WO2024218254A1

WO2024218254A1 - Method and device for breath pathogen detection

Info

Publication number: WO2024218254A1
Application number: PCT/EP2024/060631
Authority: WO
Inventors: Douglas T. Gjerde; Daniel Bollinger
Original assignee: Vosbio, Inc.
Priority date: 2023-04-21
Filing date: 2024-04-18
Publication date: 2024-10-24

Abstract

In the device and method of the invention, pathogens can be detected in the exhaled breath of a user Breath is blown into a capillary or tube breath condensate collector and the breath condensate is deposited as a film and/or droplets on the collector inner surface. The breath collection time may be 1 sec to 60 sec. Detection reagents are introduced into the breath condensate collection device to mix and effectively collect and recover the small volume sample and prepare the sample for detection. Detection reagent liquids combine with the breath film sample to recover the collected sample for pathogen detection. In some embodiments the capillary or tube collection surface may comprise a portion of dried or lyophilized reagents for detection. The pathogen is a virus, spore, bacterium, cell, or other entity. The collected sample is recovered and mixed with a liquid detection reagent to form a mixture. RNA, DNA, protein, or another molecule related to the pathogen is detected. Detection can be performed inside the collection chamber or detection can be performed after transferring the mixture from the collection chamber to a detection chamber. Pathogen amplification may be performed before detection isothermally or by thermal cycling.

Description

Method and Device for Breath Pathogen Detection

Field of the Invention

The present invention relates to devices and methods for capturing and detecting pathogens from exhaled breath.

Background of the Invention

The detection of pathogens in breath exhaled from individuals can be difficult. While the concentration of pathogens in samples such as saliva, blood, feces, and nostril and nasopharyngeal and oropharyngeal collected swab is high in infected individuals, the concentration of pathogens exhaled from these infected individuals is very low. Nevertheless, respiratory infections can be spread from an infected individual to other individuals by pathogens found in exhaled breath.

For example, an individual exhaling shed virus at the rate of 60 viruses per minute will, on average, shed and exhale only 60 viruses in one minute and, on average, only 1 virus per second. It can be difficult to recover these low numbers of viruses collected in breath.

Even if the viruses are recovered, additional manipulation of the collected virus sample will cause degradation and loss of nucleic acid, especially RNA. Finally, it is difficult to detect low copies of pathogens even after amplification.

False positives for low concentration of sample provides another barrier to accurate detection of low numbers of pathogens. In amplification technologies, all samples will eventually amplify, whether the target nucleic acid is present or not, perhaps due to primer mismatching or other mechanisms. This appears to be true especially for LAMP and other isothermal amplification methods, but also is true for PCR. But any type of detection method including sensors and other reactions have this issue.

There exists a need to simplify and improve the detection and measurement of pathogens in exhaled breath through improved recovery of exhaled pathogens that have been collected. This includes improving and simplifying exhaled breath sample capture, sample processing and the detection process.

Summary of the Invention

In this invention, the method and device are designed to collect and process very small, exhaled breath condensate sample liquid volumes. It is counter intuitive to try collect only small breath condensate liquid volumes. One reason is that it is difficult to recover or coalesce and process small volumes of liquids. A second reason small breath condensate liquid sample volumes may contain pathogens that are not detectable pathogens because too few have been collected and cannot be detected directly or amplified and detected. Without efficient sample processing, pathogens may not be detectable even if the individual contributing the exhaled breath sample is infectious and exhaling pathogens. Or detection may be close to the detection limit and difficult to determine if a positive signal is true or false. The device and method of this invention are able to recover, process and detect pathogens that have been captured in a small capture volume amount. In some embodiments of the invention this includes liquid films deposited on the capture surface.

In some embodiments of the invention, small volumes of sample liquid are collected in a capillary or small tube. The collector may be cooled on the outside. In the device and method of the invention, breath is introduced into a collector, and breath at least a portion of breath liquid particles and vapor is deposed as a film on the surface. In the device and method of the invention, the breath sample may be collected as a film deposited on a surface or a film and droplet combination deposited on a surface. In some embodiments, at least a portion of the collected breath liquid/frost film is comprised of coalesced droplets. The amount of liquid collected may be very small and may be difficult to recover. In some embodiments of the invention, the total liquid volume of the film and possible droplets collected may be less than 100 pL, less than 50 pL, less than 20 pL, less than 10 pL, less than 5 pL or less than 1 pL.

Recovery, manipulation, and transfer of collected breath liquid sample, especially small amounts of liquid, is difficult. In the device and method of the invention all or most of the captured liquid is recovered and processed for possible detection of pathogen detection. In some embodiments of the invention, a liquid containing detection reagents is introduced into the capillary or tube collection structure, where it is combined and mixed with the breath film to form a mixture of the entire breath sample and detection reagents. The detection reagents are introduced into the capillary or small tube collection device to mix and effectively collect and recover the small volume sample and prepare the sample for detection. In some embodiments of the invention, the detection mixture may contain lysing reagent. In some embodiments of the invention, the mixture may be removed and processed for detection. In some embodiments of the invention, the collection capillary or tube chamber is the detection chamber. In other embodiments, the sample detection reagent mixture is transferred to a separate chamber prior to the detection step. In some embodiments of the inventions, the mixture may be heated, either isothermally or by thermal cycling in the collection structure to amplify nucleic acid. In some embodiments of the invention, the amplified nucleic acid may be detected and measured in the capillary or tube collection structure. In some embodiments of the invention, the mixing solvent may contain an amplification or detection mixture and/or may contain a nucleic acid transcription and/or amplification master mix.

In some embodiments of the invention, the amplification method is isothermal. In some embodiments, the isothermal amplification is LAMP or RPA. In some embodiments, the isothermal amplification is Loop-Mediated Isothermal Amplification (LAMP), Whole Genome Amplification (WGA), Strand Displacement Amplification (SDA), Helicase-Dependent Amplification (HDA), Recombinase Polymerase Amplification (RPA), or Nucleic Acid Sequences Based Amplification (NASBA). In some embodiments of the invention, the detection process is by PCR or qPCR. In some embodiments the detection is by a sensor. This can be done with or without amplification.

Brief description of the Figures

Embodiments of the invention are discussed in more detail below with reference to the accompanying drawings, in which:

Figure 1 shows a multi-capillary or small tube device for capturing breath condensate liquid;

Figure 2 illustrates how captured breath condensate liquid in the capillary or tube is mixed with the detection reagents in the capillary or tube so that detection may be performed in the capillary or small tube; and

Figure 3 shows a multi-capillary/tube device comprised of one or more capillary or tubes for capturing breath condensate liquid. The capillary/tube serves for multiplex detection and positive or negative controls for pathogen measurement in exhaled breath.

Definitions

Pathogens: For the purpose of this invention, pathogens include any type of analyte that can be exhaled from breath that is treated with a detection reagent to optionally amplify and detect the analyte. This includes cells including cancer cells, fungi, virus, and bacteria.

Breath film: Breath condensate containing liquid, or frost condensed from exhaled breath deposited on a collection surface. Film is comprised of a thin layer of liquid or frost and droplets. The terms breath film, breath liquid, breath frost, breath droplets and similar terms are used interchangeably in this invention.

Breath liquid: Liquid condensate deposited in a capture collector device. The terms breath film, breath liquid, breath frost, breath droplets and similar terms are used interchangeably in this invention. Breath condensate collector: A device used for capture, recovery, and detection. Any collection structure used to first capture breath sample condensate liquid and then recover the sample liquid through the addition of detection reagents to consolidate the sample and detection reagents liquid. Then the mixed sample volume in the collection structure is processed to detect pathogens.

Capture and recover structure: Any capillary or tube collection structure that is used to first capture breath sample condensate liquid and then recover the sample liquid through the addition of detection reagents to construct and consolidate sample liquid into a mixture. Optionally the recovered sample liquid and detection reagent mixture is further moved to a detection pathogen chamber.

Collection device: In the device of the invention, capillary or tube collection device is a device that captures exhaled breath condensate liquid.

Collection capillary or tube: Capillary or tube in which breath film condensate is collected. The capture tube may be any material including glass, metal, ceramic, or plastic. The capillary may be glass. The capture tube may be a small syringe. The terms collection capillary, capillary, capture tube and tube are used interchangeably in this application.

Capillary: The terms capillary and tube are used interchangeably in this invention. Detection may be performed after the sample detection reagent mixture is formed in the capillary or tube. Any suitable detection method may be used.

Tube: The terms tube and capillary are used interchangeably in this invention. Detection may be performed after the sample detection reagent mixture is formed in the capillary or tube. Any suitable detection method may be used.

Sample time: The time in which exhaled breath is introduced to the device.

Template control amplification: Non pathogen positive control to confirm detection reagents are active and working. The process confirms that the positive detection of a pathogen that occurs if the pathogen is present.

Recovered sample: Sample liquid that has been mixed with a liquid detection reagent. Recovered sample liquid can be manipulated and processed.

Recovery reagent: Liquid detection reagent or part of a detection reagent introduced into a capture collection device of the invention to mix and recover breath condensate liquid. Mixing and recovery reagent: The liquid portion of a detection reagent is added to a collection device of the invention to mix with collected exhaled breath film and droplets and recover the sample into a larger volume mixture.

Detection reagents: Anything needed or used for detection of a pathogen. Detection reagents are generally liquid as used in this invention for the mixing recovery of sample in the collection device. This could include or be comprised of lysing, transcription, amplification, hybridization, primer and tagging reagent(s). Detection reagents may be added separately or in a group in any order required by the method. In this invention, detection reagents are used to mix breath sample liquid to recover the sample for pathogen detection. In the invention, liquid detection reagents are used to mix and recover sample liquid. Detection reagents that are lyophilized and solid are not used for recovery and mixing in the device and method of the invention but may be used for detection processing.

Lysing reagent: Reagent that lyses cells or envelops cells or causes release of cellular materials including nucleic acid. A lysing reagent may be added separately or with other detection reagents.

Reverse transcription reagent. Reagent that transcribes RNA into DNA. A reverse transcription reagent may be added separately or with other detection reagents.

In collector detection: Detection is performed in the capillary where the breath film is captured and to which liquid detection reagents had been added to mix and recover breath film into an analyzable mixture.

Detection in separate chamber: Exhaled breath sample liquid is collected and then mixed with liquid detection reagents in the collector where the breath film is captured to recover breath condensate and detection reagents into a volume of liquid. The liquid is transferred to another chamber to perform detection of pathogens present in exhaled breath.

Detailed Description of the Invention

The device and method of this invention are able to collect, process and detect pathogens present in a film or droplets on a surface or in very low capture volume amount. The device and method of this invention are able to collect, process and detect pathogens present even if very few pathogen entities have been captured and/or the total capture liquid volume is very small. In an embodiment of the invention, the collection device is one or more capillaries or tubes. Film and/or droplets are collected by optional cooling of the capture surface or collector surface. The amount of liquid collected from the breath sample is in the 0.01 - 20 pL range, 0.05 - 10 pL range, 0.01 - 10 pL range or the 0.01 - 5 pL range. In some embodiments, the amount of liquid collected from the breath sample is larger and in the 0.01 - 100 pL range, 0.1 - 100 pL range, 0.1 - 100 pL range, 0.02 - 50 pL range. In some embodiments, the amount of liquid collected from the breath is less than 100 pL, less than 50 pL, less than 25 pL, less than 10 pL, less than 5 pL or less than 1 pL.

In the invention, a user exhales into a breath condensate collector. The captured breath liquid film is deposited or spread over a collection surface or inner surface and cannot be manipulated to coalesce into a single definable, movable liquid volume. In the device and method of the invention, the collected exhaled breath condensate sample may be a film, or some droplets deposited in the collection chamber on the inner surface of the collector. The collected sample may be primarily a film with some droplets collected on a surface.

Collected film is difficult to coalesce. Collected droplets may be difficult to coalesce. Due to the collector diameter, internal space, or shape, it may be difficult to introduce a scraper or plunger. The breath collector shape and configuration may not be suitable to place in a centrifuge. But even using a plunger, scraper, or a centrifuge, with sample liquid volumes less than 100 pL, it is likely that at least part of the sample liquid volume is not recovered and is left behind in the collection device.

In some embodiments of the invention the capillary or tube is 0.1 mm, 0.3 mm, 0.5 mm, 0.75 mm, 1 mm, 2 mm, 3 mm, 4mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 12 mm, 16 mm, 20 mm or more. In some embodiments of the invention, the collection capillary or tube diameter is in the range of 0.5 - 2 mm. In some embodiments of the invention, the collection capillary tube diameter is in the range of 1 - 5 mm.

In some embodiments of the invention, vacuum, capillary action, gravity, or pressure may be used to introduce detection reagents into the capillary or tube of the invention. In some embodiments of the invention, a plunger may be used to pull detection reagents into the capillary tube of the invention.

In some embodiments of the invention, after filling or introducing the detection reagents, the capillary or tube may be capped. In some embodiments of the invention, the capillary may not be capped after introduction of the detection reagents. After introduction of the detection reagents, in some embodiments, the capillary or tube may be inverted and introduced into a heater open or capped side up. In some embodiments, both sides of the capillary or tube are capped or sealed when placed in a heating application device. In some embodiments of the invention, capping, sealing, or closing either or both the capillary or tube ends may be performed by a plunger. In some embodiments of the invention, capping, sealing, or closing either or both the capillary or tube ends may be performed by a slip-on cap or slip inside cap or by a plastic or wax sealant. In the process and device of the invention, detection reagents in liquid form are introduced into the collection device to create a sample detection reagent mixture. Sample liquid is not removed from the collection capillary or tube device before the detection reagent liquid is mixed with the sample. The collection device is configured so that liquid detection reagents can be introduced into the collection device and mix the breath condensate film to process the collected sample in a single liquid volume comprised of sample and detection reagents. In some embodiments of the invention, detection reagents may be introduced into the collection chamber by depositing, gravity, capillary action, vacuum, or pressure.

In some embodiments of the invention, the breath sample is collected in one or more capillaries. In some embodiments of the invention, the capillaries are 10 to 200 pL volume. In some embodiments of the invention, small volume capillaries are used in parallel. In some embodiments of the invention, the capillaries are glass, metal, or plastic. In some embodiments of the invention, one or more plastic or glass syringes are used to collect sample. In some embodiments of the invention, the capillary or tube syringes are in the volume range of 0.1 mL to 5 mL.

The volume of the combined sample liquid and detection reagent liquid mixture are large enough to manipulate the combined liquid volume. In practice this volume can be defined as the volume of liquid detection reagent. In some embodiments of the invention, the volume of detection reagents that is suitable for mixing and recovering the breath sample is 5% or 10% or more of collector chamber volume. In some embodiments of the invention, the volume of the detection reagent liquid is 20%, 30%, 40%, 50%, 75% or 100% of the collection chamber volume. Low volume amounts of detection reagent may not be enough to collect the breath sample film and/or droplets to consolidate into a combined sample detection reagent volume. The volume of detection reagents used to recover the sample can be a function of the collection chamber volume. In some embodiments of the invention, the detection regent volume is equal or greater than the collection chamber volume. In some embodiments of the invention, the detection reagent volume is less than the volume of the collection chamber. In some embodiments of the invention the detection reagent volume is in the range 1-100% of the collection capillary or tube chamber volume. In some embodiments of the invention the detection reagent volume is in the range 1- 50% of the collection chamber volume. In some embodiments of the invention the detection reagent volume is in the range 1-25% of the collection chamber volume. In some embodiments of the invention the detection reagent volume is in the range 1-10% of the collection chamber volume. Adding the detection reagent to the sample allows complete recovery of the sample regardless of the volume of breath sample liquid that was collected. Adding the detection reagent to the sample allows complete processing of recovered the sample liquid regardless of the volume of sample liquid that was collected. Sample processing includes detection of a pathogen if present. In some embodiments the collector of the invention is a capillary or tube. In some embodiments of the invention, dried or lyophilized detection reagents or standards may be contained in the collection device where breath film is captured. In this case, buffer or water may be added to the collected sample and reagents and mixed. In some embodiments, the entire sample film is collected with lyophilized reagents and is processed.

The device and method of the invention is intended to collect breath condensate liquid, add, and mix detection reagents in the collection capillary or tube device with the collected sample. The mixed sample is processed to detect any pathogens that may have been exhaled from the breath sample.

In some embodiments of the invention, pathogens are optionally amplified and detected in the collection device. In some embodiments of the invention, the mixed sample and detection reagents are deposited or transferred, and the pathogen detection is performed in a separate detection chamber.

Exhaled respiratory pathogens are typically located in the breath condensate of exhaled shed breath. Breath condensate should be collected in order to collect the pathogen or virus. The sampling times of the device and method of the invention can be short while still collecting detectable pathogens. This is because with the device and method of the invention, even very small sample liquid volumes can now be recovered and processed. The sampling times for the collection device of the invention can be up to 1 second or more. Sample collection times may be up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or 30 seconds. Sample collection times may be 60 seconds or less. Sample collection times may be up to 60 seconds, up to 120 seconds or more. Sampling times may be in the range of 1 second to 30 seconds or in the range of 2 seconds to 15 seconds, the range of 1 second to 45 seconds, the range of 1 second to 60 seconds, or in the range of 1 to 10 seconds.

In some embodiments of the invention, breath liquid collection is into a capillary or tube by exhaling breath and blowing through a capillary or tube. In some embodiments of the invention, the outside of the capillary or tube is cooled. Breath liquid condensate is collected on the inside wall of the collector. In some embodiments, the collected breath liquid may be a film. In some embodiments, the collected breath liquid is frost film that melts to film or droplets. In some embodiments, at least a portion of the collected breath liquid/frost film is comprised of coalesced droplets.

In some embodiments of the invention a yes/no statement on whether an individual is shedding and exhaling a pathogen such as a virus relies on having collected enough pathogens to be detectable if they are present. Because of the efficient recovery through reagent mixing of the device and method of the invention, even very small numbers of pathogens can be detected. In some embodiments of the invention a thin film of liquid is collected. However, this is only a portion of the liquid collected that is contained in the exhaled breath sample. It is preferred to capture as much of the liquid condensate from the breath as possible during the sampling time. However, in some embodiments of the invention, 1 - 2 % of the pathogens are collected, recovered, and processed. In some embodiments of the invention, 0.1 - 1%, 1 - 5%, 0.5 - 10% and 1 - 10% of the pathogens are collected, recovered, and processed. In some embodiments of the invention, up to 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to 100% of the exhaled breath pathogens are collected from the total breath sample passing through the device.

In some embodiments of the invention a minimum pathogen shed rate exhaled from an individual can be calculated. In some embodiments of the invention a minimum pathogen shed rate can be calculated. For example, if a single virus or pathogen is detected from 5 seconds of sampling this indicates 12 virus/min or greater pathogen shed rate. If a 10 virus or pathogen is detected in 1 second of sample this indicates 600 virus or pathogen per minute or greater shed rate. The calculation may include the estimated pathogen collection and recovery efficiency of the device and method. Since the device and method may not collect all of the breath condensate liquid from exhaled breath and therefore may not collect all of the pathogens exhaled, the detection shed rate calculated may only be a minimum pathogen shed rate.

Collection of breath condensate can be monitored and measured by measurement of the film on the collection surface. A clear tube becomes cloudy or opaque as the breath condensate is deposited. This can be measured by an optical device. A minimum amount of film measured can indicate that sufficient sample has been collected. The amount of liquid collected may be less than 1 pL, less than 5 pL or only a few pL. After collection, a detection reagent may be added directly to the amplification capillary tube, sometimes by capillary action, to mix the collected sample with the detection reagents.

Existing breath condensate capture devices are designed to capture all or nearly all of the breath condensate. However, in cases where only a yes or no answer is desired on whether an individual is shedding and exhaling a pathogen, partial capture is acceptable. Another characteristic of the device and method of the invention is the ability to process the entire sample captured. This increases the ability to detect pathogens if they are present.

In some embodiments of the invention the collection chamber and the recovery/mixing chamber is also the detection chamber. Detection is performed in the capillary where the breath film is captured and to which liquid detection reagents had been added to mx and recover breath film into an analyzable mixture in the collection chamber.

In some embodiments of the invention, detection of the pathogens is in separate chamber: Exhaled breath sample liquid is collected and then mixed with liquid detection reagents in the collector where the breath film is captured to recover breath condensate and detection reagents into a volume of liquid. The liquid is transferred to another chamber to perform detection of pathogens present in exhaled breath.

In some embodiments of the invention the pathogen is a virus. In some embodiments of the invention the pathogen is a spore. In some embodiments of the invention the pathogen is bacteria. In some embodiments of the invention the pathogen is a cell. In some embodiments of the invention, the pathogen is cancer cell analyte.

There may be variations of the configuration of the collection capillary or tube. The collection I amplification chamber may be small and have low surface area and volume. A small collection chamber can allow complete manipulation of the recovered sample for in situ detection. There may be thermal control of the wall for breath film collection or to control liquid temperature and may detect the pathogen or virus in situ. Thermal control of the capillary tube may be used to control the liquid temperature of the amplification mixture.

In some embodiments of the invention, the collection device is a capillary or tube. Figure 1 depicts how breath film condensate may be collected. An individual 10 may exhale breath 12 into a capillary or tube 14. Breath 12 exits the capillary or tube and leaves breath condensate liquid inside the capillary or tube. In some embodiments of the invention, the outside of the capillary or tube may be cooled. In some embodiments of the invention, the collection surface area is less than 20 cm², less than 15 cm², less than 10 cm², less than 8 cm², less than 7 cm², less than 7 cm², less than 5 cm², less than 4 cm², less than 2 cm², or less than 1 cm². In some embodiments of the invention, one or more capillaries are used for collection with surface areas of the individual capillary of less than 1 cm². Detection reagents are added after collection and the mixture is processed and detected.

Parallel operation may be performed. Operation of different samples can start and stop independent of each other. Results may be reported by graph, number, chart, work, or color. Isothermal monitoring may be performed via light absorption, light emission, heat, or cloudiness or may be performed with any type of transducer specific for pathogen or product of pathogen.

The capillary tube may be a single capillary tube, a few capillary tubes held in parallel, or several capillary tubes held in parallel. Detection may be in situ. Detection may be by Isothermal reactor with real time addressable, independent channels. The reactors may be run in parallel with serial operation. Parallel operation may be of different samples that start and stop independently of each other. Capillaries may be arranged in parallel and processed in parallel. Results may be reported by graph, number, chart, work, or color. Isothermal monitoring may be performed via light absorption, light emission, heat, or cloudiness or may be performed with any type of transducer specific for pathogen or product of pathogen.

In some embodiments of the invention, the breath collection chamber or surface may be shaped as a tube or chamber that allows addition and mixing of the detection reagent mixture with breath condensate film and/or droplets. Collected liquid volumes that are low and are difficult to coalesce and collect are easily captured into manipulable volumes of liquid by the mixing mixture.

Figure 2 shows the use of this type of tube and how detection reagents may be introduced into a capillary or tube to consolidate and collect the sample containing the pathogen. Reagents 24 from LAMP/PCR vial 22 are introduced into capillary 20 by vacuum. Vacuum can be introduced by plunger 26 or externally. The capillary serves the functions of breath collection, mixing detection reagents and optionally amplification and detection.

Figure 3 shows a multiple capillary device 30 for capturing breath condensate film from exhaled breath 32. By splitting the entire sample into segments, some capillaries may contain the pathogen target and will be detected. Other capillaries may contain no pathogen. Because a very small sample volume can be collected and utilized, it is possible to directly digitize from the point of breath sampling. Essentially, a breath condensate sample is collected into a capillary array. Then the master mix is drawn into the array. The array is inserted into an instrument for amplification detection where each capillary is detected individually. Using the ratio and the sampling time, the exhaled shed rate of an individual can be measured.

In other methods, multiplexing of several diseases can be performed in parallel, each capillary detecting a specified disease. In addition, a separate capillary may serve as a positive control in exhaled breath matrix having a plasmid or other internal positive control standard deposited and dried inside the capillary prior to breath introduction. In one example, two clear glass capillaries having dimensions of 1 mm inside diameter and 129 mm long are fitted with two mouth pieces made of 200 pL pipette tips. Breath is exhaled through the two parallel capillaries for 1 - 5 seconds. The capillaries were tested several times. Liquid film formed almost immediately inside the capillary. The depth and amount of film could be measured with a light transmission device. The film deposition formed first on the last 30% of the capillary, but with continued breath introduction film formed almost to the intake of the capillary. The capture was performed at room temperature, approximately 70 degrees Fahrenheit. Then, a detection mixture is drawn into the two capillaries, the bottoms are capped, and the capillaries are inserted into a LAMP detection instrument. After several minutes, the positive control amplifies in a separate capillary and is detected. The sample capillary will amplify if virus is detected in 10 - 20 minutes. The sample capillary is said to be pathogen free if it does not amplify and detect virus in 30 minutes or more.

The capillary array collection may be detected after isothermal or, thermal cycling treatment of sample mixed with detection reagents. The detection reagent may contain reagents for the lysis step, RT step, isothermal amplification, or other amplification or detection. After collection the single capillary or array of capillaries or tubes may be filled with detection reagents by a self-filling operation or by gravity or vacuum. Vacuum may be introduced externally or with a plunger. After filling, the ends may be covered. The number of chambers may be in the range of 10 - 5,000,000 or in the range of 10 - 10,000 or 10 - 1,000. The number of arrays may be 2 - 5, 2 - 10, 5 - 100, 5 - 50, 10 - 100 or in the range of 10 - 1 ,000.

The detectable signal in a technology is based on the signal to noise ratio of the technology. The background signal or noise will limit the ability to detect a positive signal. If an amplification process is used to detect, no-template negative controls can be used.

Additional manipulation of the sample can result in degradation and loss of nucleic acid, especially RNA. Pathogen concentration in exhaled breath is likely to be low. Sample manipulation e.g. purification, digestion, etc. can result in lowering the already limited pathogen nucleic acid concentration. This in turn makes more difficult the processing of sufficient nucleic acid to be detected. The lower concentration of a sample due to lost nucleic acid may prevent pathogen detection.

In some embodiments of the invention, some transducers for detection temperature control detection are not required. In some embodiments of the invention cooling the collection wall isn’t required. In some embodiments of the invention the collection wall is cooled for collection. In some embodiments of the invention, detection for multiple diseases can be performed by using tagged primers specific to the desired targets.

Lysing can be performed by heat or protein digestion or with the use of chemicals or solvent release of nucleic acid. After mixing the sample with a master mix, amplification and detection can be performed.

Thermal cycling detection may be performed via light absorption, light emission, heat, or cloudiness or may be performed with any type of transducer specific for a pathogen or product of pathogen.

Reporting results may be performed by light indicators. Lights may cycle until a minimum value is reached and then may report a solid color. In some cases, there may be no indicated light or a yellow light (waiting) until green (safe) or red (infected) is reported. Infectiousness results are reported faster than negative results. In some embodiments of the invention breath sampling with in-situ sample mixing and isothermal detection and infected results can be reported in 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min.

In some embodiments of the invention, the detection reagents, master mix or buffer/water is used to recover and mix the collected breath film. The mixture may be detected in situ or directed into an amplification and detection chamber tube. In some embodiments of the invention the solution used for mixing is less than 500 pL, less than 200 pL, less than 100 pL, less than 50 pL, less than 25 pl, less than 20 pL, less than 15 pL, less than 10 pL, less than 5 pL, or less than 1 pL.

Using standard of killed virus from ZeptoMetrix (California) the results show that as little of 5 killed virus could be detected in 20 min with this method. In this example, of the invention, the mixing solvent, i.e. the solvent containing detection reagents, contains the lysing reagent such an organic solvent or other lysing reagent. By introducing the detection reagent into the collection device, the entire sample that is collected as a film is mixed with detection reagent and detected in a PCR thermal cycler instrument. In other examples the sample is detected with LAMP isothermal amplification. In this example, detection is performed inside the capillary or tube. In some embodiments of the invention, after mixing with the lysing reagent and/or detection reagents, the liquid is dispensed and detected in a separate chamber.

Detection of the pathogen may be performed directly or after amplification. Detection may be accomplished by a sensor. Detection may involve a chemical reaction. Detection may be accomplished by hybridization, electrochemical methods, fluorescence, CRISPR or any other technology. Detection may be electrochemical or optical. Detection may be isothermal, step temperature gradients or thermal cycling. Detection may be of nucleic acid, protein, carbohydrate, or any chemical associated or connected to a pathogen of interest.

In the invention, lysing and/or detection reagents are used to coalesce the breath condensate liquid that has been collected. In some embodiments of the invention the internal volume of the capillary/tube is determined by the amount of detection reagent employed. In some embodiments the collection capillary and/or tube has an internal volume of 1.5 mL or 1.0 mL or less. In some embodiments of the invention, the detection reagent volume is 50, 40, 30, 20, 10, 5 or 1 % of the collection capillary and/or tube volume.

In some embodiments of the inventions the collection syringe size is 2.5 mL 1.5 mL, 1 mL, 0.5 mL, 0.2 mL, 0.1 mL or smaller.

In some embodiments of the invention, the collection capillary I tube contains an insert. The collector may provide a laminar breath flow or a turbulent breath flow. In some embodiments of the invention the insert may be a turbulence inducer. The collector may be straight, bent, circular, or oscillating shape. There is no restriction on the shape of the collector.

Summary of device and method

The device and method are characterized by a device and method where exhaled breath is blown into capillary or tube collector where breath condensate is deposited as a film and/or droplets on the collection surface. The sampling breath collection time is 1 sec to 60 sec. Detection reagent liquid is introduced into capillary or tube to combine with the breath film sample to recover the entire collected sample for pathogen detection. The collection surface may comprise at least a portion of dried or lyophilized reagents for detection.

The breath condensate liquid in the exhaled breath is deposited in the collector. The sample may contain a pathogen. The pathogen can be a virus, spore, bacterium, cell, or any other entity. All of the collected sample is recovered and mixed with a liquid detection reagent to form a mixture. RNA, DNA, protein, organic molecules, or any other molecules related to the pathogen can be detected. In some embodiments, detection is performed inside the collection chamber while in other embodiments, detection is performed after transferring the mixture from collection chamber to a detection chamber.

The collector can be a tube or capillary. The capillary or tube can be comprised one, two, three or more capillaries and that can be individually addressable. The capillaries can be in an array. The array can be used to quantify pathogen shed number and/or shed rate. The number of capillary chambers can be in the range of 1 - 10 for an individual sample. The number of capillaries can be in the range of 5 - 10,000 for quantification.

The detection process can be isothermal. The detection can be performed by thermal cycling. One or more of the lysis step, reverse transcription step and amplification step can be performed separately. That is, lysis, reverse transcription and amplification can be performed sequentially. Alternatively, the lysis step, RT step and amplification can be performed at the same time or simultaneously in a single mixture. Sample collection, sample recovery by detection reagent capture and mixing, sample processing and detection can be completed in 120, 60, 45, 30, 15, 5 minutes or less. Pathogen detection can be by electrochemical light absorption, light emission, heat, or cloudiness. Detection can be by isothermal reactor with real time addressable, independent channels. The collected and processed reactors can run in parallel or can run in serial operation.

Results can be reported as a graph, number, chart, work, or color. Results can be reported with a phone, tablet, or display. Instrument detection lights may cycle until minimum value is reached and then may report a solid color. Detection lights may be by colored lights. Infectiousness or positive results may be reported at the same time as negative results or may be reported before negative results. Samples may be addressable by RFID, QR code, bar code or other identifiers.

In some embodiments of the invention, a kit comprising detection reagents and capillary or tube collector or collectors can be assembled.

Examples

Example 1

Collection and mixing of breath condensate in a glass capillary.

A glass capillary having a length of 130 mm and diameter of 1 mm was fitted to a 200 pL pipette tip. The pipette served as a breath intake mouthpiece. Breath was exhaled into the mouthpiece for 2 - 3 seconds. Freeze Spray medical skin coolant (1 ,1-Difluroethane from ETL Solutions, USA) was sprayed onto the end of the capillary while breath passed through the capillary. By taring and weighing the capillary, it was determined 1 to 2 pL of breath condensate liquid was collected. In another experiment, breath was collected for 5 seconds and 9 to 10 pL breath condensate liquid was collected. In other experiments, CO2 gas used for bicycle tires was blown over the capillary end during collection and 0.5 to 1 pL of breath condensate liquid was collected.

Example 2 Mixing of breath condensate liquid with detection reagents.

Using the collector of Example 1, a pipette was fitted to the end of the capillary collector. 10 pL of detection reagent was drawn up into the capillary to mix with the condensed liquid. In some cases, back and forth flow was used to further mix the reagents.

The liquid is ready to be dispensed in an amplification well to detect nucleic acids. In some experiments, the end of the capillary was sealed with heavy grease. In some experiments, a plastic cap was fitting over the capillary end to seal and prevent leaking of the liquid from the capillary. In other experiments, a silicone plug was pushed into the inside of the capillary to prevent leaking. The capillary was ready to amplify and detect nucleic acids.

Claims

1. A method for detecting pathogens in the breath of a user, the method comprising:

(a) providing a breath condensate collector, having an inner surface, wherein the breath condensate collector is a tube or capillary;

(b) exhaling breath into the breath condensate collector, wherein the condensate from the exhaled breath is deposited as a film or droplets on the inner surface of the breath condensate collector;

(c) providing a liquid detection reagent;

(d) introducing the liquid detection reagent into the collector to form a sample detection reagent mixture; and

(e) processing the sample detection reagent mixture to detect pathogens.

2. The method of claim 1 , wherein step (e) is performed inside the breath condensate collector.

3. The method of claim 1 , wherein the sample detection reagent mixture is transferred to a separate chamber prior to step (e).

4. The method of any one of the preceding claims, wherein the detected pathogen is a virus, spore, bacteria, or cell.

5. The method of any one of the preceding claims, wherein the detection process is isothermal.

6. The method of any preceding claim, wherein the detection method is thermal cycling.

7. The method of any preceding claim, wherein the detection process involves a chemical reaction.

8. The method of any preceding claim, wherein lysis, reverse transcription, and amplification are performed sequentially in step (e).

9. The method of any preceding claim, wherein lysis, reverse transcription, and amplification are performed simultaneously in step (e).

10. The method of any preceding claim, wherein step (e) is performed using electrochemical methods, light absorption, light emission, heat, or cloudiness measurements.

11. The method of any preceding claim, wherein RNA is detected in step (e).

12. The method of any one of claims 1 to 10, wherein DNA is detected in step (e).

13. The method of one of claims 1 to 10, wherein a protein or organic molecule is detected in step (e).

14. The method of any preceding claim, wherein the breath condensate collector is a glass capillary.

15. The method of any preceding claim, wherein the breath condensate collector comprises multiple capillaries.

16. The method of claim 15, wherein one or more capillaries are individually addressable.

17. The method of claim 15, wherein the capillaries are arranged in an array.

18. The method of any preceding claim, wherein the inner surface of the breath condensate collector surface is comprised of at least a portion of the dried detection reagents.

19. The method of any preceding claim, wherein step (b) is performed in 60 seconds or less.

20. The method of any preceding claim, wherein steps (a) through (e) are performed in 60 minutes or less.

21. The method of any preceding claim, wherein less than 100 pL breath condensate is collected.

22. The method of any preceding claim, wherein less than 50 pL breath condensate is collected.

23. The method of any preceding claim, wherein less than 25 pL breath condensate is collected.

24. The method of any preceding claim, wherein less than 10 pL breath condensate is collected.

25. The method of any preceding claim, wherein less than 5 pL breath condensate is collected.

26. The method of any preceding claim, wherein less than 1 pL breath condensate is collected.

27. A device for detecting pathogens in the breath of a user, the device comprising: a breath condensate collector, having an inner surface; and a mouthpiece for directing exhaled breath into the breath condensate collector, wherein the breath condensate collector is configured to receive a liquid detection reagent to form a sample detection reagent mixture with exhaled breath is deposited as a film or droplets on the inner surface of the breath condensate collector, and wherein the breath condensate collector comprises a tube or capillary.

28. The device of claim 27, wherein the breath condensate collector comprises an array of capillaries.

29. Use of a device according to claim 27 or 28 for collecting breath of a user to detect pathogens therein.

30. A kit comprising a device according to claim 27 or 28, wherein the kit comprises a plurality of disposable elements of the device and/or reagents for processing a sample detection reagent mixture to detect pathogens.