Method for detecting urinary tract infections and sample analysis using liquid chromatography

A technology for sample analysis and urinary tract infection, applied in the field of clinical sample analysis, can solve the impractical problems of waiting for clinical results

Pending Publication Date: 2021-03-16
I A 路易斯 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the long timeframe required for UTI diagnosis makes waiting for clinical results impractical

Method used

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  • Method for detecting urinary tract infections and sample analysis using liquid chromatography
  • Method for detecting urinary tract infections and sample analysis using liquid chromatography
  • Method for detecting urinary tract infections and sample analysis using liquid chromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment IA

[0064] Example IA - Analysis of Urine Samples Using Isocratic Sequential Chromatography

[0065] Sequential isocratic chromatography was first optimized using unlabeled agmatine spiked into healthy urine control samples to construct a simulated UTI sample. The optimal isocratic solvent composition for agmatine was determined to be 86% acetonitrile with 0.1% (v / v) formic acid, which enables efficient adsorption while maintaining the mobility of agmatine on the column for fast Plug spacing. An offset of about 4% between adsorption and elution is considered to be beneficial to improve the shape of the chromatographic peaks while speeding up the column elution and maintaining the baseline separation between the sample peaks, e.g. Figure 3B shown. These conditions enable continuous injection with intervals as low as 30 seconds between peaks. Once the optimal solvent ratio has been determined, the urine sample and the isotope-labeled 13 C Agmatine.

Embodiment I

[0066] Example IB - Analysis of Two-Stage Isocratic Continuous Elution with Isotope Dilution and Injection Using Composite Columns

[0067] added to urine 13 C Agmatine and in Thermo Q-Exactive TM HF LC-MS platform using Syncronis TM HILIC column for analysis. A binary solvent system comprising 20 mM ammonium formate pH 3.00 (solvent A) and acetonitrile with 0.1% (v / v) formic acid (solvent B) was used for the chromatographic separation. Mass spectra were acquired in positive ion mode using parallel reaction monitoring. The two-stage isocratic continuous elution of isotope dilution with composite column injection mode analysis was implemented by the following steps: i) continuous injection of isotope-labeled urine sample in a continuous 86% solvent B (first mobile phase) isocratic flow, and ii) The series of sample plugs were eluted using an isocratic step with 82% solvent B (second mobile phase). Includes hardware to switch valves ( figure 1 ) ensure that step i) is pe...

Embodiment 2

[0070] Example 2 – as Figure 6 Blood culture diagnosis indicated

[0071] Although urinalysis is shown above, the method of the present invention can also be applied to other systems. For example, if Figure 6 As shown, two-stage isocratic continuous chromatography was used to differentiate nine microbial cultures of Pseudomonas aeruginosa (Pae) ​​from Escherichia coli (Eco). Pseudomonas aeruginosa does not produce putrescine, whereas Escherichia coli produces significant levels of putrescine under the microbial culture conditions of this experiment. Therefore, cultures growing E. coli can be distinguished from cultures growing Pseudomonas aeruginosa based on the levels of putrescine observed in the samples. like Figure 6 As shown, 9 peaks were detected, representing 9 urine samples, and 3 groups of peaks were eluted over time, representing 3 technical replicates performed, demonstrating the stability and reliability of the method. The microbial culture examples used he...

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PUM

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Abstract

Methods for analyzing a urine sample to determine if it contains a microorganism linked to an infection. The method comprising: providing culture-independent urine sample from patient; and analyzing the sample; where the culture-independent urine sample contains a microorganism linked to a UTI if at least one decarboxylated amino acid metabolite selected from agmatine, putrescine or cadaverine isdetected in the culture-independent urine sample. The presence of agmatine is strongly indicative of a urinary tract infection caused by a majority of UTI-causing microorganisms. Another method for sample analysis, for or apart from UTI issues, employs liquid chromatography and mass spectroscopy of eluents separated using continuous chromatography of a sample spiked with an amount of isotopicallylabelled target compound. In another embodiment, the method further employs two-stage, isocratic continuous chromatography and possibly including chromatography through more than one column with elution to a common mass spectrometer.

Description

technical field [0001] The present invention relates to methods of detecting urinary tract infection (UTI). The invention also relates to the analysis of clinical samples using liquid chromatography. Background technique [0002] More than 75% of urinary tract infections (UTIs) are caused by organisms from the Enterobacteriaceae family, which includes one or more of the following microorganisms: Escherichia coli (EC); Klebsiella species such as pneumonia Klebsiella (KP) or Klebsiella oxytoca (KO); Proteus mirabillis (PM); Enterobacter species (Esp); and Citrobacter species (Csp) (hereinafter referred to as common UTI-causative microorganism). [0003] Urinary tract infections (UTIs) are so common that more than half of all women will develop a UTI during their lifetime. As a result, urine cultures are the most commonly performed tests in microbiology. Current UTI diagnostic protocols require a bacterial culture step, which results in a long diagnostic timeline. Using cu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/493G01N30/02G01N33/48G01N33/50
CPCG01N33/493G01N2800/348G01N30/88G01N30/466G01N30/7233G01N2030/884Y02A50/30G01N30/72
Inventor I·A·路易斯D·格雷格森R·格罗韦斯
Owner I A 路易斯
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