WO2016161413A1 - Biomarqueurs d'infection des voies urinaires - Google Patents

Biomarqueurs d'infection des voies urinaires Download PDF

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Publication number
WO2016161413A1
WO2016161413A1 PCT/US2016/025824 US2016025824W WO2016161413A1 WO 2016161413 A1 WO2016161413 A1 WO 2016161413A1 US 2016025824 W US2016025824 W US 2016025824W WO 2016161413 A1 WO2016161413 A1 WO 2016161413A1
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Prior art keywords
urinary tract
urine
tract infection
hnpl
kit
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PCT/US2016/025824
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English (en)
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Andrew SCHWADERER
David Hains
John David Spencer
Joshua WATSON
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Research Institute At Nationwide Children's Hospital
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Publication of WO2016161413A1 publication Critical patent/WO2016161413A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4721—Cationic antimicrobial peptides, e.g. defensins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/348—Urinary tract infections

Definitions

  • Urinary tract infection 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
  • 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.
  • 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.
  • 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.
  • the kit also includes a third antibody or binding fragment thereof specific for leukocyte esterase.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • diagnosis can encompass determining the likelihood that a subject will develop a disease, or the existence or nature of disease in a subject.
  • diagnosis 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).
  • treatment refers 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 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.
  • therapy encompasses activity carried out to treat a disease.
  • the specific activity carried out to conduct therapy can include use of antibiotics, analgesics, or probiotics.
  • terapéuticaally 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.
  • biomarkers generally encompasses biological compounds which are useful for the diagnosis, prediction, prognosis and/or monitoring of urinary tract infection as disclosed herein.
  • 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.
  • livestock e.g. , ungulates, such as swine, pigs, goats, sheep, and the like
  • 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.
  • 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.
  • HD5 human oc-defensin 5
  • HNP human neutrophil peptides
  • 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).
  • HNP Human neutrophil 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.
  • the levels of additional biomarkers or control compounds can be determined.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a urinary tract infection 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.
  • 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).
  • cystitis bladder infection
  • pyelonephritis kidney infection
  • Infection of the kidneys can result in upper back and side pain, high fever, shaking and chills, nausea, and vomiting.
  • Infection of the bladder e.g., cystitis
  • pelvic pressure can result in pelvic pressure, lower abdomen discomfort, frequent and painful urination, and blood in the urine.
  • Infection of the urethra 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.
  • a fever will be present, and possibly other associated symptoms such as shaking and chills as well.
  • 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).
  • 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.
  • 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.
  • 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).
  • urinary tract infection 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a urinary tract infection can also occur as a result of infection by pathogens other than bacteria.
  • 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 include those by BK virus, cytomegalovirus (CMV) and Epstein-Barr virus (EBV). Fungal infection is commonly caused by
  • 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.
  • a standard method for obtaining a urine sample can be referred to as the clean-catch sample method.
  • doctors usually request a so-called midstream, or clean-catch, urine sample.
  • 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.
  • urine can be collection with a catheter.
  • Some patients for example, small children, elderly patients, or hospitalized patients
  • 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.
  • 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.
  • FPLC fast performance liquid chromatography
  • 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.
  • urine samples are either chilled or frozen shortly after collection if they are being stored to prevent deterioration of the sample.
  • an analytic device is used to measure biomarker levels.
  • the analytic device can be either a portable or a stationary device.
  • the analytic device can also include additional equipment to provide physical separation of analytes prior to analysis.
  • 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.
  • Levels of biomarkers such as the antimicrobial peptides HD5 and HNP1-3, or LE, can be determined using a variety of different methods.
  • the levels of biomarkers are determined using an immunoassay.
  • the levels of the biomarker are determined using a method other than an immunoassay, such as mass spectrometry.
  • biomarkers such as HD5, HNP1-3, or LE can be detected using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF).
  • 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.
  • 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.
  • 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.
  • type e.g., IgG, IgE, IgM, IgD, IgA, and IgY
  • class e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2
  • subclass of immunoglobulin molecule e.g., immunoglobulin molecule.
  • 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).
  • tumors in vivo such as peritoneal tumors
  • the lytic complement activity of serum may be optionally inactivated, for example by heating.
  • 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).
  • 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 Fab 1 fragments. Papain digestion can lead to the formation of Fab fragments.
  • 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.
  • 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.
  • 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.
  • 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.
  • a sample obtained from a subject can be contacted with the antibody that specifically binds the biomarker.
  • 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.
  • 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.
  • the mixture is washed and the antibody- marker complex formed can be detected.
  • This detection reagent may be, e.g., a second antibody which is labeled with a detectable label.
  • 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.
  • 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.
  • an indirect assay wherein, for example, a second, labeled antibody is used to detect bound biomarker- specific antibody
  • 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.
  • 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.
  • EIA enzyme immune assay
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmune assay
  • Western blot assay a Western blot assay
  • slot blot assay a slot blot assay
  • 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 acts as a sponge and holds an excess of sample fluid.
  • 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.
  • 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.
  • lateral flow tests can operate as either competitive or sandwich immunoassays.
  • 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.
  • the levels of the biomarkers can be displayed in a variety of ways.
  • 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.
  • 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.
  • a method of diagnosing urinary tract infection in a human subject 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.
  • 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.
  • another compound e.g., creatinine
  • 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.
  • the reference cohort is the general population.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • the method also includes providing treatment for the human subject identified as having a urinary tract infection.
  • a therapeutic agent is an antibiotic.
  • suitable antibiotics include trimethoprim-sulfamethoxazole, cephalosporins, nitrofurantoin, amoxicillin, AugmentinTM, doxycycline, and fluoroquinolones. Pyelonephritis is treated more aggressively than a simple bladder infection using either a longer course of oral antibiotics or intravenous antibiotics.
  • Urinary tract infections can also be treated with analgesics to relieve the burning pain and urgent need to urinate.
  • analgesics for example, the local analgesic phenazopyridine hydrochloride (Pyridium ® ) can be used together with an antibiotic for treatment of a urinary tract infection.
  • 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 for diagnosing urinary tract infection in a human subject 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.
  • the kit also includes a third antibody or binding fragment thereof specific for leukocyte esterase.
  • 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.
  • 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.
  • the kit includes a lateral flow strip which can be used to conduct an immunoassay using the antibodies included in the kit.
  • 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.
  • the reagents include antibodies, or binding fragments thereof, capable of specifically binding to the compound they are capable of detecting.
  • the kit can include an antibodies specific for HD5, HNP1-3, or LE.
  • 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.
  • a standard curve can be generated by preparing dilutions of a single antibody solution provided in the kit.
  • 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.
  • a particular structure e.g., an antigenic determinant or epitope
  • 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.
  • 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.
  • 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.
  • electronic storage media e.g., magnetic discs, tapes, cartridges, chips
  • optical media e.g., CD ROM
  • AMPs antimicrobial peptides
  • 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.
  • HNP Human neutrophil peptides
  • Table 1 Demographic Characteristics and Documented Clinical Signs and Symptoms of Study Population a
  • 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.
  • 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).
  • 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.
  • 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).
  • HNP1-3 did not improve specificity when combined with LE, perhaps because LE and HNP1-3 are both neutrophil markers and therefore indicate pyuria.
  • HD5 is expressed throughout the urothelium of the lower urinary tract and in the nephron and collecting tubules of the kidney.
  • epithelial-derived AMP 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.
  • 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.
  • AssayAssureTM validation The stability of AMPs was independently verified for up to 14 days in AssayAssureTM 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 AssayAssureTM urine collection tubes. On days 1, 2, 7, and 14, an aliquot of each sample was removed and stored at -80°C 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.
  • RNase 7 ribonuclease 7
  • 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., Madison 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 log 1Q 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).
  • Urine culture was the reference standard for evaluating test characteristics of the urine dipstick and AMPs.
  • 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).
  • 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.
  • the sensitivities and specificities of LE, HD5, HNPl-3, and combinations of the aforementioned tests were compared.
  • Antimicrobial peptides 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.
  • Study Protocol The 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.
  • Urine was collected using standard clinical procedures (clean-catch or catheter). Both urine and serum were centrifuged and stored at -80°C. 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.
  • a positive urinary culture was defined as >10 4 CFU/mL of one to two organisms from a female clean-catch specimen or >10 3 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.
  • 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).
  • 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).
  • 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.
  • 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.

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Abstract

L'invention concerne un procédé de diagnostic d'une infection des voies urinaires chez un sujet. Le procédé comprend l'obtention d'un échantillon d'urine auprès du sujet ; la détermination de la teneur en α-défensine humaine 5 (HD5) et/ou en peptides neutrophiles humains (HNP)1-3 dans l'échantillon d'urine et leur comparaison à une valeur témoin correspondante ; et le diagnostic du sujet comme présentant une infection des voies urinaires si la teneur en HD5 et/ou en HNP1-3 est supérieure à la valeur témoin. L'invention concerne également des kits de diagnostic d'une infection des voies urinaires chez un sujet au moyen d'anticorps spécifiques de HD5 et HNP1-3.
PCT/US2016/025824 2015-04-02 2016-04-04 Biomarqueurs d'infection des voies urinaires WO2016161413A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110506210A (zh) * 2017-03-01 2019-11-26 莫洛迪克有限公司 尿道感染诊断
US20220017941A1 (en) * 2020-05-27 2022-01-20 Board Of Regents, The University Of Texas System Electroanalytical determination of leukocyte esterase
CN114058695A (zh) * 2021-12-09 2022-02-18 广东省科学院微生物研究所(广东省微生物分析检测中心) 泌尿道菌群检测在女性泌尿道结石诊断中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6174664B1 (en) * 1997-02-05 2001-01-16 University Of Pittsburgh Screening method for inflammatory diseases using neutrophil defensins and lactoferrin
US20080051333A1 (en) * 2005-11-30 2008-02-28 Jishu Shi Human alpha-defensins inhibit interleukin-1beta release
US20100274156A1 (en) * 2005-12-27 2010-10-28 Gorres Geoffrey H Device for Monitoring a Patient for a Urinary Tract Infection
US20140322722A1 (en) * 2011-09-30 2014-10-30 The Research Institute At Nationwide Children's Hospital Compositions and methods for detection of defensins in a patient sample

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6174664B1 (en) * 1997-02-05 2001-01-16 University Of Pittsburgh Screening method for inflammatory diseases using neutrophil defensins and lactoferrin
US20080051333A1 (en) * 2005-11-30 2008-02-28 Jishu Shi Human alpha-defensins inhibit interleukin-1beta release
US20100274156A1 (en) * 2005-12-27 2010-10-28 Gorres Geoffrey H Device for Monitoring a Patient for a Urinary Tract Infection
US20140322722A1 (en) * 2011-09-30 2014-10-30 The Research Institute At Nationwide Children's Hospital Compositions and methods for detection of defensins in a patient sample

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WATSON ET AL.: "Evaluation of novel urinary tract infection biomarkers in children.", PEDIATRIC RESEARCH (2016, 26 May 2016 (2016-05-26), XP055317065 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110506210A (zh) * 2017-03-01 2019-11-26 莫洛迪克有限公司 尿道感染诊断
CN110506210B (zh) * 2017-03-01 2023-07-07 莫洛迪克有限公司 尿道感染诊断
US20220017941A1 (en) * 2020-05-27 2022-01-20 Board Of Regents, The University Of Texas System Electroanalytical determination of leukocyte esterase
CN114058695A (zh) * 2021-12-09 2022-02-18 广东省科学院微生物研究所(广东省微生物分析检测中心) 泌尿道菌群检测在女性泌尿道结石诊断中的应用
CN114058695B (zh) * 2021-12-09 2024-05-10 广东省科学院微生物研究所(广东省微生物分析检测中心) 泌尿道菌群检测在女性泌尿道结石诊断中的应用

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