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EP-4739787-A1 - METHOD AND DEVICE FOR DETECTING PRESENCE, CONCENTRATION, AND ANTIBIOTIC RESISTANCE OF BACTERIA IN URINE SAMPLES

EP4739787A1EP 4739787 A1EP4739787 A1EP 4739787A1EP-4739787-A1

Abstract

An automated system that determines the presence of bacteria in a urine sample, concentration of the bacteria, and the bacteria's resistance to a panel of antibiotics, in around one hour instead of days. An automated assay to detect cell viability in mammalian cell cultures using reduction of alamarBlue through oxidation by NADH to NAD+. The assay is conducted within a fluidic chip, wherein a sample is mixed with alamarBlue, a weakly fluorescent indicator dye. Its rate of conversion to a highly fluorescent compound is monitored using a kinetics fluorimeter for 15 minutes, and metabolic activity is shown by an increase in fluorescence. A sample with live bacteria displays a consistent and rapid increase in fluorescence, whereas the negative control exhibits little to no increase over time.

Inventors

  • AL-ADHAMI, Mustafa
  • CAVIN, Courtney

Assignees

  • Astek Diagnostics Inc.

Dates

Publication Date
20260513
Application Date
20240708

Claims (7)

  1. 1. A method for rapid diagnosis and antibiotic susceptibility testing of urinary tract infections, comprising the steps of: a. collecting a urine sample from a patient, b. transferring the urine sample into a fluidic cartridge, c. performing a buffer exchange to isolate and concentrate bacteria present in the urine sample, d. introducing a panel of antibiotics to the concentrated bacterial suspension, e. incubating the bacteria-antibiotic mixtures for a predetermined period, f. introducing a reagent to assess bacterial metabolic activity, g. determining bacterial concentration in the original urine sample based on fluorescence readings, and h. determining antibiotic susceptibility by comparing fluorescence intensities with predetermined threshold values.
  2. 2. The method of claim 1, wherein the buffer exchange process involves passing the urine sample through a series of filters and buffer solutions to separate bacteria from other cellular debris and contaminants.
  3. 3. The method of claim 1, wherein the panel of antibiotics includes a range of antibiotics commonly used to treat urinary tract infections.
  4. 4. The method of claim 1, wherein the reagent for assessing bacterial metabolic activity is alamarBlue.
  5. 5. A fluidic cartridge for use in the method of claim 1, comprising: a. a sample input for receiving the urine sample, b. a buffer exchange module for isolating and concentrating bacteria present in the urine sample, c. a plurality of microchambers for accommodating bacteria-antibiotic mixtures, and d. a reagent introduction module for introducing the reagent to assess bacterial metabolic activity.
  6. 6. An analyzer for use in the method of claim 1, comprising: a. a solid-state laser for excitation, b. a photodiode with a bandpass filter for detecting emitted light, and c. a processor for calculating bacterial concentration in the urine sample and determining antibiotic susceptibility based on fluorescence readings.
  7. 7. The method of claim 1, wherein filtration, incubation and analysis takes place within a total duration of less than 60 minutes.

Description

Method and Device for Detecting Presence, Concentration, and Antibiotic Resistance of Bacteria in Urine Samples FIELD OF THE I NVENTION [0001] The invention relates to diagnostic methods and devices for detecting bacteria in urine. BACKGROU ND OF THE INVENTION [0002] Traditional UTI treatment consists of three steps: 1) determining if the patient has a UTI through clinical evaluation and urinalysis; 2) identification of the bacteria causing the infection, typically achieved through culturing methods, which can take days; 3) antibiotic sensitivity testing to select the most effective antibiotic for the specific bacterial strain. SU MMARY OF THE INVENTION [0003] The present invention is an automated system that determines the presence of bacteria in a urine sample, concentration of the bacteria, and the bacteria's resistance to a panel of antibiotics, skipping the days-long second step, allowing healthcare professionals to directly proceed with antibiotic sensitivity testing without the need for bacterial identification. This shortens the time required to obtain antibiotic sensitivity results from days to just one hour, enabling faster and more accurate treatment decisions. The expedited process not only benefits patients by delivering quicker relief but also helps in the fight against antibiotic resistance, as it minimizes the use of broad-spectrum antibiotics and promotes targeted therapy. [0004] The detection system of the invention automates an assay commonly used to detect cell viability in mammalian cell cultures: reduction of alamarBlue through oxidation by NADH to NAD+. The conversion rate is proportional to the bacterial metabolic rate and concentration. The assay is conducted within a fluidic chip, wherein a sample is mixed with alamarBlue, a weakly fluorescent indicator dye. Its rate of conversion to a highly fluorescent compound is monitored using a kinetics fluorimeter for 15 minutes, and metabolic activity is shown by an increase in fluorescence. A sample with live bacteria displays a consistent and rapid increase in fluorescence, whereas the negative control exhibits little to no increase over time. [0005] The difference of the slopes (sample - control) is proportional to the live bacterial concentration in the sample. This technique was shown to be capable of detecting bacterial infections at a level of 10 CFU (Colony Forming Units)/mL with clinically-relevant bacteria. [0006] To transform the alamarBlue assay from a process that typically takes several hours to one that only takes 15 minutes, the following optimizations and modifications are implemented: 1. Enhanced penetration: Use sodium salicylate or other agents to shrink the polysaccharide shell of bacteria, allowing for better diffusion of the alamarBlue dye and other assay components into the cells, thus accelerating the reduction of resazurin to resorufin. 2. Closed analysis chamber: The analysis chamber is closed to the atmosphere, which increases the sensitivity of the device by maintaining a stable and controlled environment for the assay. 3. Methyl Viologen: Methyl Viologen Dichloride Hydrate is introduced to the assay to increase NADH production, enhancing the reduction of resazurin to resorufin and speeding up the overall assay process. 4. Enhanced detection system: a sensitive single-excitation, single-emission kinetics fluorometer is used with a carefully selected filter to measure the fluorescence signal of resorufin accurately and efficiently, allowing for real-time monitoring of the assay progress. BRI EF DESCRI PTION OF DRAWINGS [0007] Figure l is a flow chart showing the steps performed according to one embodiment of the invention. [0008] Figure 2 is a perspective view of a multi-channel fluidic cartridge analysis device according to an embodiment of the invention. [0009] Figure 3 is a perspective view of a multi-channel fluidic cartridge analysis device according to another embodiment of the invention. [0010] Figure 4 is a perspective view of a disposable multi-channel fluidic cartridge according to an embodiment of the invention. [0011] FIGURE 5 is a representation of a cross-flow membrane chip according to an embodiment of the invention. DETAI LED DESCRIPTION OF TH E I NVENTION [0012] Sample Introduction: The user begins by transferring a patient's urine from a urine collection cup to a standard Vacutainer or other sterile vacuum-sealed container with a rubber cap designed to minimize contamination risks. Once the sample is collected, the user inserts the Vacutainer into the designated slot or holder on a multi-channel fluidic cartridge analysis device. The holder is specifically designed to securely accommodate the Vacutainer, ensuring proper alignment with the sample transfer needles in the device to facilitate seamless sample transfer. The multi-channel fluidic cartridge analysis device features a removable, preferably disposable, cartridge containing a plurality of separate channels, each channel preferably h