculated area under the curve (AUC) with 95% confidence intervals (CIs). Results are also reported for the older adult subgroup. Because this was the first study of its kind and AMP levels were unknown, we were unable to perform a priori power calculations, but chose a sample size of 40 subjects based on capacity of the ELISA plates used in the study. RESULTS

[0098] Forty patients were enrolled with mean (SD) age of 57 (21) years. Twenty-three (58%) were > 65 years, 25 were female (64%), and 33 (83%) were white. Comorbidities included diabetes in 12 (31%), cardiac disease (myocardial infarction or heart failure) in eight (20%), stroke in five (12%), and immunosuppression in 18 (45%), including 11 with cancer and three with organ transplant. Thirty (75%) reported at least one urinary symptom, including fever (n = 11, 27%), urgency (18, 45%), frequency (13, 32%), suprapubic pain or dysuria (15, 38%), flank pain (six, 29%), hematuria (9, 22%), incontinence (8, 20%), or confusion (12, 30%). Thirty-eight patients had microscopic urinalyses: 20 (53%) had leukocyte esterase, three (7.9%) had nitrites, and 14 (27%) had bacteria. Cultures were positive in 13 of 40 (32%), one with two organisms. Seven of 23 older adults had positive cultures. Identified organisms included Escherichia coli (three), Citrobacter (two), Klebsiella pneumoniae (two), Morganella morganii (one), Lactobacillus-like (one), Staphylococcus aureus (two), Staphylococcus saprophyticius (one), Enterococcus faecalis (one), and Gardnerella vaginalis-like (one). Patients > 65 years accounted for seven of the 13 positive cultures (54%). Urinary symptoms were present in 12 of 13 with positive cultures.

[0099] HNP 1-3, HD5, and hBD-2 levels were significantly higher in those with positive than negative urine cultures, and AUCs were > 0.75 (Figures 3 and 4). In patients with positive compared to negative cultures, median HNP1-3 was 5.39 ng/mg (IQR = 2.74 to 11.09) versus 0.81 ng/mg (IQR = 0.06 to 3.87; p = 0.001). Median HD5 was 4.75 pg/mg (IQR = 1.6 to 22.7) versus 0.00 pg/mg (IQR = 0 to 2.60; p = 0.002). Median hBD-2 was 0.13 pg/mg (IQR = 0.08 to 0.17) versus 0.02 pg/mg (IQR = 0 to 0.04; p = 0.001). hBD-2 was analyzed in only 35 patients due to a technical problem with the assay.

[0100] Findings were similar in the older adult subgroup. Median HNP1-3 was 5.39 ng/mg (IQR = 2.70 to 12.68) in positive versus 0.71 ng/mg (IQR = 0.02 to 4.42) in negative cultures (p = 0.032). Median HD5 was 4.75 pg/mg (IQR = 1.6 to 38.20) versus 0.06 pg/mg (IQR = 0.00 to 7.60; p = 0.056). Median hBD-2 was 0.13 pg/mg (IQR = 0.08 to 0.17) versus 0.01 pg/mg (IQR = 0.00 to 0.09; p = 0.032). Although HD5 was nonsignificant, given the mean HD5 and assuming an alpha of 0.05, we had a power of only 0.21 for this comparison. [0101] LL-37 was not significantly higher in patients with positive cultures. Inadequate serum 25(OH)D levels were present in 16 of the 22 subjects (72%) in whom serum was obtained, including eight < 20 ng/mL and eight 20 to 30 ng/mL.

DISCUSSION

[0102] The findings provide the first evidence in an adult, ED population that AMPs may be markers of positive urine culture. If confirmed, AMP levels could result in more timely and accurate UTI diagnosis in acute care, leading to decreases in morbidity, unnecessary antibiotics, and health care costs. These findings represent the recommended first step in the evaluation of novel biomarkers: demonstration of a difference in levels between subjects with and without the outcome. Hlatky et al, Circulation 119:2408-16 (2009).

[0103] Consistent with prior work in other populations, urine levels of HNP 1-3, HD5, and hBD-2 in patients with positive urine culture were five times or more greater than in culture- negative patients. In one study, urine levels of HNP1-3 increased eightfold in chronic pyelonephritis versus both controls and patients with glomerulonephritis. Urinary HD5 increases in children with bacterial UTI, and hBD-2 becomes detectable in chronic pyelonephritis. The extension of these findings to the ED provides novel evidence of the potential use of AMPs as a diagnostic marker in acute care settings.

[0104] Diagnosis of UTI in older adults is a particular challenge due to frequent absence of UTI symptoms. In addition, in older ED patients, 30% of reagent strips in the presence of positive culture are negative for both nitrites and leukocyte esterase, and > 50% of positive strips are associated with negative cultures. Ducharme et al., CJEM 9:87-92 (2007). In our subset of older adults, AMP levels were also greater in those with positive cultures.

[0105] LL-37 was not increased with positive cultures, which is inconsistent with prior findings where urinary LL-37 increased with bacteriuria. van der Starre et al., PLoS One 10:e0121302 (2015). However, LL-37 expression is vitamin D dependent, and 67% of study patients had 25(OH)D < 30 ng/mL. Further study is required to understand the relationship between urinary LL-37, vitamin D status, and UTI. CONCLUSIONS

[0106] Urinary levels of human neutrophil peptides 1-3, human oc-defensin 5, and human beta defensin 2 are significantly greater in the presence of positive urine cultures in ED patients with suspected urinary tract infection. These findings were maintained in the subgroup of older adults.

[0107] The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

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1 *WATSON ET AL.: "Evaluation of novel urinary tract infection biomarkers in children.", PEDIATRIC RESEARCH (2016, 26 May 2016 (2016-05-26), XP055317065
Classifications
International ClassificationG01N33/68
Cooperative ClassificationG01N2800/348, G01N2333/4721, G01N33/6893
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