URINARY TRACT INFECTION DIAGNOSTIC

Abstract

Method for detecting a urinary tract infection (UTI) in a subject comprising determining levels of one or more biomarkers selected from MMP8, HNE, Cystatin C, MMP9, HSA, IL-8, interleukin-6 (IL-6), interleukin-1 beta (IL-1b), fibrinogen, RBP4, active MMP9 and MMP2, NGAL, Desmosine, MPO and CRP in a urine sample obtained from the subject. The determined levels may then be compared with a threshold level, wherein increased levels of at least one of the biomarkers in the urine sample relative to the threshold level is indicative of the presence of a urinary tract infection. Methods for monitoring a UTI and monitoring treatment of a UTI are also provided as are companion systems or test kits.

Claims

1.-39. (canceled)

40. A method for detecting and/or monitoring a urinary tract infection in a subject comprising: i) determining levels of one or more biomarkers selected from MMP8, HNE, Cystatin C, MMP9, HSA, IL-8, interleukin-6 (IL-6), interleukin-1 beta (IL-1b), fibrinogen, RBP4, active MMP9 and MMP2, NGAL, Desmosine, MPO and CRP in a urine sample obtained from the subject; and ii) comparing each determined level with a threshold level; wherein increased levels of at least one of the biomarkers in the urine sample relative to the threshold level is indicative of the presence of a urinary tract infection; and/or wherein the continued presence of non-decreased or increased levels of at least one of the biomarkers relative to the threshold level or relative to the levels measured in a sample taken from an earlier time point is indicative that the urinary tract infection persists and/or that treatment has not been effective and/or decreased levels of at least one of the biomarkers relative to the threshold level or relative to the levels measured in a sample taken from an earlier time point is indicative of recovery from, or successful treatment of, a urinary tract infection.

41. A method for monitoring treatment of a urinary tract infection in a subject comprising: i) determining levels of one or more biomarkers selected from MMP8, HNE, Cystatin C, MMP9, HSA, IL-8, interleukin-6 (IL-6), interleukin-1 beta (IL-1b), fibrinogen, RBP4, active MMP9 and MMP2, NGAL, Desmosine, MPO and CRP in a urine sample obtained from the subject prior to treatment of the urinary tract infection in order to set a threshold level; ii) determining levels of one or more biomarkers selected from MMP8, HNE, Cystatin C, MMP9, HSA, IL-8, interleukin-6 (IL-6), interleukin-1 beta (IL-1b), fibrinogen, RBP4, active MMP9 and MMP2, NGAL, Desmosine, MPO and CRP in a further urine sample obtained from the subject following treatment of the urinary tract infection; wherein non-decreased or increased levels of at least one of the biomarkers relative to the threshold level is indicative that the treatment has not been effective and/or decreased levels of at least one of the biomarkers relative to the threshold level is indicative of successful treatment of the urinary tract infection.

42. The method of claim 40 wherein the levels of at least two or three of the biomarkers are determined.

43. The method of claim 40 comprising determining the levels of: a. at least one of MMP8 or HNE, optionally both MMP8 and HNE; b. at least one of MMP8, HNE, fibrinogen or CRP, and optionally: (i) MMP8, HNE and fibrinogen; or (ii) MMP8, HNE and CRP; or c. at least one of NGAL, MMP9, Desmosine or MPO, and optionally all of (i) NGAL, MMP9 and Desmosine or (ii) NGAL, MMP9, Desmosine and MPO, and preferably further comprising determining the levels of Cystatin C.

44. The method of claim 40 wherein the biomarkers are: (i) MMP8, HNE and Cystatin C; or (ii) MMP8, HNE and fibrinogen; or (iii) MMP8, HNE and CRP.

45. The method of claim 40 wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more samples are taken from the subject at different times and the levels of the one or more biomarkers is determined, preferably wherein the samples are taken every 6 to 24 hours, such as daily, or every 3, 4, 5, 6, 7 or 14 days.

46. A method according to claim 40, further comprising selecting the subject for treatment with an antibiotic where a urinary tract infection is detected or persists, or predicting responsiveness of the subject to treatment with an antibiotic where a urinary tract infection is detected or persists, wherein the antibiotic is optionally selected from aminoglycoside, a cephalosporin, a glycopeptide, a penicillin, a quinolone, aztreonam, clindamycin, imipenem-cilastin, linezolid, metronidazole, rifampin, an antifungal and an antiviral.

47. A method of treating a urinary tract infection comprising administering an antibiotic to the subject suffering from a urinary tract infection, wherein the subject has been selected for treatment by performing the method of claim 40, wherein the antibiotic is optionally selected from aminoglycoside, a cephalosporin, a glycopeptide, a penicillin, a quinolone, aztreonam, clindamycin, imipenem-cilastin, linezolid, metronidazole, rifampin, an antifungal and an antiviral.

48. A system or test kit for detecting a urinary tract infection in a subject, comprising: a. One or more testing devices for determining levels of one, two, three or more biomarkers selected from MMP8, HNE, Cystatin C, MMP9, HSA, IL-8, interleukin-6 (IL-6), interleukin-1 beta (IL-1b), fibrinogen, RBP4, active MMP9 and MMP2, NGAL, Desmosine, MPO and CRP in a urine sample obtained from the subject; b. A processor; and c. A storage medium comprising a computer application that, when executed by the processor, is configured to: i. Access and/or calculate the determined levels of each biomarker in the sample on the one or more testing devices; ii. Calculate a test score from the levels of the biomarkers in the sample that detects a urinary tract infection; and iii. Output from the processor the detected result for the subject.

49. A system or test kit for monitoring a urinary tract infection in a subject, comprising: a. One or more testing devices for determining levels of one, two, three or more biomarkers selected from MMP8, HNE, Cystatin C, MMP9, HSA, IL-8, interleukin-6 (IL-6), interleukin-1 beta (IL-1b), fibrinogen, RBP4, active MMP9 and MMP2, NGAL, Desmosine, MPO and CRP in a urine sample obtained from the subject at multiple time points; b. A processor; and c. A storage medium comprising a computer application that, when executed by the processor, is configured to: i. Access and/or calculate the determined levels of each biomarker in the sample on the one or more testing devices; ii. Calculate a test score from the levels of the biomarkers in the sample, optionally including a comparison of the levels with those taken at one or more earlier time points, that detects a urinary tract infection; and iii. Output from the processor the detected result for the subject.

50. The system or test kit of claim 48 wherein the biomarkers comprise: a. at least one of MMP8, HNE, fibrinogen or CRP, optionally both MMP8 and HNE; b. at least one of MMP8, HNE, fibrinogen or CRP, and optionally: (i) MMP8, HNE and fibrinogen; or (ii) MMP8, HNE and CRP; or c. at least one of NGAL, MMP9 or Desmosine, and optionally all of (i) NGAL, MMP9 and Desmosine, or (ii) NGAL, MMP9, Desmosine and MPO, and preferably wherein the biomarkers further comprise Cystatin C.

51. The system or test kit of claim 48 wherein the biomarkers are: (i) MMP8, HNE and Cystatin C; or (ii) MMP8, HNE and fibrinogen; or (iii) MMP8, HNE and CRP.

52. A testing device, testing kit or testing composition of matter comprising: a. A sample receiving zone to which a urine sample from a subject is added; b. A conjugate zone comprising at least one, two or three labelled binding reagents, each of which specifically binds to one of the biomarkers selected from MMP8, HNE, Cystatin C, MMP9, HSA, IL-8, interleukin-6 (IL-6), interleukin-1 beta (IL-1b), fibrinogen, RBP4, active MMP9 and MMP2, NGAL, Desmosine, MPO and CRP; c. A solid support defining a liquid flow path for the sample and comprising corresponding test lines for each of the at least one, two or three biomarkers, each test line comprising: i. an immobilised further binding reagent that also specifically binds to one of the at least one, two or three biomarkers thereby immobilising the biomarker at the test line to produce a signal via the labelled binding reagent also specifically bound to the biomarker; or ii. an immobilised version of one of the at least one, two or three biomarkers or an analogue thereof able to compete with the biomarker in the sample for specific binding to the labelled binding reagent.

53. The testing device, testing kit or testing composition of matter of claim 52 further comprising: d. At least one labelled control binding reagent that binds to a binding partner immobilised at a control line downstream of the test lines for the at least one, two or three biomarkers and thus confirms that the test has completed successfully; and optionally further comprising: e. An absorbent material downstream of the test (and control, where present) lines to absorb excess sample.

54. The testing device, testing kit or testing composition of matter of claim 52 wherein the solid support comprises a chromatographic medium and/or a capillary flow device.

55. The testing device, testing kit or testing composition of matter of claim 52 which is a test strip.

56. The testing device, testing kit or testing composition of matter of claim 52 further comprising a vessel for collecting a urine sample and/or a visual aid displaying different test line intensity patterns from which the user can interpret the observed test line results.

57. The testing device, testing kit or testing composition of matter of claim 52 further comprising a reader to determine levels of the markers at the respective test lines wherein the reader preferably comprises: a. A processor; and b. A storage medium comprising a computer application that, when executed by the processor, is configured to: i. Access and/or calculate the determined levels of each biomarker in the sample; ii. Calculate a test score from the levels of the biomarkers in the sample that detects a urinary tract infection; and iii. Output from the processor the detected result for the subject; or i. Access and/or calculate the determined levels of each biomarker in the sample on the one or more testing devices; ii. Calculate a test score from the levels of the biomarkers in the sample by comparing the levels with those taken at one or more earlier time points to thereby detect and/or monitor a urinary tract infection; and iii. Output from the processor the detected result for the subject.

58. The testing device, testing kit or testing composition of matter of claim 52 wherein the biomarkers comprise: a. at least one of MMP8 or HNE, optionally both MMP8 and HNE; b. at least one of MMP8, HNE, fibrinogen or CRP, and optionally: (i) MMP8, HNE and fibrinogen; (ii) MMP8, HNE and CRP; or c. at least one of NGAL, MMP9 or Desmosine, and optionally all of (i) NGAL, MMP9 and Desmosine, or (ii) NGAL, MMP9, Desmosine and MPO and preferably wherein the test line for each biomarker respectively comprises an immobilised further binding reagent that also specifically binds to each biomarker respectively thereby immobilising each biomarker at the respective test line to produce a signal via the labelled binding reagent also specifically bound to each biomarker.

59. The testing device, testing kit or testing composition of matter of claim 58 wherein the biomarkers further comprise Cystatin C optionally wherein the test line for Cystatin C comprises an immobilised version of Cystatin C or an analogue thereof able to compete with Cystatin C in the sample for specific binding to the labelled binding reagent.

60. The testing device, testing kit or testing composition of matter of claim 52 wherein the biomarkers are: (i) MMP8, HNE and Cystatin C; or (ii) MMP8, HNE and fibrinogen; or (iii) MMP8, HNE and CRP.

61. The system or test kit of claim 19 wherein the testing device comprises: a. A sample receiving zone to which a urine sample from a subject is added; b. A conjugate zone comprising at least one, two or three labelled binding reagents, each of which specifically binds to one of the biomarkers selected from MMP8, HNE, Cystatin C, MMP9, HSA, IL-8, interleukin-6 (IL-6), interleukin-1 beta (IL-1b), fibrinogen, RBP4, active MMP9 and MMP2, NGAL, Desmosine, MPO and CRP; c. A solid support defining a liquid flow path for the sample and comprising corresponding test lines for each of the at least one, two or three biomarkers, each test line comprising: i. an immobilised further binding reagent that also specifically binds to one of the at least one, two or three biomarkers thereby immobilising the biomarker at the test line to produce a signal via the labelled binding reagent also specifically bound to the biomarker; or ii. an immobilised version of one of the at least one, two or three biomarkers or an analogue thereof able to compete with the biomarker in the sample for specific binding to the labelled binding reagent.

Description

DESCRIPTION OF THE FIGURES

[0183] FIG. 1Concentration of MMP8 (FIGS. 1A and 1B) and HNE (FIGS. 1C and 1D) in samples taken at a first visit (i.e. at the time of presenting with acute symptomsSuspected UTI) and a second visit (i.e. typically after symptoms have been resolved) from 112 patients. Samples taken at Visit 1 and Visit 2 from each patient were assessed for statistical significance in relation to the levels of MMP8 in each sample using a Wilcoxon matched-pairs signed rank test. The same data is shown using two different statistical plots (1A and 1C are scatter plots) and (1B and 1D respectively are box plots).

[0184] FIG. 2Concentration of MMP8 (FIG. 2A), MMP9 (FIG. 2B), active MMP (FIGS. 2C and 2D), HNE (FIG. 2E), NGAL (FIG. 2F), RBP4 (FIG. 2G), HSA (FIG. 2H), desmosine (FIG. 2I), Fibrinogen (FIGS. 2J and 2K), IL-6 (FIG. 2L), IL-8 (FIG. 2M), IL-1b (FIG. 2N) in samples taken at a first visit (i.e. at the time of presenting with acute symptomsSuspected UTI) and a second visit (i.e. typically after symptoms have been resolved) from over 200 patients. Samples taken at Visit 1 and Visit 2 from each patient were assessed for statistical significance in relation to the levels of each marker in each sample using a Wilcoxon matched-pairs signed rank test.

[0185] FIG. 3Receiver operating curve for MMP8, MMP9, HNE, HSA and IL-8 as biomarkers for a UTI in samples from 112 patients that had both a visit 1 and a visit 2 sample taken. Blue=MMP8; Green=MMP9; Dark yellow=HNE; Purple=HSA; Light yellow=IL-8. Diagonal segments are produced by ties. Area under the curve (AUC) values are shown for all 5 biomarkers.

[0186] FIG. 4Receiver operating curve for MMP8, MMP9, HNE, HSA and IL-8 as biomarkers for a UTI in samples from 93 patients for whom the visit 1 sample gave a positive HNE result with respect to detection of a UTI. Blue=HNE; Green=MMP8; Dark yellow=MMP9; Purple=HSA; Light yellow=IL-8. Diagonal segments are produced by ties. Area under the curve (AUC) values are shown for all 5 biomarkers.

[0187] FIG. 5Correlation between the lateral flow assay and ELISA assay for HNE detection

[0188] FIG. 6Correlation between the lateral flow assay and ELISA assay for MMP8 detection

[0189] FIG. 7Correlation between the lateral flow assay and ELISA assay for Cystatin C detection

[0190] FIG. 8MMP8: Wilcoxon matched-pairs signed rank test for UTI positive visit 1 and visit 2 patients (FIG. 8A-C) along with ROC analysis and AUC values (FIG. 8D).

[0191] FIG. 9MMP8: Wilcoxon matched-pairs signed rank test for UTI negative visit 1 and visit 2 patients (FIG. 9A-C) along with ROC analysis and AUC values (FIG. 9D).

[0192] FIG. 10HNE: Wilcoxon matched-pairs signed rank test for UTI positive visit 1 and visit 2 patients (FIG. 10A-C) along with ROC analysis and AUC values (FIG. 10D).

[0193] FIG. 11HNE: Wilcoxon matched-pairs signed rank test for UTI negative visit 1 and visit 2 patients (FIG. 11A-C) along with ROC analysis and AUC values (FIG. 11D).

[0194] FIG. 12Cystatin C: Wilcoxon matched-pairs signed rank test for UTI positive visit 1 and visit 2 patients (FIG. 12A-C) along with ROC analysis and AUC values (FIG. 12D).

[0195] FIG. 13Cystatin C: Wilcoxon matched-pairs signed rank test for UTI negative visit 1 and visit 2 patients (FIG. 13A-C) along with ROC analysis and AUC values (FIG. 13D).

[0196] FIG. 14MMP8+HNE: Wilcoxon matched-pairs signed rank test for UTI positive visit 1 and visit 2 patients (FIG. 14A-C) along with ROC analysis and AUC values (FIG. 14D).

[0197] FIG. 15MMP8+HNE: Wilcoxon matched-pairs signed rank test for UTI negative visit 1 and visit 2 (FIG. 15A-C) patients along with ROC analysis and AUC values (FIG. 15D).

[0198] FIG. 16MMP8, HNE+Cystatin C: Wilcoxon matched-pairs signed rank test for UTI positive visit 1 and visit 2 (FIG. 16A-C) patients along with ROC analysis and AUC values (FIG. 16D).

[0199] FIG. 17MMP8, HNE+Cystatin C: Wilcoxon matched-pairs signed rank test for UTI negative visit 1 and visit 2 patients (FIG. 17A-C) along with ROC analysis and AUC values (FIG. 17D).

[0200] FIG. 18MMP8: Wilcoxon matched-pairs signed rank test for UTI positive and negative samples (confirmed by microbiological testing) (FIGS. 18A and 18B) along with ROC analysis and AUC values (FIG. 18C).

[0201] FIG. 19HNE: Wilcoxon matched-pairs signed rank test for UTI positive and negative samples (confirmed by microbiological testing) (FIGS. 19A and B) along with ROC analysis and AUC values (FIG. 19C).

[0202] FIG. 20Cystatin C: Wilcoxon matched-pairs signed rank test for UTI positive and negative samples (confirmed by microbiological testing) (FIGS. 20A and B) along with ROC analysis and AUC values (FIG. 20C).

[0203] FIG. 21MMP8+HNE: Wilcoxon matched-pairs signed rank test for UTI positive and negative samples (confirmed by microbiological testing) (FIGS. 21A and B) along with ROC analysis and AUC values (FIG. 21C).

[0204] FIG. 22MMP8, HNE+Cystatin C: Wilcoxon matched-pairs signed rank test for UTI positive and negative samples (confirmed by microbiological testing) (FIGS. 22A and B) along with ROC analysis and AUC values (FIG. 22C).

[0205] FIG. 23Exemplary test strip of the invention and use thereof. Blue arrows indicate direction of flow along the test strip. (A) Cystatin C immuno-capture test line. (B) Anti-HNE antibody immuno-capture test line. (C) Anti-MMP8 antibody immuno-capture test line. (D) Gold particles bearing anti-MMP8 antibody, anti-HEN antibody or anti-Cystatin C antibody. (E) MMP8, HNE or Cystatin C present in a urine sample obtained from a subject. (F) Antibodies bound to gold particles capture Cystatin C and prevent it binding to the Cystatin C immuno-capture test line. (G) Antibodies bound to gold particles capture HNE and bind to the anti-HNE antibody immuno-capture test line. (H) Antibodies bound to gold particles capture MMP8 and bind to the anti-MMP8 antibody immuno-capture test line. (I) Anti-HNE and anti-MMP8 antibodies bound to gold particles but which have not bound to HNE and MMP8 respectively escape the capture test line. (J) Anti-Cystatin C antibodies bound to gold particles and also bound to Cystatin C escape the capture test line. BSAbovine serum albumin.

[0206] FIG. 24(A) An exemplary test strip is placed in an adaptor for a reader. (B) A reader is placed above the test strip appropriately housed in the adaptor for the reader.

[0207] FIG. 25Standard curves for (A) MMP8, (B) HNE and (C) Cystatin C present in a sample determined according to the invention.

[0208] FIG. 26Quantification of MMP8 in fresh and frozen urine samples according to the methods and apparatus of the invention.

[0209] FIG. 27Quantification of HNE in fresh and frozen urine samples according to the methods and apparatus of the invention.

[0210] FIG. 28Quantification of Cystatin C in fresh and frozen urine samples according to the methods and apparatus of the invention.

[0211] FIG. 29UTRiPLEX assay variability with urine samples taken over 5 days from a first healthy volunteer

[0212] FIG. 30UTRiPLEX assay variability with urine samples taken over 5 days from a second healthy volunteer

[0213] FIG. 31UTRiPLEX assay variability with urine samples taken over 5 days from a third healthy volunteer

[0214] FIG. 32UTRiPLEX assay variability with urine samples taken over 5 days from a fourth healthy volunteer

[0215] FIG. 33UTRiPLEX assay variability with urine samples taken over 5 days from a fifth healthy volunteer

[0216] FIG. 34UTRiPLEX assay variability with urine samples taken over 5 days from a sixth healthy volunteer

[0217] FIG. 35UTRiPLEX assay variability with urine samples taken over 5 days from a seventh healthy volunteer

[0218] FIG. 36UTRiPLEX assay variability with urine samples taken over 5 days from a eighth healthy volunteer

[0219] FIG. 37UTRiPLEX assay variability with a urine sample from a first healthy volunteer stored either at room temperature or 4 C.

[0220] FIG. 38UTRiPLEX assay variability with a urine sample from a first healthy volunteer stored either at room temperature or 4 C. as shown in FIG. 37 shown per each biomarker individually

[0221] FIG. 39UTRiPLEX assay variability with a urine sample from a second healthy volunteer stored either at room temperature or 4 C.

[0222] FIG. 40UTRiPLEX assay variability with a urine sample from a second healthy volunteer stored either at room temperature or 4 C. as shown in FIG. 39 shown per each biomarker individually

[0223] FIG. 41UTRiPLEX assay variability with a urine sample from a third healthy volunteer stored either at room temperature or 4 C.

[0224] FIG. 42UTRiPLEX assay variability with a urine sample from a third healthy volunteer stored either at room temperature or 4 C. as shown in FIG. 41 shown per each biomarker individually

[0225] FIG. 43Daily monitoring of a subject suffering from a UTI (based on positive culture results) over 43 days. The levels of MMP8, HNE and Cystatin C as measured via UTRiPLEX are shown. Also shown are leukocyte levels and nitrite levels in each sample measured using known Multistix tests. Leukocyte levels were graded: negative (neg), Trace, Small and Large. Nitrite levels were graded: + (present) and (absent).

[0226] FIG. 44Results of HNE stability studies at 4 C. (FIG. 44A), room temperature (FIG. 44B) and 37 C. (FIG. 44C); represented as average calculated reader values.

[0227] FIG. 45Results of MMP8 stability studies at 4 C. (FIG. 45A), room temperature (FIG. 45B) and 37 C. (FIG. 45C); represented as average calculated reader values.

[0228] FIG. 46Results of Cystatin C stability studies at 4 C. (FIG. 46A), room temperature (FIG. 46B) and 37 C. (FIG. 46C); represented as average calculated reader values.

[0229] FIG. 47ROC curves for each of CRP, HNE, MMP-8 and fibrinogen when used to detect a urinary tract infection pre-boric acid addition (FIG. 47A) and post-boric acid addition (FIG. 47B).

[0230] FIG. 48Decision tree analysis for detecting a urinary tract infection using the combination of CRP+HNE+MMP-8 pre-boric acid addition (FIG. 48A) and post-boric acid addition (FIG. 48B).

EXAMPLES

[0231] The invention will be further understood with reference to the following experimental examples.

Example 1Selection of Biomarkers

[0232] Samples were collected as part of an 141 funded project, whereby a total of 189 urine samples from adult women presenting with suspected uncomplicated UTI were fully analysed with respect to 60 biomarkers.

[0233] There were two objectives: [0234] i. To define a panel of biomarkers to distinguish between negative and positive culture [0235] ii. To select a biomarker that provided the best discrimination between the suspected UTI samples and those from patients who have recovered (healthy).

[0236] In order to distinguish between patients presenting with different types of infection, binary logistic regression was used to predict which biomarkers are significant for outcome on their own, without interaction with other parameters. The markers that were significant in this analysis were then analysed further using the Stuttgart Neural Network Simulation (RSNNS) in R where the biological markers significant on their own were weighed against each other and interactions taken into consideration. The results were visualised by drawing Receiver operating curves (ROC), and areas under the curve (AUC) were calculated as a measure of how well the selected markers predict outcome in a given case.

[0237] First-generation fingerprints are available for the discrimination of UTI samples according to their microbiological profile (2 distinct outcomes: negative, positive). Whilst other combinations were possible, the combinations selected for further study were based on available reagents.

Marker 1=HNE

Marker 2=Cystatin C

Marker 3=MMP8

[0238] AUCs were measured for different combinations of markers 1-3 and are shown in the table below:

TABLE-US-00001 Combination of Markers AUC Markers 1 + 2 0.857826 Markers 1 + 3 0.915173 Markers 1 + 2 + 3 0.922939

[0239] These statistical analyses suggest that a minimum number of 2 biomarkers is sufficient to constitute diagnostically relevant immune fingerprints able to detect a UTI.

[0240] In an independent analysis, we also looked at biomarkers that would be able to discriminate between urine samples taken at visit 1 (i.e. at the time of presenting with acute symptomssuspected UTI) and two weeks later at visit 2 (i.e. typically after symptoms have been resolved).

[0241] A cross sectional analysis of the population, not taking into account individual personalised threshold values was conducted. Non parametric testsMann-Whitney and unpaired t-tests were used and receiver operator characteristic to determine the discrimination accuracy.

Visit 1-202 samples
Visit 2-222 samples

TABLE-US-00002 Assay Method of testing Unit Unpaired t test Mann-Whitney AUC MMP9 ELISA ng/mL 8.09987E20 <0.0001 0.7559 MMP8 ELISA ng/mL 5.60E11 <0.0001 0.75 IL1b ELISA pg/mL 4.40954E10 <0.0001 0.7344 Fibrinogen ELISA ng/mL 1.92498E16 <0.0001 0.7304 H.S.A ELISA ng/mL 3.62118E21 <0.0001 0.7302 NGAL ELISA ng/mL 1.29098E18 <0.0001 0.7264 IL8 ELISA pg/mL 2.6059E11 <0.0001 0.7222 HNE ELISA ng/mL 1.49814E12 <0.0001 0.7173 Active MMP Zymography Arbitrary Unit 1.73818E10 <0.0001 0.677 IL6 ELISA pg/mL 2.87862E05 <0.0001 0.6238 RBP4 ELISA ng/mL 0.012862881 0.0001 0.6076 Desmosine ELISA ng/mL 0.020936351 0.1453 0.5427

[0242] Urinary biomarkers were assessed in 112 patients that had both a visit 1 and a visit 2 sample and were analysed using a paired t test and receiver operator characteristic (see FIGS. 1-3). Amongst the markers that were highly significant in this context featured marker 1 and 3, with p<0.0001 in paired t-tests and with AUC values of 0.75-0.80 on their own suggesting that, in fact, a minimum number of at least one of the selected biomarkers is sufficient to detect a UTI.

[0243] The AUC for each biomarker shown in FIG. 3 was as follows: MMP8=0.756; MMP9=0.795; HNE=0.756; HSA=0.778; IL-8=0.757. In a further analysis, a ROC was plotted basing the results on HNE i.e. omission of samples with a negative HNE visit 1 (n=93). This is shown in FIG. 4. The AUC for each biomarker shown in FIG. 4 was as follows: MMP8=0.809; MMP9=0.871; HNE=0.842; HSA=0.826; IL-8=0.837.

[0244] Amongst the markers that were highly significant between acute symptoms and recovery, Human Neutrophil Elastase (HNE) and Matrix Metalloproteinase-8 (MMP8) was selected with p<0.0001 from a paired t test and an ROCAUC of 0.76.

[0245] Based on this agreement between the two different analyses, one looking at discrimination between negative and positive samples at visit 1 and one looking at discrimination between pre-treatment and post-treatment, it was decided to take biomarkers forward that were identified by both methods.

Example 2Development of Lateral Flow Assays

[0246] The three selected biomarkers HNE, MMP8 and Cystatin C were taken forward for lateral flow development. The three lateral flow assays were developed and optimised enclosed into a single well plastic housing supplied by Bibby sterylin using a standard lateral flow form.

HNE Lateral Flow

[0247] Preparation of materials: The capture line, anti-HNE BSA-PEG Fab (Alere San Diego, Cat No 01241) and control line BSA-Biotin were immobilised onto the nitrocellulose membrane (Sartorius, CN140) at 1 mg/mL in PBS+1% Sucrose using the Imagene Isoflow dispenser. Membranes were subsequently dried in a tunnel dryer (Hedinair) at 60 C. and stored with desiccant prior to use.

[0248] The gold conjugates were prepared according to known protocols. Anti-HNE BSA-PEG Fab (Alere San Diego, Cat No 01871) was conjugated to 40 nm gold colloid in a suspension buffer of 20 mM MES pH5.3 to a final concentration of 15 g/mL. Following a 10-minute incubation, any unbound colloid was blocked with a final concentration of 2 mg/mL bovine serum albumin (200 mg/mL stock in PBS). Both test conjugate and control gold: Goat anti Biotin gold 40 nm (BBI) were mixed together in the presence of a gold drying buffer, Tris with BSA, 5% (w/v) sucrose and 2% (v/v) TritonX100. The gold conjugate was sprayed onto Ahlstrom 8951 glass fibre pads using the Imagene Isoflow dispenser deposited at 0.8 l/mm. The sprayed conjugate pads were dried in a tunnel dryer (Hedinair) at 60 C. and stored with desiccant prior to use.

[0249] Both prepared membranes and conjugate pads were assembled into one-well plastic cassettes.

[0250] Running of the assay: The sample was diluted 1 in 100 in sample diluent (Tris with BSA, 1% (v/v) Tween20). After dilution, 80 L of the sample was added to the sample well of the device and the result was read after 10 minutes.

[0251] The HNE lateral flow assay performance was compared to a HNE ELISA (Mologic BHNEV1) by running a standard curve and 40 UTI urine samples run at a 1:100 dilution in TBST (tris-buffered saline with Tween 20). Data was analysed according to a Four Parameter Logistic Fit. The correlation between the lateral flow assay and ELISA assay in terms of HNE detection is shown in FIG. 5. A strong positive correlation (R.sup.2=0.9684) was observed.

MMP8 Lateral Flow

[0252] Preparation of materials: The capture line, anti-MMP8 BSA-PEG Fab (Alere San Diego, Cat No 99611) and control line BSA-Biotin were immobilised onto the nitrocellulose membrane (Sartorius, CN140) at 1 mg/mL in PBS+1% Sucrose using the Imagene Isoflow dispenser. Membranes were subsequently dried in a tunnel dryer (Hedinair) at 60 C. and stored with desiccant prior to use.

[0253] The gold conjugates were prepared according to known protocols. Anti-MMP8 BSA-PEG Fab (Alere San Diego, Cat No 99651) was conjugated to 40 nm gold colloid in a suspension buffer of 20 mM BES pH6.6 to a final concentration of 15 g/mL. Following a 10-minute incubation, any unbound colloid was blocked with a final concentration of 2 mg/mL bovine serum albumin (200 mg/mL stock in PBS). Both test conjugate and control gold: Goat anti Biotin gold 40 nm (BBI) were mixed together in the presence of a gold drying buffer, Tris with BSA, 5% (w/v) sucrose and 2% (v/v) TritonX100. The gold conjugate was sprayed onto Ahlstrom 8951 glass fibre pads using the Imagene Isoflow dispenser deposited at 0.8p1/mm. The sprayed conjugate pads were dried in a tunnel dryer (Hedinair) at 60 C. and stored with desiccant prior to use.

[0254] Both prepared membranes and conjugate pads were assembled into one-well plastic cassettes.

[0255] Running of the assay: The sample was diluted 1 in 10 in sample diluent (Tris with BSA, 1% (v/v) Tween20). After dilution, 80 L of the sample was added to the sample well of the device and the result was read after 10 minutes.

[0256] The MMP8 lateral flow assay performance was compared to the commercially available R&D duoset MMP8 ELISA by running a standard curve and 40 UTI urine samples run at a 1:10 dilution in TBST. Data was analysed according to a Four Parameter Logistic Fit. The correlation between the lateral flow assay and ELISA assay in terms of MMP8 detection is shown in FIG. 6. A strong positive correlation (R.sup.2=0.9819) was observed.

Cystatin C Lateral Flow

[0257] Preparation of materials: The capture line, anti-Cystatin C BSA-PEG Fab (Alere San Diego, Cat No 180) and control line BSA-Biotin were immobilised onto the nitrocellulose membrane (Sartorius, CN140) at 1 mg/mL in PBS+1% Sucrose using the Imagene Isoflow dispenser. Membranes were subsequently dried in a tunnel dryer (Hedinair) at 60 C. and stored with desiccant prior to use.

[0258] The gold conjugates were prepared according to known protocols. Anti-Cystatin C (BBI, Cat No BP234-3) was conjugated to 40 nm gold colloid in a suspension buffer of 20 mM TAPS pH8.5 to a final concentration of 15 g/mL. Following a 10-minute incubation, any unbound colloid was blocked with a final concentration of 2 mg/mL bovine serum albumin (200 mg/mL stock in PBS). Both test conjugate and control gold: Goat anti Biotin gold 40 nm (BBI) were mixed together in the presence of a gold drying buffer, Tris with BSA, 5% (w/v) sucrose and 2% (v/v) TritonX100. The gold conjugate was sprayed onto Ahlstrom 8951 glass fibre pads using the Imagene Isoflow dispenser deposited at 0.8p1/mm. The sprayed conjugate pads were dried in a tunnel dryer (Hedinair) at 60 C. and stored with desiccant prior to use.

[0259] Both prepared membranes and conjugate pads were assembled into one-well plastic cassettes.

Running of the assay: The sample was diluted 1 in 20 in sample diluent (Tris with BSA, 1% (v/v) Tween20). After dilution, 80 L of the sample was added to the sample well of the device and the result was read after 10 minutes.

[0260] The Cystatin C lateral flow assay performance was compared to the commercially available R&D duoset Cystatin C ELISA by running a standard curve and 40 UTI urine samples run at a 1:20 dilution in PBST 1% BSA (phosphate buffered saline with Tween 20+1% bovine serum albumin). Data was analysed according to a Four Parameter Logistic Fit. The correlation between the lateral flow assay and ELISA assay in terms of Cystatin C detection is shown in FIG. 7. A strong positive correlation (R.sup.2=0.8234) was observed.

Conclusion

[0261] The three biomarkers HNE, MMP8 and Cystatin C were selected based on testing of suspected UTI samples and subsequent recovery samples with reference ELISA's (Mologic in-house assays and R&D duoset ELISA). Lateral flow assays have been developed that produce equivalent results to the ELISA's (40 UTI samples tested (different samples used for each assay)) as determined by acceptable R.sup.2 values.

Example 3Statistical Analysis and Algorithm Development

[0262] In the Cardiff POETIC study (Bates 2014), 424 samples were collected in total: 204 were from a visit 1 and 220 were from a visit 2 samples (not all of these were matched samples). There were 93 matched visit 1 and visit 2 samples in total.

[0263] Sample information was available for a total of 194 samples; 175 from a visit 1 and 19 from a visit 2 samples. Of the 93 matched samples, 81 of the visit 1 samples had accompanying clinical information.

[0264] From the 81 visit 1 samples, 32 of the samples were UTI positive and 49 were UTI negative. The status was based on the microbiology results: [0265] No growth=No growth found [0266] No significant growth=growth of pure isolate <10.sup.5 [0267] Mixed 2 orgs=Growth of 2 orgs at 10.sup.5 cfu/mL or more [0268] Mixed>2 orgs=Growth of 3 or more isolates at 10.sup.5 cfu/mL or more [0269] POS=either a pure growth at 10.sup.5 cfu/mL or more OR a growth of a predominant isolate at 10.sup.5 or more with growth of other isolates at 3log 10 less

[0270] The urine samples were analysed by the inventors and logistic regression analysis was used to generate a statistical model for correlation of the levels of the three biomarkers HNE, MMP8 and Cystatin C with UTI status.

[0271] The model was derived from the positive UTI visit 1 and subsequent visit 2 samples. It was assumed that at the time of collection of the visit 2 sample the patient had fully recovered. Two logistic regression models were generated.

TABLE-US-00003 MMP8 only classification table Predicted MMP8 Only Visit 1 Visit 2 % correct Observed Visit 1 27 5 84.4 Visit 2 3 29 90.6 Overall % 87.5

TABLE-US-00004 HNE only classification table Predicted HNE Only Visit 1 Visit 2 % correct Observed Visit 1 24 8 75.0 Visit 2 2 30 93.8 Overall % 84.4

TABLE-US-00005 Cystatin C only classification table Predicted Cystatin C Only Visit 1 Visit 2 % correct Observed Visit 1 23 9 71.9 Visit 2 26 6 18.8 Overall % 45.3

TABLE-US-00006 MMP8 and HNE only classification table - MODEL 1 Predicted MMP8 and HNE only Visit 1 Visit 2 % correct Observed Visit 1 28 4 87.5 Visit 2 1 31 96.9 Overall % 92.2

TABLE-US-00007 MMP8, HNE and Cystatin C classification table - MODEL 2 Predicted MMP8, HNE and Cystatin C Visit 1 Visit 2 % correct Observed Visit 1 28 4 87.5 Visit 2 1 31 96.9 Overall % 92.2

[0272] These 2 models were used for the remaining analysis. It is noted that whilst Cystatin C did not increase the predictive accuracy when combined with MMP8 and HNE, it is expected that Cystatin C will increase the predictive accuracy when a larger number of biomarkers are analysed. The inclusion of Cystatin C is also predicted to diagnose renal infection.

UTI Visit 1 and Visit 2

[0273] The individual biomarker levels and the 2 statistical models were evaluated for the ability to detect a UTI. Wilcoxon matched-pairs signed rank test was used to determine the discrimination between positive and negative detection of a UTI as they were paired samples. ROC analysis and AUC values were determined for each biomarker and the 2 statistic models. The results are shown in FIGS. 8-17. As shown in FIG. 16, the combined markers produced an excellent AUC of 0.96 and thus are able to discriminate between culture positive confirmed UTI patients at visit 1 and visit 2 post treatment. This result is further strengthened by the data in FIG. 17 whereby a poor AUC of 0.65 was obtained with the culture negative UTI patients indicating that the biomarkers were not significantly different pre and post treatment.

UTI Positive and Negative

[0274] Although the algorithm was initially created using the visit 1 and visit 2 data, it was also applied to analyse the UTI confirmed positive and negative data. The individual biomarker levels and the 2 statistical models were evaluated. A Mann-Whitney test was used to determine the discrimination between the 2 groups. ROC analysis and AUC values were determined for each biomarker and the 2 statistical models. The results are shown in FIGS. 18-22.

Conclusion

[0275] The samples with suspected UTI were poorly characterised according to the reference method used. Eighty-one patients with suspected samples were given antibiotics to treat UTI but, in fact, only 32 of these were positive based on microbiology results and 49 were negative. Thus 60% of these patients had been misdiagnosed. Using the combination of MMP8, HNE and Cystatin C, 28 of the 32 patients would have been correctly diagnosed with a sensitivity of 88% and 34 of the 49 negative UTI would have been given a negative result (specificity of 69%), therefore 19% of the patients would have been given unnecessary treatment compared to 60%. Analysis into the causative organisms may provide further insight. As there was no clinical information for the visit 2 samples, it is possible that some of these patients may have had a lingering infection.

Example 4Exemplary Test Strip of the Invention

[0276] The exemplary test strip detects MMP8, HNE and Cystatin C if present in a urine sample taken from a subject. The test strip first comprises a sample receiving zone to which the urine sample is added. This is connected via a liquid flow path to a conjugate zone comprising an anti-HNE antibody (bovine serum albumin, BSA linked) and an anti-MMP8 antibody (BSA linked) which are each complexed to gold particles and an anti-Cystatin C polyclonal antibody (complexed to gold particles). Alternatively, the sample receiving zone and conjugate zone may be combined as a single zone. Connected to the conjugate zone is a solid support defining a liquid flow path for the sample and comprising corresponding test lines for each of MMP8, HNE and Cystatin C. The HNE and MMP8 test lines comprise an immobilised anti-HNE antibody (BSA linked) and an immobilised anti-MMP8 antibody (BSA linked) respectively. The Cystatin C test line comprises immobilised Cystatin C or an analogue thereof to which the anti-Cystatin C polyclonal antibody is still able to bind. All three test lines are perpendicular to the direction of flow of the sample along the test strip flowing away from the sample receiving zone.

[0277] The HNE and MMP8 tests are sandwich assays. When HNE and MMP8 are present in a urine sample added to the sample receiving zone, they travel in the liquid along the liquid flow path to the conjugate zone and are bound by the anti-HNE antibody (bovine serum albumin, BSA linked) and an anti-MMP8 antibody (BSA linked) which are each complexed to gold particles. The HNE-antibody-gold complexes and MMP8-antibody-gold complexes then travel along the liquid flow path and are bound at the HNE and MMP8 test lines by the immobilised anti-HNE antibody (BSA linked) and immobilised anti-MMP8 antibody (BSA linked) respectively. As a consequence, a coloured test line appears on the test strip by virtue of the immobilised gold particles to indicate the presence of HNE and MMP8 respectively in the sample.

[0278] The Cystatin C test is a competition assay. When Cystatin C is present in a urine sample added to the sample receiving zone, it travels in the liquid along the liquid flow path to the conjugate zone and is bound by the anti-Cystatin C polyclonal antibody (complexed to gold particles). The Cystatin C-antibody-gold complexes then travel along the liquid flow path. As the anti-Cystatin C polyclonal antibody is already complexed with Cystatin C present in the sample it cannot bind to the Cystatin C or an analogue thereof to which the anti-Cystatin C polyclonal antibody is still able to bind immobilised at the test line. Thus, when Cystatin C is present in the sample, a weak or no test line appears on the test strip proportional to the amount of Cystatin C present in the sample.

[0279] This is shown in FIG. 23. This exemplary test strip may be referred to as the UTRiPLEX test strip assay, UTRiPLEX test strip, UTRiPLEX assay or simply UTRiPLEX. The sequential order of HNE, MMP8 and Cystatin C along the test strip is not important and can be in any order.

[0280] A procedural control line (C) may be included downstream of the test lines (relative to the flow of sample) to indicate that the assay has been performed correctly, that is to say the control line provides a positive signal once contacted with the sample indicating that the sample has flowed past, and therefore been in contact with, the test lines. Thus, when a negative result on the test lines indicating an absence of MMP, HNE and/or Cystatin C in the sample is observed, the user can have confidence that this is a true result and not simply because for some reason the sample did not flow along the flow path and come into contact with the test lines. An exemplary control line comprises immobilised BSA-Biotin. The conjugate zone further comprises an anti-Biotin antibody complexed with gold particles.

Reagents and Pilot Manufacture

[0281] The test lines Cystatin C antigen (BBI P713-1), anti-HNE BSA-PEG Fab (Alere San Diego, Cat No 01241), anti-MMP8 BSA-PEG Fab (Alere San Diego, Cat No 99611) and BSA-Biotin (Mologic) were immobilised onto a nitrocellulose membrane at 0.75 mg/mL, 0.5 mg/mL, 0.5 mg/mL and 0.5 mg/mL respectively in PBS+1% w/v Sucrose using the Imagene Isoflow dispenser. Cystatin C, HNE, MMP8 and BSA-Biotin lines were plotted at 12 mm, 16 mm, 20 mm and 24 mm onto a 40 mm CN95 nitrocellulose membrane and subsequently dried in a tunnel dryer (Hedinair) at 60 C. and stored with desiccant prior to use.

[0282] The gold conjugates' preparation: [0283] Anti-Cystatin C goat polyclonal (BBI, BP234-3) was conjugated to 40 nm gold colloid in a suspension buffer of 20 mM aqueous borate at pH8.5, to a final concentration of 15 g/mL. Following a 10 min incubation, any unbound colloid was blocked with a final concentration of 2 mg/mL bovine serum albumin (200 mg/mL stock in PBS). [0284] Anti-HNE BSA-PEG Fab (Alere San Diego, Cat No 01871) was conjugated to 40 nm gold colloid in a suspension buffer of 20 mM aqueous MES at pH5.3 to a final concentration of 15 g/mL. Following a 10 min incubation, any unbound colloid was blocked with a final concentration of 2 mg/mL bovine serum albumin (200 mg/mL stock in PBS). [0285] Anti-MMP8 BSA-PEG Fab (Alere San Diego, Cat No 99651) was conjugated to 40 nm gold colloid in a suspension buffer of 20 mM aqueous BES at pH6.6 to a final concentration of 15 g/mL. Following a 10 min incubation, any unbound colloid was blocked with a final concentration of 2 mg/mL bovine serum albumin (200 mg/mL stock in PBS). [0286] Control gold: Goat anti Biotin gold 40 nm (BBI)

[0287] The gold conjugates were mixed together with a final optical density of 5 (Cystatin C), 2 (HNE), 3 (MMP8) and 1 (Control) in gold drying buffer 1M Tris pH 9, 3% (w/v) BSA, 5% (w/v) sucrose and 2% (v/v) TritonX100. The gold conjugate was sprayed onto 27 mm Ahlstrom 8951 glass fibre pads using the Imagene Isoflow dispenser deposited at 0.8p1/mm. The sprayed conjugate pads were dried in a tunnel dryer (Hedinair) at 60 C. and stored with desiccant prior to use.

[0288] Both prepared membranes and conjugate pads were assembled into strips and laminated onto a backing card. The strip includes a terminal region downstream of the control line which the user can grip by hand or otherwise. Thus, the test strip is very convenient for the user to use.

Sample Collection and Storage

[0289] Collect a sample of urine mid-stream in a sterile screw-top container (avoid the first urine of the day).

[0290] The sample can be tested directly from the collection pot or transferred to a clean dry, container.

[0291] If testing cannot be done within an hour, refrigerate the specimen and let it return to room temperature before testing.

[0292] For storage of samples, aliquot and store at 20 C./4 F. or below. Avoid repeated freeze-thaw cycles.

Assay Procedure

[0293] Equilibrate urine to room temperature prior to use.

[0294] Testing should be done at room temperature (15-30 C./59-86 F.)

1) Collect a midstream urine sample in a clean, dry container as described above
2) Remove one UTRiPLEX test strip from the container and immediately replace the cap. Do not touch areas of the test strip apart from the terminal grip region indicated (this may be a red coloured terminal region of the strip that says hold)
3) Dip the sample receiving zone portion of the strip into the sample for approximately 10 seconds. The boundary of the sample receiving zone may be marked for the user's convenience; for instance, with arrows. The user should dip the sample receiving zone portion of the strip into the sample up to the arrows.
4) Place strip in adaptor for readerensure correct orientation (see FIG. 24A)
5) Place reader on top of the adaptor and read after 10 minutes (see FIG. 24B)

Reader Instructions

[0295] The instructions are for single use mode (as proposed for use by healthcare professional) using the Cube reader's automatic timer. [0296] To turn the test reader on, press the button briefly. A beep sounds and the display shows ON. If notsee error guide for instructions on how to change the batteries. [0297] Press and hold the button for longer than 1 second. The display will show RFID. [0298] Place the supplied RFID card onto the top of the reader. A beep sounds and the display shows TEST. (Remove the RFID card after the beep has been heard). [0299] Start the timer by pressing the button. The display will show a countdown from 10 minutes. [0300] After the countdown has finished a beep sounds and the result is displayed and saved automatically. For the single use mode, an alarm will indicate when the 10 minutes is up. The strip can be disposed of once complete.

Standard Curves

[0301] Standard curves can be used to quantify unknown levels of each biomarker in urine samples.

[0302] Cystatin C, HNE and MMP8 were diluted in the sample diluent (with PBS at pH7.2), to give concentrations between:

MMP8: 1.95-125 ng/mL
HNE: 62.5-2000 ng/mL
Cystatin C: 78.12-2500 ng/mL

[0303] The results were as follows:

TABLE-US-00008 MMP8 Reader HNE Reader Cystatin C Reader (ng/mL) value (ng/mL) value (ng/mL) value 125 255.25 2000 352.55 2500 23.45 62.5 230.35 1000 272.625 1250 54.775 31.25 200.1 500 169.65 625 106.15 15.625 151.975 250 83.075 312.5 154.775 7.8125 100.5 125 41.4 156.25 188.525 3.90625 56.275 62.5 18.1 78.125 204.1 1.953125 30.725 0 2.2 0 231.125 0 0.925

[0304] The results are shown graphically in FIG. 25.

Example 5Reference Assay Validation

MMP8

[0305] The reference ELISA for MMP8 (Human Total MMP8 Duoset ELISA, R&D Systems) was validated according to FDA standards and specifications were met for the following criteria: [0306] Sensitivity (lowest limit of detection; LLOD) [0307] Standard curve [0308] Intra-plate reproducibility [0309] Inter-plate reproducibility [0310] Spike recovery [0311] Linearity [0312] Cross reactivity
HNE
Cystatin C

Example 6UTRiPLEX Validation

Sensitivity

[0313] For HNE and MMP8 the lowest level of detection (LLOD) was determined by adding two standard deviations to the mean reader value of twenty zero standard replicates and calculating the corresponding concentration.

[0314] For Cystatin C the lowest level of detection (LLOD) was determined by subtracting two standard deviations from the mean reader value of twenty zero standard replicates and calculating the corresponding concentration.

LLOD is typically 64.8 ng/mL for Cystatin C, 29.2 ng/mL for HNE and 0.16 ng/mL for MMP8.

Precision

Intra-Assay Precision

[0315] Six or seven samples of known concentrations were tested twelve times on one batch of strips by one operator.

TABLE-US-00009 MMP8 HNE MMP8 HNE concen- concen- tration % tration % (ng/mL) % CV accuracy (ng/mL) % CV accuracy 125 5.8 106.4 2000 7.9 92.2 62.5 3.7 93.8 1000 8.0 110.8 31.25 1.4 102.4 500 4.8 94.0 15.63 3.1 101.7 250 3.6 101.1 7.81 3.3 96.8 125 5.3 113.1 3.91 1.4 98.9 62.5 6.7 113.9 1.95 8.3 109.4 Cystatin C Cystatin C concen- tration % (ng/mL) % CV accuracy 2500 2.4 99.2 1250 3.6 100.8 625 0.9 99.1 312.5 10.8 105.0 156.3 14.8 90.7 78.0 11.2 105.6

MMP8

[0316] Average CV: 3.9% (1.4-8.3) [0317] Average accuracy: 101.3% (93.8-109.4)

HNE

[0318] Average CV: 6.0% (3.6-8.0) [0319] Average accuracy: 104.2% (92.2-113.9)

Cystatin C

[0320] Average CV: 7.3% (0.9-14.8)
Average accuracy: 100.1% (90.7-106.6)

Inter-Assay Precision

[0321] Six or seven samples of known concentrations were tested four times on three different batches of strips by two different operators.

TABLE-US-00010 MMP8 HNE MMP8 HNE concen- concen- tration % tration % (ng/mL) % CV accuracy (ng/mL) % CV accuracy 125 10.5 105.5 2000 4.2 97.4 62.5 10.8 95.4 1000 5.4 103.7 31.25 3.0 102.3 500 4.8 97.6 15.63 2.3 100.3 250 6.4 97.4 7.81 2.2 100.2 125 7.9 116.3 3.91 2.1 95.1 62.5 8.2 113.3 1.95 4.7 107.2 Cystatin C Cystatin C concen- tration % (ng/mL) % CV accuracy 2500 2.4 98.7 1250 3.7 101.6 625 3.2 101.1 312.5 1.9 94.3 156.3 8.8 104.1 78.0 6.0 113.7

MMP8

[0322] Average CV: 5.1% (2.1-10.8) [0323] Average accuracy: 100.9% (95.1-107.2)

HNE

[0324] Average CV: 6.2% (4.2-8.2) [0325] Average accuracy: 104.3% (97.4-116.3)

Cystatin C

[0326] Average CV: 4.3% (1.9-8.8) [0327] Average accuracy: 102.3% (94.3-113.7)

Recovery

[0328] The recovery of MMP8 spiked into 6 urine samples was evaluated. [0329] Standard added value: 50 ng/mL [0330] Recovery %: 84.9-110.9 [0331] Average recovery %: 98.0

[0332] The recovery of HNE spiked into 6 urine samples was evaluated. [0333] Standard added value: 750 ng/mL [0334] Recovery %: 86.0-115.4 [0335] Average recovery %: 101.2

[0336] The recovery of Cystatin C spiked into 6 urine samples was evaluated. [0337] Standard added value: 800 ng/mL [0338] Recovery %: 106.7-117.0 [0339] Average recovery %: 111.9

Specificity

[0340] The specificity of the assay was evaluated by measuring the degree of cross-reactivity to various compounds.

TABLE-US-00011 Cross Concentration % cross reactivity reactant (ng/mL) MMP8 HNE Cystatin C Cystatin C 2500 0 0 N/A HNE 2000 0 N/A 0 MMP8 500 N/A 0 0 A1AT 500 0 0 0 MMP9 500 0 0 0 TIMP1 500 0 0 0 TIMP2 500 0 0 0 CRP 500 0 0 0

Example 7Verification Studies

Fresh and Frozen Samples

[0341] Twelve samples freshly collected were tested on the UTRIPLEX test and then frozen and tested after 3 days. The results were as follows:

TABLE-US-00012 Reader value Concentration ng/mL MMP8 Fresh Frozen Fresh Frozen Sample 1 29.5 17.1 3.35 1.585 Sample 2 67 21.1 7.467 2.019 Sample 3 136.2 83.3 18.75 9.543 Sample 4 214.3 187.5 63.52 57.52 Sample 5 227 201 91.17 91.72 Sample 6 126 61.8 16.52 6.589 Sample 7 48.4 44.3 4.989 4.526 Sample 8 63.2 76.3 6.765 8.516 Sample 9 45.5 59.4 4.66 6.291 Sample 10 111.5 108.8 14.62 14.05 Sample 11 111.3 118.4 14.58 16.19 Sample 12 10.7 14.3 0.8512 1.272

[0342] The results are shown graphically in FIG. 26.

TABLE-US-00013 Reader value Concentration ng/mL HNE Fresh Frozen Fresh Frozen Sample 1 29.5 17.3 121.4 53.84 Sample 2 51.5 22.4 175.4 71.93 Sample 3 7.3 4.3 0 0 Sample 4 8.7 3.1 0 0 Sample 5 30.4 19.1 124 60.45 Sample 6 3.1 3.5 0 0 Sample 7 3.9 1 0 0 Sample 8 3.3 4.1 0 0 Sample 9 3.3 4.8 0 0 Sample 10 3.9 1 0 0 Sample 11 12.4 24.3 33.73 78.24 Sample 12 2 1.3 0 0

[0343] The results are shown graphically in FIG. 27.

TABLE-US-00014 Reader value Concentration ng/mL Cystatin C Fresh Frozen Fresh Frozen Sample 1 119 106 370.1 248.2 Sample 2 168.5 104.6 218.3 256.2 Sample 3 124.5 86.5 352 381.7 Sample 4 156.5 116.6 254.3 194.9 Sample 5 161.5 110 239.4 226.8 Sample 6 151 95.1 270.6 316.1 Sample 7 148.5 140 85.07 108.5 Sample 8 85.8 84.5 387.6 398.8 Sample 9 65 81.6 615.2 425 Sample 10 161 159 56.5 60.65 Sample 11 143.5 180 98.41 24.09 Sample 12 140.4 148 107.3 86.35

[0344] The results are shown graphically in FIG. 28.

[0345] In general, the results appear to be largely reproducible and stable when frozen.

UTRiPLEX Assay Variability with Healthy Urine Over 5 Days with Multiple Operators

[0346] Samples were collected from every urination by 8 healthy local volunteers over 5 days, and were tested using UTRiPLEX strips to explore test performance and results. The results are shown in FIGS. 29-36 corresponding to volunteers 1-8 respectively. Volunteers 1, 2, 6 and 7 were female. The rest were male. Some variability observed throughout the day, in particular late evening. All variability within acceptable limits.

Stability of Urine Over 1 Day with Multiple Operators

[0347] Urine samples may be collected off-site and shipped for next day testing. Therefore an investigation into the stability of the urine samples and the biomarkers levels contained therein was conducted over a 24-hr period from sampling. Urine was collected from 3 local volunteers in the morning and measured straight away using the UTRiPLEX test assay. The sample was then split into 2 aliquots, and stored either at room temperature or at 4 C. (fridge). The samples were tested throughout the day. The results are shown in FIGS. 37 and 38, 39 and 40, and 41 and 42 corresponding to volunteers 1-3 respectively. All 3 volunteers were female. Samples were deemed stable over a 24 period at room temperature. Next day testing is acceptable including overnight shipping of samples at RT or 2-8 C.

Stability of the UTRiPLEX Test Strip Over Time in Storage

[0348] One batch of UTI strips was manufactured. The strips were stored in sealed pouches for up to 104 weeks at 3 storage temperatures, 4 C., Room Temperature and 37 C. More strips were placed in the sealed pots which will be used as packaging: one pot opened and then re-sealed until the next test time point and other pots opened only at one specific time point. These pots were stored at room temperature. The data obtained were used to confirm the shelf life of the UTRiPLEX test strips and evidence >6 month stability.

Conclusion

[0349] The UTRiPLEX test has been successfully validated and is ready for clinical studies. It is a 5-10 minute dip test measuring three biomarkers: Cystatin C, MMP8 and HNE. In the current state it is accompanied with a cube reader until the statistical model is validated.

[0350] The table below summarizes the performance of the UTRiPLEX test.

TABLE-US-00015 Cystatin C HNE MMP8 Standard curve 78.12-2500 ng/mL 62.5-2000 ng/mL 1.95-125 ng/mL LLOD 64.8 ng/mL 29.2 ng/mL 0.16 ng/mL Intra-assay CV: 7.3% CV: 6.0% CV: 3.9% precision Accuracy: 100.1% Accuracy: 104.2% Accuracy: 101.3% Inter-assay CV: 4.3% CV: 6.2% CV: 5.1% precision Accuracy: 102.3% Accuracy: 104.3% Accuracy: 100.9% Spike recovery 111.9% 101.2% 98.0% (6 urine samples) (106.7-117.0) (86.0-115.4%) (84.9-110.9%) Cross reactivity 0% 0% 0% Stability 2 months at RT 2 months at RT 2 months at RT

[0351] With the combined three markers, the sensitivity of the test was 87.7% and the specificity of the test was 96.9%. Four samples from 32 samples that were culture positive were missed using the diagnostic. Only one sample of the 32 negative samples gave a false positive result, whereas all 32 samples gave a false positive result with the method that was used to put them in the suspected UTI group (presumed to be Multistix urinalysis strips and clinical symptoms). Using ROC analysis an AUC of 0.96 was obtained and a p value of <0.0001 using Wilcoxon matched-pairs signed rank test.

Example 8UTI Monitoring Using the UTRiPLEX Test Assay

[0352] From 1 volunteer, samples were tested daily using UTRiPLEX alongside known Multistix leukocyte and nitrite assays. The results are shown in FIG. 43. All samples were deemed to be UTI positive based on culture results tested periodically throughout the period. At day 43, UTRiPLEX markers and nitrite/leukocyte results were negative indicating the end of infection. Throughout the 3-4 week period, the UTRiPLEX markers indicated that the infection was not resolved whilst leukocytes and nitrite levels were positive for a UTI only sporadically. Correlation was good between the UTRiPLEX and Leukocytes (small and large) with all 3 biomarkers positive for these samples.

[0353] Thus, the UTRiPLEX assay more reliably and easily detects a UTI than known methods.

Example 9UTRiPLEX Assay Stability Studies

HNE

[0354] Seven standards at known concentrations (2000 ng/mL down to 62.5 ng/mL) and one negative were run in duplicate for each storage condition 4 C., Room Temperature (RT) and 37 C. and at each time point (weeks). The average reader values were calculated and graphically represented in FIG. 44.

MMP8

[0355] Seven standards at known concentrations (500 ng/mL down to 7.8 ng/mL) and one negative were run in duplicate for each storage condition 4 C., Room Temperature (RT) and 37 C. and at each time point (weeks). The average reader values were calculated and graphically represented in FIG. 45.

Cystatin C (competition)

[0356] Seven standards at known concentrations (5000 ng/mL down to 78.125 ng/mL) and one negative were run in duplicate for each storage condition 4 C., Room Temperature (RT) and 37 C. and at each time point (weeks). The average reader values were calculated and graphically represented in FIG. 46.

Conclusion

[0357] Results met all stability specifications: Accuracy, non-specific binding and signal reduction over time (within acceptable limits). The data obtained were used to confirm the shelf life of the UTRiPLEX test strips for 9 months at up to 37 C.

Example 10Performance Evaluation of the UTRiPLEX Strips in a Clinical Setting

[0358] The scope of this performance evaluation was to determine the sensitivity and specificity of the UTRiPLEX test with urines collected from suspected UTI patients, clinically confirmed by microbiological culture (Gold Standard). Results obtained from the UTRiPLEX test (using the combined results from cystatin C, HNE and MMP8) for both reader values and visual readings after the recommended time of 6 minutes were used to determine the performance characteristics. Multistix results were used as GP surgery reference standard.

[0359] For this performance evaluation, urines obtained from patients with suspected UTI, that were subsequently clinically confirmed, were evaluated with the UTRiPLEX strip. The urines were collected from individuals presenting at a GP surgery. These urines were then transferred to a central lab and tested on the UTRiPLEX MUV1 strips and sent off for lab confirmatory tests including culture.

[0360] The UTRiPLEX strips used in this study were manufactured under controlled production processes and conditions which met the QC release specifications.

[0361] A total of 38 urines from patients with suspected UTI were available to evaluate the UTRiPLEX strip [0362] 38 urines have clinical status confirmed by bacterial culture. [0363] 10 (26%) had no significant bacterial growth i.e. UTI negative [0364] 28 (74%) had bacterial growth i.e. UTI positive

TABLE-US-00016 Fresh sample testing at Central lab UTRiPLEX positive negative culture positive 25 3 28 negative 3 7 10 sensitivity % 89 specificity % 70 Relative agreement 84

TABLE-US-00017 Fresh sample testing at central lab Multistix positive negative culture positive 26 2 28 negative 7 3 10 sensitivity % 93 specificity % 30 Relative agreement 76

[0365] Preliminary results show improved specificity with UTRiPLEX compared to the multistix results and comparable sensitivity.

Example 11Further Biomarker Development

[0366] 25 urine samples taken from subjects with culture positive results and 24 urine samples from subjects with culture negative results were analysed with 2 different test strips (per the approach described herein), each consisting of different biomarker combinations;

a. MMP8+HNE+Fibrinogen
b. MMP8+HNE+CRP

[0367] Both test strips measure three biomarkers, all three biomarker assays are in a sandwich assay format (as described elsewhere herein). In summary, one anti analyte antibody is positioned on the capture line and the second anti analyte antibody is conjugated to gold particles (which may, for instance, be sprayed onto a conjugate glass fibre pad depending on the structure of the specific test strip). Presence of analyte in the sample creates a complex on the capture line which can be seen visually as a red line or measured by a line reader device.

[0368] Visual readings were taken by 1 operator. Reader values obtained using a lateral flow reader (Cube, Optricon)

[0369] In the culture positive group, 12% were males. In the culture negative group, 38% were males.

[0370] Samples were tested pre- and post-boric acid addition.

Results

Individual Biomarker Analysis (Using OD Readings Provided by a Immunochromatographic Reader)

[0371] FIG. 47A shows (pre-boric acid addition to the samples) the Individual ROC curve for each biomarker. The corresponding AUC was 0.702, 0.769, 0.869 and 0.788 for CRP, HNE, MMP8 and Fibrinogen respectively.

[0372] FIG. 47B shows (post-boric acid addition to the samples) the Individual ROC curve for each biomarker. The corresponding AUC was 0.756, 0.837, 0.872 and 0.773 for CRP, HNE, MMP8 and Fibrinogen respectively.

Combined Biomarker AnalysisVisual Readings

[0373] For these specific experiments, in order to increase confidence in the result, to be considered a UTI positive result, 2 of the 3 lines needed to show a positive result (i.e. presence of a detectable line). To be considered a UTI negative result, 2 of the 3 lines needed to show a negative result (i.e. absence of a detectable line).

[0374] The table below shows the diagnostic accuracy of the MMP8+HNE+Fibrinogen test strip (pre-boric acid)visual scoring

TABLE-US-00018 MMP8 + HNE + Fibrinogen (pre-boric acid) Lateral flow strip Lateral flow strip positive ( negative ( biomarkers) biomarkers) Total Culture positive 21 4 25 samples Culture negative 6 18 24 samples Total 27 22 49

[0375] The results demonstrate that this combination of markers (pre-boric acid addition) has:

TABLE-US-00019 Sensitivity 84% Specificity 75% Relative agreement 80% Negative Predictive Value (NPV) 82% Positive Predictive Value (PPV) 78%

[0376] The table below shows the diagnostic accuracy of the MMP8+HNE+Fibrinogen test strip (post-boric acid)visual scoring

TABLE-US-00020 MMP8 + HNE + Fibrinogen (post-boric acid) Lateral flow strip Lateral flow strip positive ( negative ( biomarkers) biomarkers) Total Culture positive 22 3 25 samples Culture negative 5 19 24 samples Total 27 22 49

[0377] The results demonstrate that this combination of markers (post-boric acid addition) has:

TABLE-US-00021 Sensitivity 88% Specificity 79% Relative agreement 84% NPV 86% PPV 81%

[0378] The table below shows the diagnostic accuracy of the MMP8+HNE+CRP test strip (pre-boric acid)visual scoring

TABLE-US-00022 MMP8 + HNE + CRP (pre-boric acid) Lateral flow strip Lateral flow strip positive ( negative ( biomarkers) biomarkers) Total Culture positive 21 4 25 samples Culture negative 6 18 24 samples Total 27 22 49

[0379] The results demonstrate that this combination of markers (pre-boric acid addition) has:

TABLE-US-00023 Sensitivity 84% Specificity 75% Relative agreement 80% NPV 82% PPV 78%

[0380] The table below shows the diagnostic accuracy of the MMP8+HNE+CRP test strip (post-boric acid)visual scoring

TABLE-US-00024 MMP8 + HNE + CRP (post-boric acid) Lateral flow strip Lateral flow strip positive ( negative ( biomarkers) biomarkers) Total Culture positive 21 4 25 samples Culture negative 6 18 24 samples Total 27 22 49

[0381] The results demonstrate that this combination of markers (post-boric acid addition) has:

TABLE-US-00025 Sensitivity 84% Specificity 75% Relative agreement 80% NPV 82% PPV 78%

Combined Biomarker AnalysisDecision Tree Analysis Using OD Readings Provided by a Immunochromatographic Reader

[0382] Using decision tree analysis, combination of all three biomarkers CRP+HNE+MMP8 give a sensitivity and specificity of 91.7% and 76% respectively pre-boric acid (FIG. 48A) and 83.3% and 84% respectively post-boric acid addition (FIG. 48B).

Summary of the Results

[0383] ROC AUC for the individual biomarkers ranges from 0.702-0.872. The ROC AUC is an indication of the utility of each marker in its ability to discriminate between culture negative and culture positive urine samples. This supports the validity of the use of these biomarkers individually and in combination within the UTI diagnostic panel.

[0384] Using a simple visual positive or negative score to provide a diagnostic result, both combinations of biomarkers (MMP8+HNE+Fibrinogen & MMP8+HNE+CRP) provide results with good sensitivity and specificity >75% and NPV >80%. In this current study, presence of boric acid has little effect on the final results.

[0385] The Decision tree analysis indicates the cut-off levels for each marker when separating them into culture negative and culture positive groups. This analysis further strengthens the diagnostic utility of these markers in UTI using complex statistical analysis and reader values.

[0386] For the avoidance of doubt, the term comprises is used in this specification to mean that the subject of the phrase to which comprises relates has the following features but may include other features not stated. That is to say, it is used as an open term. Conversely, the term consists of is used in this specification to mean that the subject of the phrase to which consists of relates has the following features and only those features. That is to say, it is used as a closed term.

[0387] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Moreover, all aspects and embodiments of the invention described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, including those taken from other aspects of the invention (including in isolation) as appropriate. Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.