EARLIEST PRIORITY DATE:

This Application claims priority from a provisional patent application filed in India having Patent Application No. 202241004593 dated 27th May, 2022, and titled “DEVICE AND METHOD FOR PHENOTYPIC DETECTION FORCELL VIABILITY TO DRUGS USING PAPER

MICROFLUIDICS”

FIELD OF INVENTION

Embodiments of the present disclosure relate to the field of medical diagnosis, and more particularly, a device and method for phenotypic detection of antimicrobial resistance to drugs using paper microfluidics.

BACKGROUND

The emergence of antibiotic-resistant microorganisms, along with the development of antibiotic resistance has become a serious health crisis worldwide. Antibiotic resistance occurs when bacteria, viruses, or fungi become resistant to the drugs that are designed to kill or stop their growth, rendering these drugs ineffective. This means that infections that were once treatable with antibiotics are becoming more difficult, if not impossible, to cure, leading to prolonged illness, increased healthcare costs, and even death.

According to the centers for disease control and prevention, over 2.8 million people acquired the antibiotic-resistant infection, with over 38,000 deaths. Amongst all the consumed antibiotics, between 80-90% are prescribed in the primary care setting (antimicrobial resistance: risk associated with antibiotic overuse and initiatives to reduce the problem). This is a cause for concern since many of these prescriptions are unnecessary, particularly for viral infections that do not respond to antibiotics. Currently, testing methods such as routine testing and AST (Antibiotic Susceptibility Testing) are employed to identify the presence of pathogens, especially in cases of urinary tract infections. However, these testing methods are time-consuming, taking approximately 12-24 hours for routine testing and 36-72 hours for AST testing. This delay in obtaining accurate results poses a significant challenge for effective treatment.

Further, there is a lack of accurate, low-cost, rapid, and point-of-care diagnostic devices in the market that can better equip physicians in primary care settings to identify the type of infection quickly and accurately, as well as determine the need for antibiotics. Furthermore, the available diagnostic methods often require highly skilled individuals to operate them.

Hence, there is a need for an improved device and method for phenotypic detection of antimicrobial resistance to drugs using paper microfluidics which addresses the aforementioned issue(s).

OBJECTIVE OF THE INVENTION

An objective of the invention is to provide a rapid, and low-cost device for phenotypic detection of antimicrobial resistance (AMR) in microbes suspected of exhibiting resistance against drugs and other biologies.

Another objective of the invention is to develop a device that can be used at the point of care, specifically in primary care settings for enhancing accessibility to diagnostic testing.

Yet Another objective of the invention is to develop the device that may be operated by non-skilled individuals.

BRIEF DESCRIPTION

In accordance with an embodiment of the present disclosure, a device for phenotypic detection of antimicrobial resistance to drugs using paper microfluidics is provided. The device includes a top layer enclosed within a casing. The top layer is adapted with a plurality of reaction zones on a porous membrane. The top layer is adapted to receive a predetermined volume of a sample liquid composed of a plurality of pathogens on the plurality of reaction zones. The top layer is further adapted to concentrate the plurality of pathogens on the plurality of reaction zones by pulling the sample liquid through the plurality of reaction zones into an absorber positioned beneath the top layer via a driving force. The device includes a lid positioned on the top layer. The lid is configured with a plurality of reagent storage zones that aggregates to the plurality of reaction zones in the top layer. Each of the plurality of reagent storage zones in the lid includes a mixture of antibiotics, culture media, and colorimetric dyes. Further, the top layer (120) and the lid (136) allow the plurality of pathogens (128) to incubate for a predefined time period and at a predetermined temperature thereby treating the plurality of pathogens (128) to the one or more antibiotics and detection reagents to yield a colorimetric output (132) wherein the colorimetric output (132) signifies if the sample liquid (126) exhibits resistance against the one or more antibiotics.

In accordance with another embodiment of the present disclosure, a device for a two-dimensional paper network for single-step detection for phenotypic detection of antimicrobial resistance to drugs using paper microfluidics is provided. The device includes a medium configured to receive a sample liquid from the repository. The device also includes a filter paper membrane arranged beneath the medium wherein the filter paper membrane is configured to flow the sample liquid horizontally towards a first region deposited with sugar wherein the sugar dissolves in response to receiving the sample fluid. Further, the filter paper membrane is configured to flow the sample liquid horizontally towards a second region wherein the one or more antibiotics and detection reagents dissolve and reacts with the plurality of pathogens to produce the colorimetric output. Furthermore, the filter paper membrane is configured to flow the sample liquid vertically towards the absorber.

In accordance with another embodiment of the present disclosure, a method for phenotypic detection of antimicrobial resistance to drugs using paper microfluidics is provided. The method includes receiving, by a plurality of reaction zones, a predetermined volume of a sample liquid composed of a plurality of pathogens. The method includes concentrating, by the plurality of reaction zones, the plurality of pathogens by pulling the sample liquid through the plurality of reaction zones into an absorber positioned beneath the top layer via a driving force. The method includes sealing, by a lid, the top layer during the incubation with a lid comprising of a plurality of reagent storage zones that aggregates to the plurality of reaction zones in the top layer, wherein each of the plurality of reagent storage zones in the lid comprises a mixture of antibiotics, culture media, and colorimetric dyes. The method includes allowing, by the plurality of zones, the plurality of pathogens to incubate for a predefined time period and at a predetermined temperature thereby treating the plurality of pathogens to the one or more antibiotics and detection reagents to yield a colorimetric output wherein the colorimetric output signifies if the sample liquid exhibits resistance against the one or more antibiotics.

To further clarify the advantages and features of the present disclosure, a more particular description of the disclosure will follow by reference to specific embodiments thereof, which are illustrated in the appended figures. It is to be appreciated that these figures depict only typical embodiments of the disclosure and are therefore not to be considered limiting in scope. The disclosure will be described and explained with additional specificity and detail with the appended figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will be described and explained with additional specificity and detail with the accompanying figures in which:

FIG. 1 is a schematic representation of a workflow of a device for phenotypic detection of antimicrobial resistance to drugs using paper microfluidics in accordance with an embodiment of the present disclosure;

FIG. 2(a)- FIG. 2(d) are schematic representation of an exemplary embodiment of anti-microbial resistance detection of FIG. 1 in accordance with an embodiment of the present disclosure;

FIG. 3 is a schematic representation of a 2-D paper network for single-step detection in accordance with an embodiment of the present disclosure; and

FIG. 4 illustrates a flow chart representing the steps involved in a method for phenotypic detection of antimicrobial resistance to drugs using paper microfluidics in accordance with an embodiment of the present disclosure.

Further, those skilled in the art will appreciate that elements in the figures are illustrated for simplicity and may not have necessarily been drawn to scale. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the figures by conventional symbols, and the figures may show only those specific details that are pertinent to understanding the embodiments of the present disclosure so as not to obscure the figures with details that will be readily apparent to those skilled in the art having the benefit of the description herein.

DETAILED DESCRIPTION

For the purpose of promoting an understanding of the principles of the disclosure, reference will now be made to the embodiment illustrated in the figures and specific language will be used to describe them. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Such alterations and further modifications in the illustrated system, and such further applications of the principles of the disclosure as would normally occur to those skilled in the art are to be construed as being within the scope of the present disclosure.

The terms “comprises”, “comprising”, or any other variations thereof, are intended to cover a nonexclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such a process or method. Similarly, one or more devices or subsystems or elements or structures or components preceded by "comprises... a" does not, without more constraints, preclude the existence of other devices, sub-systems, elements, structures, components, additional devices, additional subsystems, additional elements, additional structures or additional components. Appearances of the phrase "in an embodiment", "in another embodiment" and similar language throughout this specification may, but not necessarily do, all refer to the same embodiment.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. The system, methods, and examples provided herein are only illustrative and not intended to be limiting.

In the following specification and the claims, reference will be made to a number of terms, which shall be defined to have the following meanings. The singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.

Embodiments of the present disclosure relate to a device for phenotypic detection of antimicrobial resistance to drugs using paper microfluidics. The device includes a top layer enclosed within a casing. The top layer is adapted with a plurality of reaction zones on a porous membrane. The top layer is adapted to receive a predetermined volume of a sample liquid composed of a plurality of pathogens on the plurality of reaction zones. The top layer is further adapted to concentrate the plurality of pathogens on the plurality of reaction zones by pulling the sample liquid through the plurality of reaction zones into an absorber positioned beneath the top layer via a driving force. The device includes a lid positioned on the top layer. The lid is configured with a plurality of reagent storage zones that aggregates to the plurality of reaction zones in the top layer. Each of the plurality of reagent storage zones in the lid includes a mixture of antibiotics, culture media, and colorimetric dyes. Further, the top layer (120) and the lid (136) allow the plurality of pathogens (128) to incubate for a predefined time period and at a predetermined temperature thereby treating the plurality of pathogens (128) to the one or more antibiotics and detection reagents to yield a colorimetric output (132) wherein the colorimetric output (132) signifies if the sample liquid (126) exhibits resistance against the one or more antibiotics.

FIG. 1 is a schematic representation of a workflow of a device (100) for phenotypic detection of antimicrobial resistance to drugs using paper microfluidics in accordance with an embodiment of the present disclosure. In one embodiment, the device (100) is adapted to the size of a credit card. It will be appreciated to those skilled in the art that the device (100) may also be adapted to a suitable dimension and shape that facilitates the detection of antimicrobial resistance to drugs.

The device (100) includes a top layer (120) enclosed within a casing. The top layer (120) is adapted with a plurality of reaction zones on a porous membrane. In an embodiment, the plurality of reaction zones (122) are composed of a nitrocellulose membrane or a filter paper as the top layer (120). The plurality of reaction zones (122) includes culture media, antibiotics, certain salts, and buffers appropriate to the reaction. The one or more detection reagents are dried on the plurality of the reaction zones. The examples for detection reagents includes, but are not limited to, Mean Transmit Time (MTT), Resazurin, WST-8, or the like. The plurality of reaction zones (122) includes, but not limited to, a no-antibiotic control as a positive control for microbial growth, a nicotinamide adenine dinucleotide phosphate (NADPH) control as a positive control for metabolic reagent activity, and a hydrogen peroxide control as a negative control for microbial growth. Further, the top layer (120) is adapted to receive a predetermined volume of a sample liquid (126) composed of a plurality of pathogens (128) on the plurality of reaction zones. The plurality of pathogens (128) are concentrated on the plurality of reaction zones by pulling the sample liquid (126) through the plurality of reaction zones (122) into an absorber (134) positioned beneath the top layer (120) via a driving force. The driving force (130) for the sample liquid (126) is achieved by at least one of a capillary force, centrifugal force, and gravity. The driving force (130) allows the sample liquid (126) containing the pathogens to be drawn and distributed across the porous membrane, thereby receiving the plurality of pathogens (128) on the plurality of zones. The size of the porous membrane is smaller than that of a bacterium, ensuring efficient capture and concentration of the plurality of pathogens (128). In one embodiment, the predetermined volume of the sample liquid (126) is approximately 5 mL.

In a preferred embodiment, the sample liquid (126) may be a urine sample of the patient, as the patient is suspected to be infected with the plurality of pathogens (128). Examples for the plurality of pathogens (128) include but are not limited to Escherichia coli, Staphylococcus aureus, influenza viruses, Plasmodium falciparum and the like. Urine, commonly used in medical diagnostics, contains various pathogens and indicators of antimicrobial resistance. Further, the top layer (120) is adapted to concentrate the plurality of pathogens (128) on the porous membrane.

Furthermore, the top layer (120) is adapted to allow the plurality of pathogens (128) to incubate for a predefined time and at a predetermined temperature. During this incubation period, the plurality of pathogens (128) is exposed to one or more antibiotics and detection reagents present in the reaction zones that results in a colorimetric output (132). The colorimetric output (132) signifies if the sample liquid (126) exhibits resistance against the one or more antibiotics. The one or more detection reagents are used to test the antimicrobial resistance of the plurality of pathogens (128). The colorimetric output (132) indicates a color change of the plurality of reaction zones (122) upon completion of the incubation period.

In an embodiment, various combinations of membranes can be used to introduce the reagents into the reaction zone. Further, the plurality of reaction zones (122) are placed within a plastic casing, wherein the plurality of reaction zones (122) are separated from each other.

In a specific embodiment, the device (100) may incorporate additional accessories such as a pump, syringe, vacuum chamber, or similar components to accelerate sample fluid flow. Further, the device (100) includes an absorber (134) positioned beneath the top layer (120). Typically, the absorber (134) is a wi eking pad. In one embodiment, the wicking pad may be made of a porous material such as, without limitations, cellulose fibers (CFSP), nitrocellulose, paper, silica, cotton, glass (for example, glass fiber), or synthetic material (for example, polyester, polyethylene, polymers, rayon, nylon and the like). The absorber (134) is adapted to absorb the sample liquid (126) that drops from the plurality of zones. Furthermore, the device (100) includes a lid (136) positioned over the top layer (120). The lid (136) is adapted to seal the top layer (120) during the incubation to prevent evaporation and exposure of the plurality of zones to the atmosphere.

It must be noted that the lid (136) is configured with a plurality of reagent storage zones that aggregates to the plurality of reaction zones (122) in the top layer (120). Each of the plurality of reagent storage zones in the lid (136) comprises a mixture of antibiotics, culture media, and colorimetric dyes. Further, the mixture of antibiotics comprises of distinct concentrations pertaining to a same antibiotic. For instance, antibiotic ‘A’ is deposited with a volume of 5mg, lOmg and 15mg across the plurality of reaction zones (122).

The incubation process typically lasts approximately for 45 minutes at a temperature of 37°C. Upon completion of the incubation process, the plurality of pathogens (128) in the filter paper membrane is prepared for phenotypic or genotypic detection. In one embodiment, the phenotypic detection includes using bacterial live or dead assays to concentrate the plurality of pathogens (128) over the filter paper membrane. It must be noted that several other qualitative and quantitative insights are drawn from the phenotypic detection. Examples of such qualitative and quantitative insights include, but are not limited to, viability, the total number of pathogens, presence or absence of drug resistance. The membranes may also be used directly for Polymerase Chain Reaction (PCR), Loop-mediated Isothermal Amplification (LAMP), Recombinase Polymerase Amplification (RPA), and other genotypic detection techniques. Typically, the occurrence of metabolic activity is reported by the detection reagents as a color change in the plurality of reaction zones (122) upon completion of the incubation period. Darkened reaction zones indicate a positive test (210), indicating the presence of the resistance against the one or more antibiotic, while lighter reaction zones indicate a negative test (212), indicating the absence of the resistance against the one or more antibiotic. In one embodiment, the device (100) includes a positive control zone (214) and a negative control zone (216). The positive control zone (214) always shows a color change, indicating that the reaction between the pathogens and the detection reagents is working properly. The negative control zone (216) remains lighter, indicating no bacterial growth.

FIG. 2(a) - FIG. 2(d) are schematic representations of an exemplary embodiment of anti-microbial resistance detection in accordance with an embodiment of the present disclosure. A predetermined volume of a sample liquid (126) containing a plurality of pathogens (128) is added to the top layer (120) of the device (100). This can be achieved by using a microfluidic distributor or other means to dispense the sample onto the plurality of zones. The porous membrane allows the pathogens in the sample liquid (126) to concentrate onto its surface, effectively capturing them (shown in FIG 2(a)). Further, the device (100) allows the microbes on the membrane to incubate for 45 Minutes at 37° C. This allows the drugs or biologies to interact with the microbes and elicit a response. To prevent evaporative losses and exposure to the ambient atmosphere during incubation, the device (100) is equipped with a lid (136) for further sealing (Shown in FIG. 2(b)). After the incubation period, the reaction between the pathogens and the detection reagents leads to a color change in the reaction zones. This color change serves as a visual indication of the presence or absence of drug resistance. The colorimetric output (132) can be interpreted to determine if the sample liquid (126) exhibits resistance against the antibiotics (shown in FIG. 2(c)). The result interpretation is shown in FIG. 2(d). The result interpretation may be similar to the one explained in FIG. 1. Darkened reaction zones indicate a positive test (210), indicating the presence of the resistance against the one or more antibiotic, while lighter reaction zones indicate a negative test (212), indicating the absence of the resistance against the one or more antibiotic. The device (100) includes a positive control zone (214) and a negative control zone (216). The positive control zone (214) always shows a color change, indicating that the reaction between the pathogens and the detection reagents is working properly. The negative control zone (216) remains lighter, indicating no bacterial growth.

FIG. 3 is a schematic representation of 2-D paper network for single-step detection in accordance with an embodiment of the present disclosure. In one embodiment, a 2D paper network for a single- step concentration of bacteria, antibiotic treatment and assessment of antimicrobial resistance may be achieved. A reservoir (136) includes the sample containing the microbe to be tested for drug resistance. The sample reservoir could be a simple solid structure that holds the fluid. The fluid is introduced to a membrane such as a glass fiber membrane (GF) (138). The fluid wicks through the glass fiber membrane (138) and the wicked fluid, are introduced to filter paper (140) vertically. Further, the fluid wicks into the wicking pad (134). Parallelly, the fluid in filter paper moves horizontally to the zone marked by a specific color, for example, green, wherein sugar (142) is deposited on filter paper. The sugar (142) dissolves after some duration upon contact with an aqueous sample, to allow the fluid then to move further horizontally. As the sugar (142) gets dissolved the aqueous sample further dissolves the dried reagents on to the dark region (144). Herein the components important for the assay such as antibiotics, culture medium, and antimicrobial resistance assay (MTT, WST-8) are dried. The dissolved components further diffuse into the filter paper (140) wherein the microbes reacts with the components. An incubation step may be included at this point at which the components react with the microbes to produce a color change for a test result. The result interpretation may be similar to the one explained in FIG 1 and FIG.2(d). In one embodiment, if the filter paper membrane (138) turns green, the sample is considered negative. Likewise, if the filter paper membrane (138) turns orange, then the sample is considered positive.

FIG. 4 illustrates a flow chart representing the steps involved in a method for phenotypic detection of antimicrobial resistance to drugs using paper microfluidics in accordance with an embodiment of the present disclosure.

The method (300) includes receiving, by a plurality of zones, a predetermined volume of a sample liquid composed of a plurality of pathogens on the plurality of zones via a driving force in step 310. The driving force for the sample liquid is achieved by at least one of a capillary force, centrifugal force and gravity to enable the sample liquid to flow and distribute across the plurality of zones.

In one embodiment, the sample liquid may be a urine sample of the patient, as the patient is suspected to be infected with the plurality of pathogens. Examples for the plurality of pathogens include but are not limited to Escherichia coli, Staphylococcus aureus, influenza viruses, Plasmodium falciparum and the like. The method (300) includes concentrating the plurality of pathogens on the plurality of reaction zones by pulling the sample liquid through the plurality of reaction zones into an absorber positioned beneath the top layer via a driving force in step 320. The porous membrane acts as a filter, allowing the plurality of pathogens to be captured and retained on its surface, effectively concentrating them in one location.

The method (300) includes sealing, by a lid, the top layer during the incubation with a lid comprising of a plurality of reagent storage zones that aggregates to the plurality of reaction zones in the top layer, wherein each of the plurality of reagent storage zones in the lid comprises a mixture of antibiotics, culture media, and colorimetric dyes in step 330. This lid effectively seals the device, creating a controlled incubation chamber where the plurality of pathogens and the detection reagents can interact without interference. The lid stores different substances in dry form. These substances include drugs, antibiotics, buffers and culture reagents. They are arranged in specific zones that match the reaction zones in a membrane below the lid. When the lid is closed, the dry substances comes in contact with the moist zones and dissolve and flow down to the reaction zones, where they interact with the sample.

The method (300) includes allowing, by the plurality of zones, the plurality of pathogens to incubate for a predefined time and at a predetermined temperature thereby treating the plurality of pathogens to the one or more antibiotics and detection reagents to yield a colorimetric output wherein the colorimetric output signifies if the sample liquid exhibits resistance against the one or more antibiotics in step 340. This output provides providing valuable information about the viability of the pathogens and their response to the drugs or the antibiotics.

Various embodiments of the device and method for phenotypic detection of antimicrobial resistance to drugs using paper microfluidics as described above provide a rapid, low-cost, point- of-care diagnostic device for phenotypic detection of microbes exhibiting resistance against drugs and other biologies The diagnostic process takes less than one hour, ensuring quick turnaround times and accurate results, thereby allowing healthcare professionals to promptly identify the type of infection and determine the need for antibiotics without significant delays. Moreover, the device is designed to be user-friendly, with simple operation that even individuals as young as children less than 10 years old can use effectively. This eliminates the requirement for highly skilled personnel, simplifies the diagnostic process, and enhances access to accurate testing. The use of paper-based microfluidics contributes to the affordability and accessibility of the device, making it an ideal solution for healthcare facilities with limited resources.

It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the disclosure and are not intended to be restrictive thereof.

While specific language has been used to describe the disclosure, any limitations arising on account of the same are not intended. As would be apparent to a person skilled in the art, various working modifications may be made to the method in order to implement the inventive concept as taught herein.

The figures and the foregoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, the order of processes described herein may be changed and are not limited to the manner described herein.

Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts need to be necessarily performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples.