Anti-microbial sensitivity testing of microbes in a urine sample

Abstract

The invention relates to a method of conducting an anti-microbial susceptibility test (AST) of microbes in a urine sample from a subject, wherein the microbes are cultured in the presence of one or more anti-microbial agents; and associated methods.

Description

[0001] Anti-microbial sensitivity testing of microbes in a urine sample

[0002] The present invention relates to methods to determine anti-microbial susceptibility of microbes in a urine sample.

[0003] Urinary tract infections (UTIs) are among the most common bacterial infections, affecting millions of individuals annually. Accurate and timely detection of bacterial concentration in urine samples is critical for diagnosing UTIs and guiding appropriate antimicrobial therapy. Traditional culture-based techniques remain the gold standard for bacterial detection and quantification. These involve inoculating urine samples on selective media, incubating them for 24-48 hours, and counting bacterial colonies to determine concentration. Although these methods are highly sensitive and specific, they are time-consuming and require significant pre-processing steps.

[0004] Antibiotic Susceptibility tests (AST) requires a further overnight incubation step from pure cultures isolated in the first step. Leading to a minimum of 48 h before antibiotic susceptibility is known, this leads to empirical prescribing and potential delay for inappropriate use of antibiotics.

[0005] In addition to culture techniques, other methods of bacterial detection and quantification, such as flow cytometry, polymerase chain reaction (PCR), and microscopy, have been explored. Flow cytometry allows for rapid bacterial enumeration by analyzing suspended particles, but it often requires pre-treatment steps like sample staining or filtration to isolate bacteria from the urine matrix. Similarly, PCR provides high sensitivity and specificity by detecting bacterial DNA, yet it necessitates complex sample preparation steps such as DNA extraction and amplification. Microscopic methods, including direct counting with a hemocytometer or specialized imaging systems, demand careful sample preparation to minimize debris and enhance visualization of bacteria. These pre-processing requirements can introduce additional delays, costs, and variability into the diagnostic workflow.

[0006] The dependence on pre-processing steps, coupled with the intrinsic time demands of these methods, underscores the need for more efficient approaches. Delays in obtaining bacterial sensitivity results can hinder timely diagnosis and treatment, potentially leading to prolonged patient discomfort, disease progression, or inappropriate antimicrobial use.

[0007] Aim of the InventionThe present invention seeks to overcome the limitations of existing anti-microbial susceptibility test (AST) methods by developing a technique that minimizes or eliminates pre-processing steps while providing rapid, accurate, and reliable measurement of microbial growth and antibiotic susceptibility directly in samples. This innovation aims to significantly reduce the time to diagnosis, streamline laboratory workflows, and support the early and effective management of antibiotic use for UTIs.

[0008] According to a first aspect of the present invention, there is provided a method of conducting an antimicrobial susceptibility test (AST) of microbes in a urine sample from a subject, wherein the microbes are cultured in the presence of one or more anti-microbial agents, the method comprising:

[0009] providing a urine sample comprising the microbes to be tested;

[0010] placing the urine sample into at least three microcapillaries of a multiplexed microcapillary device, wherein a first microcapillary is a control comprising no anti-microbial agents, a second microcapillary comprises an anti-microbial agent at a first concentration and a third microcapillary comprises:

[0011] a) the same anti-microbial agent at a second concentration that is different from the first concentration, and / or

[0012] b) a different anti-microbial agent; and

[0013] i) wherein the urine sample further comprise a growth indicator dye, and measuring a change in colour and / or fluorescence of the growth indicator dye over a period of time which indicates microbial growth kinetics in the microcapillaries; and

[0014] ii) detecting any increase in turbidity in the microcapillaries over a period of time which indicates microbial growth kinetics in the microcapillaries; and

[0015] determining whether growth of the microbes in the microcapillaries of steps i) and ii) is inhibited by the anti-microbial agent or a particular concentration of the anti-microbial agent relative to the control, wherein any growth inhibition indicates a susceptibility of the microbes to a particular anti-microbial agent or a particular concentration of the anti-microbial agent, and

[0016] wherein agreement between the susceptibility results from the growth kinetics of steps i) and ii) indicates an accurate susceptibility result; and disagreement between the susceptibility result from the growth kinetics of steps i) and ii) indicates decreased confidence in one of the steps resulting in a susceptibility result.

[0017] The method may further comprise repeating (serially or in parallel) the method of the invention with a diluted urine sample, for example with one or more increasing dilution factors, until a dilution is identified that provides agreement between the susceptibility results from the growth kinetics of stepsi) and ii), thereby indicating an accurate susceptibility result, or at least higher confidence in the result from both i) and ii) steps.

[0018] The method of the invention may be used to determine the minimum inhibitory concentration of an anti-microbial and / or the presence of anti-microbial resistance of microbes in the urine sample. Antibiotic susceptibility may be confirmed by an inhibition of growth of microbes in a microcapillary in the presence of an antimicrobial. The MIC of the antibiotic may be determined as the lowest concentration at which the microbes are inhibited from growing. In one embodiment, where growth of the microbes is detected in the control and / or in the presence of a concentration of the antimicrobial agent and growth of the microbes is inhibited at a higher concentration of the anti-microbial agent, the higher concentration may be an indication of the minimum inhibitory concentration (MIC) of the anti-microbial agent.

[0019] Advantageously, the accuracy of the measurement of anti-microbial susceptibility can be improved whilst not having to pre-process the sample prior to testing. Advantageously, this removes the need for time consuming and costly pre-processing of the sample prior to its testing. For example, the maximum wait time for a threshold infection of a Gram-negative bacterial infection could be in the order of about 6h, which is a significant improvement from the >48 h under the current standard clinical laboratory testing pathways. For testing antibiotic resistance using a metabolic dye, high bacterial densities can cause false resistance results by rapidly converting dye before antibiotics affect the metabolism of the bacteria. This can potentially be avoided simply by testing serial dilutions and ignoring dilutions where rapid detection indicates high starting cell density. Furthermore, fluorescence signals such as resorufin may not be stable over long time periods. For example, resorufin, can be further reduced to dihydroresorufin, a colourless and non-fluorescent product. In this case, a single time point (for example following 18h incubation) cannot be used to determine the antibiotic susceptibility as the endpoint result may indicate false susceptibility. In this case a non-fluorescent result may be mistaken for growth inhibition whereas the bacteria have grown, metabolised resazurin and further metabolised resorufin. However, other methods of bacterial cell measurements are less sensitive to bacterial inoculums and have more stable signals over time. For example, light scatter measurements are not affected by the rapid conversion of the metabolic dye at high cell densities. The light scatter signal is innately more stable, as this is a direct measure of bacterial cells (particles) present in the sample. Combining light scatter and metabolic dye conversion measurements allows for rapid confirmation of bacteria in the sample (by rule in) and fewer dilutions needed for accurate monitoring (such as monitoring of antibiotic susceptibility). Advantageously, determining theminimum inhibitory concentration of an antimicrobial and / or antimicrobial resistance of the microbes in the sample can allow the selection of an antimicrobial or an antimicrobial combination, that is capable of treating a subject's infection, such as a UTL

[0020] The multiplexed microcapillary device

[0021] The multiplexed microcapillary device may comprise a multiplexed microcapillary film. In a preferred embodiment, the multiplexed microcapillary device comprises a fluoropolymer microcapillary film (MCF) that contains a parallel array of multiple microcapillaries, such as about 5-10 microcapillaries. The multiplexed microcapillary device may comprise a hydrophilic polymer coating, for example to modify the internal walls of the capillaries allowing the sample uptake by capillary action. Suitable microcapillary film is described, for example, by Edwards et al. (Lab Chip, 2011, 11 , 4267 —4273), which is herein incorporated by reference.

[0022] The multiplexed microcapillary device may comprise a multiplexed microcapillary film, for example with a range of different antibiotic concentrations in the capillaries or a range of different antibiotics. Each capillary may have one anti-microbial agent, or more than one type of anti-microbial agent, such as two or more antibiotic combinations.

[0023] The capillaries may be microcapillaries (i.e. having a diameter in the 1-1000 micrometer range). The multiplexed microcapillary device may comprise microcapillary film. The volume of the sample in each capillary may be about 0.5-3 microliters, preferably 1 microliter.

[0024] In a preferred embodiment, the multiplexed microcapillary device comprises a multiplexed microcapillary film comprising a first control capillary having no anti-microbial agent, and a second capillary comprising an anti-microbial agent at a first concentration. The multiplexed microcapillary film may comprise a third capillary with an increased concentration of the same anti-microbial agent as the second capillary. A fourth, fifth, sixth, or more, capillary may be provided wherein each additional capillary comprises an increased concentration of the anti-microbial agent relative to the previous capillaries (or vice versa with decreasing concentrations of the anti-microbial agent. The increasing / decreasing concentrations may be used to determine the minimum inhibitory concentration (MIC) of the anti-microbial agent for microbes in the sample.

[0025] The anti-microbial agent may be pre-loaded in the microcapillaries prior to the sample addition.The anti-microbial agent

[0026] The anti-microbial agent may comprise a test agent that may be tested for potential as an antimicrobial agent. Therefore, in one embodiment, the term "anti-microbial agent" may be substituted for "test agent" herein.

[0027] The anti-microbial agent may comprise any agent that is capable of, or potential for, inhibiting the growth of the microbes in the sample. The anti-microbial agent may comprise an antibiotic. In an embodiment wherein the sample comprises yeast, the antimicrobial agent may comprise an antifungal.

[0028] The antibiotic may comprise any selected from the group comprising cephalexin, ampicillin, amoxicillin, amoxicillin-clavulanic acid, nitrofurantoin cefoxitin, cefotaxime, ciprofloxacin, cefuroxime, ceftazidime, co-trimoxazole, meropenem, ofloxacin, nitrofurantoin, amikacin, gentamicin, Fosfomycin, trimethoprim, ertapenem, penicillins, cephalosporins, carbapenems, monobactams, fluoroquinolones, aminoglycosides, tetracyclines; or combinations thereof.

[0029] Advantageously, determining the minimum inhibitory concentration of an antimicrobial and / or antimicrobial resistance of the microbes in the sample can allow the selection of an antibiotic or antibiotic combination, and / or the dose of antibiotic, that is capable of treating a subject's UTL

[0030] The Urine Sample

[0031] Preferably the sample is diluted before placing it into the microcapillaries. The sample may be diluted 1:10, or more. Alternatively, the sample may be diluted 1:100, or more. In one embodiment, the sample may be diluted 1:200, or more. In another embodiment, the sample may be diluted 1:1000, or more. In another embodiment, the sample may be diluted 1:2000, or more. In another embodiment, the sample may be diluted 1:4000, or more. In one embodiment, the sample may be diluted up to 1:5000. In another embodiment, the sample may be diluted between 1:2 and 1:10,000. In another embodiment, the sample may be diluted between 1:10 and 1:4000. In a preferred embodiment, the sample may be diluted between 1:10 and 1:1000. Multiple serial dilutions of different factors may be provided, for example multiple serial dilutions of the sample may be provided in different microcapillaries. The sample may be diluted by at least two different dilution factors, toprovide at least three different dilutions of the sample. In another embodiment, the sample may be diluted by at least three different dilution factors, to provide at least three different dilutions of the sample. In another embodiment, the sample may be diluted by at four different dilution factors, to provide at least four different dilutions of the sample. In another embodiment, the sample may be diluted by at 2-6 different dilution factors, to provide at least 2-6 different dilutions of the sample. In one embodiment, the sample may be diluted about 1:5 to 1:20, about 1:100 to 1:500, and about 1:3000 to 1:10,000. In one embodiment, the sample may be diluted about 1:10, about 1:200 and about 1:4000.

[0032] Higher factor dilutions in series may be provided to give ever more confidence in the antimicrobial sensitivity result relative to the lower-factor dilutions. Additional serial dilution factors may advantageously increase the likelihood of providing a dilution that can provide a high confidence result within 18 hours, or less, such as 6 hours or less.

[0033] In a preferred embodiment, the sample is of unknown microbial concentration (e.g. prior to conducting the method herein). The sample or dilutions of the sample loaded into the microcapillaries may be of unknown microbial concentration. The microbial concentration may not be determined prior to dilution and / or prior to measuring growth of the microbes in the microcapillaries. The provision of dilutions of a urine sample of unknown microbial density may provide at least one sample dilution that is within a 105to 107cfu / ml range of microbial cell density, which is likely to provide an accurate result for AST. The microbial cell density in the sample, neat and / or as diluted, may be determined as part of or during the method of the invention, for example by use of the microbial growth kinetics and standard calibration curves for growth or metabolic dye conversion.

[0034] The dilution may be with a microbial growth medium. The dilution may be with a bacterial growth medium, such as Mueller Hinton broth or a functionally equivalent medium thereof. The growth medium may be suitable for maintaining a culture of bacteria, such as Gram-negative bacteria. The growth medium may be suitable for maintaining a culture of Gram-negative and / or Gram-positive bacteria. The growth medium may be capable of maintaining a culture of Escherichia spp. (such as E. coli), Klebsiella spp., Pseudomonas spp. (such as Pseudomonas aeruginosa), Enterococcus spp., Proteus spp., Enterobacter spp., Staphylococcus spp. (such as coagulase-negative staphylococci), or Acinobacter spp.; or combinations thereof.

[0035] In another embodiment, the microbial growth medium may be a yeast growth medium.The sample may be diluted with a growth indicator dye in addition to the medium.

[0036] Preferably, the sample is diluted with Mueller Hinton broth. More preferably, the sample is diluted with Mueller Hinton broth and growth indicator dye.

[0037] Preferably the sample is diluted, but otherwise not pre-processed prior to loading it into the microcapillaries. Preferably the sample is diluted, but otherwise not pre-processed prior to the addition of the metabolic dye. The microbes in the sample may not be subject to one or more of staining, filtration, inoculation, pelleting, resuspending or incubation prior to being added to the microcapillaries. The sample may be diluted and the microbes in the sample may not be subjected to one or more of staining, filtration, inoculation, or incubation prior to being added to the microcapillaries. The microbes in the sample may be diluted and the microbes in the sample may not be subjected to pelleting and resuspension, for example to adjust their concentration prior to dilution.

[0038] The sample may be from a subject who is a mammal, preferably a human. In another embodiment the sample is from a subject who is a non-human animal, such as a non-human mammal. The sample may be from a subject who is suffering from or suspected to be suffering from a disease or condition, such as an infection. The sample may be from a subject known or suspected to be suffering from a UTL The UTI may comprise cystitis.

[0039] The sample may be a fresh sample (i.e. not stored or frozen). Alternatively the sample may be a stored sample, for example stored in a universal or boric acid container. In one embodiment, the sample may have been stored in a container comprising boric acid. The sample may comprise or not comprise a bacteriostatic agent, such as boric acid. In one embodiment, the sample is in the presence of a bacteriostatic, such as boric acid.

[0040] Advantageously, the dilutions of the present method have been found to overcome the presence of a bacteriostatic agent, such as boric acid used in sample containers. In particular, a linear relationship can be established at higher dilutions where the boric acid has been diluted out sufficiently to not interfere with the antimicrobial sensitivity testing.

[0041] Measuring GrowthIn a preferred embodiment, the colour / fluorescence change measurement and the irradiation measurement steps are provided on the same sample in the same microcapillary. The colour / fluorescence change measurement and the turbidity measurement steps may be provided on the same sample in the same microcapillary sequentially (i.e. one before the other). The colour / fluorescence change measurement and the turbidity measurement steps may be immediately after each other, such as within less than 5 minutes, preferably within 1 minute. The colour change / fluorescence measurement and the turbidity measurement steps may be within 30 seconds of each other.

[0042] Each of the colour / fluorescence change and the turbidity measurement steps maybe repeated at least once (i.e. there are at least two measurements of the colour / fluorescence and the turbidity properties of the sample in the microcapillaries). Such repeated detections / measurements may be separated by the period of time. The colour / fluorescence change and the turbidity measurement steps may be repeated periodically, such as about every 1-5 minutes, or more. The colour / fluorescence change and the turbidity measurement steps may be repeated about every 30 seconds, or more. The colour / fluorescence change and the turbidity measurement steps may be repeated about every 1 minute, or more. The colour / fluorescence change and the turbidity measurement steps may be repeated about every 5 seconds, or more.

[0043] The period of time for measuring the microbial growth may be 0 minutes to 18 hours (i.e. the time between at least two measurements). The period of time for measuring the microbial growth may be 10 minutes to 18 hours. The period of time for measuring the microbial growth may be 1 to 18 hours. The period of time for measuring the microbial growth may be 18 hours, or less. The period of time for measuring the microbial growth may be 6 hours, or less.

[0044] The period of time for measuring the change in colour / fluorescence of the growth indicator dye may be 10 minutes to 18 hours (i.e. the time between at least two measurements). The period of time for measuring the change in colour / fluorescence of the metabolic dye may be 1 to 18 hours. The period of time for measuring the change in colour / fluorescence of the metabolic dye may be 18 hours, or less. The period of time for measuring the change in colour / fluorescence of the metabolic dye may be 6 hours, or less.

[0045] The period of time for detecting any increase in the turbidity from the microbes in the microcapillaries with the light detector may be 10 minutes to 18 hours (i.e. the time between at least twomeasurements). The period of time for detecting any increase in the turbidity from the microbes in the microcapillaries with the light detector may be 1 to 18 hours. The period of time for detecting any increase in the turbidity from the microbes in the microcapillaries with the light detector may be 18 hours, or less. The period of time for detecting any increase in the turbidity from the microbes in the microcapillaries with the light detector may be 6 hours, or less.

[0046] The period of time for measuring the change in colour / fluorescence of the growth indicator dye and detecting any increase in the light scatter from the microbes in the microcapillaries may be 18 hours, or less. The period of time for measuring the change in colour / fluorescence of the growth indicator dye and detecting any increase in the light scatter from the microbes in the microcapillaries may be 6 hours, or less. The period of time for measuring the change in colour / fluorescence of the growth indicator dye and detecting any increase in the turbidity from the microbes in the microcapillaries may be from 10 minutes to 18 hours.

[0047] The growth indicator dye

[0048] The growth indicator dye may comprise any molecule that has a detectable change in colour and / or fluorescence intensity upon metabolization by microbes. The growth indicator dye may comprise metabolic dye. In a preferred embodiment, the metabolic dye comprises or consists of resazurin.

[0049] Resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) is a phenoxazine dye that is weakly fluorescent, nontoxic, cell-permeable, and redox-sensitive. Resazurin is blue to purple in colour and weakly fluorescent, and it is irreversibly reduced to resorufin, which is pink in colour and strongly fluorescent, by microbial metabolism.

[0050] In another embodiment, the growth indicator dye may comprise a pH indicator dye, such as phenol red, methylene blue or fluorescein. pH indicator dyes will change colour based on the pH of the media which is affected by microbial metabolism. In another embodiment, the growth indicator dye may comprise a sugar conjugate dye, such as MUG (4-Methylumbelliferyl-a-D-galactopyranoside).

[0051] Advantageously, growth indicator dye conversion, such as resazurin to resorufin, is proportional to the number of microbes present. The higher the microbial concentration the faster the reaction which can be calibrated to a calibration curve.The growth indicator dye may, such as resazurin, may be provided in sufficient concentration to provide a detectable colour and / or fluorescence change in the presence of microbial metabolism. The growth indicator dye may, such as resazurin, may be provided in sufficient concentration to provide a detectable colour and / or fluorescence change in the presence of microbial metabolism, but at a concentration that is not toxic to microbes in the sample. The growth indicator dye may, such as resazurin, may be provided at a concentration of at least 5 pg mL-1. The growth indicator dye may, such as resazurin, may be provided at a concentration of at least 50 pg mL-1. The growth indicator dye may, such as resazurin, may be provided at a concentration of at least 0.1 mg mL-1. The growth indicator dye may, such as resazurin, may be provided at a concentration of at least 0.2 mg mL-1. The growth indicator dye may, such as resazurin, may be provided at a concentration of about 0.005 mg mL-1to about 1 mg mL-1. The growth indicator dye may, such as resazurin, may be provided at a concentration of about 0.25 mg mL-1.

[0052] The light detector may be any means that can measure the intensity and wavelength of light emanating from the microcapillaries. The light detector may be any means that can detect and measure a change of colour and / or fluorescence in the sample in the microcapillaries, for example from blue to pink, and / or from lower fluorescence to higher fluorescence. The change in colour and / or fluorescence of the metabolic dye may be determined by measuring a change in fluorescence intensity. The detected wavelength may be at the appropriate, such as maximal, emission for the metabolic dye or its converted product, such as resorufin. In a preferred embodiment, the light detector is a camera. Preferably the camera is a digital camera. The camera may be a smartphone camera.

[0053] The same light detector, such as the same camera, may be used for both the colour / fluorescence change measurement and the turbidity measurement steps described herein.

[0054] The light detector, such as a camera, may determine the colour / fluorescence intensity of the growth indicator dye and the determined colour / fluorescence intensity may be converted to a microbial concentration value, such as CFU / ml, for example by reference to a standard. The standard may be a predetermined standard, such as a chart or table, or the standard maybe one or more microcapillaries comprising microbes of a known concentration and measured under the same conditions as the sample (i.e. the test sample).

[0055] In one embodiment, the detection of an increase in the turbidity from the microbes in the microcapillaries is an indication of growth and concentration of the microbes wherein theconcentration of the microbes is determined in CFU / ml relative to a standard. The level of turbidity may be converted to a microbial concentration value, such as CFU / ml, for example by reference to a standard. The standard may be a predetermined standard, such as a chart or table, or the standard may be one or more microcapillaries comprising microbes of a known concentration and measured under the same conditions as the sample (i.e. the test sample).

[0056] The turbidity detection may otherwise be known to the skilled person as an irradiation and light scatter measurement. The light may be irradiated at an optimum wavelength and intensity for detection of microbial cells. In one embodiment, the detection of light scatter is at 600 nm (ODgoo), optionally at an intensity capable of allowing the detection of light scatter. Preferably, the light is irradiated at a wavelength and intensity that does not excite the metabolic dye.

[0057] The level of turbidity may be determined by a turbidimeter or a nephelometer. Nephelometric detection is based on a diode laser that directs a light beam through the sample. If there are no insoluble particles in solution, the light beam will simply pass through. The detector, set to the side of the path of the laser light beam (usually at a 90° angle), will measure no turbidity. In case a solid, insoluble matter is present in solution, the laser beam will be scattered in different directions by the particles and an optic system will collect and direct the scattered light to the detector. The higher the amount of particles scattering the light beam, the more light reaches the detector, the more Nephelometric Turbidity Units (NTU) will be measured. Nephelometry, particle density is a function of the scattered light directed to the detector.

[0058] The light source may be a laser or LED and this may be collimated or not.

[0059] In a preferred embodiment, the same camera, such as a digital camera, is used to determine the colour / fluorescence intensity of the sample and the level of turbidity.

[0060] The microbes

[0061] The microbes in the sample may be single cell microbes, such as bacteria or yeast.

[0062] The microbes in the sample or potentially in the sample may be bacteria, such as Gram-negative and / or -positive bacteria. The bacteria in the sample or potentially in the sample may be any of the group consisting of Escherichia spp. (such as E. coli), Klebsiella spp., Pseudomonas spp. (such as Pseudomonasaeruginosa), Enterococcus spp., Proteus spp., Enterobacter spp., Staphylococcus spp. (such as coagulase-negative staphylococci), and Acinobacter spp.; or combinations thereof. In one embodiment, the bacteria in the sample or potentially in the sample is E. coli. The bacteria in the sample or potentially in the sample may be antibiotic-resistant bacteria. The antibiotic-resistant bacteria may be resistant to multiple antibiotics.

[0063] In one embodiment, the microbes comprise yeast. The yeast may be Candida spp., such as one or more of Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, and Cryptococcus neoformans.

[0064] Other Aspects

[0065] According to another aspect of the present invention, there is provided a method of selecting an antimicrobial for treatment of a subject with an infection, the method comprising determining the antimicrobial sensitivity or resistance of microbes in a urine sample of the subject in accordance with the invention herein, and selecting an antimicrobial or antimicrobial combination that is capable of treating the infection.

[0066] According to another aspect of the present invention, there is provided a method of determining the incidence of antimicrobial sensitivity of a microbial infection in a subject, the method comprising the determination of the antimicrobial sensitivity of microbes in a urine sample from the subject in accordance with the invention herein.

[0067] The incidence of antimicrobial sensitivity may comprise or consist of one or more of the presence of antimicrobial sensitivity, the level of antimicrobial sensitivity, or type of antimicrobial sensitivity, of microbes in a urine sample from the subject.

[0068] The subject may have a UTI.

[0069] Such determination may inform treatment options for the infection, such as a UTI.

[0070] The methods herein may further comprise the treatment of the subject for the infection, such as a UTI, such as by administration of a therapeutic agent for treatment of the infection (such as treatment for a UTI).According to another aspect of the present invention, there is provided a method of treatment of a subject for an infection, the method comprising the determination of the antimicrobial sensitivity or resistance of microbes in a urine sample from the subject in accordance with the invention herein; and administration of a therapeutic agent for treatment of the infection.

[0071] In one embodiment, the subject has a UTL

[0072] The therapeutic agent may be an antimicrobial, such as an antibiotic. The choice (e.g. type or combination) of the antimicrobial may be determined by the method of the invention herein. The therapeutic agent may be an antimicrobial or antimicrobial combination to which the microbe is susceptible, e.g. as determined by the methods herein.

[0073] The term "agreement between the susceptibility results" is intended to mean that the determined antimicrobial sensitivity is substantially the same, for example where there is no significant difference in the result. For example, when the result is expressed as an MIC of an antimicrobial, results may be in agreement if the results are within no more than + / - 1 Iog2 difference, for example between the two methods of susceptibility testing. Reference to a known reference standard of a known quality control organism and cfu / ml may be used to confirm quality of result. Categorical agreement may also be used in which results may be in agreement if the organism is determined to be susceptible or resistant in both the microcapillary tests i) and ii)..

[0074] The skilled person will appreciate that preferred features of any one embodiment and / or aspect of the invention may be applied to all other embodiments and / or aspects of the invention.

[0075] Examples embodying an aspect of the invention will now be described with reference to the following figures:

[0076] Figure 1 - Example workflow for dual detection. A sample containing bacteria is diluted at least two times, in this case three times in Mueller-Hinton broth containing 20 pg / mL resazurin. The microcapillary AST devices are placed in a reader with monitors both the fluorescence conversion of resazurin by bacterial metabolism and light scatter caused by bacterial cells. These measurements are made in the same capillaries, with image capture one after the other.The change in fluorescence and light scatter is monitored over time. Conversion of resazurin to resorufin indicates active metabolism.

[0077] Figure 2 - Ciprofloxacin minimum inhibitory concentration (MIC) of E. coli isolate at an inoculum of 5x 105CFU / mL. a) Ciprofloxacin concentrations ranged from 125 ng / mL - 0.3 ng / mL in two-fold serial dilution with the 1stcapillary containing no antibiotic (growth control), b) Light scatter results following an 18h incubation indicates an MIC result of 30 ng / mL. c) The same capillaries measured using resazurin conversion following an 18h incubation also indicates an MIC of 30 ng / mL. Images are displayed in the red channel.

[0078] Figure 3 - Indirect methods for detecting bacterial growth are not always stable. Resazurin is irreversibly converted to resorufin, however, resorufin can be reversibly converted to a non-fluorescent product. Furthermore, pH of solutions which will change during bacterial growth may also affect the solubility and fluorescence of indirect substrates.

[0079] Figure 4 - Resazurin conversion can be detected rapidly. A) Side by side images of light scatter and resazurin conversion of E. coli 25922 over time. Resazurin conversion can be clearly seen at 4 h before the emergence of light scatter. B) Indicates the end point MIC for both light scatter and resazurin based measurements, indicating a variation of + / - 1 Iog2 difference which is within essential agreement. The quality control organism E. coli 25922 has an acceptable range of 2-8 mg / L for the antibiotic ampicillin. Fluorescence images are displayed in the red channel and light scatter in the green channel.

[0080] Figure 5 - AST of multiple antibiotic agents within a single test strip with multiple test strips tested simultaneously. Test strips indicate the AST of a uropathogenic E. coli (UPEC) tested against 9 antibiotics. The following acronyms indicate DOX: doxycycline, CIP: ciprofloxacin, CFX: cefoxitin, FAZ: cefazolin, TET: tetracycline, AMI: amikacin, IMI: imipenem, CXM: cefuroxime, AXO: ceftriaxone. Concentrations in mg / L for each antibiotic are read left to right.

[0081] Figure 6 - Ciprofloxacin minimum inhibitory concentration (MIC) of E. coli isolate at different inoculums. A) Ciprofloxacin concentrations ranged from 125 ng / mL - 0.3 ng / mL in two-fold serial dilutions dilution with the 1stcapillary containing no antibiotic (growth control). B) MIC results using fluorescence conversion of resazurin to resorufin to indicate bacterial growth show no inhibition of bacterial growth even at the highest concentration of ciprofloxacin at107CFU / mL. This inoculum effect disappears at 10sand 105CFU / mL which indicates an MIC of <0.5 ng / mL. C) MIC results using light scatter indicate and MIC of <0.5 ng / mL regardless of the starting inoculum. Blue arrows indicating the first capillary which shows growth inhibition and therefore the MIC result. Images are displayed in the red channel.

[0082] Figure 7 - Boric acid delays growth detection in microcapillaries. Urine samples recruited from a UK emergency department were collected in sterile universals and split into samples to contain boric acid at an approximate concentration of 2 g / L. Data indicates the time for resazurin conversion of these matched sample types when diluted in Mueller-Hinton Broth and resazurin mix. In the absence of boric acid, there is a linear relationship between the log CFU / mL and time to resazurin conversion. Boric acid delays time to resazurin conversion in proportion to the amount of boric acid present.

[0083] Figure 8 - Boric acid does not affect AST. Antibiotic susceptibility test using light scatter based detection performed directly from urine using urine collected within 4 h and stored in a sterile Universal or Boric Acid container. A) indicates the antibiotics and concentrations used. Urine samples were processed as per Figure 1. B-c) indicate light scatter detection following 18h incubation with corresponding intensity plots below. The samples indicate growth in the presence of Trimethoprim (TRM), Cefalexin (CFX) and growth under some conditions in Fosfomycin (FOS). This may indicate an MIC near the breakpoint for Fosfomycin. The samples show total growth inhibition in the presence of Ciprofloxacin (CIP) regardless of storage condition. Blue arrows indicate capillaries in which bacterial growth was inhibited. Images are displayed in the red channel.

[0084] Example 1

[0085] Analysis of AST directly from urine samples:

[0086] Antibiotic susceptibility testing

[0087] Antibiotic susceptibility can be measured directly from urine. The urine sample can be diluted in Mueller-Hinton Broth and resazurin solution. The microcapillary AST device is dipped into the sample which is loaded into the device by capillary action. The sample is then incubated in a reader that is capable of monitoring the fluorescence conversion of resazurin to resorufin and the light scatter ofbacterial cells in the capillaries (Figure 1). Resazurin (low fluorescence) conversion to resorufin (high fluorescence) indicates bacterial growth and no inhibition by antibiotics in the capillaries. Light scatter is seen as an increase in signal as bacteria grow and numbers increase, indicating no inhibition by antibiotics in the capillaries. Low fluorescence or low scatter signal in the presence of antibiotics indicates growth inhibition. Low fluorescence or low scatter signal in the absence of antibiotics indicates poor bacterial growth or no bacteria present in the urine sample.

[0088] Under controlled conditions, ie a known inoculum of a pure culture, the fluorescence determination and light scatter measurement of antibiotic susceptibility are nearly identical (Figure 2).

[0089] However, resazurin conversion and light scatter detection of bacteria can vary depending on organism size and structure. Indirect methods, such as resazurin, can be affected by subsequent breakdown products and pH of media which will be affected by bacterial growth (Figure 3). At high inoculums, the bacteria in the capillaries may already be visible by light scatter or resazurin conversion may start immediately upon mixing. Resazurin conversion is more uniform along and across the capillaries due to the solubility of the dye used. Light scatter however, can have more variation in appearance due to characteristics of the bacteria, for example, motility and biofilm formation may affect the appearance and intensity in each capillary. This may allow a moderate increased speed of detection. Typically we see resazurin conversion earlier than light scatter so confirm the presence of bacteria in a sample (Figure 4).

[0090] The microcapillary test strips used for AST may comprise the same anti-microbial agent at a different concentrations to read a minimum inhibitory concentration (MIC) for that specific antibiotic (Figure 4) or a different anti-microbial agents can be present (Figure 5). This allows multiple antibiotic susceptibility tests to be performed in a single test strip.

[0091] Antibiotic susceptibility testing in non-standard inoculum preparation

[0092] Antibiotic susceptibility tests are highly standardised methods which require a specific starting inoculum to output a valid result. Urine samples however have a range of bacterial inoculum (103-1011CFU / mL), which is laborious to accurately quantify for every urine sample that requires culture. Monitoring several dilutions of urine can allow rapid identification of urine samples with a highbacterial load and cover a wide dynamic range of cell densities to measure accurate antibiotic susceptibility.

[0093] Resazurin conversion is a non-reversible, indirect measurement of bacterial growth. Using this method at high cell densities, resazurin conversion is rapid which can be faster than inhibition by antibiotics. This results in a false resistant result (Figure 6b). This is reduced by dilution of the sample to the standardised method inoculum (2-8xl05CFU / mL) which correctly indicates susceptibility results for the samples.

[0094] Light scatter is a direct measurement of bacterial cells in the sample and is less susceptible to false resistant results due to rapidly converted dyes at high inoculums, although the inoculum effect (IE), a well characterised effect that increases MIC when the inoculum increases can still occur. In the same conditions we see the light scatter-based AST indicates the same MIC result recorded for all inoculums from 105-107CFU / mL (Figure 6c).

[0095] Analysis of AST direct from urine samples stored in boric acid containers:

[0096] Boric acid is a bacteriostatic agent which is used in urine sample transport to avoid overgrowth of bacteria present in a sample. In the absence of boric acid the relationship between log CFU / mL and growth detection is linear. However, boric acid delays the detection of growth, resulting in a loss of linearity (Figure 7). While this can interfere with the inferring of bacterial density until further dilutions are used, the antibiotic susceptibility pattern determined in the presence or absence of boric acid was found to be the same (Figure 8).

Claims

CLAIMS1. A method of conducting an anti-microbial susceptibility test (AST) of microbes in a urine sample from a subject, wherein the microbes are cultured in the presence of one or more anti-microbial agents, the method comprising:providing a urine sample comprising the microbes to be tested;placing the urine sample into at least three microcapillaries of a multiplexed microcapillary device, wherein a first microcapillary is a control comprising no anti-microbial agents, a second microcapillary comprises an anti-microbial agent at a first concentration and a third microcapillary comprises:a) the same anti-microbial agent at a second concentration that is different from the first concentration, and / orb) a different anti-microbial agent; andi) wherein the urine sample further comprise a growth indicator dye, and measuring a change in colour and / or fluorescence of the growth indicator dye over a period of time which indicates microbial growth kinetics in the microcapillaries; andii) detecting any increase in turbidity in the microcapillaries over a period of time which indicates microbial growth kinetics in the microcapillaries; anddetermining whether growth of the microbes in the microcapillaries of steps i) and ii) is inhibited by the anti-microbial agent or a particular concentration of the anti-microbial agent relative to the control, wherein any growth inhibition indicates a susceptibility of the microbes to a particular anti-microbial agent or a particular concentration of the anti-microbial agent, andwherein agreement between the susceptibility results from the growth kinetics of steps i) and ii) indicates an accurate susceptibility result; and disagreement between the susceptibility result from the growth kinetics of steps i) and ii) indicates decreased confidence in at least one of the steps resulting in a susceptibility result.

2. The method according to claim 1, further comprise repeating, serially or in parallel, the method of claim 1 with a diluted urine sample with one or more increasing dilution factors, until a dilution is identified that provides agreement between the susceptibility results from the growth kinetics of steps i) and ii), thereby indicating an accurate susceptibility result.

3. The method according to claim 1 or 2, wherein the minimum inhibitory concentration of the antimicrobial and / or the presence of anti-microbial resistance of microbes in the sample is determined byconfirming inhibition of growth of microbes in a microcapillary in the presence of a particular antimicrobial or antimicrobial concentration4. The method according to claim any preceding claim, wherein the multiplexed microcapillary device comprises a fluoropolymer microcapillary film (MCF) that contains a parallel array of multiple microcapillaries.

5. The method according to any preceding claim, wherein the volume of the sample in each capillary is 0.5-3 microliters.

6. The method according to any preceding claim, wherein the multiplexed microcapillary device comprises a multiplexed microcapillary film comprising a first control microcapillary having no antimicrobial agent, and a second microcapillary comprising an anti-microbial agent at a first concentration; andoptionally the multiplexed microcapillary film comprises a third microcapillary with an increased concentration of the same anti-microbial agent as the second microcapillary; and further optionally a fourth, fifth, sixth, or more, microcapillary is provided wherein each additional capillary comprises an increased concentration of the anti-microbial agent relative to the previous microcapillaries.

7. The method according to any preceding claim, wherein the sample is diluted by at least one factor prior to placing the sample into the at least three microcapillaries.

8. The method according to any preceding claim, wherein the anti-microbial agent comprises a test agent that is to be tested for potential as an anti-microbial agent.

9. The method according to any preceding claim, wherein the anti-microbial agent comprises an antibiotic or antibiotic combination; orWherein the antimicrobial agent comprises an anti-fungal.

10. The method according to any preceding claim, wherein the sample is a urine sample.

11. The method according to any preceding claim, wherein sample is of unknown microbial concentration.

12. The method according to any preceding claim, wherein the sample is in the presence of a bacteriostatic.

13. The method according to any preceding claim, wherein the sample is diluted 1:10, or more.

14. The method according to any preceding claim, wherein sample is diluted between 1:10 and 1:4000.

15. The method according to any preceding claim, wherein the sample is diluted by at least three different dilution factors, to provide at least three different dilutions of the sample.

16. The method according to any preceding claim, wherein the sample is diluted about 1:5 to 1:20, about 1:100 to 1:500, and about 1:3000 to 1:10,000.

17. The method according to any preceding claim, wherein period of time for measuring microbial growth is less than 18 hours.

18. A method of selecting an antimicrobial for treatment of a subject with an infection, the method comprising determining the antimicrobial sensitivity or resistance of microbes in a sample of the subject in accordance with any of claims 1-17, and selecting an antimicrobial or antimicrobial combination that is capable of treating the infection.

19. A method of determining the incidence of antimicrobial sensitivity of a microbial infection in a subject, the method comprising the determination of the antimicrobial sensitivity of microbes in a sample from the subject in accordance with any of claims 1-17.

20. A method of treatment of a subject for an infection, the method comprising the determination of the antimicrobial sensitivity or resistance of microbes in a sample from the subject in accordance with any of claims 1-17; andadministration of a therapeutic agent for treatment of the infection.

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