Patent Application
20250050326
This invention relates to hydrogels and equipment and devices incorporating them for use in microfluidic diagnostic assays and rapid assays for detecting microbial presence and activity in biological samples. The invention is particularly suited for assessing antimicrobial susceptibility in biological samples from patients suspected of suffering from a microbial infection, and in particular for detecting antimicrobial susceptibility of bacteria in urine samples from patients suffering a Urinary Tract Infection (“UTI”).
Progress in the development of medical devices used for testing patient samples has accelerated in recent years due to advances in computing and the miniaturisation of electrical components. However, medical devices deployed in point of care settings still present an issue as the patient sample typically must be processed a certain way in order to prepare the sample before being presented to, and correctly analysed by, the device. The patient sample may be processed by adding the sample to reagents in a mixing vessel to form a mixture before presenting the mixture to the device for analysis. Frequently, the patient sample is not processed correctly by the healthcare professional. This either renders the sample unusable for analysis (requiring further samples to be taken from the patient) or can result in the wrong or incomplete diagnosis being given by the device. Furthermore, the act of mixing patient samples with reagents can pose a biohazard threat to the healthcare professional if adequate personal protective equipment is not (or incorrectly) deployed.
There are a number of medical conditions where a quick and reliable point of care medical device could make a dramatic difference to the correct diagnosis and result in the prescription of the correct medicine the first time. Microbial infections is one such area, where a physician often prescribes a course of a particular antimicrobial drug, despite not knowing whether the infection is of viral or bacterial origin and indeed whether it is a bacterial infection to which the antimicrobial drug is effective. The patient may return to physician after the course of antimicrobials if they have not worked, where the physician will then proceed to prescribe a different course of antimicrobials which may be effective. This approach not only wastes the time of both the patient and physician, but puts the patient at risk of the infection worsening and also contributes to the rise of resistance to antimicrobials.
What would be desirable would be a very rapid means of knowing, even before a patient left a doctor's surgery, that a particular antimicrobial was indeed capable of killing the organism causing the infection. While genotypic (whole-genome-sequencing) methods hold out some promise for this, what is really desired is a phenotypic assay that assesses the activity of anti-infectives in the sample itself.
Urinary tract infections (“UTIs”) are a worldwide patient problem. Other than in hospital-acquired infections, they are particularly common in females, with 1 in 2 women experiencing a UTI at some point in their life. Escherichia coli is the most common causative pathogen of a UTI. However, other Enterobacteriaceae such as Proteus mirabilis, Klebsiella spp. and Pseudomonas aeruginosa, and even Gram-positive cocci such as staphylococci and enterococci, may also be found (Kline and Lewis 2016; Tandogdu and Wagenlehner 2016).
E. coli cells in all conditions are highly heterogeneous (Kell et al. 2015), even if only because they are in different phases of the cell cycle (Wallden et al. 2016), and in both ‘exponential’ and stationary phase contain a variety of chromosome numbers (Åkerlund et al. 1995; Skarstad et al. 1986; Skarstad et al. 1985; Steen and Boye 1980; Stokke et al. 2012). To discriminate them physiologically, and especially to relate them to culturability (a property of an individual), it is necessary to study them individually (Kell et al. 1991; Taheri-Araghi et al. 2015), typically using flow cytometry. Flow cytometry has also been used to count microbes (and indeed white blood cells) for the purposes of assessing UTIs. Single cell morphological imaging has also been used, where in favourable cases antibiotic susceptibility can be detected in 15-30 minutes or less (Baltekin et al. 2017; Choi et al. 2014).
WO2020/109764 discloses method for rapidly determining the susceptibility of a microorganism to an antimicrobial agent using flow cytometry. Whilst the method was successful in quickly determining the susceptibility of a microorganism to an antimicrobial agent, flow cytometry is currently expensive and requires complex instrumentation and a significant amount of reagents to evaluate cell growth.
One object of the present invention is to provide a device for performing an assay on an aqueous sample which controls the flow of the biological sample and also allow the assay reagents to be released evenly.
It is another object of the present invention is to provide a device and media which can be used in rapid assays for detecting microbial presence and activity in biological samples. It is desirable that the device and media is simple to use and facilitate a fast and accurate assay which would enable a healthcare worker or physician to quickly assess microbial presence and activity in biological samples, the antimicrobial susceptibility of the microbe to a range of antimicrobials, and allow the prescription of an antimicrobial therapy that would successfully treat the microbial infection. Ideally, the device should be able to be used on a range of whole bodily fluids, such as blood, urine, mucus or saliva. It would also be desirable to provide a device for detecting antimicrobial susceptibility in urine from patients suffering from UTIs.
In accordance with a first aspect of the present invention, there is provided, a device for performing an assay on an aqueous biological sample, the device comprising:
The desiccated assay reagent will preferably be distributed evenly on, and/or throughout, the desiccated hydrogel.
Advantageously, the hydrogel controls the release of the reagent or regents. A common issue in devices employing microfluidics is that pre-coated reagents are dissolved too quickly when the microfluidic chamber is filled. That means the first volume of liquid entering the chamber dissolves more reagents than fluid entering later resulting in a concentration gradient. Distributing the desiccated assay reagent evenly on, and/or throughout, the desiccated hydrogel allows for a much improved controlled release of the reagent.
A further advantage of the hydrogel is when at least partially hydrated, it swells in volume, concentrating the solid and particulate matter in the chamber and also helping to expel bubbles from the chamber.
In certain embodiments, the device comprises a second chamber comprising a desiccated hydrogel, wherein the hydrogel incorporates the same desiccated assay reagent or reagents.
The first and second chamber may be connected to one another in a parallel arrangement. Alternatively, the first and second chamber are connected to one another in a series arrangement. It is preferred that the at least partial hydration of the hydrogel results in the hydrogel substantially preventing the flow of the aqueous biological sample between chambers. The prevention of flow of the aqueous biological sample between the chambers may be effected by the patrial hydration of the hydrogel blocking one or more conduit openings and essentially forming a plug.
Providing the hydrogel in the different chambers advantageously allows for the control of diffusion of regents between chambers in microfluidic applications, since the regents are only released by the hydrogel there is less chance of convective mixing occurring between neighbouring chambers.
The device may comprise an array of chambers.
The device may further comprise a heating arrangement adapted to apply heat to one or more chambers.
The imaging device may comprise a microscope.
The device may further comprises at least one transparent cover to cover a chamber and the imaging device may be adapted to be focused on the area of the chamber corresponding to the surface of hydrogel once partially or fully hydrated. Furthermore, the imaging device may be adapted to be focused on the area of the chamber between the transparent cover and the surface of hydrogel once partially or fully hydrated.
The imaging device may have a field of view of about 500 μm× about 700 μm or smaller.
The chamber may further comprise an optically contrasting filter paper on the desiccated hydrogel. Such a filter paper may be black. Alternatively, the chamber may further comprise low auto-fluorescence paper on the desiccated hydrogel.
The first desiccated assay reagents may comprise at least one bacterial growth medium, at least one fluorescent dye, and optionally, an antimicrobial agent.
In accordance with a second aspect of the present invention, there is provided a method for performing an assay on an aqueous biological sample, the method comprising the steps:
In accordance with a third aspect of the present invention, there is provided a kit of parts for forming a device for performing an assay on an aqueous biological sample, the kit comprising:
The kit may be for use in producing a device herein above described with reference to the first aspect or for use in the method herein above described with reference to the third aspect.
In accordance with a fourth aspect of the present invention, there is provided, a device for rapidly determining the susceptibility of a microorganism in an aqueous biological sample to an antimicrobial agent comprising:
The provision of the combination of the growth medium, dye and desiccated hydrogel (and antimicrobial agent if present) in a single chamber, enables aqueous biological sample to be quickly tested as it contains all the reagents in the chamber for assessing growth, visualisation and antimicrobial susceptibility. Furthermore, the hydration of the hydrogel concentrates and presents the bacterial cells for imaging in a set focal plane for all chambers, which results in more accurate imaging and identification of bacterial cells and greatly reduced background signals. Indeed, the imaging device can utilise a relatively standard microscope if desired. The device of the present invention is relative simple and inexpensive to produce and enables the bacteria in a aqueous biological sample to be quickly identified and/or the susceptibility of the microorganism to multiple antimicrobial agents assessed. In a clinical setting, this would enable the physician to quickly identify which antimicrobial agent would be most successful in treating an infection.
In certain embodiments of the present invention, the desiccated hydrogel incorporates or is coated with a desiccated growth medium and/or a desiccated first dye.
The term, “aqueous biological sample” is intended to mean a sample derived from (or is) any bodily fluid (such as urine, blood, mucus or saliva for example), any environmental fluid (such as river or lake water or soil slurry), or any food or drink sample, which includes a component of water which is available for the hydration of the hydrogel.
The term, “hydrogel” is intended to mean any natural or synthetic hydrophilic polymer which does not dissolve in water.
In certain embodiments, the hydrogel comprises sodium polyacrylate gel or agar. Other hydrogels which are compatible with the invention may include sodium alginate, gelatine methacrylate, sodium polyacrylate, Matigel, synthetic peptide gels, agrose gel, polyacrylamide, fibrin gel and Laminin gels. It will of course be apparent to the skilled addressee that other hydrogels may also be employed. Preferably, the hydrogel hydrates to the desired volume within about 5 minutes, within about 4 minutes, within about 3 minutes, within about 2 minutes or within about 1 minute when in contact with the biological sample.
Most preferably, the hydrogel hydrates to the desired volume within about 2 minutes.
The device may further comprise a heating arrangement adapted to apply heat to one or more chambers or the whole of the plate.
The imaging device will preferably comprises a microscope. The microscope may additionally comprise or incorporate filters. Such filters could be either optical filters or software filters applied to the image data or a combination of both.
The device may further comprise one or more transparent covers to cover one or more chambers.
The imaging device may be adapted to be focused on the area of the chamber corresponding to the surface of hydrogel once partially or fully hydrated. Preferably, the imaging device is adapted to be focused on the area of the chamber between the transparent cover and the surface of hydrogel once partially or fully hydrated. The imaging device preferably has a field of view of about 750 μm× about 750 μm, about 500 μm× about 700 μm, about 400 μm×about 600 μm, about 300 μm× about 500 μm, about 200 μm× about 400 μm, or 100 μm×about 100 μm.
The dimensions of the chamber will vary depending upon application of the device. In some embodiments, the chamber may be in the range of about 10 mm×about 10 mm. In other embodiments, the chamber may be in the range of about 9 mm×about 9 mm, in the range of about 8 mm×about 8 mm, in the range of about 7 mm×about 7 mm, in the range of about 6 mm×about 6 mm or in the range of about 5 mm×5 mm. It will be obvious to the skilled address that the chamber may be any shape and not necessarily square in profile, Each chamber may further comprise an optically contrasting filter paper on the desiccated hydrogel. It is preferred that the filer paper is of a dark colour, such as black. Alternatively, each chamber may comprise low auto-fluorescence paper. In some embodiments, rather than providing optically contrasting filer paper or low auto-fluorescence paper, the hydrogel or the surface of a chamber may themselves be of a dark colour, such as black.
Advantageously, the present inventors found that using a black filter greatly reduced the background fluorescence and enhanced the signal to noise ratio by reducing the noise and enhances the fluorescent signal intensity for those bacterial cells which were ‘in focus’ and no fluorescent signal was produced for cells which were ‘out of focus’. The low background fluorescence with black filter paper therefore significantly assists in identifying the cells.
The dye will preferably comprise a fluorescent dye. In certain embodiments, a chamber may comprises two or more fluorescent dyes.
The plate may further comprise:
The imaging device may be operably coupled to an image analysis device. The imaging device may continuously or periodically analyse two or more chambers for bacterial cell number and/or bacterial morphology and/or fluorescence signal so as to determine the growth vs inhibition of growth or proliferation of a microorganism between two chambers containing the same growth media and dye and where only one of those chambers contains an antimicrobial agent.
The biological sample will preferably be derived from an individual believed to be suffering from a microorganism infection. The biological sample may be derived directly from potentially any body fluid, such as urine, blood, mucus or saliva. The biological samples may be whole or pre-treated with reagents or buffers or filtered prior to being contacted with chamber. In certain embodiments, the biological sample is urine.
The device as herein above described, may be for use in identifying the type or strain of microorganism infection in a biological sample. Preferably, the microorganism infection is a UTI.
In accordance with a fifth aspect of the present invention, there is provided a method for rapidly determining the susceptibility of a microorganism in an aqueous biological sample to an antimicrobial agent comprising the steps:
The chambers may be heated during incubation. If the chambers are heated, it is preferred that the chambers are heated to a temperature in the range of about 35° C. and about 40° C. and preferably at a temperature of about 37° C.
Incubation step b) may be up to about 1 hour, up to about 55 minutes, up to about 50 minutes, up to about 45 minutes, up to about 40 minutes, up to about 35 minutes, up to about 30 minutes, up to about 25 minutes or up to about 20 minutes.
The chambers may be imaged using a microscope.
After the sample has been placed in contact with the chambers, the method may further comprise applying one or more transparent covers to cover the chambers.
The microscope may be adapted to be focused on the area of the chambers corresponding to the surface of the hydrogel once partially or fully hydrated.
The microscope may be adapted to be focused on the area of the chambers between the transparent cover and the surface of hydrogel once partially or fully hydrated. The microscope may have a field of view of about 750 μm× about 750 μm, about 500 μm× about 700 μm, about 400 μm× about 600 μm, about 300 μm× about 500 μm, about 200 μm× about 400 μm, or of about 100 μm× about 100 μm.
The dimensions of the chamber will vary depending upon application of the device. In some embodiments, the chamber may be in the range of about 10 mm×about 10 mm. In other embodiments, the chamber may be in the range of about 9 mm×about 9 mm, in the range of about 8 mm×about 8 mm, in the range of about 7 mm×about 7 mm, in the range of about 6 mm×about 6 mm or in the range of about 5 mm×5 mm. It will be obvious to the skilled address that the chamber may be any shape and not necessarily square in profile.
The method may further comprise applying optically contrasting filter paper over the hydrogel prior to contacting the samples with the chamber. Preferably, the filter paper is of a dark colour, such as black. Alternatively, the hydrogel or the surface of a chamber is of a dark colour, such as black.
The dye preferably comprises a fluorescent dye. In certain embodiments, the chamber comprises two or more fluorescent dyes.
Step a) of the method may further comprises contacting:
The chambers may be continuously or periodically imaged and analysed for bacterial cell number and/or bacterial morphology and/or fluorescence signal so as to determine the growth vs inhibition of growth or proliferation of a microorganism between two chambers containing the same growth media and dye and where only one of those chambers contains an antimicrobial agent. Alternatively, the chambers may simply be imaged and analysed after incubation step b).
The biological sample may be derived from an individual believed to be suffering from a microorganism infection. The biological sample may be urine and the microorganism infection may be a Urinary Tract Infection (UTI).
The method may be used for determining the antimicrobial agent for use in the treatment of a microorganism infection in an individual, wherein the method identifies which antimicrobial agent to administer to the individual based on comparing the bacterial cell number and/or bacterial morphology and/or fluorescence signal of the bacterial cells in two chambers containing the same growth media and dye and where only one of those chambers contains an antimicrobial agent.
In accordance with a sixth aspect of the present invention, there is provided a kit for rapidly determining the susceptibility of a microorganism in an aqueous biological sample to an antimicrobial agent comprising:
The kit may further comprise:
The kit may be for use in producing a device herein above described with reference to the fourth aspect or for use in the method herein above described with reference to the fifth aspect.
Features, integers, characteristics, compounds, described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. For example, the features, integers, characteristics of the fourth, fifth and sixth aspects will largely be applicable to, and interchangeable with the first, second and third aspects. All of the features disclosed in this specification (including any accompanying claims, abstract and figures), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
Embodiments of the invention are described below, by way of example only, with reference to the accompanying figures in which:
This example outlines embodiments which may be employed in accordance with the present invention.
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In use, a urine sample 18 from a patient suspected of suffering from a urinary tract infection is transferred to the upper portion 12 of the chamber 10. Immediately upon transferring the urine sample 18 to the chamber 10, the desiccated hydrogel 16 starts to absorb the liquid from the urine sample 18 and in doing so swells and expands, reducing the volume of liquid of the urine sample and concentrating the bacterial cells 20 into a much smaller volume of liquid at the top of the chamber 10. The image capture device 26 can then more accurately image the number and morphology of the bacterial cells 20 in order to help diagnose what type or strain of bacteria is causing the urinary tract infection.
The chamber 10 as shown in
In order to allow the image capture device to accurately count and assess the morphology of the bacterial cells 20, the desiccated hydrogel 16 incorporates media, dyes and other reagents which may support (i.e. growth media) and/or inhibit (i.e. an antibiotic) the growth of a pre-determined bacterial strain in order to enable the assay to be performed.
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Experiments were conducted to see whether the use of a hydrogel could result in improved identification of the number and morphology of bacterial cells. The experiments, investigated the image resolution using Green Fluorescent Protein (GFP) (488/530 nm) beads 1-3 μm in size (which are similar in size to the bacterial strains of interest): (i) in liquid media without using a hydrogel; (ii) embedded inside a hydrogel; and (iii) on top of a hydrogel. The hydrogels investigate were sodium polyacrylate and agar.
The following protocol was undertaken during these experiments.
Masks measuring 0.25 cm×0.25 cm were prepared with double-sided tape on a glass microscope slide so as to form chambers. One mask was not modified further, where sodium polyacrylate hydrogel was added to four masks and two covered with black filter paper.
10 μL of an aqueous mixture containing the GFP beads stained with 10× SYBR Green I was then applied to masks with coverslips, so as to form (i) a chamber containing GFP beads in liquid media without hydrogel; (ii) a chamber containing GFP beads in liquid media embedded inside the sodium polyacrylate hydrogel without black filter paper; (iii) a chamber containing GFP beadsin liquid media embedded inside the sodium polyacrylate hydrogel with black filter paper; (iv) a chamber containing GFP beads in liquid media on top of the sodium polyacrylate hydrogel without black filter paper; and (v) a chamber containing GFP beads in liquid media on top of the sodium polyacrylate hydrogel with black filter paper. For those cambers utilising a hydrogel, the gel was left for a period of approximately 2 minutes so as to absorb the bead mixture while the beads remain on the filter paper if present.
Each of the chambers were then visualised using a fluorescent microscope with the appropriate filters and images of the bacteria recorded.
In practice, it is envisaged that an automated fluorescence microscope with a movable stage will be utilised and this will take sequences of images across all chambers which will be placed on a heater block to keep them at 37° C. Software for automated cell counting will also be employed in order to help automate the diagnosis for healthcare workers.
The experiments of Example 2 were repeated utilising a 1.5% agar hydrogel in place of the sodium polyacrylate gel and similar results were obtained and confirmed that utilising a chamber containing GFP beads in liquid media on top of a hydrogel with a black filter resulted in all beads being in focus and showing an enhanced fluorescent signal.
The forgoing embodiments are not intended to limit the scope of the protection afforded by the claims, but rather to describe examples of how the invention may be put into practice.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2118945.1 | Dec 2021 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2022/053395 | 12/23/2022 | WO |
