阅读说明：本技术 检测尿路感染的方法和使用液相色谱的样本分析方法 (Method for detecting urinary tract infection and sample analysis method using liquid chromatography ) 是由 I·A·路易斯 D·格雷格森 R·格罗韦斯 于 2019-05-31 设计创作，主要内容包括：用于分析尿样以便测定其是否含有与感染有关的微生物的方法。该方法包括：提供来自患者的免培养尿样；和分析样本；其中,如果在免培养尿样中检测到选自胍基丁胺、腐胺或尸胺的至少一种脱羧氨基酸代谢物,那么该免培养尿样含有与UTI有关的微生物。存在胍基丁胺强烈地表明了由大多数UTI-致病微生物引发的尿路感染。用于或不用于UTI组织的另一样本分析方法采用洗脱液的液相色谱和质谱法,该洗脱液是对加入了一定量同位素标记的目标化合物的样本进行连续色谱分析而分离得到的。在另一实施方案中,该方法还采用了两阶段等度连续色谱法,可能包括超过一根色谱柱的色谱法,洗脱至普通色谱仪。(Methods for analyzing urine samples to determine whether they contain microorganisms associated with infection. The method comprises the following steps: providing a culture-free urine sample from a patient; and analyzing the sample; wherein the culture-free urine sample contains microorganisms associated with UTI if at least one decarboxylated amino acid metabolite selected from agmatine, putrescine, or cadaverine is detected in the culture-free urine sample. The presence of agmatine strongly indicates urinary tract infections caused by most UTI-pathogenic microorganisms. Another sample analysis method, used or not for UTI tissue, employs liquid chromatography and mass spectrometry of an eluent that is separated by sequential chromatographic analysis of a sample to which an amount of an isotopically labeled target compound is added. In another embodiment, the method also employs two-stage isocratic continuous chromatography, possibly involving chromatography on more than one chromatographic column, eluting to a common chromatograph.)

1. A method of diagnosing a Urinary Tract Infection (UTI) in a patient, the method comprising:

receiving a culture-free urine sample from a patient;

analyzing the culture-free urine sample;

wherein the patient is diagnosed with UTI if at least one of agmatine, putrescine or cadaverine is detected in the culture-free urine sample.

2. The diagnostic method of claim 1, wherein the presence of agmatine represents UTI.

3. The diagnostic method of claim 1 or 2, wherein analyzing the sample comprises separating analytes in urine using Liquid Chromatography (LC).

4. The diagnostic method of claim 3, wherein analyzing comprises eluting at least one of agmatine, putrescine or cadaverine using isocratic solvent running liquid chromatography.

5. The diagnostic method of any one of claims 1-4, wherein at least one of agmatine, putrescine or cadaverine is detected using mass spectrometry.

6. The diagnostic method of any one of claims 3-5, wherein analyzing further comprises a two-stage isocratic continuum LC comprising: in a first phase, a plurality of samples are injected into the LC column using a first mobile phase, followed by a second phase in which analytes are eluted from the LC column using a second mobile phase different from the first mobile phase.

7. The diagnostic method of claim 6, wherein analyzing further comprises two-stage isocratic continuous LC provided with a composite column injection pattern comprising a plurality of LC columns.

8. The diagnostic method according to any one of claims 1 to 7, wherein the analysis further comprises adding a known amount of an isotopically labeled form of agmatine to the non-cultured urine sample for determining the concentration of agmatine.

9. The diagnostic method of claim 1, further comprising diagnosing a plurality of patients and analyzing a plurality of samples in series using an LC-MS apparatus, wherein the LC is run using an isocratic solvent.

10. The diagnostic method of any one of claims 5 to 9, wherein the isocratic solvent is acetonitrile and formic acid.

11. The diagnostic method of any one of claims 5 to 9, wherein a threshold concentration of agmatine of greater than 170nM is indicative of UTI.

12. A rapid sample analysis method of analyzing a plurality of samples for the presence of a target compound, the method comprising:

sequentially injecting a plurality of samples one at a time into an LC column with an isocratic solvent that substantially provides for stepwise elution of the target compound; and

one or more analytes are received into a mass spectrometer for analyte detection to determine whether the one or more analytes include a target compound.

13. The rapid sample analysis method of claim 12, further comprising reconditioning the LC column with a reconditioning solvent to prevent clogging of the LC column.

14. The rapid sample analysis method of claim 12, further comprising adding a known amount of an isotopically labeled form of the target analyte to each of the plurality of samples, wherein the signal from the isotopically labeled compound is used to calculate the concentration of the target compound to minimize chromatographic variations or quantitative errors resulting from ionization of the target compound in the sample.

15. The rapid sample analysis method according to any one of claims 12-14, further comprising a two-stage isocratic continuous LC-MS to separate chromatographic peaks, the two-stage isocratic continuous LC-MS comprising: in a first phase, a plurality of samples are injected into the LC column using a first mobile phase, followed by a second phase in which the analytes are eluted from the LC column using a second mobile phase.

16. The rapid sample analysis method of claim 15, further comprising a two-stage isocratic continuous LC provided with a composite column injection pattern comprising a plurality of LC columns.

17. The rapid sample analysis method according to any one of claims 13-16, wherein the plurality of samples are free urine samples.

18. The rapid sample analysis method of any of claims 13-16, wherein the plurality of samples are microbial cultures.

19. An antibiotic for use in treating a patient suffering from UTI associated with the detection of the presence of at least one of agmatine, putrescine or cadaverine in a culture-free urine sample; wherein the antibiotic is selected to be active against a species of the Enterobacteriaceae family; wherein at least one of agmatine, putrescine or cadaverine is detected in the patient's culture-free urine sample.

20. The antibiotic for use in treating a patient with UTI of claim 19, wherein the antibiotic is selected based on a metabolic susceptibility test comprising: culturing the patient in a growth medium a culture-free urine sample; after culturing, analyzing the cultured growth medium using a chemical assay to determine the level of each of the one or more metabolites in the cultured growth medium; identifying the cell type by comparing the level of each of the one or more metabolites to a reference metabolite curve and matching the level of each of the one or more metabolites to the reference metabolite curve representing the cell type.

21. Use of an antibiotic for treating a patient suffering from UTI associated with the presence of at least one of agmatine, putrescine or cadaverine in a free-culture urine sample from the patient; wherein the antibiotic is selected to be active against a species of the Enterobacteriaceae family; wherein the metabolite is detected in a patient's free-culture urine sample.

22. An LC-MS apparatus for rapid sample analysis for detection of a target compound, the apparatus comprising:

a first column configured for liquid chromatography;

a second column configured for liquid chromatography and in parallel with the first column;

a detector for detecting a compound comprising a target compound, the compound eluting from the first and second columns;

a sample injector for sequentially injecting a plurality of samples alternately into the first column and the second column together with a first mobile phase;

a solvent pump for alternately injecting a second flow phase different from the first flow phase into the first column and the second column; and

a valve switch for alternately connecting the sample injector and the solvent pump to the first column and the second column.

23. An LC-MS apparatus for rapid sample analysis for sequential elution of a plurality of culture-free samples for detection of a compound of interest, the apparatus comprising:

an LC column;

a first mobile phase for injecting a sample into the LC column;

a second mobile phase having a different composition than the first mobile phase and configured to elute a sample from the LC column;

a detector for detecting a compound comprising a target compound, said compound eluting from said LC column;

a sample injector configured to sequentially inject a plurality of culture-free samples into the LC column; and

a valve switch for switching between the first and second flow phases;

wherein the valve switch is configured to switch from the first flow phase to the second flow phase after injecting the plurality of samples into the LC column, and the valve switch is configured to switch from the second flow phase to the first flow phase after eluting the plurality of samples from the LC column.

24. The LC-MS apparatus for rapid sample analysis according to claim 23, wherein the injector injects the plurality of samples into the LC column with the first mobile phase flowing continuously.

25. The LC-MS apparatus for rapid sample analysis according to claim 23, further comprising a modification phase configured to elute compounds from the LC column, preventing clogging of the LC column.

26. Use of the device of claim 23 or 24 for rapid sample analysis, wherein the injection of a plurality of samples and elution of compounds occurs in a continuous manner.

27. A method of analyzing a sample of bodily fluid to determine whether it contains a microorganism associated with an infection, the method comprising:

receiving a sample of body fluid from a patient;

analyzing the sample on a liquid chromatography-mass spectrometry (LC-MS) device, wherein the sample is analyzed with the LC-MS device and the Liquid Chromatography (LC) is run with an isocratic solvent;

wherein the body fluid sample comprises a microorganism associated with the infection if a metabolite of the microorganism is detected in the body fluid sample using isocratic continuous LC-MS.

28. The method of claim 27, wherein the sample is a culture-free urine sample, the method being for determining whether it contains microorganisms associated with Urinary Tract Infection (UTI), and wherein the culture-free urine sample comprises microorganisms associated with UTI if at least one of agmatine, putrescine, or cadaverine is detected in the culture-free urine sample.

29. The method of claim 27, wherein the presence of agmatine represents UTI.

Technical Field

The present invention relates to methods of detecting Urinary Tract Infection (UTI). The invention also relates to clinical sample analysis using liquid chromatography.

Background

More than 75% of Urinary Tract Infections (UTIs) are caused by organisms from the Enterobacteriaceae family (Enterobacteriaceae family) which include one or more of the following microorganisms: escherichia Coli (EC); klebsiella, such as Klebsiella Pneumoniae (KP) or Klebsiella Oxytoca (KO); proteobacteria Mirabilis (PM); enterobacter (Enterobacter species, Esp); and Citrobacter (Csp) (hereinafter referred to as common UTI-pathogenic microorganism).

Urinary Tract Infections (UTIs) are very common, with more than half of women developing urinary tract infections during their lifetime. As a result, urine culture is the most commonly done assay in microbiology. Current UTI diagnostic protocols require bacterial culture steps, which can lead to long diagnostic schedules. Using current technology, it takes about two days to identify UTI-pathogenic organisms and characterize their antibiotic susceptibility.

Ideally, organisms responsible for UTI would be identified and their antibiotic susceptibility measured before the physician prescribes the antibiotic in order to ensure proper infection management. However, the long timeframes required for UTI diagnosis make waiting for clinical results impractical. As a result, many patients are given antibiotic therapy that does not properly match their symptoms. Faster diagnostic methods may reduce complications arising from improperly treated UTIs.

Disclosure of Invention

Methods of detecting Urinary Tract Infection (UTI) have been invented.

Methods have been devised for analyzing urine samples to determine whether they contain microorganisms associated with urinary tract infections. The urine sample may be analyzed to determine whether it contains at least one decarboxylated amino acid metabolite selected from agmatine (agmatine), putrescine (putrescine) or cadaverine (cadeverine). The presence of agmatine, putrescine or cadaverine in the urine indicates urinary tract infection by any common UTI-pathogenic microorganism. The presence of agmatine strongly suggests urinary tract infections caused by any common UTI-pathogenic microorganism.

Thus, according to one aspect of the present invention, there is provided a method of diagnosing a Urinary Tract Infection (UTI) in a patient, the method comprising: obtaining a culture-free urine sample from the patient; analyzing the culture-free urine sample; wherein the patient is diagnosed with UTI if at least one of agmatine, putrescine or cadaverine is detected in the culture-free urine sample.

According to another broad aspect of the invention, there is provided an antibiotic for use in treating a patient suffering from UTI associated with the detection of the presence of at least one of agmatine, putrescine or cadaverine in a culture-free urine sample; wherein an antibiotic is selected having activity against a species of the Enterobacteriaceae family; wherein at least one of agmatine, putrescine or cadaverine is detected in a patient's free urine sample.

According to another broad aspect of the invention, there is provided the use of an antibiotic for treating a patient suffering from UTI associated with the presence of at least one of agmatine, putrescine or cadaverine in a culture-free urine sample from the patient; wherein the antibiotic is selected to be active against a species of the Enterobacteriaceae family; wherein the metabolite is detected in a patient's free-culture urine sample.

Although urine samples can be analyzed for at least one of agmatine, putrescine or cadaverine using a variety of methods, rapid sample analysis methods are very valuable because there are hundreds to thousands of samples to analyze per day in the laboratory due to the frequent occurrence of UTI. Therefore, sample analysis methods have also been invented with or without UTI tissue. The method enables rapid analysis of complex samples such as body fluids, e.g. urine samples in cases of UTI and blood samples in cases of bloodstream infections. The method employs liquid chromatography and mass spectrometry for chemical substances, in which a sample is injected into a liquid chromatography system in the form of a series of consecutive sample plugs (sample plugs) under conditions where the target substance is eluted under isocratic continuous conditions. In another embodiment, the method employs multi-stage isocratic continuous elution, with continuous injection in one stage and rapid elution from the column in a second stage.

Thus, according to a further aspect of the present invention, there is provided a method of rapid sample analysis of a plurality of samples for the presence of a compound of interest, the method comprising: sequentially injecting a plurality of samples into the LC column one at a time, together with an isocratic solvent (isocratic solvent) which essentially provides for stepwise elution of the target compound; the one or more analytes are received into a mass spectrometer for analyte detection to determine whether the one or more analytes include a target compound.

According to another broad aspect of the present invention there is provided an LC-MS instrument for rapid sample analysis for detection of a compound of interest, the instrument comprising: a first column configured for liquid chromatography; a second column configured for liquid chromatography and parallel to the first column; a detector for detecting a compound comprising a target compound, which elutes from the first column and the second column; a sample injector for successively and alternately injecting a plurality of samples into the first column and the second column together with the first mobile phase; a solvent pump for alternately injecting a second flow phase different from the first flow phase into the first column and the second column; a valve switch for alternately connecting the sample injector and the solvent pump to the first column and the second column.

According to another broad aspect of the present invention, there is provided an LC-MS instrument for rapid sample analysis providing sequential elution of a plurality of culture-free samples for detection of a compound of interest, the instrument comprising: an LC column; a first mobile phase for injecting a sample into the LC column; a second mobile phase having a different composition than the first mobile phase configured to elute the sample from the LC column; a detector for detecting compounds eluted from the LC column, including the target compound; a sample injector configured to continuously inject a plurality of culture-free samples into the LC column; a valve switch to switch between a first mobile phase and a second mobile phase; wherein the valve switch is configured to switch from the first flow phase to the second flow phase after injecting the plurality of samples into the LC column, and the valve switch is configured to switch from the second flow phase to the first flow phase after eluting the plurality of samples from the LC column.

According to another broad aspect of the present invention, there is provided a method of analyzing a sample of bodily fluid to determine whether it contains a microorganism associated with an infection, the method comprising: providing a sample of bodily fluid from a patient; analyzing the sample in a liquid chromatography-mass spectrometry (LC-MS) device, wherein the sample is analyzed with the LC-MS device and the Liquid Chromatography (LC) is run with an isocratic solvent; wherein the body fluid sample contains a microorganism associated with an infection if a metabolite of the microorganism is detected in the body fluid sample by isocratic continuous LC-MS.

In one embodiment, a known amount of an isotopically labeled substance, such as decarboxylated amino acids, is added to a complex sample to enable quantification of the target substance by measuring the isotopic ratio.

It is understood that other aspects of the present invention will become readily apparent to those skilled in the art from the following detailed description, wherein various embodiments of the invention are shown and described by way of example. It is to be understood that the invention is capable of other and different embodiments and that several details of its design and implementation can be modified in various other respects, all within the scope of the appended claims. Accordingly, the detailed description and examples are to be regarded as illustrative in nature and not as restrictive.

Drawings

For a better understanding of the invention, the following figures are attached:

FIG. 1 is a schematic diagram of two-stage isocratic continuous chromatography (two-stage isocratic continuous chromatography) provided with a multiple column injection scheme (two columns) in which two columns are used.

Fig. 2A to 2D are timing schemes (TI) for multiple injection LC analysis (TD) for four different chromatography methods: a conventional 3 minute gradient (fig. 2A); isocratic continuous chromatography (fig. 2B); two-stage isocratic continuous chromatography (fig. 2C); and composite (multiplexed) two-stage isocratic continuous chromatography, in which there are two chromatography columns, each operating in two-stage isocratic continuous chromatography (fig. 2D). Isocratic continuous chromatography (fig. 2B) involves repeated feeding (arrows) for time TI followed by sample elution with isocratic solvent to detect TD, possibly followed by column repair TR using selected mobile phase to wash the column. Two-stage isocratic continuous chromatography (fig. 2C) comprises a first stage comprising injection of TI using solvent B (i.e. a first mobile phase) and a second stage comprising sample elution using a solvent different from solvent B (i.e. a second mobile phase), during which the separated molecules elute in peak form and are detected for TD, possibly followed by column repair TR using a selective mobile phase in order to wash the column. A composite two-stage isocratic continuous chromatography (fig. 2D) comprises two columns, wherein, when column 1 is in the second stages TD and TR, column 2 is in the first stage TI and the column stages alternate with each other-as such, more samples can be analyzed over time and more efficient use of the detector can be achieved.

Fig. 3A to 3C are comparisons of sample peaks eluted per unit time of the laboratory: conventional 3 minute gradients (fig. 3A), isocratic continuous chromatography (fig. 3B) and two-stage isocratic continuous chromatography (fig. 3C). The eluted samples were generated from duplicate injections of urine samples containing 5uM agmatine standards.

Figure 4 shows the effect of error caused by different ion suppression (differentiation administration) during an isocratic continuous chromatographic sample run (upper curve) and an isocratic continuous chromatographic sample run using isotopically labeled agmatine C13 (lower curve), indicating that the ratio of agmatine to isotopically labeled agmatine can be used to correct the error.

Figure 5 shows two graphs demonstrating that accurate measurement of agmatine can be achieved using isocratic continuous chromatography with isotopically labeled agmatine added as an internal reference for each sample, the concentration being calculated by the ratio of agmatine to isotopically labeled agmatine. The upper curve shows the addition to a solution containing 100nM¹³In urine samples of C agmatine¹²Calibration curve for C agmatine standards. The lower curve shows an independently quality controlled urine sample,¹²the amount of C agmatine compared to the calculated agmatine concentration from the observed isotope ratio is known.

FIG. 6 is a graph of 9 E.coli cultures and 9 P.aeruginosa cultures analyzed by isocratic continuous chromatography¹²C and¹³comparison of the level of C putrescine (3 technical replicates). The culture methods used to produce these samples are matched with established methods for detecting bloodstream infections.

FIG. 7 shows 96 urine samples from human beings which were negative for bacterial growth by isocratic continuous chromatography (upper left panel) versus 96 urine samples which were positive for E.coli infection by isocratic continuous chromatography (upper right panel)¹²C and¹³comparison of C agmatine levels. By¹²C/¹³Multiplying the C ratio by¹³The known concentration of the C agmatine standard substance is calculated¹²C agmatine concentration (lower curve shows isotopically labelled agmatine standards).

FIG. 8 shows that 96 human urine samples (upper left) negative in bacterial growth detected by two-stage isocratic continuous chromatography using a composite column injection mode (using two columns) are more positive in Escherichia coli infection detected by 96 human urine samples (upper right) positive in Escherichia coli infection detected by two-stage isocratic continuous chromatography using a composite column injection mode (using two columns)¹²C and¹³comparison of C agmatine levels. By¹²C/¹³Multiplying the C ratio by¹³The known concentration of the C agmatine standard substance is calculated¹²C guanidino radicalButylamine concentration (lower curve shows isotopically labelled agmatine standard).

FIG. 9 shows E.coli (EC), a bacterium from a healthy subject (Neg) and with 6 different types of bacteria; klebsiella, such as Klebsiella Pneumoniae (KP) or Klebsiella Oxytoca (KO); proteus Mirabilis (PM); enterobacter (Esp); and agmatine concentration observed in urine samples from patients with urinary tract infections caused by Citrobacter sp. The upper squares represent the observed agmatine concentration in the sample and the lower squares show the same data on a log10 scale. The dashed line in the second box represents the threshold of 58nM, which is determined by the isotope ratio and corresponds to the UTI detection threshold with a sensitivity of 0.94 and a specificity of 0.99.

FIG. 10 is a schematic showing the metabolic preference test (MPA) used to determine the sensitivity of 8 clinical E.coli isolates (rows 11-18) to a range of concentrations of 4 antibiotics. Metabolic activity was monitored by succinate levels (metabolites secreted by e.coli in culture). An increased metabolite production rate (light grey) relative to control medium indicates that the metabolic activity is characterized by resistance to a given concentration of antibiotic. A decrease in metabolite yield (dark grey) relative to control indicates antibiotic sensitivity. Each isolate was subjected to 3 technical replicates under each condition. The black bars represent the antibiotic susceptibility profile of each isolate as determined by conventional culture-based methods in the regional diagnostic laboratory (CLS). The concentration unit is μ g/mL. Wherein T/S refers to trimethoprim-sulfamethoxazole.

Figure 11 is a schematic showing agmatine can be used as a marker for the presence of UTI. 519 samples were obtained from Alberta Public Laboratories (Alberta Public Laboratories) and analyzed. Agmatine concentration was closely correlated with the culture positive samples containing Enterobacteriaceae (Enterobacteriaceae). In the schematic, the samples are divided into subgroups according to the speciation results.

Detailed Description

The detailed description and examples set forth below are intended to describe various embodiments of the present invention and are not intended to represent the only embodiments contemplated by the inventors. The detailed description includes specific details for a thorough understanding of the invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced without these specific details.

Analysis of human urine samples shows that, unexpectedly, elevated levels of agmatine, putrescine or cadaverine are present in urine samples in at least 75% of Urinary Tract Infections (UTIs), which is not the case in urine samples of healthy subjects. Studies have shown that the concentration of agmatine, putrescine or cadaverine in urine of human subjects without UTI is undetectable in view of the detection limit of the assay (50 nM).

It is understood that analogs of the metabolites, such as adducts, isotopes, fragments, multiple charge states, and the like, are also considered metabolites. It should be noted that the metabolite signal in the spectroscopic analysis may be a complex (complex) and in fact may comprise more than one signal per molecule. Overall, the signature set of the molecule can be broken down and considered as a single metabolite signature. For example, mass spectrometers detect 10-50 signals per molecule, including the original molecule (parent) and various analogs of the original molecule, including fragments, adducts (chemical binding that occurs in the instrument), multiple charge states, and isotopes (naturally occurring forms of molecules with 1 or more additional neutrons). Detection of any of these signals can represent a molecule, and in the present application, any analogue of agmatine, putrescine or cadaverine is included in the metabolite of interest: agmatine, putrescine or cadaverine.

This study unexpectedly concluded that: UTI caused by microorganisms of the enterobacteriaceae family can be diagnosed by detecting agmatine, putrescine and/or cadaverine in the urine of a patient. Microorganisms of the enterobacteriaceae family include: escherichia coli, proteus mirabilis, Citrobacter (including Citrobacter buchneri, Citrobacter freundii, Citrobacter acidanulatus (Citrobacter ammonianatriens), Citrobacter faeri (Citrobacter farmi), and Citrobacter cruzi (Citrobacter koseri)), Enterobacter (including Enterobacter aerogenes (now classified as Klebsiella aerogenes) and Enterobacter cloacae (Enterobacter cloacae cplx.), Klebsiella (including Klebsiella oxytoca and/or Klebsiella pneumoniae)). All enterobacteria studied produced agmatine, putrescine and/or cadaverine. Thus, regardless of whether the infection is caused by E.coli, Klebsiella, Proteus, Enterobacter, Citrobacter, the infection can be identified by analyzing the concentration of one or more of agmatine, putrescine or cadaverine in a urine sample from the patient. A threshold of 170nM agmatine or greater in the urine indicates Enterobacter infection.

Agmatine is identifiable from a sample of a patient infected with at least one of the following microorganisms: escherichia coli, Proteus mirabilis, Citrobacter (including Citrobacter buchneri, Citrobacter freundii, Citrobacter malonate-free, Citrobacter faeri, and Citrobacter cruzi), Enterobacter (including Enterobacter aerogenes and Enterobacter cloacae), Klebsiella (including Klebsiella oxytoca and Klebsiella pneumoniae). For example, referring to fig. 9 and 11, a urine sample from a human subject is analyzed, and a sample from a subject infected with a microorganism comprising agmatine: coli (EC), Proteus Mirabilis (PM), citrobacter (Csp) (including citrobacter buchneri, citrobacter freundii, citrobacter malonate-free, citrobacter faeri, and citrobacter cruzi), enterobacter (Esp) (including enterobacter aerogenes and enterobacter cloacae), klebsiella (including Klebsiella Oxytoca (KO) or Klebsiella Pneumoniae (KP)), and samples of subjects who did not suffer from any UTI (i.e., UTI-negative) had no detectable level of agmatine. Therefore, unexpectedly, a test against agmatine in urine could be used to reliably indicate that a patient suffers from an infection caused by any of the common UTI-pathogenic microorganisms. In some cases, putrescine (fig. 6) and cadaverine can also be used as indicators to diagnose UTI in human subjects.

Enterobacteriaceae, such as escherichia coli, proteus mirabilis, citrobacter, enterobacter, and klebsiella, produce agmatine and sometimes putrescine or cadaverine, but the human body does not produce any agmatine, putrescine, or cadaverine in the urinary tract. The breakdown of proteins, particularly amino acids, produces agmatine, putrescine and cadaverine. Agmatine is aminoguanidine produced by the decarboxylation of chemical arginine. Putrescine or tetramethylenediamine (tetramethylenediamine) is a metabolite of ornithine or agmatine. Cadaverine, also known as pentamethylene diamine, is produced by chemical lysine decarboxylation.

Thus, in one embodiment, a method of determining whether a patient has a urinary tract infection may comprise: and analyzing the urine sample of the patient for the presence of agmatine, putrescine or cadaverine, wherein a positive result indicates that the patient has urinary tract infection caused by at least one of escherichia coli, klebsiella pneumoniae, klebsiella oxytoca, proteus mirabilis, enterobacter or citrobacter. Since the assay detects metabolites of the microorganism rather than directly detecting the microorganism, sample handling is simplified, e.g., the sample can be used without any incubation, thus avoiding the materials and time required for incubation.

In one embodiment, urine samples from patients can be analyzed directly after collection. The sample does not need to be incubated, so an incubation-free sample can be used and incubation-free analysis can be performed.

After collection, urine samples can be detected directly using this method. Alternatively, the human urine sample may be preserved using a preservative, such as a boric acid buffer solution, which prevents the growth of any microorganisms in the human urine sample. Alternatively, the urine sample may be fixed after collection using a fixative (e.g., methanol) to stop microbial activity. Preparation of human urine samples may also include removal of solids, for example by filtration or centrifugation. Thereafter, the liquid portion of the urine sample (e.g., the supernatant in the case of centrifugation) is analyzed for the presence of agmatine, putrescine, or cadaverine.

Optionally, the sample can be enriched using solid phase extraction after fixing and removing the solids. Solid phase extraction is used to purify biological samples. A stationary phase (e.g., silica) is used in conjunction with a high pH mobile phase to capture molecules in the urine sample. Then, when the pH of the mobile phase is lowered, the stationary phase elutes molecules, thus purifying the biological sample. The method of solid phase extraction is included in example 8.

If the sample analysis gives a positive result for at least one of agmatine, putrescine or cadaverine, indicating that the patient has a urinary tract infection, the sample may be further analyzed to determine more information about the cause of UTI, if desired. For example, urine samples can be tested for antibiotic resistance in order to determine the appropriate antibiotic that can be used to treat UTI (fig. 10). If the sample gives a positive result, a metabolic preference test can be performed to determine antibiotics that cannot be used to treat UTI due to antibiotic resistance. The metabolic tests may also indicate the type of pathogen present, e.g., gram negative or positive, genus, species, etc., and thus, instead of the usual antibiotics, specific, appropriate courses of antibiotic therapy may be administered to the patient.

The metabolic preference test identifies a cell type in a positive result sample, the test comprising: culturing the sample in a growth medium; after culturing, analyzing the cultured growth medium using a chemical assay to determine metabolite levels in the cultured growth medium; and identifying the cell type by comparing the metabolite level to the reference metabolite map and matching the metabolite level to the reference metabolite map representing the cell type. The cell type of an organism may be a general classification of the organism (i.e., gram-negative, gram-positive, etc.), genus or species of origin (i.e., species of bacteria, etc.), or strain or distinguishing characteristic (i.e., resistance or sensitivity to an antibiotic).

For metabolic preference testing, the growth medium in which the cells are grown provides nutrients and accumulates waste products, called metabolites of the microorganism. Over time, the microbial metabolic signals are amplified by accumulated changes in growth medium composition. The incubation period allows the cells in the positive result sample to metabolize by consuming their preferred nutrients and secreting waste products. The nutrients present in the medium are recorded and after analysis the metabolites are analysed in order to obtain metabolic data of the organism in the growth medium. A reference metabolic map, which is a known metabolic result of a microbial community or individual species, subspecies or strain, is compared to metabolic data obtained from sample analysis in order to classify unknown organisms. This method is described in more detail in WO 2018/165751, published 2018, 9, 20, which is incorporated by reference into the present application.

Due to the rapid diagnosis of the present application, if agmatine, putrescine or cadaverine is detected in the urine sample, a more specific antibiotic (such as ampicillin) can be administered to the patient. Other possible treatments include ceplatra, cefamycin, nitrofurantoin, fosfomycin and fluoroquinolone. However, rapid detection would reduce the need for a broader spectrum of agents, such as fluoroquinolones or broad spectrum cephamycins. The other antibiotic susceptibility tests and/or metabolic preference tests described above may allow for a more precise and targeted selection of antibiotics for actual cell types within the enterobacteriaceae family.

Analysis of urine samples from patients for the presence of agmatine, putrescine or cadaverine can be achieved by a variety of techniques.

Spectroscopic analysis techniques may be used. In some cases, the spectral analysis may be preceded by a sample separation, for example by chromatographic separation.

In particular, a detector can be used to detect agmatine, putrescine or cadaverine in a urine sample from a patient. Useful detectors include mass spectrometers, UV-Vis spectrophotometric detectors, array diode detectors or nuclear magnetic resonance spectrometers.

These detectors may be used to receive the sample after it is separated or, in some cases, directly. In particular, in some methods, urine samples can be analyzed without separation (such as chromatography), for example, using a direct injection strategy in combination with isotope dilution. This strategy sometimes applies to the following cases: the target analyte has been purified from the urine sample or the ion suppression effect is controlled by isotopic dilution. One current practice is to analyze samples by direct injection techniques that connect an autosampler directly to a mass spectrometer, thereby eliminating the chromatographic separation step. This direct injection strategy circumvents the time required for chromatographic methods, but is only roughly quantitative when analyzing low complexity solutions. Since diagnostic applications typically involve analysis of complex samples, current practice may include repeated sample extraction and molecular purification steps, such as Solid Phase Extraction (SPE). These methods enable analysis of complex samples by rapid direct injection methods and minimize the quantitative error caused by ion suppression, but introduce other sources of experimental error.

Most commonly, complex samples such as urine are analyzed by separation followed by detection. Liquid Chromatography (LC) is well recognized as a useful method for separating analytes from complex samples. Conventional liquid chromatography typically uses two or more solvent gradients to separate the component compounds from each sample, wherein the type and concentration of solvent changes over time after sample injection. Each sample was injected into the column and then eluted using a solvent stream (mobile phase) whose composition changed over time as shown in fig. 2A. While traditional chromatography is effective, the length of time required to inject a sample, complete a gradient, and rebalance a column is not ideal for analysis where multiple samples are analyzed in the shortest amount of time. With conventional LC, it is difficult to achieve effective data quality using LC run times of less than 3 minutes when analyzing complex samples such as urine (fig. 3). While such time frames may be acceptable in some cases, the large number of clinical samples associated with UTI that are processed by most clinics emphasizes the importance of sample throughput. Complex biological samples often require extensive LC gradients or multi-step extraction protocols to minimize quantitative problems associated with ion inhibition. However, time-intensive methods are not ideal for clinical applications that typically take hundreds of samples per day. Therefore, clinical LC must balance throughput and quantitative characteristics.

To increase sample throughput without requiring additional sample purification processes, it is desirable to shorten the time for molecular chromatographic separation. In one embodiment, the present invention provides for the continuous injection of multiple samples through an LC system into a continuous flow of solvent to reduce sample run time, thereby increasing sample throughput. This is called isochromatography. In this embodiment, the sample may be analyzed using a mobile phase chromatography solvent that maintains a substantially constant composition over time (fig. 2B). Thus, multiple samples can be injected into the column without substantially changing mobile phase solution conditions and without using a solvent gradient. To enable separation of the analyte of interest, the composition of the mobile phase is chosen so as to enable a modest exchange rate between the bound and unbound states of the analyte. The constant composition of the solvent is capable of preferentially retaining the target analyte. When the mobile phase conditions are appropriately adjusted, the non-target analytes will still bind to the stationary phase of the column or will elute from the column rapidly and the target molecules will be retained for a period of time sufficient to achieve separation between the target and non-target molecules. Since the result of this strategy is a continuous gradual migration of target analytes through the column, multiple samples can be injected into a single gradient and the target analytes for each sample will elute as multiple peaks resolved in time, the order of the peaks matching the sample injection order (fig. 3B). Finally, the column requires a repair step, wherein the elution is selected with a new solvent composition, thereby clearing the bound molecules from the column.

Isocratic continuous elution is unexpectedly useful for identifying the presence of a compound of interest and analyzing complex biological samples (e.g., urine samples). The isocratic continuous LC allows a sample-to-sample interval of approximately 1 minute. The time interval between samples is affected by the following factors: the physical dimensions of the column, the flow rate of the solvent, the chemical nature of the solvent, the chemical composition of the stationary phase of the column, the temperature of the column, the fluid pressure within the column, and the operational capabilities of the autosampler (e.g., the rate at which the instrument can perform repeated injections). Under typical analysis conditions, the time interval between target analyte elution peaks ranges from 30 to 90 seconds. This is a small fraction of the time required for conventional gradient chromatography, as shown by a comparison of fig. 3A and 3B.

The molecules eluted from the chromatographic apparatus are analyzed using spectroscopy (such as NMR), mass spectrometry, UV-Vis, array diodes, and the like. In one embodiment, Liquid Chromatography (LC) may be used in conjunction with Mass Spectrometry (MS). The combination of LC-MS is a powerful tool for medical sample analysis.

As shown in fig. 4, an inherent problem encountered when analyzing complex samples using LC-MS is that there is a differential ion suppression where different compounds (such as salt forms) elute from the column to the detector at different rates. Differential ion suppression refers to ionization of analytes within an ion sourceThe signal changes due to contention. Different ion suppression may even cause the exact same sample to appear at different intensities upon multiple injections, limiting the accuracy and precision of LC-MS. For example, in FIG. 4, 5uM is contained¹²Cyagmatine and 0.5uM¹³A sample of cyagmatine (isotopically labelled) 12 identical samples were injected sequentially into a column flowing solvent at equal flow rates, giving rise to a difference in MS signal of 35-40%. In addition to affecting signal intensity, retention of interfering compounds to varying degrees can alter the shape of chromatographic peaks and the residence time of target analytes. When the compound contains carbon (C:¹³C) or nitrogen (a)¹⁵N), these quantitative and qualitative changes in chromatographic and ionization characteristics are largely unaffected. In order to control the variation of ionization and chromatographic properties, the present invention may apply isotopically labeled target compounds agmatine, cadaverine or putrescine, which allow for accurate and reliable detection of the target compounds.

Thus, in one embodiment, isocratic continuous chromatographic elution is combined with the following: in this method, a known concentration of an isotopically labeled form of the target analyte is added to each sample, as observed¹²C/¹³The C signal ratio calculates the concentration of the target analyte. This isotopic normalization strategy, also known as isotopic dilution, corrects for errors caused by different ion suppression because the target analyte and the isotopically labeled target analyte co-elute and thus experience the same ionization and chromatographic conditions. For example, as shown in FIG. 4, although there is a signal-to-signal variation for the same sample, 5uM¹²C agmatine with 0.5uM¹³The ratio of the signal intensities of successive analyses of C agmatine remained essentially the same in each sample. Despite the inherent variable ion suppression of LC-MS, fig. 5 shows the known concentrations using this isotopic normalization method¹²Quantification of C-agmatine and indicates that stable quantification of target analyte levels can be achieved over a wide range of concentration values, including clinically relevant concentration values. In this example, the quantitative performance of the method was evaluated using a panel of healthy urine samples to which a series of additions were made¹²C agmatine labelStandard and 100nM U-¹³C agmatine is used as an internal reference. The injected samples were analyzed using isocratically run columns and the concentrations were calculated using the calibration curve shown in figure 5. Quantitative performance was evaluated using a set of independent quality controls.

The urine sample analysis may incorporate a known concentration of isotope of the target analyte, which in the method of detecting UTI of the present invention is one or more of agmatine, cadaverine or putrescine, and the signal intensity of the known concentration of isotope labeled target analyte may be used to normalize the quantitative variation and calculate the concentration of the target analyte. The method comprises the following steps: detecting the target analyte and the simultaneously eluted isotopically labeled form of the target analyte, and comparing their peak intensities. Using isocratic serial elution and isotopic dilution, samples can be accurately analyzed even with variability in ion suppression. The isotope dilution strategy enables accurate quantification of target analytes in multiple urine samples injected sequentially into a continuous isocratic gradient.

In another embodiment, chromatographic separation of analytes may include two stages, with isocratic continuous elution to achieve good separation and close spacing of chromatographic peaks. Fig. 2C illustrates this method. For example, referring to fig. 2C, two-stage isocratic continuous chromatography comprises: in a first phase, a first mobile phase is used to inject a plurality of samples into the column, followed by a second phase, in which a second mobile phase is used to elute analytes from the column for analyte detection, e.g., using MS. The second mobile phase has a different composition than the first mobile phase. Depending on the type of sample, the nature of the target compound, and the physicochemical properties of the column stationary phase, the mobile phase may differ in hydrophobicity, solvent ionic strength, solvent composition, pH, etc. In the case of agmatine, the first mobile phase differs in its hydrophobicity from the second mobile phase, wherein the first mobile phase is more hydrophobic than the second mobile phase. The first mobile phase is selected so as to elute the target analyte from the LC column. Thus, when the sample is loaded onto the column in the first stage using the first mobile phase, the target analyte is substantially retained on the LC column. Once loaded, the second mobile phase allows for accelerated elution of the target analyte.

Two-stage isochromatography enables analysis of a target analyte from multiple samples by loading the multiple samples onto a column in a first stage and then rapidly eluting in a second stage. Elution of all samples in the second phase may occur in less (sometimes much less) time than the first phase sample injection. The first mobile phase is selected for the analyte of interest to provide adequate binding of the analyte to the stationary phase of the chromatography column such that the analyte in the plurality of samples migrates through the chromatography column in a series of successive peaks. The second mobile phase is selected so as to rapidly elute the sample from the chromatography column, but substantially retain binding of non-target analytes. This two-stage strategy separates target molecules in multiple samples while minimizing the time required to wash the target analyte column. The second mobile phase is capable of eluting analytes from a series of consecutive samples in a much shorter time than is possible with single-stage chromatography. Thus, quantitative analysis of a series of samples loaded onto the column can be accomplished within a fraction of the time required to load the samples.

In order to maintain the flash chromatographic separation of target analytes for a large number of samples, the two-stage isocratic continuous chromatography may further include a complex column injection mode in which a plurality of chromatography columns are used (fig. 1 and 2D). Each column is capable of receiving a number of samples that can be loaded sequentially until the column reaches its capacity. This loading depends on the physicochemical properties of the column (for example, the loading of the column used in fig. 8 is 12 samples). Any potential time savings through two-stage isochromatography is limited to batch analysis of samples equal to or less than the column loading. However, multiplexing multiple columns can extend time savings to a larger number of samples by loading and then eluting the target analytes from a series of chromatography columns in sequence. Furthermore, an apparatus that enables multiple columns to be loaded while simultaneously eluting from other columns is capable of continuously analyzing large numbers of samples.

For example, as shown in fig. 1, an LC system may include two or more columns 10a, 10b configured to be connected to the same detector, such as mass spectrometer 12. This provides for the continuous elution of sample from the plurality of columns 10a, 10b, resulting in a substantially stable and high frequency flow of analyte into the mass spectrometer for detection and analysis (fig. 3C). The column may receive a sample from the same source, such as autosampler 14, the sample being the first mobile phase (dashed line). Both columns 10a, 10b may also receive a second mobile phase (dotted line) from other sources, such as elution pump 16. Valve switch 18a can be used to switch communication between the sample injector and each of the two columns, while communication between the elution pump and the two columns is also switched. Another valve switch 18b is provided at the output of the column, alternately connecting the detector to each of the first and second columns. The valve switch appropriately directs the eluted phase to either the mass spectrometer 12 or the waste 20. The mobile phase flowing from the column can be directed to waste during the reconditioning/equilibration stage, and at any time, the eluted mobile phase need not be analyzed by a detector. If more mobile phase is needed for repair, it may be supplied to valve switch 18a and upstream thereof.

In operation, a first flow phase may be used to inject multiple samples into the first column 10a, while a second flow phase may be used to elute analytes from the samples. The sample units are injected into each column at different times. The basic premise of this aspect of the method is to inject a series of samples into a first column and then elute the samples from a second column, where the injection and elution occur on separate columns in an alternating or sequential manner. In other words, the first column is in the first stage and the second column is in the second stage. The process is cyclic, with one column eluting the sample to the mass spectrometer, and the other column re-equilibrating and loading the sample, for example, with an autosampler. There may be a plurality of columns in the first stage, wherein the sample cells are injected continuously into the columns. There may be multiple columns in the second stage, where the sample is rapidly eluted from the columns. Thus, in the method according to the invention, the analysis comprises two stages, isocratic continuous elution and composite column injection mode. This enables high sample throughput and good utilization of laboratory time and resources for the detection of target compounds in complex samples on the LC-MS platform.

In a possible complex column injection hardware set up as shown in fig. 1, valve switch 18a can be used to rapidly switch the mobile phase running on the column between two mobile phases: a first mobile phase (dashed line) from the autosampler, having a composition selected to be suitable for injecting the sample; a second mobile phase (dotted line) from elution pump 16 configured to rapidly elute the target compound from the column. In fig. 1, state a shows a first stage column 1 and a second stage column 2; state B has been switched by valve switch 18a, which shows column 1 in the second stage and column 2 in the first stage. Valve switch 18b on the elution end of the column alternately sends the eluted analytes from both columns to the mass spectrometer one at a time. Although fig. 1 shows two columns, it should be understood that the process can employ more than two columns.

While chromatography has other uses besides methods for diagnosing UTI, the combination provides very high sample throughput, particularly useful in medical laboratories. Considering that the conventional gradient method achieves an elution time of about 2 to 4 minutes from peak to peak, an isocratic system can typically achieve an elution time of about 0.5 to 1.5 minutes from peak to peak, and a 2-stage isocratic system can achieve an elution time of less than 1 minute from peak to peak, for example, about 0.25 to 1 minute (fig. 3A to 3C). The complex 2-stage system has generally greater throughput because two or more columns can receive samples and deliver them to one detector at the same time. Thus, in another embodiment, isocratic continuous chromatography is used to detect the presence of at least one decarboxylated amino acid metabolite selected from agmatine, putrescine or cadaverine for diagnosing a patient with UTI, wherein an incumbent urine sample from the patient is analyzed using isocratic continuous chromatography. In another embodiment, a two-stage isocratic continuous chromatography is used to detect the presence of at least one decarboxylated amino acid metabolite selected from agmatine, putrescine or cadaverine for diagnosing a patient with UTI, wherein a culture-free urine sample from the patient is analyzed using the two-stage isocratic continuous chromatography. In another embodiment, a composite column injection mode employing multiple columns and a two-stage isocratic continuous chromatography are used to detect the presence of at least one decarboxylated amino acid metabolite selected from agmatine, putrescine, or cadaverine for diagnosing a patient with UTI, wherein a culture-free urine sample from the patient is analyzed using the two-stage isocratic continuous chromatography.

In another embodiment, the analysis of the urine sample of the patient is achieved by emphasising the detection of agmatine using LC-MS. Agmatine has shown the highest level of ionization and the highest predictive power over putrescine and cadaverine.

Furthermore, although methods of analyzing UTI and in particular with respect to the target analytes agmatine, putrescine or cadaverine are described herein, it will be appreciated that the LC methods described herein may be used for other applications of target compound detection in a sample.

Examples

Example IA-analysis of urine samples Using isocratic continuous chromatography

Unlabeled agmatine was added to healthy urine control samples to construct simulated UTI samples, which were used to first optimize continuous isochromatography. The optimal isocratic solvent composition of agmatine was determined as 86% acetonitrile with 0.1% (v/v) formic acid, which enabled efficient adsorption while maintaining the mobility of agmatine on the column, thus enabling fast plug spacing. An offset of about 4% between adsorption and elution (offset) is believed to be beneficial in improving the shape of the chromatographic peak while enabling faster column elution and maintaining baseline separation between sample peaks, as shown in fig. 3B. These conditions enable continuous injection, and the interval between peaks can be as low as 30 seconds. Once the optimal solvent ratio is determined, a urine sample and isotopically labeled¹³C agmatine.

Example IB-two-stage isocratic continuous elution with isotope dilution and analysis using composite column injection

Adding into urine sample¹³C agmatine in Thermo Q-active^TMUse of Syncronis by HF LC-MS platform^TMHILIC column was analyzed. The binary solvent system, which contained 20mM ammonium formate pH 3.00 (solvent a) and acetonitrile with 0.1% (v/v) formic acid (solvent B), was used for chromatographic separation. Mass spectra were acquired using parallel reaction monitoring in positive ion mode. The two-stage isocratic continuous elution of isotope dilution with composite column injection mode analysis is performed by: i) at 86% solvent B (first mobile phase) in succession, etcSerial injection of isotope-labeled urine samples in a gravity flow, and ii) elution of this series of sample plugs with 82% solvent B (second mobile phase) using isocratic steps. Hardware including switching valves (fig. 1) ensures that step i) is performed on the first column and step ii) is performed on the second column, and that the solvent is immediately changed when the elution is changed from the first mobile phase to the second mobile phase and back to the first mobile phase in an alternating manner. By co-eluting with a metabolite of natural agmatine¹³The isotopic ratios between the C standards were quantified to control the different ion inhibitions.

Example 1C-error correction as shown in FIG. 4

Using isocratic continuous chromatography with 12 consecutive injections of 5uM¹²C and 500nm U-¹³Urine sample of C agmatine. Note how the signal errors (illustrated as the variance between the signals) for the two target compounds, in particular, agmatine and isotopically labeled agmatine were identical for each sample. Thus, by observation¹²C and¹³c ratio is calculated by removing the error term¹²C analyte concentration, error terms are for example different ion suppression, which may be caused by ionization or chromatographic variations between samples. The method provides accurate determination of analyte concentration even in the presence of significant sample-to-column variation in the performance of the detection system.

Example 2 blood culture diagnostics as shown in FIG. 6

Although urinalysis is shown above, the method of the present invention is also applicable to other systems. For example, as shown in FIG. 6, two-stage isocratic continuous chromatography was used to distinguish nine microbial cultures of Pseudomonas aeruginosa (Pae) from Escherichia coli (Eco). Pseudomonas aeruginosa did not produce putrescine, whereas Escherichia coli produced significant levels of putrescine under the microbial culture conditions of this experiment. Thus, cultures grown for E.coli can be distinguished from cultures grown for P.aeruginosa according to the levels of putrescine observed in the samples. As shown in figure 6, 9 peaks were detected, representing 9 urine samples, and 3 sets of peaks eluted over time, representing 3 technical replicates performed, demonstrating the stability and reliability of the method. The microbial culture examples used herein illustrate the general applicability of this method to microbial analysis. In addition, the microbial culture conditions used in this study were similar to those used in the analysis of Blood Stream Infection (BSI), indicating that the method is suitable for other clinical applications, including BSI detection.

Example 3-UTI detection-fig. 7 and 8

After optimization, 96 urine samples from patients with and without Urinary Tract Infection (UTI) were analyzed under two conditions: i) example IA, i.e. isotope-labeled isocratic continuous chromatography (fig. 7); and ii) example IB, i.e. isotopically labelled two-stage isocratic continuous chromatography, provided with a complex column injection pattern (figure 8). This study gave satisfactory results. The error rate obtained with the quantitative method of isotope ratio is equivalent to that observed in the more traditional 15 minute linear gradient.

In particular, 96 urine samples from patients with UTI (in particular e.coli) compared to 96 urine samples from healthy subjects are shown. The bottom views of FIGS. 7 and 8 show isotopically labeled¹³Detection of C agmatine, the upper left hand graph of fig. 7 and 8 shows 96 urine samples detected as UTI negative, and the upper right hand graph of fig. 7 and 8 shows 96 urine samples detected as e. In fig. 7, the samples were analyzed by isocratic continuous chromatography according to the method described in example IA, and in fig. 8, the samples were analyzed by two-stage isocratic continuous chromatography provided with a composite column injection pattern according to the method described in example IB. Both FIG. 7 and FIG. 8 show in the upper left panel a negative urine sample (not infected by UTI-pathogenic microorganisms) and in the upper right panel a urine sample infected with E.coli.

These data indicate that isocratic continuous chromatography and two-stage isocratic continuous chromatography equipped with a composite column injection mode are both viable strategies for high-throughput quantitative analysis of selected biomarkers. Furthermore, these methods can be directly applied to UTI detection.

By comparing the results of fig. 7 and 8, we noticed that the two-stage isocratic continuous chromatography provided with the composite column injection mode (fig. 8) performed a shorter run time for sample detection than the isocratic continuous chromatography (fig. 7). We note that the run time for sample detection using isocratic continuous chromatography is one sample per minute to 30 seconds, while using two-stage isocratic continuous chromatography provided with a composite column injection pattern can further compress the sample elution/detection time.

Example 4 Blind clinical study

To validate the method of the invention, a set of random urine samples from patients was subjected to a two-stage isocratic continuous chromatography equipped with a complex column injection pattern in order to detect the natural agmatine content. Urine samples from patients were analyzed separately by conventional UTI diagnostic methods. Using only agmatine, the data indicate that the presence of agmatine in the uncultured or uncultured urine samples was able to diagnose approximately 85% of patients clinically exhibiting bacterial urinary tract infections. In this study, 192 patient samples were subjected to multi-column, biphasic and isocratic continuous chromatography as shown in example IB. The total run time was 430 minutes, including 192 patient samples with two technical replicates and some quality control injections, for a total of 448 injections. This averages an average run time of 1.1 minutes per patient sample and an average time of 0.96 minutes per injection. In 192 patient samples, only one false positive was observed. False positives may be due to differences between the internal positive threshold and the threshold for traditional microbial detection. All false negatives were expected from unknown microorganisms that produce agmatine in the urine. Overall, this is an extremely rapid and efficient screen compared to conventional detection methods, which, once implemented, has a profound impact on the speed of UTI diagnosis and can obviate the need to incubate 85% of patient samples. Only negative samples need to be cultured to confirm bacterial UTI.

Example 5 urine and boric acid experiments

Urine samples are typically collected in a urine culture tube containing a boric acid buffer solution that can reduce microbial growth during transport of the sample to a microbiological laboratory. To evaluate the effect of growth preservatives on agmatine production, 7 E.coli strains (MG1665, ATCC 25922, ESBL ATCC BAA-196 and 4 clinical isolates) were inoculatedTo Mueller Hinton medium, overnight culture, subculture in filter-sterilized urine with or without preservatives (boric acid, 2.63 mg/mL; sodium borate, 3.95 mg/mL; sodium formate, 1.65 mg/mL). Press 10 on 96-well plate⁵CFU/mL bacterial samples were inoculated separately into culture media in the presence of 5% CO at 37 ℃₂And (5) standing and culturing. Growth (OD) was monitored using Microskan GO plate reader (Thermo Scientific, waltham, massachusetts)_600nm). The samples for MS were centrifuged (4200g, 10 min, 4 ℃) to remove the microorganisms. The supernatant was then diluted 1:1 in methanol and stored at-20 ℃. The samples were thawed and immediately centrifuged again (4200g, 10 min, 4 ℃) to precipitate any residual protein, diluted with 50% methanol to a final dilution of 1: 20. In Q active^TMSamples were analyzed by high frequency mass spectrometry (Thermo Scientific, waltham, massachusetts). LC-MS analysis showed that the agmatine concentration of the samples with preservative after incubation time was virtually the same as before the preservative exposure; no significant microbial growth or agmatine production was detected in the samples containing the boric acid preservative solution throughout the incubation, whereas significant growth and agmatine production was observed in the samples not including the preservative. Thus, we have found that boric acid preservative solutions substantially inhibit microbial growth and agmatine production beyond the time of sample storage consistent with clinical practice. It was determined that the collection of a standard urine sample in a boric acid tube did not affect the agmatine concentration in the sample.

Example 6 Metabolic preference detection

A batch of E.coli positive urine samples with different sensitivity curves was analyzed using metabolic bias detection. The bacteria in these samples were pelleted, washed and resuspended in an equal volume of Mueller Hinton medium. 96-well plates were seeded with 10% washed cells, cultured for 4 hours in the presence or absence of clinically relevant concentrations of antibiotics, and analyzed by LC-MS (FIG. 10). Diagnosis based on metabolic preference detection approximately reproduces the antibiotic sensitivity curve reported by traditional culture methods. Breakpoints determined using a metabolic preference-based assay for antibiotics were substantially identical to breakpoints determined based on traditional culture methods. The metabolic preference detection strategy correctly determined antibiotic susceptibility in 97% of the samples.

Example 7-Gaminobutamine as an indicator of UTI

To evaluate the performance of agmatine as a predictor of UTI, a data set of 519 urine samples obtained from eberta public laboratory was analyzed. The data show that the natural agmatine concentration correlates well with culture positive samples containing enterobacteriaceae (fig. 11). The 11 enterobacteriaceae species observed in the data set produced agmatine at a median concentration of 2.1 μ M, whereas no detectable agmatine was present in culture negative samples or samples classified as clinically inconclusive by the eberta public laboratory (fig. 11). Although most UTIs are caused by enterobacteriaceae, a small fraction of UTIs are caused by other microorganisms. The data set included a limited number of these organisms, none of which produced detectable levels of agmatine. Using the data set, the diagnostic threshold for agmatine to distinguish culture negative from culture positive samples was calculated to be about 0.17 μ M. Correcting this threshold corresponds to a diagnosis of enterobacteriaceae-associated UTI of approximately 97% specificity and 94% sensitivity.

Example 8 solid phase extraction of agmatine from urine samples

A urine sample in 50% methanol (1.2mL) was centrifuged at 14,800g for 5 minutes. From these samples, 800. mu.L of urine was added to 200. mu.L of an isotope dilution solution of 1. mu.M [ U-13C ]]A solution of agmatine and 100mM ammonium bicarbonate (pH 8.0) in 50% methanol to a final concentration of 200nM [ U-13C ]]Agmatine and 20mM ammonium bicarbonate. Using 400. mu.L HPLC-grade methanol followed by 400. mu.L 50% HPLC-grade methanol/H₂O Pre-equilibration Thermo Scientific HyperSep silica 96-well plates (25mg bed volume, 1mL column capacity). After equilibration, 1mL of the prepared sample was added to the column and dropped through (skip through). Next, a methanol wash was performed with 1mL of HPLC methanol, followed by a water wash with 1mL of HPLC water. All solutions used in the washing step were passed through the column by means of centrifugal force (20g, 5 min). The column was then prepared by adding 250 μ L of 99.9% methanol and 0.1% formic acid. To elute the target fraction125 μ L of H with 2% formic acid₂O was dropped through the column. Ammonium bicarbonate (pH 8.0) was added to a final concentration of 100mM to raise the solution pH to pH prior to LC-MS/MS analysis>3.0. Note that: 196 samples of the first group included a 13C agmatine concentration of 100nM and a final volume of 500 μ L.

The foregoing description and examples enable those skilled in the art to better understand the present invention. The invention is not limited to these descriptions and embodiments, but is to be given broad interpretation based on the appended claims.