WO2023038582A2

WO2023038582A2 - Exhaled breath condensate collection kit

Info

Publication number: WO2023038582A2
Application number: PCT/SG2022/050648
Authority: WO
Inventors: Ngeow Khing CHIA
Original assignee: National University of Singapore
Current assignee: National University of Singapore
Priority date: 2021-09-10
Filing date: 2022-09-12
Publication date: 2023-03-16
Anticipated expiration: 2024-03-10
Also published as: WO2023038582A3

Abstract

Disclosed is a kit for collecting exhaled breath condensate. The kit includes an outer member comprising at least one inner chamber and at least one orifice for receiving exhaled breath from a user into the at least one inner chamber. The kit also includes at least one inner member disposable in the outer member for cooling the at least one inner chamber for condensing the exhaled breath into exhaled breath condensate on condensing surfaces of the at least one inner chamber and/or at least one inner member. At least one detachable collector attachable to the outer member for collecting the exhaled breath condensate from the condensing surfaces. Moreover, the collected exhaled breath condensate is subsequently assayable for detection of biomarkers in the exhaled breath condensate.

Description

EXHALED BREATH CONDENSATE COLLECTION KIT

Field

The present disclosure relates to a kit for collecting exhaled breath condensate.

Background

There are various types of tests available for screening and diagnosing diseases including respiratory diseases such as COVID-19. Polymerase chain reaction (PCR) tests and antigen rapid tests (ART) are commonly used to screen for diseases in the population. The PCR test is widely regarded as the gold standard to detect biomarkers (e.g. viruses and viral proteins) in samples (e.g. mucous secretions) due to its high accuracy and high specificity (e.g. low levels of false positives and negatives). However, the efficiency of this approach is hindered by slow testing and delivery of the PCR results - mostly 1 or 2 days after sampling. Rapid tests, typically based on lateral flow assays or enzyme-linked immunosorbent assay (ELISA) technologies, therefore are routinely used as pre-screening methods. The results of these tests are available in 10-30 minutes, and their sensitivity is up to 90%. Antigen tests and antibody tests are often immunoassays (lAs) of one kind or another, such as dipstick lAs or fluorescence immunoassays, however ART is an immunochromatographic assay which gives visual results that can be seen with the naked eye.

Current PCR tests and ART for respiratory diseases require nasal swabs using swab sticks inserted into the nostril. The PCR test requires insertion of a nasopharyngeal swab stick about 4 cm into the nostril, which is invasive and can sometimes be painful. For the ART, the depth of insertion is only about 2 cm into the nostril. So, the ART is less intrusive and more comfortable than the nasopharyngeal swabs taken for PCR tests. However, both PCR tests and ART for respiratory diseases are invasive tests and users would likely experience at least some discomfort from these nasal swabs.

Exhaled breath condensate (EBC) can be a source of biomarkers of respiratory disease. EBC collection is largely non-invasive. An infected person may shed virus in the exhaled aerosol, that can be present in the condensate subsequently collected. It is important to note that EBC is not a biomarker, but rather a matrix of biomarkers. EBC may be thought of either as a body fluid or as a condensate of exhaled gas (Volatile organic compound (VOC) that is soluble in condensate and therefore not a body fluid). When a person is infected with virus such as SARS-CoV-2 causing COVID-19, viral shedding will occur when the person coughs or sneezes. The viral shedding can be atomized in breath aerosols.

Using EBC for detection of the viral load or biomarkers is non-invasive since the user does not need to insert swab sticks into the nostril. However, the environment can dramatically alter the volume of EBC collected or the amount of virus per mL. For example, humid environments can dilute a sample or result in more sample needing to be collected to detect virus when viral shedding is low. Particularly in Point-Of-Care- Testing (POCT) facilities, where EBC collection devices are stored at ambient temperature, the condensation rate during EBC collection can be slow as the collection device has the same temperature as its surrounding environment. Moreover, existing systems for analysing breath are not suitable for POCT. For example, gas chromatography mass spectrometry (GC MS) is widely viewed as the gold standard for detecting biomarkers in breath. However, GC MS requires complex systems and can only be done in clinical facilities.

It would be desirable therefore to provide a kit that can overcome one or more of the disadvantages of the prior art.

Summary of the invention

The devices and kits described herein use EBC collection to detect illness. EBC from alveolar exhalation has sufficient virus load for detection using existing tests like PCR and ART. Biomarker interest lies both in the non-volatile constituents mostly derived from the airway lining fluid particles and in water-soluble volatile constituents which are found in substantially higher concentrations and are therefore more readily assayed than the non-volatile compounds. EBC collection kits disclosed herein therefore seek to collect non-volatile constituents and condensed water containing the water-soluble volatile constituents.

Disclosed herein is a kit for collecting exhaled breath condensate, the kit comprising: an outer member comprising at least one inner chamber and at least one orifice for receiving exhaled breath from a user into the at least one inner chamber; at least one inner member disposable in the outer member for cooling the at least one inner chamber (i.e. by providing a surface that is colder than the air or gas in the inner chamber and thus transfers heat from that air or gas) for condensing the exhaled breath into exhaled breath condensate on condensing surfaces of the at least one inner chamber and/or at least one inner member; and at least one detachable collector attachable to the outer member for collecting the exhaled breath condensate from the condensing surfaces, wherein the collected exhaled breath condensate is subsequently assayable for detection of biomarkers in the exhaled breath condensate.

In various embodiments described herein, the kit is in the form of a device or assembly of devices.

Brief description of the drawings

Some embodiments will not be described, by way of non-limiting example only, with reference to the accompanying drawings in which:

Figure 1 is an EBC collection kit in accordance with present teachings;

Figures 2a and 2b show parts of an alternative EBC collection kit with three inner members and three detachable collectors;

Figure 3 illustrates the process of mixing buffer solution with EBC in a EBC collection kit; Figures 4a and 4b show the detachable collector of Figure 1 , in use in a lateral flow assay test and antigen raid test, respectively; and

Figures 5a and 5b show an alternative embodiment of a EBC collection kit or device in accordance with present teachings.

Description

Described are kits for collecting EBC, which is the exhalate from breath that has been condensed.

With reference to Figure 1 , the EBC collection kit 100, which may also be referred to as a device or assembly of devices, comprises an outer member 102, an inner member 104, an inner chamber 106, an orifice presently in the form of inlet 108, and a detachable collector 1 10. The orifice can communicate fluid to (i.e. is fluidically communicative with) the inner chamber 106. The fluid in this circumstance is an exhaled breath. In use, a breath is exhaled into the orifice 108 and passes into the inner chamber 106. On contact with an outer surface 1 12 of the inner member 104, the breath cools and forms EBC. The EBC may be formed on the outer surface 1 12 or on any other surface within the kit 100. The EBC runs down the surface on which it has formed, to the detachable collector 1 10.

The outer member 102 is a cylindrical tube, though other shapes and cross-sections are possible. The outer member 102 together with inlet 108 form a unitary T-shaped body. In other embodiments, the inlet 108 connects to the outer member 102 - e.g. by friction fit, mating screw threads or another suitable mechanism known to the skilled person.

In some embodiments, the inner member 104 is integrally formed with the outer member 102. In this case, the inner chamber 106 is defined between the inner member 104 and outer member 102. In other embodiments, the inner member 104 is attachable to the outer member 102. In this case, the inner chamber 106 may, as dictated by context, be defined by the outer member 102 alone (e.g. be the internal volume in the outer member 102) or between the inner member 104 and outer member 102. In each case, the inner chamber 106 is open in the direction of the detachable collector 1 10.

Presently the inner member 104 and outer member 102 comprise complementary fittings, to enable the inner member 104 to be at least partially inserted into the outer member 102 and to engage therewith. In the embodiment shown in Figure 1 , the outer member 102 and inner member 104 comprises complementary screw threads such that the inner member 1404 engages the outer member 102 by engaging the screw threads. Such an embodiment works best for cylindrical inner and outer members 104, 102. For other shapes - e.g. square cross-section devices - a friction fit or other fitting may be used, such as a Luer connector.

Inner member 104 may be in the form of a tube (i.e. an inner tube with respect to the outer member) or plunger. The inner member 104 comprises or is a heat sink for absorbing heat from exhaled breath or other gases within the inner chamber 106. Presently, the heat sink is a reservoir 1 14 that contains cooling agent when in use. The reservoir comprises a closure 1 16, to prevent cooling agent from leaking from the reservoir 1 14. The present closure 116 comprises a plug or plunger 1 18 and cap 120. The cap 120 comprises a plug driver 122 the forces the plug 1 18 into the top of the reservoir 1 14. The plug 1 18 remains in position in the reservoir 1 14 under friction fit. In other embodiments, the cap and plug or plunger are an integral body, or a simply bottle cap type fitting is provided, with no plug or plunger. Moreover, the inner member may be enclosed within the outer member by the cap, and thus be disposed entirely within the outer member when in use.

In some embodiments, the inner member does not contain any reservoir but instead the heat sink comprises a solid (i.e. not hollow) insert disposed within the outer member during use. The solid insert may be formed from a material with high thermal mass - i.e. thermal mass sufficient to cool surface 1 12, any other internal surfaces and/or inner chamber 106 to promote breath condensation. The inner member is configured to cool the at least one inner chamber for condensing the exhaled breath into EBC on condensing surfaces of the at least one inner chamber 106 and/or at least one inner member 102. Since the inner chamber 106 is defined by other components, such as the inner member 104, outer member 102 and, depending on context, the inlet 108, reference made herein to "inner surfaces of the inner chamber" are references to the inner surface of those components that define the inner chamber. The kit further comprises a detachable collector 110 (e.g. test tube or vial) attachable to the outer member 104 for collecting the EBC from the condensing surfaces. The collected EBC is subsequently assayable for detection of biomarkers in the EBC.

While the detachable collector 1 10 is attached to the outer member 102, it may similarly be attached to the inner member 104 or elsewhere, provided the detachable collector is arranged to collect EBC draining from the inner chamber 106.

The inner member is preferably aerodynamically shaped to facilitate or guide the downward flow of liquid EBC. For example, the inner member may taper towards the detachable collector - e.g. may be cylindrically shaped with a tapered tip similar to a bullet shape or teardrop shape. The inner member may also be shaped to maximize collection of volatile compounds that come from the end tidal breath. For example, the inner member may comprise nano dots disposed on the outer surface (i.e. cooling surface) thereof, to increase surface area of the inner member. Incidentally, in some embodiments the internal surfaces of the device - i.e. the outer surface of the inner member and the inner surface of the outer member - may be ribonuclease-free.

There may be multiple inner members. Figure 2a shows an embodiment in crosssection, comprising a cap 200 and inner members 202. The view is looking toward the cap 200 (i.e. upwardly) from the end of the inner members 202 that is further from the cap 200 - i.e. the end closest to the detachable collectors 206. Similarly, each

Each inner member 202 tapers towards a respective detachable collector 206, shown in Figure 2b in side view. This enables multiple EBC samples to be taken for different analyses. It will be understood that different numbers of inner members and detachable collectors may be used. The side view in Figure 2b shows a supporting skirt or housing 208 so that the detachable collectors 206 can be stably placed on a table top after being detached from the outer member. The same applies to housing 124 of Figure 1. In each case, part of the housing 124, 208 has been removed for illustration purposes.

The detachable collector of Figure 1 may connect to the outer member by friction fit or screw thread. Similarly, the detachable collectors of Figure 2a may form an integral set, connected to the outer member by friction fit or screw thread provided by collar 204.

There are three main types of components that the EBC can contain. Accordingly, each detachable collector 206 may be used for testing or analysing for a respectively different type of component. Similarly, a single inlet may trifurcate into three separate inner chambers each of which is associated with a corresponding or respective inner member and detachable collector. This is illustrated in broken lines in Figure 2a, where inlet 210 trifurcates to separately deliver exhaled breath to each of three internal chambers 212, 214 - internal chamber 214 is the volume around chambers 212 and thus does not need a separate channel, such as channels 216, since any exhaled breath that does not enter channels 216 will flow into the outer member and thus into internal chamber 214. Different samples of EBC are collected in the detachable collectors and they are subsequently tested for the different types of compounds.

The first type of components are variable-sized particles or droplets that are aerosolized from the airway lining fluid. These are particulates containing the virus that are shed by the infected person during the infectious period and when the person is symptomatic. The detachable collector may contain a buffer solution (virus transport medium) that contains suitable reagents for detection of certain biomarkers in these type of compounds in the EBC. The detachable collector may be pre-loaded with the buffer solution - e.g. viral transport medium. In some embodiments, the kit includes a bottle of the buffer solution for the user to load the detachable collector with the buffer solution. In other embodiments, no buffer solution is used or, where there is more than one detachable collector, each detachable collector may contain buffer solution, be empty or contain a respectively different solution as necessary. This kit would be suitable as a POCT for infected persons to use at home.

The second type of component is distilled water that condenses from gas phase out of the nearly water-saturated exhalate.

The third type of components are water-soluble volatiles that are exhaled and absorbed into the EBC, particularly during the pre-infectious period when the person may be asymptomatic. The EBC is collected in the collector without the buffer solution depending on the types of analysis for proteins, H2O2, etc. Biomarkers in the EBC can be evaluated by other methodology using GC mass spec, proton transfer reaction (PTR) mass spec, Raman Spectrometer etc. These are normally done in clinical facilities so the collected EBC has to be frozen below 0 °C to maintain viability of the EBC.

To use the collection kit, the user attaches the detachable collector to the outer member. The user then exhales breath from the user’s mouth to the orifice. Inlet 108 is shaped to fit comfortably into the mouth - e.g. may itself be or comprise a mouthpiece. Alternatively, the inlet 108 may be shaped to connect to a mouthpiece 126. To that end, the kit 100 may include a mouthpiece 126 fluidically communicative with the orifice or inlet 108 to facilitate exhalation from the user’s mouth. The mouthpiece 126 is an elongated component that fits into the user’s mouth. The mouthpiece may be integrally formed with the outer member or inlet, or may be a separate component attachable to the outer member or inlet. Present mouthpiece 126 fits over the inlet 108 by friction fit.

The EBC flows down along the condensing surfaces into the detachable collector that has been attached to the outer member. As the EBC flows from the cooling surface into the detachable collector, the EBC mixes with the buffer solution (if required) in the detachable collector. Optionally, the user can swirl the kit to mix the EBC with the buffer solution. As shown in Figure 3, the mouthpiece or inlet is capped with cap 300 to seal the kit 302 and prevent leakage of fluids while the kit is being swirled. The kit 302 is then swirled. After mixing, the detachable collector 304 is removed and capped with cap 306 for subsequent testing. The remaining parts of the kit 302 are sealed (e.g. with another cap on the outer member, in place of the detachable collector, once the detachable collector has been detached) and disposed of.

As mentioned, the exhaled breath is communicated from the orifice towards the inner chamber and inner member and condenses on the condensing surfaces. The inner member cools the condensing surfaces such that the temperature of the condensing surfaces is lower than the temperature of the exhaled breath, thus promoting condensation. Preferably, the temperature of the condensing surfaces is below room temperature, such as 15 °C or down to -80 °C. The EBC collection from the aerosols (with virus load for patients with respiratory disease, particularly symptomatic patients) will flow down upon accumulating sufficient weight while attached on the condensing surfaces and will be mixed with the buffer solution for testing. Any suitable tests can be used, such as rapid tests (e.g. antigen rapid tests) and lateral flow assays strips, PCR lab tests or reverse transcription loop-mediated isothermal amplification. The EBC is also suitable for detection of early infection when the person is asymptomatic.

As mentioned above, condensation is promoted by a temperature difference between that of the exhaled breath and that of the condensing surfaces. Surface wettability is another property that can be used to promote condensation on condensing surfaces. Wettability relates to the water contact angle on a surface. Water contact angle is the main parameter that characterizes the shape of droplets on the solid surface. Similarly, water contact angle, with derives directly from hydrophobicity or hydrophilicity, relates to the ease with which droplets move across a surface. Higher hydrophobicity results in easier movement and thus easier collection of EBC and the opposite happens with higher hydrophilicity. Wettability depends on surface roughness, surface charge and chemical composition. Changes of these parameters can adjust the contact angle and, therefore, wettability. The condensing surfaces may be hydrophobic, such as being formed from or coated with a material having a water contact angle >=85°. Surfaces can be modified to be hydrophobic such as using plasma discharge treated surfaces and/or hydrophobic coatings, while bulk properties remain unchanged. In this case, the condensing surfaces can be hydrophobic, by surface treatment, surface coating or formation from hydrophobic material, to facilitate communication of the EBC into the detachable collector that has been attached.

As mentioned, the cooling surface of the inner member needs to be cooled to a lower temperature than the exhaled breath so that the exhaled breath can condense. The cooling surface should be cold enough to enable sufficient EBC to form. A lower temperature may increase the volume collected, but may alter the relative concentration of the biomarkers of interest. The temperature also should not be too cold such that the EBC freezes. The inner member may be cooled to a predetermined temperature, the predetermined temperature depending on the test intended to be conducted and/or the biomarkers or antibodies sought to be detected through testing. Various tests were performed to evaluate the condensation of the exhaled breath with respect to temperature of the cooling surface. In general, these tests involved 5 minutes of exhalation into the device, from which 2ml of condensate was collected. In these tests, the inner member was cooled using a gel (<= 15 °C), wet ice (0 °C), or dry ice (-20 °C). From these tests, the volume of the EBC collected per unit time was found to be affected by thermal conductivity I diffusivity of the material medium (cooling agent) particularly for inner members having only a thin wall around the reservoir, as well as the concentration of biomarkers or viral load, total amount of protein, hydrogen peroxide concentration, and nitrite/nitrate concentration.

In some embodiments, the inner member can be kept in the refrigerator or freezer (individually or together with the outer member particularly if they are integrally formed) to cool the inner member and thus the cooling surface. The inner member may contain a cooling agent disposed therein to facilitate cooling, such as a liquid (e.g. water) or a gel.

In some embodiments, the inner member comprises one or more cooling agents disposed therein - i.e. in the reservoir. Where there are two or more cooling agents, the cooling agents may endothermically react to cool the cooling surface.

In some embodiments, the inner member comprises a first cooling agent and a second cooling agent disposed therein (i.e. in the reservoir), wherein the first and second cooling agents are endothermically reactive to cool the cooling surface. Specifically, when the first and second cooling agents are mixed, they undergo an endothermic reaction that absorbs heat from the environment, thereby cooling the cooling surface. Preferably, the two cooling agents produce an endothermic reaction that changes the temperature gradient (temperature differential) between the exhaled breath and condensing surfaces by at least 5 °C, and preferably lowers the temperature of the exhaled breath by 10 °C - i.e. the surface is 10 °C lower in temperature than the exhaled breath. The cooling agents may be in powder and/or liquid form. One example for an endothermic reaction in the inner member is to contain the two reagents in two separate compartments (such as top and bottom). For example, a twist mechanism, such as that used in glowsticks that breaks upon twisting or bending of the casing may break or release the separator to enable the reagent in the top compartment to flow into the bottom compartment, the two reagents thereby mixing to form a cooling agent by an endothermic reaction. In the present case, the inner member could be formed from a flexible material with a frangible divider between two chambers of the reservoir, such that bending of the inner member causes the divider to break to enable the two reagents to mix. Once the separator is released or broken, the cooling agents will mix with each other and an endothermic reaction commences. Temperature can drop from room temperature to -20 °C. For example, the first cooling agent may comprise ammonium chloride (NH4CI) and the second cooling agent may comprise barium hydroxide (Ba(OH)2). Mixing 1 1 g of NH4CI with 32 g of Ba(OH)2 can reduce the temperature of the cooling surface to -20 °C in less than 3 minutes. Use of endothermic cooling agents predisposed in the inner member (i.e. within the inner member prior to use of the kit) may be suitable for outfield mass testing where there are limited cooling facilities.

Typically, water condensate freezes at around 0 °C. It is undesirable for EBC to freeze on the condensing surfaces. The freezing point of water condensate can be altered by changing the charge on the surface of materials. Negative charge lowers the freezing point of water condensate below zero. Positive charges raise the freezing point of water condensate above zero. Thus, it is desirable to have water condensate not to form ice on the condensing surfaces if the inner member has lower than freezing point temperature. Additives or coatings can be applied to the surface 1 12 of the inner member to control the condensate freezing temperature. By adjusting the surface charge to form liquid condensation at a temperature that might typically freeze EBC, the rate of condensation and thus sampling is increased, and the overall time to testing can be reduced. This reduces the degradation of VOCs from exhalation to testing.

Thermal diffusivity of the walls of the outer member and inner member affects the temperature gradient between the exhaled breath and condensing surfaces. Cooling endothermic agents can be applied to the condensing surfaces by reaction of the reagents within the inner member, to collect the EBC based on this temperature gradient. More EBC volume can be collected with lower temperature relative to the exhaled breath temperature. To that end, one or more of the condensing surfaces (including the inner surface of the outer member, the inner surface of the inlet, and the outer surface of the inner member) may comprise a filler to vary thermal diffusivity. The thermal diffusivity (a) of two polymer materials - low density polyethylene (LDPE) and polypropylene (PP) are 6.92x1 O’⁷ m²/s and 5.53x1 O’⁷ m²/s, respectively. By the addition of fillers such as metal and oxide particles to plastics, thermal transport properties, heat capacity, and density of polymers can be varied systematically to increase thermal diffusivity. Composite samples of polypropylene (PP) with various fillers in different fractions (up to 50 vol%) can be prepared with an injection moulding process to vary the properties as a function of filler content. Filler materials like magnetite, barite, talc, copper, strontium ferrite and glass fibres can be used. Higher thermal diffusivity allows more heat to be removed from the condensing surfaces so the exhaled breath condensate will form more readily with multiple droplets forming.

Thermal diffusivity is the measure of thermal inertia. In a material such as PP with high thermal diffusivity due to the use of fillers, heat moves rapidly through it because the substance conducts heat quickly relative to its volumetric heat capacity or thermal bulk. Talc and copper at 30% filler volume fraction shows higher thermal diffusivity at 1.3 mm²/s for talc and 2.4 mm²/s for copper compared with no fillers PP at 0.25 mm²/s. The wall thickness of the PP for example as a material for the outer member and inner member also affects the thermal diffusivity but to a lesser extent than adding fillers for varying the thermal gradient between the exhaled breath and condensing surfaces. The average wall thickness of the inner member with endothermic cooling agents is 0.8-1 mm depending on the temperature required in ambient environment. In any event, the inner member may comprise a reservoir and a wall between the reservoir and inner chamber, wherein the thermal diffusivity of the wall is selected to set an in use temperature (i.e. the temperature during use of the kit) of the outer surface, by at least one of selection of a filler and volume fraction of the filler and/or by forming the wall of a predetermined thickness.

The volume of EBC that can be collected by the detachable collector is small, such as around 100-200 pL, but this is typically sufficient for most rapid tests. Since the end tidal breath volume from the lungs is generally constant for the person - about 400- 600 ml per exhale depending on individual lung capacity - the total surface area of the condensing surfaces can be increased to increase the EBC volume collected. It has been shown that increasing the EBC volume leads to increase in the number of biomarkers detected. One or more of the condensing surfaces may have surface structures to increase total surface area and increase the EBC volume. These surface structures make the surface undulate, to increase the surface area. The surface structures may be fins or fin-like structures such as ribs. In a prototype, the surface area of the inner chamber with no fin design is approximately 1300 mm² and that of the outer surface of the inner member is 1000 mm². Forming surface structures as micro-sized fins can increase the total surface area by multiples - e.g. 2 to 10 times the total surface area of the surface on which the structures are formed. The surface energy for droplet adhesion can also be changed by the fin design. The surface structures are not limited to a fin design, and can include other designs such as semisphere, pyramids, needle like etc.

For condensing surfaces with slow EBC condensation, EBC containing proteins and other biomarkers, the collection volume is about 1 -3 mL of EBC collected from resting adult subjects (~100 pL min^-1 EBC with range of 40-300 pL min^-1) at 5 °C and relative humidity of 70% with 10 minutes of tidal breathing. For asymptomatic patients, the EBC collected can be used to detect early infection.

Different components in EBC are differentially sensitive to cold temperatures, and the concentration of some constituents depends on the condensing temperature. To that end, the thermal diffusivity of the wall of the inner member may be predetermined so that the in use temperature (or operating temperature) of the outer surface of the inner member is optimised for collection of a particular biomarker, protein or antibody - e.g. optimised for collection of biomarkers or antibodies from which SAVS-CoV-2 can be detected. Efficacy is improved by the use of a closed condenser design with breath recirculation in design. The embodiment 500 shown in Figures 5a and 5b shows a one-way valve 502 where inner member 503 is seated on, or formed integrally with, seats 504. The seats 504 create a discontinuous seat to hold the inner member 503 into the outer member 505, while providing gaps 506 between the inner member 503 and outer member 505 for gas (e.g. breath) to pass through. That gas displaces diaphragm 508 of valve 502 from seat 510, allowing gas to escape from the device 500. Notably, by controlling the displacement pressure of the diaphragm 508 from seat 510 increase in dwell time of gas in the device 500 can be achieved. This can similarly increase condensation collection.

The embodiment 500 of Figure 5a is an EBC kit for use with a reverse transcription loop mediated isothermal amplification (RTLAMP) test. To facilitate isothermal amplification, the kit 500 further comprises a temperature controlled member 51 1 for controlling a temperature of the detachable collector 513. This ensures the contents of the detachable collector 513 are maintained at a temperature conducive for isothermal amplification - e.g. 65 °C. The temperature controlled member 51 1 presently comprises a sleeve 512 surrounding the detachable collector 513 and thereby in thermal contact with the detachable collector 513 (i.e. in contact to facilitate heat transfer from the sleeve 512 to the detachable collector 513). The sleeve 512 may be connected to the outer member 505, such that the detachable collector 513 is inserted into the sleeve 512 and into contact with the outer member 505 for use. Alternatively, the sleeve 512 may be attached to the detachable collector 513. In this case, the detachable collector 513 can be detached from the outer member 505 and sealed (if necessary) to await completion of amplification for application in a RTLAMP test produced in the usual manner. In other embodiments, the temperature controller member 51 1 may comprise one or more heated plates, or may be incorporated into the detachable collector to heat the contents of the detachable collector. The temperature controlled member 511 includes an controller 514 which may include one or more of a battery (or power connection) for supplying power to heat the sleeve 512 (or plate or other device as the case may be), and a temperature sensor and controller for respectively sensing a temperature of the detachable collector 513 (or proxy of that temperature, such as a temperature of the sleeve 512) and controlling a temperature of the temperature controlled member (e.g. of the sleeve 512) based on the temperature measurements from the sensor.

The detachable collector may be preloaded with a medium 518. The medium 518 may comprise one or more agents (primers, which may include a stabiliser for room temperature storage) for amplifying the nucleic acid sequences in the collected sample. The medium 518 may also, or instead, comprise a colorimetric reagent that changes colour based on occurrence of nucleic acid amplification. The colorimetric agent may comprise a pH indicator. To facilitate viewing of any color change, the detachable collector 513 may comprise a transparent portion or window 520 through which color changes of liquid in the detachable collector can be observed. The window 520, and all internal surfaces of the detachable collector 513, may be ribonuclease-free.

To ensure the medium 518 remains within the detachable collector 513 prior to use, the detachable collector 513 comprises a seal 516. The seal 516 may be removed prior to use, or may break as the detachable collector 513 is attached to the outer member 505. In the case where the seal 516 is removed, the seal 516 may be a cap that can then be placed back onto the detachable collector 513 during amplification.

For symptomatic patients, the EBC collected can be used to detect infection through the virus loaded breath condensate with lateral flow assays. The user can exert more force to exhale the aerosols from the end tidal breath for EBC collection. Typically, about 100 to 200 pL volume with virus is sufficient to mix with the buffer solution for antigen rapid testing for detection of COVID-19 for example. Other respiratory detection for infection such as influenza can also be collected at <=5 °C and relative humidity of 70% with 1 -2 minutes of severe aerosol emitting tidal breathing or “blowing”.

Various embodiments herein describe a kit for collecting EBC, that includes a test for testing for a particular condition or disease. The EBC may contain virus loaded aerosol for test with antigen tests and antibody tests using immunoassays (lAs) of one kind or another, such as lateral flow dipstick lAs or fluorescence immunoassays or PCR. The EBC and virus loaded aerosol in the EBC is collected in a detachable collector or chamber for POCT test use such as Rapid Antigen test for example, or chromatography analysis or mass spectrometer measurements for EBC biomarkers such as those that correlate to respiratory disease. Collection of EBC is quick and non- invasive and can be used for fast screening of positive cases.

There are various types of tests with which kit 100 can be combined, for detecting biomarkers in the EBC. Two of these are discussed below.

The first type of test is shown in Figure 4a and involves inserting a lateral flow test strip 400 into the collected mixture of EBC and buffer solution in the detachable collector 1 10. The test strip will show a colour indication. For example, appearance of a pink colour indicates a positive test result, i.e. there is presence of the relevant biomarkers (virus) in the EBC.

The second type of testing is using an antigen test cassette 402. The detachable collector 1 10 is capped with a nozzle cap 404 that allows the user to dispense the mixture of EBC and buffer solution onto the cassette. The user adds 2-3 drops (about 50-75 pL) of the mixture into the specimen well of the cassette by gently squeezing the nozzle cap. The cassette is incubated for about 15-20 minutes before reading the test result. If two coloured bands appear within 15-20 minutes with one coloured band in the Control Zone (C) and another in the Test Zone (T), the test result is positive and valid. No matter how faint the coloured band is in the Test Zone (T), the test result should be considered as positive. If one coloured band appears in the Control Zone (C) and no coloured band appears in the Test Zone (T) within 15-20 minutes, the test result is negative and valid. A negative result does not exclude the respiratory diseases or SARS-CoV-2 viral infection, and should be confirmed by a molecular diagnostic method if COVID-19 disease is suspected.

EBC is different from the typical exhaled breath volatile organic compound (VOCs).

EBC is typically obtained by cooling exhaled breath through contact with a cold surface or “condenser”. EBC samples are collected as fluid or frozen material and can be analysed immediately or later for volatile and non-volatile macromolecules.

The collection surface can have different coating materials, such as Teflon, polypropylene, glass, silicone or aluminium, depending on the desired water contact angle and other properties. Surface or coating materials have a significant influence on different biomarkers. Thus, the material of the entire collection system including sample vials or test tubes should be inert or must be standardised for each EBC component of interest.

EBC collection devices work at different cooling temperatures ranging from 15 °C to below -80 °C. Pre-cooled devices are sensitive to higher ambient temperatures. The efficacy of condensation for disease detection mainly depends on:

1 . Exhaled breath volume passed through the system over time;

2. Condensing surface area;

3. Temperature gradient between exhaled breath and sampling system;

4. Virus load in aerosol exhaled or blown onto the cooled surface that then detaches from the surface; and

5. Surface energy where the condensates form and where the condensates can roll and fall off to the virus transport medium if use as a POCT detection kit.

Duration of collection versus volume:

For EBC, it is common that subjects are asked to breathe tidally over a defined period of time (such as 1 -15 minutes) depending on the nature or contributor of EBC to be collected. However, this mode of sampling results in a widely variable volume of exhaled breath. Presuming constant condenser conditions, the volume exhaled per time (i.e. minute volume) has been identified as the most important factor for EBC volume collected per time. Consequently, the volume of exhaled breath, the volume of condensate collected from the exhaled volume and the time of collection after any interference activity are usually reported to assess efficacy of EBC collection.

Breathing pattern and lung function: Tidal breathing sampling does not affect lung function, but variables in the spontaneous breathing pattern may significantly influence EBC collection and composition. Low airflows are advantageous because the collection becomes increasingly inefficient with increasing expiratory flow rates. Hence, it is advised that subjects refrain from exercise for at least 1 hour preceding EBC collection. Slow breathing cycles, i.e. quiet tidal breathing, are recommended because low tidal volumes and high dead-space ventilation in relation to alveolar ventilation lead to EBC samples that mainly derive from conducting airways rather than from peripheral ones. Different origins may remarkably affect EBC composition, and a larger proportion of dead-space ventilation contributes to EBC dilution (by condensed water) and to a greater influence of ambient (inspired) air.

Significant differences in EBC composition have been demonstrated between mouth and nose breathing, e.g. with respect to exhaled biomarkers. To that end, the kit may further comprise a nose clip. When mouth breathing is performed, the use of a nose clip is advised because it: 1 ) prevents inhalation of air through the nose and, therefore, contamination with possible biomarkers from the nasal epithelium; 2) prevents leakage from lower airways via the nose; and 3) prevents mixing of nasal and bronchial air. Salivary contamination should be limited by periodic swallowing. Microbial activity in the oropharyngeal tract significantly contributes to the concentration of nitrogen oxides in EBC, and may be prevented by mouth rinsing, e.g. with chlorhexidine. However, a mouth rinsing agent will bring a different types of interference and thus EBC sample collection should be avoided within 2 hours from mouthwashing or mouth rinsing.

Ambient conditions:

Ambient temperature and relative humidity may contribute to the variability of EBC results, as have been shown in various studies for pH. When EBC collection is implemented in field studies, it should be taken into account that breath temperature can significantly change between seasons such as winter and summer, which will influence the temperature gradient between exhaled breath and the collecting system. In climates with high humidity RH>60%, such as in Singapore, the condensate may be diluted by the in chamber water vapour during condensation. In addition, the EBC kit of each embodiment may comprise a purge member - e.g. a bag - containing a gas - e.g. N2 or CO2 - with predetermined moisture (i.e. desiccated or otherwise with lower humidity than the air in the device) and purity, for purging the device. This ensures the device contains low humidity air which avoids or reduces sample dilution.

Storage and processing of exhaled breath condensate samples:

EBC also contains unstable volatiles. During and immediately after collection, volatile substances can be released (evaporation), and EBC composition can change owing to ongoing biochemical processes. For example, storage for only 1 hour at room temperature can cause a significant decrease in the partial pressure of CO2 and increases EBC pH. Also, data on the stability of hydrogen peroxide (H2O2) in frozen EBC samples can range from 2 days to 2 months. Thus, measurements of at least pH and H2O2 have to be performed in real time or immediately after collection without freezing or storing EBC. Different lines of data suggest that the most important confounder of pH measurement in EBC is the presence of CO2 in the samples. Standardisation of pH measurements in EBC requires the elimination of the confounding effect of CO2. In one approach, EBC pH is measured after removing CO2 from the sample by de-gassing (e.g. de-aeration or degasification), using an inert gas such as argon, though, de-aeration cannot completely eliminate CO2 from EBC samples. In an alternative approach, rather than attempting to remove CO2 from EBC, samples are instead sequentially loaded with CO2 gas. At regular time points during the CO2 loading procedure, aliquots are taken for simultaneous pH and carbon dioxide partial pressure (PCO2) measurements by means of a blood gas analyser. By plotting several pH/pCO2 value pairs for each sample, EBC pH can be easily determined for any given pCO2 value using regression analysis. This method yields highly reproducible EBC pH values at the normal alveolar pCO2 of 5.33 kPa. Some researchers argue against artificial manipulation of EBC; however, they address only de-aeration, not standardisation by CO2 loading. Regarding de-aeration, it is still unknown how many volatiles besides CO2 are removed while bubbling gas through an EBC specimen, and how the complexity of EBC is changed by this procedure. There is also no standardised protocol for de-gassing so far. It has been demonstrated that different de-gassing procedures (bubbling versus surface delivery) with changing durations significantly influence both losses of EBC volume and concentrations of EBC components. On the contrary, CO2 loading has been shown to provide the least variability observed so far in EBC pH measurement. There is an approximate two order difference in the logarithmic scale of pH depending on the method used for standardisation, i.e. pH readings of de-gassed samples with CO2 concentration close to zero versus pH readings at a standardised CO2 concentration at a CO2 level of 5.33 kPa (physiological alveolar CO2 partial pressure) as determined by regression analysis in C02-loaded samples.

One of the key problems is that most of the concentrations measured in EBC have been published in the units they were measured in as raw data in the liquid sample, e.g. pg mL-1 , nmol mL-1 or pmol L-1. In fact, 1 mL EBC cannot be considered a standardised biological specimen at all, because the percentage of condensed liquid of the exhaled volume is not constant for each collection process. Instead, one has to be aware that different collection systems and procedures will generate differently diluted condensates with variable characteristics, despite similar concentrations in exhaled breath.

The degree of dilution of EBC by condensed water mainly depends on: 1 ) the efficacy of the collection system and environment relative humidity; and 2) the individual breathing characteristics. The use of different dilution factors (e.g. urea, conductivity or total cations) or calculating the analysed mediator in relation to the conductivity of the given EBC sample are proposed for better standardisation with a very wide range of reported physiological dilution rates (between 1000 and 48000).

Exhaled breath volume per time:

Owing to their solubility or reactivity, gaseous components can be assessed in the liquid EBC sample. For volatile substances, it is the quantity that has been exhaled in relation to the exhaled volume per time that is of interest. This recalculation is possible when taking into account the exhaled volume, the time of EBC collection and the volume of collected EBC. When this approach was applied to volatiles and even nonvolatiles in EBC, the quantities of lactate exhaled per minute and quantities of H2O2 or leukotriene B4 exhaled per 100 L of exhaled breath were less variable compared to concentrations assessed per millilitre of EBC. Particle-associated EBC components:

The presence of non-volatile molecules in EBC (proteins, cytokines, virus, bacterials etc.) is most likely linked to the exhalation of micro-droplets, i.e. aerosols or particles. Aerosol formation can be simply explained by the bronchiole fluid film burst model. This hypothesis states that aerosols are formed by a process of respiratory fluid film or bubble bursting during the reopening of respiratory bronchioles by inhalation and the subsequent fragmentation of droplet aerosols that are drawn into the alveoli until emission during the next exhalation. Emission of particles by exhalation is mainly dependent on individual lung physiology and respiratory pattern. Consequently, the quantity of droplet-associated EBC components differs significantly between subjects, is not normally distributed and presents significant inter-subject variability that exceeds variations caused by airway diseases. However to detect positive or negative infection, lateral flow assays only requires a minimum cut-off load of virus for the protein attachment and thus 100 to 200 pl of condensate aerosol is sufficient assuming there is sufficient virus load in the aerosol due to symptoms. For other EBC analysis, to overcome this problem, normalisation of non-volatile EBC components in relation to the emission rate of exhaled particles can be performed by software. This approach requires on-line monitoring of exhaled particles in future EBC studies such as real time extraction measurement using a PTR mass spectrometer or Raman spectrometer with surface-enhanced Raman scattering (SERS) nano precious metals.

The description of the various embodiments herein is not intended to call out or be limited only to specific or particular representations of the present disclosure, but merely to illustrate non-limiting examples of the present disclosure. Although only some embodiments of the present disclosure are disclosed herein, it will be apparent to a person having ordinary skill in the art in view of this disclosure that a variety of changes and/or modifications can be made to the disclosed embodiments without departing from the scope of the present disclosure. Therefore, the scope of the disclosure as well as the scope of the following statements is not limited to embodiments described herein.

Claims

22 Claims

1 . A kit for collecting exhaled breath condensate, the kit comprising: an outer member comprising at least one inner chamber and at least one orifice for receiving exhaled breath from a user into the at least one inner chamber; at least one inner member disposable in the outer member for cooling the at least one inner chamber for condensing the exhaled breath into exhaled breath condensate on condensing surfaces of the at least one inner chamber and/or at least one inner member; and at least one detachable collector attachable to the outer member for collecting the exhaled breath condensate from the condensing surfaces, wherein the collected exhaled breath condensate is subsequently assayable for detection of biomarkers in the exhaled breath condensate.

2. The kit according to claim 1 , wherein the at least one inner member is integrally formed with the outer member.

3. The kit according to claim 1 or 2, further comprising a mouthpiece fluidically communicative with the orifice to facilitate exhalation from the user’s mouth.

4. The kit according to claim 3, wherein the mouthpiece is integrally formed with the outer member.

5. The kit according to any one of claims 1 to 4, wherein the condensing surfaces comprise surface structures to increase total surface area for collection of the exhaled breath condensate.

6. The kit according to claim 5, wherein the surface structures comprise fins.

7. The kit according to any one of claims 1 to 6, wherein the condensing surfaces are hydrophobic to facilitate communication of the exhaled breath condensate into the detachable collector.

8. The kit according to claim 7, wherein the condensing surfaces comprise plasma discharge treated surfaces and/or hydrophobic coatings.

9. The kit according to any one of claims 1 to 8, further comprising fillers on the condensing surfaces to vary a temperature differential between the exhaled breath and the condensing surfaces.

10. The kit according to claim 9, wherein the fillers comprise talc and/or copper.

1 1 . The kit according to any one of claims 1 to 10, wherein at least one said inner member comprises a first cooling agent and a second cooling agent separated from each other, the first and second cooling agents endothermically reacting to cool the inner chamber.

12. The kit according to claim 1 1 , wherein the first cooling agent comprises ammonium chloride and the second cooling agent comprises barium hydroxide.

13. The kit according to any one of claims 1 to 12, wherein the detachable collector is pre-loaded with a buffer solution for assaying the exhaled breath condensate.

14. The kit according to any one of claims 1 to 13, further comprising a test for receiving the exhaled breath condensate from the detachable collector for detection of biomarkers in the exhaled breath condensate.

15. A method of preparing exhaled breath condensate (EBC) for testing, comprising: collecting an EBC sample using an EBC collection kit; and loading the EBC sample with CO2 to standardise a CO2 partial pressure in the EBC sample.

16. The method of claim 15, wherein the EBC collection kit is a kit according to any one of claims 1 to 14.

17. A method of testing exhaled breath condensate (EBC) for one or more biomarkers, comprising: performing the method of claim 16; and testing the EBC for the one or more biomarkers.

18. The method of claim 17, wherein performing the method of claim 15 comprises: measuring one or more of an exhaled volume, a time of EBC collection and a volume of collected EBC; and recalculating an original quantity of VOCs in the EBC based on the one or more of the exhaled volume, the time of EBC collection and the volume of collected EBC.

19. The kit according to claim 14, wherein the test comprises a reverse transcription loop mediated isothermal amplification test, the kit further comprising a temperature controlled member for controlling a temperature of the detachable collector.

20. The kit according to claim 19, wherein the temperature controlled member comprises a plate or sleeve in thermal communication with the detachable collector.

21 . The kit according to claim 19 or 20, further comprising a temperature sensor for sensing a temperature of the detachable collector, and a controller for controlling a temperature of the temperature controlled member based on a temperature measurement from the temperature sensor.

22. The kit according to claim 21 , wherein at least a part of the detachable collector is transparent, the kit further comprising a colorimetric reagent that changes colour based on occurrence of nucleic acid amplification.

23. The kit according to claim 22, wherein the colorimetric reagent comprises a pH indicator.