System and method for determining onset and disease progression

Abstract

To relate to screening and early detection of a variety of disease conditions in seemingly healthy subjects, enabling early intervention and treatment.SOLUTION: A method for determining presence of at least one disease-associated marker in a breath sample from a subject includes: exposing to a breath sample of a subject at least one sampling unit comprising one or more adsorbing regions capable of reversibly associating volatiles in the breath sample, the adsorbing regions being different from metal surfaces or metal nanoparticles; analyzing the at least one sampling unit being exposed to the volatiles to identify volatiles adsorbed onto the one or more adsorbing regions; and determining presence of the at least one disease-associated marker. The increase in amount of the marker compared to the marker measured in an early stage is indicative of existence of the disease state.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates generally to systems and methods for determining the onset and progression of a disease state. [Background technology]

[0002] Exhaled air from the lungs carries volatile (and semi-volatile) chemicals and metabolites that reach the lungs through the blood system. These chemicals and metabolites include pharmaceuticals that can indicate a person's health condition. Simple breath analyzers are configured to measure levels of alcohol or other volatile metabolites of drugs for forensic purposes, where the compounds of interest or their metabolites are known. Breath analysis to identify volatile compounds as indicators of clinical conditions is complicated by the large number of volatile compounds carried in breath and their fairly low concentrations.

[0003] Gas chromatography and EI mass spectrometry were used for such analyses. International Patent Publication No. 2019 / 173501 [1] and US Patent Application No. 2019 / 0274633 [2] describe systems for determining the presence of volatile organic compounds in breath exhaled by mechanically ventilated humans. These systems take advantage of the fact that the ventilator unit pressurizes the inhaled air, so that both the inlet and outlet lines can be pressurized, thereby collecting samples from the exhaled air on an adsorbent. The adsorbed compounds are analyzed by an analytical unit.

[0004] US Patent No. 9,733,225 [3] describes an interchangeable spectroscopic detector for use in a volatile organic compound testing device, such as a portable breath testing device for roadside drug testing or a testing device for any air treatment system, which prepares a concentrated sample in a single gas cell configured to reversibly adsorb compounds and perform spectroscopy of the contents of the cell.

[0005] U.S. Patent Application No. 2008 / 0009761[4] describes a breath condensate sampler for use with a ventilator, the breath condensate sampler comprising an airflow valve disposed in the expiratory limb of the ventilator, a condensate forming means, and a condensate collecting means. The airflow valve directs air from the expiratory limb to the breath condensate sampler, and a condensate portion of the exhaled gas is separated from a gas portion of the exhaled gas. A method for collecting a breath condensate sample is also disclosed herein.

[0006] International Patent Publication No. 2015 / 187938[5] describes a method for diagnosing, treating, and monitoring the treatment of invasive aspergillosis (IA), which may include detecting the presence of one or more volatile organic compounds (VOCs) in the exhaled breath of a subject suspected of having IA.

[0007] US Patent No. 10,261,071 [6] describes a set of volatile organic compounds for breath analysis. The description includes methods for identifying these VOCs, and also discloses the use of the method in the diagnosis and monitoring of excretory toxicity development.

[0008] Background technology [1] WO2019 / 173501, [2]US2019 / 0274633, [3] US9,733,225, [4]US2008 / 009761, [5] WO2015 / 187938, [6] US 10,261,071.

[0009] overview Early diagnosis of disease states in apparently healthy or asymptomatic patients is typically only performed in patient populations susceptible to or affected by a particular type of disease, or in patients with recurrent life-threatening symptoms. Even if methodologies exist to achieve such early diagnosis, such diagnosis may involve the use of rather expensive diagnostic systems in addition to routine clinical testing, and sometimes invasive diagnostics, making it less acceptable for early diagnosis in healthy individuals.

[0010] The inventors of the inventions disclosed herein have developed a methodology that allows for widespread screening and early detection of various disease states in apparently healthy subjects, enabling early intervention and treatment. The method of the invention is non-invasive, does not require complex medical devices or hospitalization, is low cost, and provides an efficient means for diagnosis and treatment monitoring by a physician or other medical staff at any time, such as in a physician's clinic or office, in an emergency room, in a clinical laboratory or pharmacy, or in the subject's home.

[0011] The invention disclosed herein provides methods and systems for diagnosing, predicting, or identifying early onset disease in asymptomatic subjects, or more broadly, in the general population. The methods involve detecting the presence of volatile organic compounds (VOCs) or semi-VOCs (sVOCs) (generically referred to herein as volatile compounds, VCs, or markers) in exhaled breath samples. As the inventors have demonstrated, breath samples collected from subjects in the general population have been found to contain markers, the presence and quantity of which provide an indication of a pre-existing disease state that was not apparent at the time of diagnosis. The ability to detect and monitor changes in the amount of markers over time also provides a means to evaluate or monitor the progression of a disease state over a period of time and assess the relevance and success of medical treatments.

[0012] Each disease state can be characterized by a different cluster of markers or marker fingerprints that are unique and indicate the specific disease state and can be used to distinguish individuals with one disease state from others. For example, a marker fingerprint characteristic of a bacterial infection can be different from a marker fingerprint characteristic of liver disease. Similarly, a marker fingerprint characteristic of a particular bacterial infection can be different and distinct from a marker fingerprint characteristic of another bacterial infection, thereby achieving differentiation between the two.

[0013] More uniquely, the methodology of the present invention allows for the identification of disease states in subjects exhibiting symptoms associated with different diseases, and the methodology of the present invention can distinguish between the two diseases. This unique ability is almost exclusively manifested in the serial diagnosis of evolving disease states in hospitalized patients, e.g., ventilated patients, who exhibit multiple medical complications and are highly susceptible to hospital-acquired infections, i.e., hospital-acquired infections (HAIs). Early detection of such infections can dramatically reduce the impact of potentially life-threatening complications.

[0014] Accordingly, in a first aspect thereof, the present invention provides a method for determining the presence of at least one disease associated marker in a respiratory sample from a subject, comprising: exposing at least one sampling unit to a breath sample of a subject, the sampling unit comprising one or more adsorbent regions capable of reversibly binding volatile substances in said breath sample; analyzing at least one sampling unit to identify volatile substances adsorbed to the one or more adsorption regions and determining the presence of said at least one pathogen-associated marker; A method is provided wherein the presence of said marker at a level greater than background level of said marker indicates the presence of a disease state.

[0015] A "marker" is typically a volatile compound (VC) or semi-volatile compound (VC), which can be inorganic or organic. If the compound is organic, it is called a volatile organic compound (VOC) or semi-volatile organic compound (sVOC). Within the context of the present invention, these terms are interchangeable.

[0016] Markers can be endogenous markers derived from within a subject's body that reflect its metabolism, or exogenous markers derived from external sources such as diet, prescription drugs, and environmental exposures. Because the production of endogenous markers is directly related to metabolic activity within the body, clusters or combinations of such markers are characteristic of specific disease processes, and the presence and amount (level, quantity, concentration) of such endogenous markers in a subject's breath, as well as their development over time, provide a direct indicator of the disease state. Therefore, these markers are referred to herein as "disease-related markers."

[0017] In the most general terms, markers are typically associated with the presence and / or proliferation of pathogens (e.g., bacteria, viruses, or fungi) involved in metabolism, organ function, or disease pathogenesis. For example, markers produced internally through the metabolism of cells or pathogens in the body are released into the circulatory system and then excreted through exhaled breath. Markers can include multiple compounds, some of which are gaseous and others of which are liquid (at physiological temperatures), that are released in exhaled breath and carried by respiratory gases or tiny water droplets, allowing for detection and quantification.

[0018] To distinguish microorganisms (bacteria, viruses, fungi) present at low levels in a subject and part of the subject's microbiome from microorganisms whose presence and load / amount / mass indicate pathogenesis, the method of the present invention allows for both qualitative (i.e., determining the presence of microorganisms, e.g., pathogens) and quantitative (i.e., determining the load / amount / mass of the aforementioned microorganisms) determination of pathogenesis. Thus, a marker associated with a microorganism normally present in a subject's microbiome is considered to indicate a pathogenic pathway if its measured amount at least doubles within two days after the first measurement. The first measurement is the background level measured in a healthy or monitored human. Therefore, if the amount of a marker or a cluster of markers is observed to increase by at least 50% (overall more than two-fold) every day between two consecutive measurements, it is indicated.

[0019] As used herein, a marker can be a single molecule or a combination of several molecules, and the methods of the present invention are equally applicable to diagnosis based on a single marker, a combination of markers, or a marker fingerprint. Thus, the singular form of the term also encompasses a plurality of markers.

[0020] A "marker fingerprint" refers to a collection of properties related to the marker content of exhaled breath obtained from a subject. These collective properties are unique and informative and can be considered a fingerprint or signature that indicates the onset, development, or progression of a disease. A profile that distinguishes one disease from another can also provide insight into the state of the disease or its progression, identify the onset of disease at an early stage before symptoms appear, and help determine the success of therapeutic treatment (prevention or treatment of existing symptoms). The properties can be: - the presence or absence of one or more markers indicative of disease; - the concentration (or amount) of one or more markers, - Evolution over time (increase or decrease in concentration or amount), especially in the case of exponential growth, - presence or absence of other marker combinations, - the ratio between the various markers, and - a change in the presence or amount of one or more markers over time.

[0021] The marker can be any marker known to be associated with a disease state. Some of these markers include 1,8-naphthyridine, 10-undecin-1-ol, 3-methyl-1-butanol, 1-phenyl-1H-imidazole, 2-(2-pyridinyl)-1H-indole, 8-methyl-1H-purine, 1-methoxyphthalazine, 1-nitro-2-propanol, 2-butyl-1-octanol, 3,7-dimethyl-1-octanol, 2-methyl-1-propanol, 1-undecene, 2-(2-methylpropyl)-3,5- Di(1-methylethyl)pyridine, 2,3-dimethylcyclohexylamine, 2,4-dithiapentane, 2-benzyl-1-methylpiperidine, 2-butanone, 3-propylidene-2-heptanone, 6-phenylhexanoic acid, 8-aminocaprylic acid, 2,2'-thiobis-acetic acid, acetone, O-isopropyloxime benzaldehyde, 4-nitro-benzamide, 2-carboxy-benzeneacetic acid, 2-methyl-butanal, 3-methylbutanal, 3- Methylbutanoic acid, dodecamethyl-cyclohexasiloxane, cyclohexene, D:C-Friedours-7-ene, dimethyl trisulfide, dimethyl disulfide, emorfazone (4-ethoxy-2-methyl-5-morpholin-4-ylpyridazin-3-one), ethylphenylhydantoin, gabapentin lactam, ethyl 5-oxohexanoate, 5-nitro-isoquinoline, lanostane-12-one, luminol (5-amino-2,3-di Hydrophthalazine-1,4-dione), 4-butyl-phenol, phthalic anhydride, pregabalin, 1-(ethynylsulfinyl)-propane, ribo-ribonucleic acid, S-(2-benzothiazolyl)cysteine, 2-butyl-5-ethyl-thiophene, cyclopropylcarbinol, 2-pyridinecarbonitrile, 2-bromo-1-(4-methylphenyl)-ethenon, 2,3,4,7-tetrahydro-1H-indene, 1-bromo-1-phenylpropane, 2,6-Dimethyldecane, N,N-dimethyl-1-dodecanamine, 3-methyl-6-(1-methylethylidene)-cyclohexene, 8-methyl-1-decene, 6-methyl-dodecane, N,N-dimethyl-1-tetradecanamine, hexanedioic acid bis(2-ethylhexyl) ester, (+)-4-carene, 2-carene, 2-methyl-1-propene, 4,11,11-trimethyl-8-methylene-bicyclo[7.2.0]undec-4-ene, 3-methylpentan-2-yl trifluoroacetate, 2-methyl-5-(1-methylethenyl)-cyclohexa nol, (Z)-4-decen-1-ol trifluoroacetate, 4-methyl-1-(1-methylethyl)-bicyclo[3.1.0]hexan-3-ol, pyruvic acid butyl ester, 2,9-dimethyl-decane, propylamine, ethylenediamine, 1-methyl-2-(3-methylpentyl)-cyclopropane, (nitromethyl)benzene, 5-ethyl-1-nonene, isopropylsulfonyl chloride, deltacyclen, 2,3,6,7-tetramethyl-octane, 1-methyl-4-(1-methylethenyl)-benzene, 3,4-dimethyl-1 -pentene, N-benzyl-N-methyl-2-methyl-β-alanine methyl ester, 2,2,4-trimethyl-pentane, trans-geranylgeraniol, 2-ethyl-4-methyl-1-pentanol, 6-methylheptyl vinyl ether, tetrahydro-6-methyl-2H-pyran-2-one, 2,3,7-trimethyl-decane, 2-decen-1-ol, (1R,4aS,8aR)-1-isopropyl-4,7-dimethyl-1,2,4a,5,6,8a-hexahydronaphthalene, 3-ethyl-2-methyl-hexane, 2-methyl-1-pentanol Then, 4,5-dimethyl-undecane, 4-methylene-1-methyl-2-(2-methyl-1-propen-1-yl)-1-vinyl-cycloheptane, 7-methyl-(E)-4-decene, 1-iodo-dotriacontane, 5-dodecyldihydro-2(3H)-furanone, butyl dodecyl ester sulfuric acid, 3,4-dimethylbenzyl alcohol, 1,4-dimethyl-cyclooctane, 2,3-dimethyl-hexane, dodecanoic acid, estragole, 4-ethyl-1-octyn-3-ol, 5-methyl-2-(1-methylethyl)-1-hexanol, 3,3-dimethyl-heptane, 7-methyl-(Z)-2-decene, 2-methyl-decane, 1,2,3,4,4a,5,6,7,8,9,10,10a-dodecahydro-1,4a-dimethyl-7-(1-methylethyl)-1-phenanthrenecarboxylic acid methyl ester, dodecanal, 1-octadecanesulfonyl chloride, 4-tert-butylcyclohexyl acetate, 4-hexen-2-one, 2,5,6-trimethyldecane, 4,4-dimethyl-1-hexene, heptadecane, isobutylene epoxide, 2,2,7,7-tetramethyloctane, 2-ethyl-1-hexanol trifluoroacetate, propylcyclopropane, anethol, octane, methyl-cyclobutane, 1,12-dodecanediol, 2-methoxy-1-propene, nitrous acid, 4-(1,1-dimethylethyl)cyclohexanol acetate, 1,5-dimethyl-8-(1-methylethylidene)-(EE)-5-cyclodecadiene, 4-methyl-2-propyl-1-pentanol, octahydro-4-methyl-8-methylene-7-(1-methylethyl)-[1S-(1α,3aβ,4α,7α,7aβ)]-1,4-methano-1H-indene, 3-ethyl The alkyl esters may be selected from the group consisting of 1,2-diphenyl-(R*,R*)-1,2-ethanediol, 7-ethyl-1,2,3,4,4a,5,6,7,8,9,10,10a-dodecahydro-1,4a,7-trimethyl-methyl ester [1S-(1α,4aα,7β,10aβ)]-1-phenanthrenecarboxylic acid, 2,7-dimethyl-octane, hexyl pentyl ether, 1,2-diphenyl-(R*,R*)-1,2-ethanediol, 7-ethyl-1,2,3,4,4a,5,6,7,8,9,10,10a-dodecahydro-1,4a,7-trimethyl-methyl ester [1S-(1α,4aα,7β,10aβ)]-1-phenanthrenecarboxylic acid,

[0022] In some embodiments, the marker associated with at least one bacterial pathogen is 1,8-naphthyridine, 10-undecin-1-ol, 3-methyl-1-butanol, 1-phenyl-1H-imidazole, 2-(2-pyridinyl)-1H-indole, 8-methyl-1H-purine, 1-methoxyphthalazine, 1-nitro-2-propanol, 2-butyl-1-octanol, 3,7-dimethyl-1-octanol, 1-methyl-1H-imidazole, 2-(2-pyridinyl)-1H-indole, 8-methyl-1H-purine, 1-methoxyphthalazine, 1-nitro-2-propanol, 2-butyl-1-octanol, 3,7-dimethyl-1-octanol, 1-methyl-1H-imidazole, 2-methyl-1H-indole, 1-methyl-1H-purine, 1-methyl-1H-imidazole, 2-methyl-1H-indole ... Benzyl, 2-methyl-1-propanol, 1-undecene, 2-(2-methylpropyl)-3,5-di(1-methylethyl)pyridine, 2,3-dimethylcyclohexylamine, 2,4-dithiapentane, 2-benzyl-1-methylpiperidine, 2-butanone, 3-propylidene-2-heptanone, 6-phenylhexanoic acid, 8-aminocaprylic acid, 2,2'-thiobis-acetic acid, acetone, O-isopropyloxime benzalkonium Dehyde, 4-nitro-benzamide, 2-carboxy-benzeneacetic acid, 2-methyl-butanal, 3-methyl-butanal, 3-methyl-butanoic acid, dodecamethyl-cyclohexasiloxane, cyclohexene, D:C-freedours-7-ene, dimethyl trisulfide, dimethyl disulfide, emorfazone (4-ethoxy-2-methyl-5-morpholin-4-ylpyridazin-3-one), ethylphenylhydanto and 2-butyl-5-ethyl-thiophene, 2-(2-benzothiazolyl)-2-propane ...

[0023] In some embodiments, the marker associated with a viral pathogen is cyclopropylcarbinol, 2-pyridinecarbonitrile, 2-bromo-1-(4-methylphenyl)-ethenone, 2,3,4,7-tetrahydro-1H-indene, 1-bromo-1-phenylpropane, 2,6-dimethyldecane, N,N-dimethyl-1-dodecanamine, 3-methyl-6-(1-methylethylidene)-cyclohexene, 8-methyl-1-decene, 6-methyl-dodecane, N,N-dimethyl-1-tetradecanamine, hexanedioic acid bis(2-ethylhexyl) ester, (+)-4-carene, 2-carene, 2-methyl-1-propene, 4,11,11-trimethyl-8-methylene-bicyclo[7.2.0]undec-4-ene, 3-methylpentan-2-yl trifluoroacetate, 2-methyl-5-(1-methylethenyl)-cyclohexanol, (Z)-4-decen-1-ol trifluoroacetate, 4-methyl-1-(1-methylethyl)-bicyclo[3.1.0]hexan-3-ol, pyruvate butyl ester, 2,9-dimethyl-decane, propylamine, ethylenediamine, 1-methyl-2-(3-methylpentyl)-cyclopropane, (nitromethyl)benzene, 5-ethyl-1-nonene, isopropylsulfonyl chloride, deltacyclen, 2,3,6,7-tetramethyl-octane, 1-methyl-4-(1-methylethenyl)-benzene, 3,4-dimethyl-1-pentene, N-benzyl-N-methyl-2-methyl-β-alanine methyl ester, 2,2,4-trimethyl-pentane, trans-Geranylgeraniol, 2-ethyl-4-methyl-1-pentanol, 6-methylheptyl vinyl ether, tetrahydro-6-methyl-2H-pyran-2-one, 2,3,7-trimethyl-decane, 2-decen-1-ol, (1R,4aS,8aR)-1-isopropyl-4,7-dimethyl-1,2,4a,5,6,8a-hexahydronaphthalene, 3-ethyl-2-methyl-hexane, 2-methyl-1-pentene, 4,5-dimethyl-undecene, 4-methylene-1-methyl-2-(2-methyl-1-propen-1-yl)-1-vinyl -Cycloheptane, 7-methyl-(E)-4-decene, 1-iodo-dotriacontane, 5-dodecyldihydro-2(3H)-furanone, dodecyl ester butyl sulfonate, 3,4-dimethylbenzyl alcohol, 1,4-dimethyl-cyclooctane, 2,3-dimethyl-hexane, dodecanoic acid, estragole, 4-ethyl-1-octyn-3-ol, 5-methyl-2-(1-methylethyl)-1-hexanol, 3,3-dimethyl-heptane, 7-methyl-(Z)-2-decene, 2-methyl-decane, 1,2,3,4,4a,5,6,7,8,9,1 0,10a-dodecahydro-1,4a-dimethyl-7-(1-methylethyl)-1-phenanthrenecarboxylic acid methyl ester, dodecanal, 1-octadecanesulfonyl chloride, 4-tert-butylcyclohexyl acetate, 4-hexen-2-one, 2,5,6-trimethyldecene, 4,4-dimethyl-1-hexene, heptadecene, isobutylene epoxide, 2,2,7,7-tetramethyloctane, 2-ethyl-1-hexanol trifluoroacetate, propylcyclopropane, anethol, octane, methylcyclobutane, 1-octadecane, methylcyclobut ... ,12-Dodecanediol, 2-Methoxy-1-propene, Nitric Acid, 4-(1,1-Dimethylethyl)cyclohexanol Acetate, 1,5-Dimethyl-8-(1-Methylethylidene)-(EE)-5-Cyclodecadiene, 4-Methyl-2-Propyl-1-Pentanol, Octahydro-4-Methyl-8-Methylene-7-(1-Methylethyl)-[1S-(1α,3aβ,4α,7α,7aβ)]-1,4-Methano-1H-Indene, 3-Ethyl-2,7-Dimethyl-Octane, Hexyl Pentyl Ether, 1,2-Diphenyl-(R*,R*)-1,2-ethanediol, and 7-ethyl-1,2,3,4,4a,5,6,7,8,9,10,10a-dodecahydro-1,4a,7-trimethylmethyl ester [1S-(1α,4aα,7β,10aβ)]-1-phenanthrenecarboxylic acid.

[0024] A "breath sample" is a sample obtained actively or passively from a subject's exhaled breath. Passive sampling involves capturing volatile substances without any specific intervention. An example of active sampling is a sampling unit with an opening designed for the Venturi effect. Active sampling involves capturing volatile substances through an implementation such as a pump (mechanical, electrical, helium, etc.) or a suction unit. Depending on the type of disease being detected, such as an internal organ disease or a GI tract disease, the breath sample can be an alveolar breath sample or a non-alveolar breath sample. Typically, the sample is collected directly in the sampling unit, and the volatile substances are allowed to interact by adsorption to one or more adsorption areas. If the subject fully cooperates, the subject exhales into the sampling unit, after which the sample is processed. If the subject is unable to cooperate, the sample can be collected from the subject's oral cavity or lungs. As disclosed herein, samples from subjects on a ventilator can be obtained by connecting a sampling unit to the outlet line of the breathing unit.

[0025] In some embodiments, the method comprises obtaining a breath sample from a subject by employing any non-invasive means known in the art. A non-limiting method for collecting breath samples may involve the use of an American Thoracic Society / European Respiratory Society (ATS / ERS) approved device, see, for example, Silkoff et al., Am.J.Respir.Crit.Care Med, 2005, 171, 912.

[0026] In some embodiments, the sample can be obtained by exhaling directly into a measurement device or apparatus.

[0027] Breath samples are collected by exhaling directly into the adsorption / sampling unit. According to such embodiments, breath samples may be captured using a mouthpiece that provides a connection between the subject and the unit operated according to the method of the present invention. Because concentrations of markers in human breath can be in the ppm-ppt range, the method may include pre-concentrating the resulting breath sample prior to analysis. Breath concentrators within the scope of the present invention include, but are not limited to, those described in US 2012 / 0326092, incorporated herein by reference.

[0028] A "sampling unit" is a container, vessel, or canister of any shape or size configured to receive and hold a respiratory sample. A sample unit can be a single unit or multiple such units. Sampling units are typically made of a material that is substantially nonreactive or exhibits limited interaction with volatile substances or markers contained in a respiratory sample. Therefore, the units are typically formed of materials such as glass or stainless steel. A sampling unit includes one or more adsorbent regions, each capable of reversibly binding to a volatile substance in the respiratory sample. The adsorbent regions can be configured to fill the volume of the sampling unit, formed on the inner surface of the wall of the sampling unit, or present in any interior region of the sampling unit. Nevertheless, the regions can be configured for selective binding of certain volatile substances, in which case other volatile substances are not adsorbed and may therefore not be removed or detected, or they can be configured to adsorb any material present in the sample. Nevertheless, such nonselective configurations can prevent the binding of background gases, such as water and oxygen or carbon dioxide, that may be present in the sample and complicate the collection, detection, and measurement of the marker and its concentration.

[0029] The distribution of adsorbent regions or adsorbent materials in each of the sampling units may vary in relative position and / or relative cooperation and / or packing configuration. Solid adsorbents are selected for sampling specific compounds in air because they (1) have the ability to capture and retain the compound of interest even in the presence of other compounds, (2) do not chemically alter the compound of interest, and (3) allow the adsorbed compound to be easily desorbed or extracted for analysis.

[0030] Adsorption regions consist of materials configured to physically capture volatile materials. Materials may be characterized by the presence of surface pores, surface roughness, increased surface area, and the like. Regardless of structural characteristics, adsorption surfaces or materials can be tailored for selective or non-selective adsorption, as disclosed herein.

[0031] In some embodiments, the adsorbent regions are formed from materials selected from organic porous polymers such as poly(vinylidene chloride) or poly(2,6-diphenyl-p-phenylene oxide (PPPO)) prepared by controlled pyrolysis such as sulfonated polymers, ion exchange resins, and carbon molecular sieves.

[0032] In some embodiments, the adsorbent material is selected from carbon allotropes or carbonaceous materials such as carbon nanotubes, graphene, fullerenes, carbon black, activated carbon, etc.; cellulosic materials such as cellulose nanocrystals, cellulose fibers, nanofibrillated cellulose; silica gel, and others.

[0033] In some embodiments, the adsorbent is a carbon adsorbent such as Carbotrap F, Carbotrap C, Carbotrap Y, Carbotrap B, Carbotrap X, Carbopack F, Carbopack C, Carbopack Y, Carbopack B, Carbopack X, Carboxen 1016, Carboxen 569, Carboxen 1021, Carboxen 1018, Carbosieve S-III, Carboxen 1003, Carbosieve G, Carboxen 1000, and Carboxen 1012.

[0034] In some embodiments, the carbon adsorbent may also be selected from among graphitized carbon black with a 20 / 40 mesh, graphitized carbon black with a 60 / 80 mesh, and carbon molecular sieves.

[0035] In some embodiments, the adsorbent is between 5 and 1500 m 2 / g surface area, a density of 0.2 to 0.7, and / or a pore diameter of 4 to 300 Å.

[0036] Alternatively, the adsorption region can be made of a substrate material to which a plurality of binding molecules capable of reversibly binding to volatile substances are applied. These binding molecules are at least bifunctional (i.e., can have two or more functionalities) molecules, with at least one functionality that allows the molecule to associate with the surface and at least one other functionality that allows it to reversibly bind to volatile substances. The bond can be any reversible chemical bond, including ionic, covalent, hydrogen, or complexation.

[0037] Excluded from this invention are any metal-based adsorbent domains, including any kind of metal nanoparticles, metal surfaces, metal matrices, etc. In other words, the adsorbent domains are free or do not include or exclude any of the above metal forms.

[0038] Exposing at least one sampling unit to the subject's breath can be accomplished by placing the unit in the path of the exhaled breath. This can be accomplished, for example, by having the subject breathe directly into the unit through a mouthpiece, by connecting the unit to a ventilation unit to which the subject is connected, or by withdrawing a sample from the subject's lungs, as disclosed herein. Regardless of the means, the exposure can be a single exposure, multiple exposures, continuous exposure over a period of time, or a timed exposure, e.g., the unit is exposed at a given time point for a predetermined period of time. To determine the presence of a marker in the breath, the sampling unit can be exposed in different sessions, i.e., at different times and for different or the same duration, and at each exposure, the unit can be analyzed to determine the presence of the marker, as disclosed herein. The duration of each exposure session can be the same or different and can vary from a few minutes to several hours or more. The first session that identifies the presence of a marker can be considered disease onset, as discussed further below.

[0039] The exposure session can also be configured (time and duration) to provide information regarding changes (increases or decreases) in marker concentration. An increase in marker concentration over time, which can be, for example, an exponential increase, typically indicates, for example, an increase in the amount or mass or load of bacteria or vials. Such an increase can be used to determine initiation of therapy, success of medical procedures, adjustment of therapy, etc., as further disclosed herein.

[0040] After exposure of the adsorption region to the breath sample, as disclosed, various volatiles are adsorbed to the surface or features of the region and trapped / bound until analyzed. To enable analysis of the adsorbed volatiles, the adsorption region is treated to cause desorption or dissociation of the volatiles from the surface, after which the volatiles are analyzed. Desorption or dissociation of volatiles from the adsorption region can be achieved thermally, under a flow of inert gas, under vacuum, or by employing any means capable of releasing the volatiles to an analyzer. Thus, as used herein, the phrase "analyzing at least one sampling unit to identify volatiles adsorbed to one or more adsorption regions to determine the presence of" refers to analyzing volatiles adsorbed and subsequently released from the adsorption region. The analysis need not identify all substances or volatiles in the sample, but should provide identification of desired markers. Thus, the analytical system or analyzer utilized can be any instrument or device capable of chemical or spectroscopic identification of volatiles. Analysis may be accomplished by any one or more of gas chromatography (GC), GC-lined mass spectrometry (GC-MS), proton transfer reaction mass spectrometry (PTR-MS), electronic nose device (E-nose), quartz crystal microbalance (QCM), infrared spectroscopy (IR), or ultraviolet spectroscopy (UV). Analysis may be configured to identify single marker molecules or clusters of molecules. In some cases, chemical identification of the marker may not be necessary, but rather, determination of a chemical profile (fingerprint), as defined herein, may be required.

[0041] In some embodiments, the analytical system utilized for analyzing the desorbed volatiles is selected from GC, MS, and GCMS. In some embodiments, the analytical system is selected from GCMS with TOF, Market BenchTOF-HD, GC Agilent 7890 with the option to simultaneously collect different collision energies and time-of-flight mass spectrometers for GCxGC modulators, quadrupole GCMS, Agilent GC 6890 with Agilent MSD5975, Agilent GC 7890B with Agilent MSD 5977B, quadrupole GCMS, Agilent GC 6890 with Agilent MSD 5973, GCMS, Agilent 7250 GC / Q-TOF, GCMS, Agilent 7010B triple quadrupole GC / MS, GCMS, Thermo Scientific Q Exactive™ GC Orbitrap™ GC-MS / MS, etc.

[0042] In some embodiments, the analytical system comprises a first (main non-polar) column selected from SGEPN99054140 (SN:073438A23), 20Mx0.18mmID-BPX5x0.18μm df with a He flow rate of 0.5ml / min (constant flow / pressure), and a second column (which is a polar column) selected from Agilent DB5-ms 30Mx0.25mmIDx0.50μm df with a He flow rate of 1.5ml / min (constant flow / pressure).

[0043] Depending on the method used for analysis, in some embodiments, identification of markers is achieved by their retention times and relative retention times. Thus, in some cases, disease-specific or pathogen-specific markers have a characteristic fingerprint of released compounds or combinations of compounds, the presence and concentration of which is indicative of that particular disease or pathogen. Thus, for example, two different bacteria may release the same marker at different concentrations. Thus, in some cases, only the complete fingerprint, including the identity and concentration of the marker, serves as evidence of the presence of the disease or pathogen. In other cases, a single marker may be used.

[0044] The subject to be diagnosed according to the method of the present invention can be a human or non-human subject.In the most general terms, as disclosed herein, the subject can be a healthy subject, i.e., a subject not known to suffer from a chronic or acute medical condition.A healthy subject can also be a subject suffering from a chronic medical condition, but at the time of diagnosis according to the methodology disclosed herein, the subject does not exhibit symptoms associated with a chronic disease.A subject can also be a diseased subject that exhibits a state of active disease at the time of diagnosis, i.e., has symptoms associated with a disease.

[0045] In some embodiments, the subject is an asymptomatic subject.

[0046] In some embodiments, at the time of diagnosis, the subject is afflicted with a disease and the diagnosis is aimed at determining the onset of different disease states.

[0047] In some embodiments, the subject is a ventilated subject. In some embodiments, the method is for detecting ventilator-associated pneumonia (VAP) in a subject connected to a respiratory system (a ventilated or intubated subject).

[0048] Ventilator-associated pneumonia is pneumonia that develops at least 48 hours after endotracheal intubation. This disease differs from hospital-acquired pneumonia, which is pneumonia that develops at least 48 hours after hospitalization in hospitalized patients not receiving mechanical ventilation. Diagnosis of both conditions is imperfect. In practice, ventilator-associated pneumonia is often suspected when new symptoms or signs, such as fever, increased secretions, worsening hypoxemia, or leukocytosis, are present, or when new infiltrates are detected on a chest radiograph taken to evaluate the condition. Because symptoms, signs, or radiographic findings lack sufficient sensitivity or specificity for diagnosis, let alone early diagnosis, diagnosis is often initiated only at the onset of symptoms. Broad-spectrum or pathogen-specific antibiotic treatment in the post-symptomatic stage does not necessarily improve the patient's condition.

[0049] The methods of the present invention offer the possibility of early detection of the presence of various pathogens in the respiratory system of a subject, before the pathogens spread from the affected organ, e.g., the lungs, to other organs and before symptoms appear. Pathogens can be hospital-acquired or transmitted through the air (airborne exposure), by direct or indirect contact, through sexual contact, or through contact with various bodily fluids such as blood, breast milk, semen, etc.

[0050] The pathogen is typically selected from among bacteria, viruses, and fungi. In some embodiments, the pathogen is a bacteria. In some embodiments, the pathogen is a virus, and in other embodiments, the pathogen is a fungus.

[0051] The methods of the present invention are directed to detecting the presence of a virus selected from any of the viral families coronavirus, adenovirus, arbovirus, arenavirus, encephalitis, orthomyxovirus, papillomavirus, paramyxovirus, picornavirus, poxvirus, retrovirus, rhabdovirus, and rhinovirus.

[0052] In some embodiments, the virus is selected from among coronaviruses, smallpox viruses and other poxviruses, arenaviruses such as Junin virus, Machupo virus, Gunarito virus, sympathetic choriomeningitis virus, and Lassa virus, hantaviruses, bunyaviruses such as Rift Valley fever virus, flaviruses such as dengue virus, filoviruses such as Ebola virus and Marburg virus, food- and water-borne pathogens such as Eastern equine encephalitis virus, Western equine encephalitis virus, Venezuelan equine encephalitis virus, La Crosse virus, Japanese encephalitis virus, Kyasanur Forest virus, California encephalitis virus, and calcivirus, hepatitis A virus, Nipah virus, yellow fever virus, influenza virus, rabies virus, and other hantaviruses.

[0053] In some embodiments, the virus is an enveloped or non-enveloped virus, hi some embodiments, the virus is a non-enveloped virus, such as a norovirus or a parvovirus.

[0054] In some embodiments, the virus is an enveloped virus, such as an influenza virus, or a coronavirus, such as SARS-CoV-2.

[0055] In some embodiments, the virus is a coronavirus.

[0056] In some embodiments, the virus is SARS-CoV-2.

[0057] If the pathogen is a bacterium, the bacterium may be selected from Bacillus anthracis, Clostridium botulinum, Francisella tularensis, Yersinia pestis, Burkholderia pseudomallei, Burkholderia mallei, Clostridium perfringens, Coxiella burnetii, Bacillus malta, Bacillus abortus, Bacillus suis and Bacillus canis, Staphylococcus aureus, Rickettsia typhi, Chlamydia psittacosis, food and waterborne pathogens such as Escherichia coli, Vibrio cholerae, Salmonella spp., Shigella spp., Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica, Mycobacterium tuberculosis, and other rickettsiae.

[0058] In some embodiments, the microorganism is one that causes so-called hospital-acquired infections (HAIs), such as methicillin-resistant Staphylococcus aureus (MRSA). Other HAI-causing microorganisms include vancomycin-resistant enterococci (VRE), Clostridium difficile, Acinetobacter baumannii, and multidrug-resistant (MDR) Acinetobacter spp.

[0059] The fungus may be selected from Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis and Stachybotrys.

[0060] In some embodiments, the fungus is selected from the group consisting of Candida albicans, Candida amfixiae, Candida antarctica, Candida argentea, Candida ascarafidarum, Candida atlantica, Candida atmosphaerica, Candida auris, Candida branchii, Candida brattae, Candida bracalensis, Candida bromeliaceum, Candida carpophylla, Candida carbahris, Candida chelumbichidarum, Candida chauliodes, Candida corydalis, Candida dossei, Candida dubliniensis, Candida ergatensis, Candida fermentati, Candida fructus, Candida glabrata, Candida guilliermondii, Candida haemuloni, Candida fumilis, Candida insectamens, Candida insectorum, and Candida intermedii. Candida jelfresi, Candida kefir, Candida cheroseniae, Candida krusei, Candida tarsitaniae, Candida ricinusophila, Candida maltose, Candida marina, Candida membranifaciens, Candida mogi, Candida oleophila, Candida oregonensis, Candida parapsisosis, Candida quercitrus a, Candida rhizoforiensis, Candida rugosa, Candida salmon, Candida charchiensis, Aspergillus fumigatus, Aspergillus flavus, Cryptococcus neoformans, Cryptococcus lorentei, Cryptococcus albidus, Cryptococcus gattii, Histoplasma capsulatum, Pneumocystis jirovecii (or Pneumocystis carinii), and Stachybotrys cartharum.

[0061] When hospital-acquired or ventilator-associated pneumonia is involved, the pathogens are Gram-negative bacteria, Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens, Enterobacter, Citrobacter, Acinetobacter, Staphylococcus aureus, and antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). The above list represents over 80% of the pathogens associated with VAP.

[0062] Thus, in another aspect thereof, the present invention provides a method for determining the presence of at least one pathogen in the body of a subject (e.g., before symptoms associated with or indicative of the presence of the pathogen develop or become apparent), the method comprising: exposing at least one sampling unit to the breath sample, the sampling unit comprising one or more adsorbent regions capable of reversibly binding volatile substances in said breath sample; analyzing at least one sampling unit to identify volatile substances adsorbed to the one or more adsorption regions to determine the presence of at least one pathogen-associated marker (or at least one marker associated with a pathogen); The presence of the aforementioned markers indicates the presence of the pathogen in the subject's body.

[0063] In some embodiments, the pathogen is a virus, bacterium, or fungus, as defined and selected herein.

[0064] In some embodiments, the subject is an asymptomatic subject, ie, a subject that does not exhibit symptoms associated with the pathogen.

[0065] In some embodiments, the subject is a ventilated subject (a subject on a ventilator system). In some embodiments, the ventilated subject does not exhibit symptoms associated with VAP. In some embodiments, the pathogen is one that causes VAP and is selected from any of the bacteria disclosed and known to cause VAP.

[0066] Thus, the present invention also provides a method for determining the presence of at least one pathogen in the body of a ventilated subject (e.g., before symptoms associated with or indicative of the presence of the pathogen develop or become apparent), the method comprising: exposing at least one sampling unit to a breath sample from a ventilated subject, wherein the at least one sampling unit comprises one or more adsorbent regions capable of reversibly binding volatile substances present in said sample; analyzing the at least one sampling unit to identify volatile substances adsorbed to the one or more adsorption regions to determine the presence of said at least one pathogen-associated marker; The presence of such markers indicates the presence of a pathogen within the body of the ventilated subject.

[0067] More specifically, the present invention also provides a method for determining the onset of VAP in a ventilated subject (e.g., before symptoms associated with or indicative of VAP develop or become evident), the method comprising: exposing a breath sample from a ventilated subject to at least one sampling unit comprising one or more adsorbent regions capable of reversibly binding volatile substances in said breath sample; analyzing at least one sampling unit to identify volatile substances adsorbed to the one or more adsorption regions to determine the presence of at least one marker of a pathogen that causes VAP; The presence of the aforementioned markers indicates the onset of VAP.

[0068] In some embodiments, the first respiratory sample is taken from a ventilated subject on day 0, i.e., the day the subject is connected to a ventilator. In other embodiments, the first sample is taken on days 1, 2, 3, 4, or 5 after being connected to the machine, and any time thereafter (i.e., second and further samples are taken hours or days after the first sample is taken).

[0069] In some embodiments, at least one sampling unit is provided within the respiratory system normally used for artificial ventilation of the subject. Thus, in some embodiments, the method comprises: exposing at least one sampling unit positioned in an outlet line of the respiratory system to a respiratory sample (e.g., an alveolar sample) exhaled by the subject, wherein the at least one sampling unit comprises one or more adsorbent regions capable of reversibly binding volatile substances present in said sample; analyzing the at least one sampling unit to identify volatile substances adsorbed to the one or more adsorption regions to determine the presence of at least one pathogen-associated marker; The presence of the aforementioned markers indicates the presence of pathogen-associated markers in the body of the ventilated subject.

[0070] In some embodiments, at least one sampling unit includes one or more adsorbent regions, eg, in the form of a vessel that allows timed dwell contact between the alveolar breath sample and the one or more adsorbent regions.

[0071] In some embodiments, one or both of the at least one sampling unit and the respiratory system outlet line are provided with a flow regulator (eg, a valve).

[0072] In some embodiments, the disease-causing pathogen is a bacterium, virus, or fungus, as defined and selected herein.

[0073] In some embodiments, the method further includes removing at least one sampling unit from the outlet line of the respiratory system and analyzing it to determine the volatile substances adsorbed to the one or more adsorption regions.

[0074] In some embodiments, volatiles adsorbed onto one or more adsorption regions are desorbed and then analyzed.

[0075] In some embodiments, the method further comprises exposing at least one other sampling unit positioned in the inlet line of the respiratory system to the ventilated air delivered to the subject.

[0076] In some embodiments, the analysis is performed by spectroscopy or spectrophotometry, as defined.

[0077] In some embodiments, the method further includes comparing the material adsorbed to the one or more adsorption regions with a database of marker substances to identify materials indicative of the presence of a disease-causing pathogen.

[0078] In some embodiments, the presence of a disease-causing pathogen is indicative of the onset of the disease.

[0079] In some embodiments, markers identified in exhaled breath are compared to markers identified in inspired breath. Exogenous markers or substances, i.e., from the subject's surroundings, may be ignored or used as background for the analysis.

[0080] As used herein, the term "disease state" refers to a medical condition for which diagnosis is desired using the methodology of the present invention. The presence of a disease state is typically concluded by determining the presence of a marker in a subject's respiratory sample that is associated with the presence and / or proliferation of a pathogen (e.g., a bacterium, virus, or fungus) involved in metabolism, organ function, or the etiology of the disease state. A disease state is any illness identified by the WHO International Classification of Diseases (http: / / apps.who.int / classifications / icd10 / browse / 2016 / en). In the most general terms, a disease state is any condition that can cause pain, impairment, suffering, social problems, and / or death in a subject. Such conditions can be or can lead to heart disease, inflammation, kidney disease, cancer, pathogen-induced illness, etc. All of these conditions are familiar to those familiar with the art.

[0081] In some embodiments, the disease state is associated with a pathogen, as defined. In some embodiments, the disease state is associated with a bacterium. The disease state can be selected from among those caused by the pathogens disclosed herein. Bacteria that cause high infectious morbidity and mortality include cholera (caused by Vibrio cholerae, an acute infection of the small intestine characterized by severe diarrhea accompanied by rapid and severe fluid and salt wasting), which tends to manifest in the lungs; diphtheria (caused by the bacillus Corynebacterium diphtheriae, usually characterized by a primary lesion in the upper respiratory tract and systemic symptoms due to the spread of bacterial toxins throughout the body); meningitis (caused by Neisseria meningitidis, H. influenzae, which occurs more frequently in infants and young children); Streptococcus pneumoniae is a common cause of meningitis in adults, though it is rare in older adults. Other bacterial causes include various strains of streptococci, pneumococci, staphylococci, and Mycobacterium tuberculosis (which can cause meningitis), tetanus (caused by a toxin produced by Clostridium tetani and characterized by rigidity and spasms of voluntary muscles), Lyme disease (caused by several closely related spirochetes, including Borrelia burgdorferi in the United States, B. mayonii in the Midwest, and B. afzelii and B. garinii in Europe and Asia; spirochetes are transmitted to the human bloodstream through the bites of various species of stray dogs), and syphilis (a systemic disease caused by Treponema pallidum). Syphilis is usually a sexually transmitted disease (STD), but it can also be transmitted through nonsexual contact with an infected person, and the fetus can become infected through maternal infection.

[0082] In some embodiments, the disease caused by the pathogen is a hospital-acquired infection, hi some embodiments, the disease state caused by the pathogen is VAP or hospital-acquired pneumonia.

[0083] In some embodiments, the disease state is cancer. Non-limiting examples of cancer include carcinoma, squamous cell carcinoma, hematopoietic tumors of myeloid or lymphoid system, tumors of mesenchymal origin, tumors of the central and peripheral nervous system, melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, corneal canthoma, follicular thyroid carcinoma, and Kaposi's sarcoma. Also included are breast cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, endometrial cancer, gastric cancer, clear cell renal cell carcinoma, glioblastoma, uveal melanoma, multiple myeloma, rhabdomyosarcoma, Ewing's sarcoma, and medulloblastoma.

[0084] In some embodiments, the disease condition is a liver or kidney disease.

[0085] In some embodiments, the disease is associated with the lungs. Non-limiting examples include bronchiectasis, emphysema, chronic bronchitis, chronic obstructive pulmonary disease (COPD), asthma, pneumonia, pleural effusion (PE) and ventilator-associated pneumonia (VAP), cystic fibrosis, ARDS (acute respiratory distress syndrome), smoking, asbestos, etc.

[0086] When referring to an asymptomatic subject, as described herein, the subject is considered to not exhibit any of the symptoms known to be associated with a disease state.In some cases, the present invention contemplates detecting the cause of disease in a subject who is free of symptoms or in the early stages of disease before symptoms become apparent or before the subject undergoes a medical examination or test to identify a medical condition.For example, in the case of a ventilated subject who suffers from VAP, it is desirable to detect pathogens in the ventilated subject before symptoms associated with infection, i.e., before symptoms such as fever or hypothermia and hypoxemia occur.In this way, the "onset of a disease state" refers to the asymptomatic stage of a disease that does not show signs and symptoms detectable by physical examination or clinical test.More specifically, the onset of disease caused by a pathogen refers to the incubation period, and in some cases, the prodromal period, as known in the art.

[0087] Also, early detection of "disease state" or onset can be considered as the time point when any increase in marker concentration over time is measured. Such an increase is directly related to the bacterial or viral load that indicates the disease state. As described herein, if a marker indicates the presence of a microorganism that is normally present in the subject's microbiome, the onset point will be considered as the time when the marker level is elevated, or at least compared to the background level, i.e., compared to the level measured in the first case or at a previous time point.

[0088] The present invention is further directed to a method for determining an injury or disease in a ventilated patient, the method comprising: contacting at least one collection or sampling unit, equipped with a valve and containing an adsorbent material, with the inlet and / or outlet lines of the respiratory system for capturing by adsorption volatile and semi-volatile compounds present in the exhaled and / or inspired air passing into the collection unit when the valve is open, wherein the valve is located between the inlet and outlet lines and the at least one collection or sampling unit, thereby allowing compounds present in the air passing through the collection or sampling unit to be adsorbed onto said adsorbent material; desorbing the adsorbed compounds to at least one collection or sampling unit and transferring them for analysis; providing an analytical unit for chemically identifying the desorbed compounds and optionally determining the relative amount of each of the detected compounds; providing an analytical system for initially creating a first list of compounds including the relative amounts of each of the detected compounds, the analytical system further comprising a database for further analysis of the identified compounds.

[0089] In some embodiments, the valve is opened for a period of time several times per day to allow exhaled and / or inhaled air to pass through at least one collection unit, and the duration of each opening and the number of times the valve is opened can be predetermined or can be done on an as-needed basis.

[0090] The present invention further provides a method for determining the success of a medical procedure in a subject undergoing the medical procedure, the method comprising: exposing at least one sampling unit comprising one or more adsorbent regions capable of reversibly binding to volatiles in a breath sample from the subject, and capable of adsorbing the volatiles in said breath sample to said one or more adsorbent regions; analyzing at least one sampling unit to determine the presence and / or amount of a marker indicative of disease; The presence or absence of said markers and / or changes in the amount of said markers relative to the amount measured at an earlier time point provides an indication of the success or failure of the medical procedure.

[0091] As used herein, a medical treatment can be any such treatment aimed at ameliorating undesirable symptoms associated with a disease, preventing the onset of such symptoms before they occur, slowing disease progression, slowing worsening of symptoms, enhancing the onset of remission periods, slowing irreversible damage caused by the progressive chronic phase of a disease, delaying the onset of said progressive phase, reducing the severity of the disease, curing the disease, improving survival or faster recovery, or preventing the onset of a disease. While a successful medical treatment can be noted in achieving any of the aforementioned objectives, according to the methods of the present invention, a successful medical treatment can be noted in one or more of the following: (1) the marker is no longer detectable in the subject's respiratory sample; (2) the amount of the marker decreases over time; (3) the amount of the marker decreases relative to other markers present in the respiratory sample; and (4) a decrease in clinical signs and symptoms associated with the diseased state as determined following initial identification by the proposed invention.

[0092] Any of the methods disclosed herein may include treating a subject identified as having a disease or condition to prevent further progression of the disease or condition. Treatment may involve the administration of an active substance or therapeutic protocol intended to ameliorate symptoms that may be associated with the disease or prevent the onset of such symptoms before they occur, slow the progression of the disease or condition, slow the worsening of symptoms that may occur, slow irreversible damage that may occur in the progressive chronic stage of the disease or slow the onset of the advanced stage, reduce the severity of an established disease or condition, improve survival, or prevent the onset of the disease.

[0093] The present invention further provides an apparatus, device, or system for carrying out the method of the present invention. The apparatus, device, or system generally comprises at least one sampling unit, as disclosed herein, including one or more adsorbent regions capable of reversibly binding to volatile substances in a breath sample from a subject, and the at least one sampling unit is optionally detachable from the device and can be handled separately. One such device is a respiratory system as disclosed herein.

[0094] If the sampling unit is removable from the device, it can be removed manually or automatically for analysis, e.g., at another location. Analysis can also be accomplished on-site. If the sampling unit is not removable or configured to exist separately from the device, the sampling unit can be provided with an opening through which the breath sample can be communicated and an opening through which post-desorption volatiles can be removed for direct analysis. In some cases, the unit has a single opening that serves both purposes. In other embodiments, the sampling unit further has two openings. Each opening can be provided with a valve or valve assembly.

[0095] As disclosed herein, a sampling unit is also provided that includes at least one adsorption region, which is formed of a porous material or a plurality of features capable of capturing or binding volatile substances in a respiratory sample. In some embodiments, the sampling unit has an opening configured and operable to allow the flow of a respiratory sample therethrough. The opening can be fitted with a valve unit that allows control of the flow of substances into and out of the unit. Optionally, the unit can further comprise or be provided with a means or port that can be adapted for tight coupling to a device or system.

[0096] The present invention also provides a respiratory system comprising a compressible air reservoir, a set of tubing, and a patient circuit comprising an inspiratory tube and an expiratory tube, the patient circuit being connected to an intubation tube, one or both of the inspiratory and expiratory tubes comprising at least one sampling unit positioned within the pathway of alveolar breath exhaled by a subject being ventilated, the at least one sampling unit comprising one or more adsorption regions capable of reversibly binding at least one volatile substance present in the exhaled alveolar breath, thereby indicating the presence of a disease state or disease-causing pathogen.

[0097] Also provided is a system configured to determine the presence of a disease-causing pathogen in a ventilated subject, the system comprising a ventilator comprising a patient circuit provided with a plurality of optionally removable collection surfaces positioned in the path of exhaled breath, each of the plurality of collection surfaces characterized by a plurality of binding regions, each of the binding regions in the form of a binding molecule and / or surface feature configured to reversibly bind at least one material indicative of the presence of the pathogen.

[0098] Any of the methods or systems of the present invention may depend to some extent on the initial knowledge and understanding of markers whose presence in a respiratory sample defines, for example, the onset of a disease or the presence of a certain pathogen. It is also important to characterize, among other things, the characteristics that distinguish one disease from another, the characteristics that define the onset of a disease state, and the characteristics that define the appropriate progression of successful medical treatment. A large number of markers are known to be present in a subject's breath and to develop along with or be caused by a disease or microorganism, thereby indicating the presence of a disease state or microorganism. Determining the relationship between such disease states or pathogens with markers that can be detected in a subject's breath can be achieved by methodologies well known in the art. Such methodologies may involve collecting markers from the headspace above abnormal cells or pathogens in a sealed vessel, or determining the identity of markers in blood or other bodily fluid samples.

[0099] Using known disease- or pathogen-specific markers, data obtained from a method of the present invention can be compared with data collected and stored in a database. For example, markers and marker concentrations, amounts, or levels measured in a breath sample can be compared with a control to determine the success of a medical treatment or to determine whether changes in a particular marker fingerprint indicate the early development of a disease state. A control refers to a subject who is not affected by a disease, i.e., a subject who has been tested and found to be unaffected by the disease, or a subject known to be disease-free, as well as any components of a marker fingerprint obtained from a subject who is affected by the disease. These can be used to define a "healthy group," i.e., a group of subjects who are not affected by the disease, and a "disease group," i.e., a group of subjects who are affected by the disease. Comparing a marker fingerprint with the marker profile of a control can determine whether the fingerprint indicates a subject who is or is not affected by the disease or pathogen. Similarly, to diagnostically determine whether a medical treatment is successful, a marker fingerprint obtained from a subject can be compared with a control sample obtained from the same subject at one or different time points before or after the start of treatment.

[0100] Control samples obtained for the purpose of determining the presence or absence of disease are typically taken from multiple (one or more) subjects identified as healthy or diseased, and the number of subjects can range from at least 10 to several thousand subjects.

[0101] As described therein, changes in VC profiles and their evolution over time, e.g., compared to symmetric, may be determined utilizing algorithms such as, but not limited to, artificial neural networks, multi-layer neural networks (MLP), generalized regression neural networks (GRNN), fuzzy inference systems (FIS), self-organizing maps (SOM), radial bias functions (RBF), genetic algorithms (GAS), neuro-fuzzy systems (NFS), adaptive resonance theory (ART), and statistical methods including, but not limited to, principal component analysis (PCA), partial least squares (PLS), multiple linear regression (MLR), principal component regression (PCR), discriminant function analysis (DFA) including linear discriminant analysis (LDA), or cluster analysis including nearest neighbor.

[0102] The target data, as disclosed, can be stored in a database compiling a qualitative and quantitative list of grouped compounds previously determined to be indicative of disease or pathogen markers. The database can further include marker combinations or fingerprints, each attributable to a particular identified pathogen, e.g., bacteria, or disease state.

[0103] As used herein, a group of markers that provides a predictive indication of a disease state or the possible presence of a pathogen is considered a fingerprint or cluster. Different microbiomes have different clusters. Thus, the detection of a specific cluster or fingerprint can define the microbiome. In addition to the list of molecules contained in the fingerprint, the algorithm analyzes the concentration changes and relationships between markers to define the contents of the cluster and / or the concentration changes (positive or negative) between consecutive patient samples. The algorithm is also configured to define the rate of change ("slope") of the cluster parameters, changes that can determine the onset of a disease, disorder, or condition, and the need and urgency of reporting to medical staff. The rate of increase (slope) of the levels of cluster components can also be attributed to the severity of the threat. The gradient defines not only the trend of a specific molecule, but also the trend of the composition. [Brief explanation of the drawings]

[0104] For a better understanding of the subject matter disclosed herein, and to illustrate how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0105] [Figure 1] FIG. 1 is a schematic diagram of a mechanical ventilator (MV) showing several locations of sampling or collection units, according to an embodiment of the present invention. [Figure 2] 1A-1C are schematic diagrams of parallel and tandem configurations of mechanical ventilators (MV) showing several locations of several sampling or collection units, according to an embodiment of the present invention. [Figure 3] 1 is a schematic diagram of an exemplary VADS (Ventilation Associated Detection System) of the present invention, where a single analytical chemistry unit is deployed and processed on a cloud server to provide the patient status in different forms. [Figure 4] 1 is a schematic diagram of an analytical chemistry unit used in the present invention. [Figure 5] FIG. 1 is a schematic diagram of a cloud server subsystem for data analysis. [Figure 6A] After processing with a data analysis software entity, it provides a computerized output of the GC-MS raw data. [Figure 6B] It provides a magnified image of specific target molecules that indicate the presence of certain bacteria in the patient's breath. [Figure 6C] The bacterial load of the patient is provided, showing characteristic markers indicative of the microbiota. [Figure 7A-7C] provide the patient's bacterial loads of Staphylococcus aureus (MRSA), Acinetobacter ( 9 ), and Klebsiella , respectively. [Figures 8A-8C] provide the patient's Staphylococcus aureus, Actinobacter, Acinetobacter, and Pseudomonas, respectively. [Figures 9A-9E] It provides detection of bacterial load by detecting "fingerprints" (clusters of compounds) previously found to be characteristic of certain bacteria. [Figures 10A-10D]The detection of functional groups previously found to be characteristic of certain bacteria provides detection of bacterial load. [Figures 11A-11D] It provides detection of bacterial load by detecting specific single compounds previously found to be characteristic of certain bacteria. [Figures 12A-12E] A method is provided for determining the concentration of a specific compound (1-ethyl-4-methylbenzene - Figure 11B) in exhaled breath. [Figures 13A-13F] The concentrations of compounds indicative of three specific bacteria are provided in patients diagnosed with pneumonia and healthy individuals. DETAILED DESCRIPTION OF THE INVENTION

[0106] The present invention relates to methods and systems as disclosed herein.

[0107] A complex medical condition was chosen for testing to demonstrate the uniqueness of the technology. VAP involves a pathogen, a typically uncooperative subject with a pre-existing active medical condition, and a potentially evolving, life-threatening disease for which early detection is highly desirable. Accordingly, the examples provided herein demonstrate: (a) early detection of pathogens (before symptoms associated with the pathogen become apparent); (b) the ability to detect pathogens or disease early and prevent potentially fatal complications if not detected early; (c) detection of pathogens in subjects already suffering from active disease; (d) the ability to distinguish markers associated with pathogens from other disease-causing factors; (e) in samples taken from uncooperative subjects; and (f) the ability to monitor treatment and correlate it with marker levels [i.e., a decrease in marker levels can be correlated with a decrease in pathogen activity (effectiveness of treatment)]. In contrast, an increase in marker levels during treatment may indicate that the treatment is not effective and that a change in treatment (such as the use of antibiotics, the use of a different antibiotic, physical therapy, or repositioning) is necessary], (g) periodically scanning ventilators for contaminants, and (h) comparing marker levels of all ventilators (MVs) within an ICU unit to detect contamination with resident bacteria, etc., demonstrating the capabilities of the technology disclosed herein.

[0108] Thus, the methods of the present invention aim to provide methods for the early detection of a bacterial condition, disease or disorder in a subject, methods for determining the onset of a bacterial condition, disease or disorder in a subject, methods for preventing the onset or delaying the progression or onset of a bacterial condition, disease or disorder in a subject, which methods are carried out in accordance with the present invention by sampling exhaled breath from a subject, the sampling being carried out by adsorbing substances / compounds present in the exhaled breath, sampling, desorbing the adsorbed substances / compounds, analyzing them and comparing them with a predetermined known database containing information on such substances / compounds released into the environment by bacteria.

[0109] The present invention also relates to a system for accurate and quantitative determination of compounds present in exhaled and / or inhaled air from the respiratory system of a living animal, preferably a mammal, preferably a human. These compounds are volatile and semivolatile compounds (VOCs, sVOCs), preferably organic compounds. The system detects such compounds by sampling the air exhaled and inhaled from the human lungs, adsorbing the compounds present in the air as the sampled air passes through a suitable unit containing a suitable adsorbent that adsorbs these compounds, preferably in a manner that does not alter their chemical properties or structure. Such sampling requires a significant volume of air to pass through the suitable unit. Therefore, according to the present invention, the system can be used with mechanically ventilated patients, as long as the pressure provided by the ventilator provides sufficient force to allow for exhaled and / or inhaled air. Passive sampling is also possible, for example, by collecting exhaled air from the nose to avoid sampling bacterial, viral, and fungal communities in the oral cavity.

[0110] Compounds adsorbed onto the adsorbent in the appropriate unit are typically desorbed by heating to a temperature above the adsorbed compound's boiling point, below which the adsorbent begins to chemically decompose. The desorbed compounds are then transferred to an analytical unit, which separates and identifies the compounds. Identification refers to identifying both the chemical nature and chemical properties of these compounds, their grouping (e.g., alkyl, alkene, alkyne, alcohol, amine, aromatic, cyclic, heteroatoms (e.g., P, N, O, S) present in any of the above groups), and their relative abundance in the sampled air. A further analytical system receives the data identified by the analytical unit and provides a chemical picture, i.e., a list of detected and identified compounds, providing an indication of the compounds present in the respiratory system of the individual whose air was sampled.

[0111] The resulting compound list contains a large number of compounds and their potencies, and the correlation with the disorder or stage of the disease is not simple and must be elucidated. To achieve this, the present invention utilizes two other features present in the analytical system: a proprietary database and a proprietary algorithm, both of which are part of the present invention.

[0112] The unique database contains previously acquired data containing a list of grouped, identified compounds released by bacteria into their surroundings, and the volatile and semi-volatile compounds produced and released into the bacterial environment are supplemented, identified, and serve as a basis for comparison. Each bacterium releases unique compounds in specific relative amounts, and the entire list of compounds is a "fingerprint" of this particular bacterium, including biomarkers. Biomarkers are useful for analysis according to the present invention.

[0113] The unique algorithm of the present invention compares a group of biomarkers, which are clusters of biomarkers present in a database, with the compound or compounds identified in the exhaled breath. Thus, a match between the compounds obtained by the individual's analyzed sampled air and the clusters in the database serves as an indication of the presence of a specific microflora indicative of specific bacteria in the individual's respiratory system, and therefore identifies a disorder or disease associated with such bacteria.

[0114] Background compounds present in an individual's respiratory system from the individual's surroundings and surrounding equipment may or may not be considered (by background subtraction). These include compounds present in the ventilator system's air source, the microbiome of a particular hospital, the clinic where the individual is located, and compounds emitted by medical equipment around the individual. Because these compounds enter the respiratory system through the air entering the lungs, sampling air through the inspiratory tube of the ventilator, characterizing the compounds as was done for exhaled breath, and subtracting them from the identified compounds present in the exhaled breath can provide a cleaner picture of compounds occurring in the respiratory system due to disorder or disease.

[0115] Thus, the present invention demonstrates and illustrates the relative portions of a group of compounds released by various bacteria, identifies the nature and relative amounts of the compounds produced by these bacteria, and generates a list of various specific compounds that indicate the presence of these bacteria in the sampled air. These compounds are called target molecules and are indicative of this specific bacterium. The group of compounds and their relative amounts are indicative of a bacterium (or bacteria) and thus function as biomarkers that specifically identify the bacterium or bacteria. According to the present invention, identifying one or more bacterial biomarkers in exhaled breath in a given measurement, and their increased concentration compared to a previous measurement, indicates a disorder, condition, or disease known to be associated with the specific identified bacteria. Identifying a bacterial biomarker or a cluster of biomarkers, i.e., a group of two or more biomarkers, indicates a disorder, condition, or disease state.

[0116] It should be understood that each bacterium is identified by its characteristic compounds. A given bacterium in a given situation may produce and release one or more compounds into the environment, and the amount of compound or compounds produced and released by one bacterium compared to another will differ. While overlap in a single compound (or compounds) released by two different bacteria is frequent, the fully identified spectrum of a particular well-identified bacterium differs from that of another bacterium by the fact that the amount and overall list of compounds of that particular bacterium differs from that of the other bacterium. Furthermore, each compound produced and released is identified by its retention time (RT) within the analytical system. Calculating the relative retention time (RRT) of each set of compounds released by a particular bacterium provides more accurate evidence of the specific bacterium. Therefore, identifying only one compound in air sampled from an individual's respiratory system is not sufficient to identify the presence of a particular bacterium, as different bacteria may produce and release the same compound (albeit at different concentrations) into the environment.

[0117] Thus, the present invention aims to identify diseases and disorders associated with bacterial infection by collecting volatile and semi-volatile compounds released by bacteria into their surroundings, identifying these compounds and their intensities, and allowing comparison with a database containing previously collected data on the nature of compounds released by known bacteria and the intensity of each compound.

[0118] In particular, the present invention is directed to identifying disorders, diseases or conditions associated with the lungs, non-limiting examples of which are bronchiectasis, emphysema, chronic bronchitis, chronic obstructive pulmonary disease (COPD), asthma, pneumonia, pleural effusion (PE) and ventilator-associated pneumonia (VAP).

[0119] 1 and 2 schematically depict a mechanical or other type of ventilator unit 1. Following proper operation of the unit, air is pumped from the unit through an inhalation tube 2 in the direction indicated by the arrow and into the patient's lungs via an intubation tube 7. Compounds present in the lungs, preferably volatile organic compounds (VOCs), or markers, are removed with exhaled air from the lungs through the intubation tube 7 and an exhalation tube 3 in the direction indicated by the arrow. Exhaled air is expelled from the lungs passively or by pressure exerted by the unit on the subject's respiratory system. Alternatively, it may be expelled by a pump. One or more sampling units 4, each optionally equipped with a valve 5 or T-connection, are placed in either or both of the inhalation tube 2 and the exhalation tube 3 to passively and / or actively adsorb VOCs.

[0120] In some cases, the location of one or more sampling units 4 may be inside the inspiratory and / or expiratory tube and / or perpendicular to the inspiratory and / or expiratory tube. When positioned perpendicular to the inspiratory and / or expiratory tube, the location is typically 1-200 cm, 1-20 cm, 10-60 cm, 50-100 cm, 100-150 cm, or 150-200 cm from the bifurcation 6 separating the inspiratory tube from the expiratory tube. The number of units within the inspiratory and / or expiratory tube may vary and may be one or more per inspiratory or expiratory tube.

[0121] The position of the sampling unit perpendicular to the inspiratory or expiratory tube is typically 1-50 mm, 1-30 cm, 1-20 cm, 1-10 cm, or 1-5 cm from the flow to minimize the Venturi effect and turbulence within the sampling unit. A flow limiter (not shown) can be positioned at the distal end of the unit to control the flow rate, increase VOC uptake, minimize variability, and therefore provide high reproducibility. An optional (and exemplary only) valve 5 is positioned to close the connection to the inspiratory or expiratory tube 2 or 3 when the unit 4 is not connected or removed. Other means are possible, such as a T-shaped connector whose tip closes when the unit is closed.

[0122] FIG. 1 shows a single sampling unit 4 positioned perpendicular to tube 2, with a single unit positioned relative to tube 3. FIG. 2 shows multiple sampling units evenly grouped on each of tubes 2 and 3. The number of sampling units can be one or more, and units can be positioned on either the inspiratory tube 2 or the expiratory tube, or both. Typically, at least one sampling unit is positioned on the expiratory tube 3. When the number of tubes is more than one, i.e., two or more, and when two or more sampling units are positioned on either the expiratory or inspiratory tube, the units can be arranged in parallel or tandem, as shown in FIG. 2. While a parallel configuration positioned on the inspiratory tube and a tandem configuration positioned on the expiratory tube are shown, this is not limiting, and any combination of configurations on the inspiratory and expiratory tubes can be used.

[0123] Turning to FIG. 3, a ventilator-associated detection system (VADS) 100 is shown. The system includes multiple sampling units (50) labeled CU1, CU2, CU3, etc. (as collection units), whose chemical content is transferred to an analytical chemistry unit 101 via a sample connection, which may be an autosampler (60). Data obtained from the analytical chemistry unit 101 is transmitted to a central server (not shown) or cloud server 201. At the central server / cloud, the data is inserted into an analytical algorithm and compared with a metadata bank and with previous patient samples. The metadata bank contains a set of "clusters," which are VOCs specific to a particular disease or pathogen (determined according to the present invention). The "clusters" can also be subdivided into specific pathogens (e.g., specific disease-causing bacteria) or specific patient subpopulations (e.g., a group of diabetic patients with VAP). The metadata also includes patient data and databases of known and hospital microbiomes, which can be used to assess the patient's condition. The algorithm determines the statistical probability of a change in the patient's status, which may indicate the onset of infection, cardiovascular status, nutritional status, cancer status, and a report that is communicated to staff. "Change" refers to a predetermined gradient (change) in the concentration of cluster elements over several consecutive sampling events. The output is then communicated for further action / treatment or archiving. The output can then be communicated to the nurse / doctor station 301, mobile application 401, hospital information system 501, and others.

[0124] In Figure 4, an analytical chemistry unit 101 is provided. It includes one or more GC-MS instruments, such as 101A and 101B, connected to a PC 102 and running software shown at 103. The contents of the collection unit 4 (shown in Figures 1 and 2), containing patient samples, are fed to the GC-MS via an autosampler for digital conversion and generation of digital raw data. The GC-MS output raw data is processed by a data analysis software entity to provide patient-specific data measurements, which are then packaged, encrypted, and transmitted over an Internet network using IP protocols. The packaged data includes the current measurements, as well as patient ID, a timestamp, and / or other relevant information. A user interface (UI) provides technicians with the ability to monitor and control the operation of the GC-MS and the analytical process.

[0125] In Figure 5, the cloud server subsystem is shown at 201A. The cloud server receives and manages information from multiple analytical chemistry units located in the system. A non-limiting embodiment of the present invention provided in this figure divides the system into two main groups 202 and 203. System 202 includes software, including an IP protocol for sending and receiving information between the cloud and chemistry units and remote stations, and a security and authorization entity for encrypting and decrypting each patient's data and providing authorization to access the information. It also includes a cloud server event management entity. These may include new data received, patient alarms, fault indications, or other system events, and statistical / analytic queries for inquiries required by users and / or other software entities. System 203 includes the main software processes related to the present invention. Cloud server database 204 includes a patient database that stores measurement history from specific patients taken at different time intervals and a cluster database related to specific characteristic molecular signals used to predict specific diseases. A data access and management software entity is an interface for storing and retrieving data from the associated databases. The cluster analysis software algorithm acquires new measurements from the patient and processes them by comparing them with information retrieved from the cluster database and previous patient measurements. The patient status assessment algorithm software is responsible for predicting indications of specific types of patient disease and / or treatment effectiveness and / or generating alarms for one or more remote stations. The algorithm determines the level of change between successive measurements, from the baseline when the patient arrived at the ICU to the most recent measurement, and based on its slope characteristics, provides a statistical prediction of the patient's status and specific disease, as well as the effectiveness of treatment based on early detection. The machine learning (ML) algorithm entity is used to continuously improve predictions made based on the slope measurements using the entire patient's historical data collected by the system. This is used to improve the system's statistical false alarms and false positives over time.

[0126] Figure 6A illustrates the computerized output after processing the raw GC-MS data with a data analysis software entity to generate patient-specific data measurements. Figure 6B provides a magnified image of specific target molecules indicating the presence of certain bacteria in the patient's breath. Figure 6C provides the patient's bacterial load and shows target molecules characteristic of the patient's microbiome.

[0127] Figures 7A, 7B, and 7C display the bacterial load of three known bacteria in a patient diagnosed according to the present invention, specifically highlighting Staphylococcus aureus (MRSA), Acinetobacter (9), and Klebsiella, respectively.

[0128] Figures 8A, 8B and 8C show the bacterial load of three known bacteria in a patient diagnosed according to the present invention, specifically highlighting Staphylococcus aureus, Actinobacter, Acinetobacter and Pseudomonas, respectively.

[0129] Turning to Figures 9A–9E, we demonstrate the detection of bacterial loads of Acinetobacter (Figures 9B and 9C), Pseudomonas (Figure 9D), and Klebsiella (Figure 9E). The reference to "Acinetobacter" in the indications for specific days, such as "Day 1" and "Day 5," refers to the finding of Acinetobacter by the traditional method, which involves collecting lavage fluid from the lungs, growing colonies on a suitable substrate, and detecting them by time-of-flight. The reason why only Acinetobacter is found by the traditional method is related to the mechanism by which it is delivered: collecting lavage fluid from a precise location in the lung, developing colonies on a growth medium, and then collecting a sample for analysis. Our results are presented as peak concentrations representing target molecules whose abundance changes over time. Therefore, of the 2,461 compounds detected by the GC-MS system (Figure 9A), the concentrations of only 41 compounds (previously characterized) are selected (Figure 9B). Figure 9C clearly shows the concentrations of 20 compounds characteristic of Acinetobacter (previously characterized) and their time course. Prior to detection of VAP by conventional means (X-ray), clear Acinetobacter growth was observed on day 4. Figure 9D demonstrates the same for Pseudomonas, and Figure 9E demonstrates the same for Klebsiella. Thus, whereas the conventional route is limited by the efficiency of lavage fluid extraction (depth and location within the lung), the present invention samples all compounds in the exhaled breath, thus providing a more complete and detailed picture of all bacteria present, as each of these bacteria is reflected by its characteristic compound, the target compound.

[0130] Turning to Figures 10A-10D, the bacterial loads of Acinetobacter (Figure 10C) and Pseudomonas (Figure 10D) are shown. The references to "Acinetobacter" or "Pseudomonas" in the designations for specific days, such as "Day 1" and "Day 5," refer to the findings of the bacteria using traditional methods, in which lavage fluid is collected from the lungs, grown on an appropriate substrate, and detected by time-of-flight. Results from the present method are given as peak concentrations representing molecules whose abundance changes over time. Figure 10A demonstrates the detection of previously characterized alcohols (277 compounds) characteristic of bacteria. Figure 10B demonstrates the detection of previously characterized alcohols (789 compounds). Figure 10C provides the detection of Acinetobacter based on its 19 previously characterized compounds, and Figure 10D provides the detection of Pseudomonas based on its 16 previously characterized compounds.

[0131] Turning to Figures 11A-11D, we demonstrate the detection of bacterial loads of three different bacteria based on specific compounds (previously characterized). The references to "Staphylococcus aureus culture" in the indications for specific days, such as "Day 1" and "Day 5," refer to the findings of bacteria obtained by the traditional method of collecting lavage fluid from the lungs, growing them on an appropriate substrate, and detecting them by time-of-flight. Thus, Figure 11A provides the concentrations of 40 target compounds previously characterized as belonging to these bacteria. Figure 11B shows the detection of 1-ethyl,4-methylbenzene, indicating the presence of Staphylococcus aureus. Figure 11C shows the detection of 1,3-dimethylbenzene, indicating the presence of Pseudomonas aeruginosa, and Figure 11D shows the detection of benzaldehyde, indicating the presence of Klebsiella.

[0132] Figures 12A-12D provide a method for determining the amount of each compound according to the present invention. Figure 12A shows a portion of a chromatogram containing multiple compounds, such as 1-ethyl, 4-methylbenzene (Figure 12E), identified according to retention time. However, it is buried under many other compounds. Figure 12B demonstrates deconvolution performed to more specifically detect the peak of this particular compound. Figure 12C demonstrates the results of the deconvolution in a more expanded view. Figure 12D shows the isolated peak of this compound, and by calculating its area (area under the curve), its concentration in exhaled breath can be determined.

[0133] Turning to Figures 13A-13F, three typical bacterial concentrations associated with pneumonia are given. Patients diagnosed with pneumonia had concentrations ranging from 0.2 to 20x10 6 The concentration of compounds that indicate specific bacteria is on a scale of 0.2 to 200x10 3 13A, 13C, and 13E illustrate patients diagnosed with pneumonia, while FIGS. 13B, 13D, and 13F illustrate healthy individuals.

[0134] experiment The sampling or collection units used contained adsorbents selected from TENAX™, i.e., poly(2,6-diphenyl-p-phenylene oxide) (PPPO), Carboxen™, i.e., sulfonated polymers, poly(vinylidene chloride), or carbon molecular sieves prepared by controlled pyrolysis of sulfonated polymers. Carbon adsorbents used in accordance with the present invention include Carbotrap F, Carbotrap C, Carbotrap Y, Carbotrap B, Carbotrap X, Carbopack F, Carbopack C, Carbopack Y, Carbopack B, Carbopack X, Carboxen 1016, Carboxen 569, Carboxen 1021, Carboxen 1018, Carbosieve S-III, Carboxen 1003, Carbosieve G, Carboxen 1000, and Carboxen 1012.

[0135] Carbon adsorbents may also be selected from graphitized carbon black having a 20 / 40 mesh, graphitized carbon black having a 60 / 80 mesh, and carbon molecular sieves. In some embodiments, the adsorbent has a surface area of 5 to 1500 m / g, a density of 0.2 to 0.7, and / or a pore size of 4 to 300 Å.

[0136] The thermal desorption units used to desorb the volatiles adsorbed on the sampling unit were selected from the following: a Markes TD100-xr (autosampler) and TD-100 cold trap, each containing a stainless steel thermal desorption sorbent tube; a Perkin Elmer Turbo Matrix 650 ATD thermal desorption system, each containing a stainless steel thermal desorption sorbent tube; a Shimadzu TD-20 or TD-30 thermal desorption system, each containing a stainless steel thermal desorption sorbent tube; a Gerstel TDS3C / TDS-A2 thermal desorption system, each containing a stainless steel thermal desorption sorbent tube; a Scientific Instrument Services (SIS) TD-5 thermal desorption system, each containing a stainless steel thermal desorption sorbent tube; a CDS9300 thermal desorption system with a CDS7550 autosampler, each containing a stainless steel thermal desorption sorbent tube; and a CDS7550S standalone 72-position thermal desorption system, each containing a stainless steel thermal desorption sorbent tube.

[0137] The analytical systems, GC, MS, and GCMS, were selected from a GCMS with TOF, a Marke BenchTOF-HD, a time-of-flight mass spectrometer for GC Agilent 7890, and a GC×GC modulator, including the option to simultaneously collect different collision energies. Quadrupole GCMS, Agilent GC 7890B with Agilent MSD 5977B, Agilent GC 6890 with Agilent MSD 5975, quadrupole GCMS, Agilent GC 7890 with Agilent MSD 5975, quadrupole GCMS, Agilent GC 6890 with Agilent MSD 5973, GCMS, Agilent 7250 GC / Q-TOF, GCMS, Agilent 7010B triple quadrupole GC / MS, GCMS, ThermoScientific QExactive™ GC Orbitrap™ GC-MS / MS.

[0138] GC Column Sample: The GC separates analytes using two capillary columns. The first (non-polar main column) column was selected from the following: SGEPN 99054140 (SN: 073438A23), 20M x 0.18mm ID - BPX5 x 0.18µm df, He flow rate 0.5ml / min (constant flow / pressure). The second column was the polar column. GC Capillary Column: Agilent DB5-ms 30M x 0.25mm ID x 0.50µm df, He flow rate 1.5ml / min (constant flow / pressure). GC Capillary Column: Zebron ZB-5, 30M x 0.25mm ID x 0.25µm df, He flow rate 1.2ml / min (constant flow / pressure). GC capillary column: Agilent DB5-ms 60Mx0.25mmIDx1.0μm df, He flow rate 1.5ml / min (constant flow / pressure). GC capillary column: Agilent DB5-ms 60Mx0.53mmIDx1.4μm df, He flow rate 5ml / min (constant flow / pressure). GC capillary column: Agilent DB1 60Mx0.32mmIDx0.5μm df, He flow rate 2ml / min (constant flow / pressure). GC capillary column: Agilent DB1 30Mx0.18mmIDx0.25μm df, He flow rate 0.6ml / min (constant flow / pressure). GC capillary column: Agilent DB1 30Mx0.15mmIDx0.15μm df, He flow rate 0.3ml / min (constant flow / pressure).

[0139] Calculating area peaks to determine bacterial / viral / fungal mass increase The compounds desorbed from the sampling unit are analyzed using a GCMS instrument after chromatographic separation on a capillary GC column. In the resulting MS chromatogram, all separated substances are displayed as chromatographic peaks ordered by their retention time. Each peak consists of a continuous line connecting several points, each of which represents the sum of the abundances of fragment ions generated by the fragmentation of a substance molecule. The peak area is determined by the time from the start point to the end point of the peak (d), as derived from Equation 1: abundance / dt).

number

[0140] It should be noted that peak area values will vary between GCMS instruments as well as depending on the integrated software used, however, the determination of disease or bacterial / viral / fungal load is indicative and conclusive as it is determined by determining the change between two consecutive measurements per subject using the same GCMS instrument.

[0141] To calibrate the peak area of each marker, a known substance is used as an internal standard (IS) and inserted into the sampling unit. The IS is used at a known concentration, volume, and pressure (e.g., 1 ml volume of standard gas with 3 ppm of IS compound at 25 psi inlet pressure). The peak area is normalized according to the IS area using known and accepted IS calculation methods. Three non-limiting examples of ISs that can be used in accordance with the present invention are shown in Table 1. [Table 1]

[0142] 1,000(10 3 The marker area size of 1,000,000 (10) is approximately equivalent to a concentration of 0.006 ppm (v / v) of IS-1. 6 ) is approximately equivalent to a concentration of 6.4 ppm (v / v) of IS-1.

[0143] Assessment of disease incidence based on calculated area Since each patient arriving / brought in for long-term hospitalization has their own medical background, by analyzing the patient's breath upon arrival in accordance with the present invention, a baseline characterizing the particular patient is created (patient baseline). The patient's baseline or background level of the marker is calculated by the sum of all areas measured for the peaks of the marker appearing in the breath.

[0144] Assessment of the onset of infection is performed by monitoring the total area of the marker peaks during the hospitalization period. These provide an indication of the change / progression of bacterial load (BL), viral load (VL), or fungal load (FL) in the patient's respiratory system, i.e., lungs. An increase of approximately 50% or more in the peak area is considered a significant change reflecting an increase in bacterial load. When the area of the marker peak increases by approximately 50%, the algorithmic description of the onset of infection is as follows:

[0145] If the total biomarker compound (TBCM) area on day 2 is greater (>50%) compared to the patient's baseline (day 1) and the TBCM area on day 3 is greater (>50%) than the TBCM area on day 2, this indicates a significant increase in bacterial / viral / fungal load (BL / VL / FL) and should be reported and continuously monitored.

[0146] Assessment of infectious disease, including VAP The algorithm, which indicates signs of an increased bacterial or viral load, can provide an assessment of the bacterial group or type of virus or fungus. The algorithm incorporates various metrics that take into account the peak area size of biomarkers, the number of biomarkers, the type of biomarkers, and the ratios between them. Thus, the algorithm includes data on, among other things, the bacterial / viral / fungal load, markers, total marker compounds, general markers, the number of bacteria / viruses / fungi, etc.

[0147] The following example is based on 36 patients enrolled in a clinical trial. The actual trial (requiring at least 3 days of sampling) included 28 patients. By using the analysis of exhaled breath samples from ventilated patients according to the present invention, 6 of the 28 patients were identified as potential VAP cases (

number

[0148] Target molecules (TM), which indicate an increase in specific bacterial burden attributable to bacteria associated with VAP, were detected in all six patients. It is important to note that TM was detected in the patients' exhaled breath 1, 2, and 3 days before the onset of standard-of-care clinical signs of VAP, as illustrated below. These standard-of-care clinical signs include new, persistent (>48 hours), or progressive radiographic infiltrates, as well as two of the following: a temperature of >38°C or <36°C; a blood white blood cell count of >10,000 cells / ml or <5,000 cells / ml; purulent tracheal secretions; reduced gas exchange; or significant bacterial growth in tracheal secretion samples. Therefore, these six patients were isolated as potential VAP cases, and VAP was subsequently confirmed by the ICU physician / medical staff through monitoring of the patients' clinical signs and symptoms throughout their ICU stay.

[0149] Example 1: A 56-year-old man, generally in good health, developed acute influenza (viral upper respiratory tract infection) and was admitted to the ICU at Sheba Hospital (Ramat Gan, Illinois) for ventilator support. The patient remained on ventilation for 9 days and was discharged on day 12. Clinical parameters are shown in Table 2. [Table 2]

[0150] Analysis of the patient's breath detected markers. In particular, four specific Pseudomonas-specific markers were detected as early as day 1, prior to the detection of clinical signs. Thus, breath analysis detected biomarkers two days prior to X-ray and two to three days prior to detection by culture. Thus, VAP was detected much earlier than routine clinical signs.

[0151] All biomarkers increased sharply on day 5. The increase in the marker designated No. 4 significantly exceeded the other markers, which is associated with exponential growth. Thus, VAP occurred in this patient during antibiotic treatment. As a result of its detection, antibiotic treatment was rescheduled after clinical signs of VAP.

[0152] Example 2: A 24-year-old man was admitted to the neurosurgical ICU at Rambam Hospital (Haifa, Illinois) after a serious fall from height, resulting in a basilar skull fracture, epidural hemorrhage, pulmonary contusion, and traumatic pneumothorax. The man received ventilator support for 13 days. The patient died on day 15. Clinical parameters are shown in Table 3. [Table 3]

[0153] Analysis of the breath samples detected biomarkers, particularly three different bacteria, which appeared at different concentrations and varied from day to day. Cultures detected only one bacterial source (MRSA). The total bacterial load detected increased sharply on day 5, 2 days before the clinical diagnosis of VAP and 2–3 days before culture (which itself required 1 or 2 days for analysis).

[0154] Example 3: [Table 4]

[0155] Breath analysis detected biomarkers. Specifically, three biomarkers were detected, i.e., three different bacteria with different concentrations and different daily occurrences. Contrary to these findings, cultures detected only one bacterial source. The total amount of bacteria detected increased sharply on the fifth day. These findings were evident two days before the X-ray diagnosis and one or two days before the culture (culture analysis requires one or two days). Additionally, note that two spikes in bacterial load were detected (days 2 and 5), with the day 2 data being unique.

[0156] Example 4: The markers identified for S. aureus were bromochloromethane, 1,4-difluorobenzene, chlorobenzene, p-bromofluorobenzene, 3-methylbutanal, 2-methylbutanal, and dimethyl trisulfide.

Claims

1. 1. A method for determining the presence of at least one disease-associated marker in a respiratory sample from a subject, comprising: exposing to the breath sample at least one sampling unit comprising one or more adsorbent regions capable of reversibly binding volatile substances in the breath sample, the adsorbent regions being different from metal surfaces or metal nanoparticles; analyzing the at least one sampling unit exposed to the volatile substance to identify the volatile substance adsorbed to the one or more adsorption regions; and determining the presence of the at least one disease-associated marker; A method wherein an increase in the amount of said marker compared to the amount of said marker measured at an earlier time point indicates the presence of a disease state.

2. 10. The method of claim 1, wherein the increase in the amount of the marker compared to the amount measured at an earlier time point is at least 50%.

3. The method of claim 1 , wherein the method is performed one or more times at different times to detect changes in the amount of the marker.

4. The method of claim 1 , wherein the at least one sampling unit is made of a glass or stainless steel material.

5. The method of any one of claims 1 to 4, wherein each of the one or more adsorbent regions is configured to reversibly bind volatile substances in the breath sample.

6. 5. The method of claim 1, wherein the one or more adsorption regions are formed from a solid adsorbent configured to physically capture the volatile material.

7. The method of claim 6 , wherein the solid adsorbent is formed from a selective or non-selective material.

8. 7. The method of claim 6, wherein the one or more adsorbent regions are formed from a material selected from an organic porous polymer, an ion exchange resin, a carbon molecular sieve, or a sulfonated polymer.

9. 7. The method of claim 6, wherein the one or more adsorbent regions are of a material selected from a carbon adsorbent, a carbon allotrope, or a carbonaceous material.

10. 10. The method of claim 9, wherein the carbon adsorbent is selected from graphitized carbon black having a 20 / 40 mesh, graphitized carbon black having a 60 / 80 mesh, and carbon molecular sieves.

11. 11. The method of claim 10, wherein the graphitized carbon black and carbon molecular sieve are selected from Carbotrap F, Carbotrap C, Carbotrap Y, Carbotrap B, Carbotrap X, Carbopack F, Carbopack C, Carbopack Y, Carbopack B, Carbopack X, Carboxen 1016, Carboxen 569, Carboxen 1021, Carboxen 1018, Carbosieve S-III, Carboxen 1003, Carbosieve G, Carboxen 1000, and Carboxen 1012.

12. The one or more adsorption regions have a length of 5 to 1500 m ２ 10. The method of claim 9, wherein the material has a surface area of 0.2 to 0.7 / g, a density of 0.2 to 0.7, and / or a pore size between 4 and 300 Å.

13. 13. The method of any one of claims 1 to 12, wherein after exposure of the one or more adsorption regions to a breath sample, the volatile materials are adsorbed onto the surface of the region and trapped until analyzed.

14. 14. The method of any one of claims 1 to 13, wherein the one or more adsorption regions are treated to cause desorption or dissociation of the volatile materials from the surface, and the desorbed volatile materials are analyzed.

15. 15. The method of claim 14, wherein the volatiles are analyzed by gas chromatography (GC), GC-lined mass spectrometry (GC-MS), proton transfer reaction mass spectrometry (PTR-MS), electronic nose device (E-nose), quartz crystal microbalance (QCM), infrared spectroscopy (IR), or ultraviolet spectroscopy (UV).

16. The method of claim 15, wherein the volatiles are analyzed by GC-MS.

17. The method of any one of claims 1 to 16, wherein the subject is asymptomatic.

18. The method according to any one of claims 1 to 16, wherein at the time of diagnosis, the subject is suffering from a disease and the diagnosis aims to determine the onset of different disease states.

19. The method of any one of claims 1 to 16, wherein the subject is a ventilated subject.

20. 20. The method of claim 19, wherein the method is for detecting ventilator-associated pneumonia (VAP).

21. 10. The method of any one of the preceding claims, wherein the disease-associated marker is a marker indicative of a bacterial, viral, or fungal disease.

22. 22. The method of claim 21, wherein the marker is associated with bacteria.

23. 22. The method of claim 21, wherein the marker is associated with a virus.

24. 24. The method of claim 23, wherein the virus is an enveloped or non-enveloped virus.

25. 25. The method of claim 24, wherein the virus is a norovirus or a parvovirus.

26. 25. The method of claim 24, wherein the virus is an influenza virus or a coronavirus.

27. 27. The method of claim 26, wherein the virus is SARS-CoV-2.

28. 22. The method of claim 21, wherein the disease is a hospital-acquired infection (HAI).

29. 29. The method of claim 28, wherein the HAI is caused by methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Clostridium difficile, Acinetobacter baumannii, or multidrug-resistant (MDR) Acinetobacter spp.

30. 1. A method for determining the presence of at least one pathogen in a subject, comprising: exposing the breath sample to at least one sampling unit comprising one or more adsorbent regions capable of reversibly binding volatile substances in the breath sample, wherein the one or more adsorbent regions are different from a metal surface or a metal nanoparticle; analyzing at least one sampling unit to identify the volatile substances adsorbed to the one or more adsorption regions to determine the presence of at least one pathogen-associated marker; wherein the presence of the marker is indicative of the presence of the pathogen in the subject.

31. 31. The method of claim 30, wherein the method is repeated one or more times to determine a change in the amount of the marker.

32. 32. The method of claim 31, wherein an increase in the amount of the marker compared to the amount of the marker measured at an earlier time point indicates the presence of a disease state.

33. 33. The method of any one of claims 30 to 32, wherein the pathogen is a virus, bacterium, or fungus.

34. 34. The method of claim 1, 30, or 33, wherein the subject is asymptomatic.

35. 34. The method of claim 1, 30, or 33, wherein the subject is ventilated.

36. 36. The method of claim 35, wherein the ventilated subject does not exhibit symptoms associated with VAP.

37. 34. The method of claim 33, wherein the pathogen causes VAP.

38. 1. A method for determining the presence of at least one pathogen in a ventilated subject, comprising: exposing at least one sampling unit to a breath sample from a ventilated subject, said at least one sampling unit comprising one or more adsorbent regions capable of reversibly binding volatile substances present in said sample; analyzing the at least one sampling unit to identify volatile substances adsorbed to the one or more adsorption regions to determine the presence of the at least one pathogen-associated marker; wherein the presence of the marker indicates the presence of the pathogen in the ventilated subject.

39. 1. A method for determining the onset of VAP in a ventilated subject, comprising: exposing a breath sample from a ventilated subject to at least one sampling unit comprising one or more adsorbent regions capable of reversibly binding volatile substances in the breath sample; analyzing the at least one sampling unit to identify volatile substances adsorbed to the one or more adsorption regions to determine the presence of at least one marker of a pathogen that causes VAP; The method, wherein the presence of the marker is indicative of the onset of VAP.

40. 40. The method of claim 39, wherein the at least one sampling unit is provided within a respiratory system normally used for ventilation of the subject.

41. The method comprises: exposing at least one sampling unit positioned in an outlet line of the respiratory system to a breath sample exhaled by the subject, the at least one sampling unit comprising one or more adsorbent regions capable of reversibly binding volatile substances present in the sample; analyzing the at least one sampling unit to identify the volatile substances adsorbed to the one or more adsorption regions to determine the presence of at least one pathogen-associated marker; 41. The method of claim 40, wherein the presence of the marker indicates the presence of the pathogen-associated marker in the ventilated subject.

42. 42. The method of claim 41, wherein the at least one sampling unit is in the form of a vessel that contains the one or more adsorption areas and allows timed dwell contact of the respiratory sample with the one or more adsorption areas.

43. 42. The method of claim 41, wherein one or both of the at least one sampling unit and the outlet line of the respiratory system are provided with a flow regulator.

44. 42. The method of claim 41, wherein the disease-causing pathogen is a bacterium, virus, or fungus.

45. 42. The method of claim 41, wherein the method comprises removing the at least one sampling unit from the outlet line of the respiratory system and analyzing it to determine volatile substances adsorbed to the one or more adsorption regions.

46. 46. The method of claim 45, wherein the volatile materials adsorbed in the one or more adsorption regions are desorbed and then analyzed.

47. 42. The method of claim 41, wherein the method comprises exposing at least one other sampling unit positioned in an inlet line of the respiratory system to ventilation air delivered to the subject.

48. 42. The method of claim 41, wherein the analysis is performed by spectroscopy.

49. 42. The method of claim 41, wherein the method includes comparing the material adsorbed to the one or more adsorption regions with a marker database to identify the material indicative of the presence of a disease-causing pathogen.

50. 42. The method of claim 41, wherein the presence of the disease-causing pathogen indicates the onset of the disease.

51. 42. The method of claim 41, wherein the markers identified in the exhaled breath are compared to markers identified in the inhaled breath.

52. 51. The method of claim 50, wherein the disease state is associated with a bacterium.

53. 51. The method of claim 50, wherein the disease is a hospital-acquired infection.

54. 1. A respiratory system comprising: a compressible air reservoir; a set of tubing; and a patient circuit comprising an inspiratory tube and an expiratory tube, said patient circuit being connected to an endotracheal intubation tube, one or both of said inspiratory tube and expiratory tube comprising at least one sampling unit positioned in the path of alveolar breath exhaled by a subject being ventilated, said at least one sampling unit comprising one or more adsorption regions capable of reversibly binding at least one volatile substance present in said exhaled alveolar breath, to indicate the presence of a disease state or a disease-causing pathogen.

55. 1. A system configured for determining the presence of a disease-causing pathogen in a ventilated subject, the system comprising a ventilator comprising a patient circuit provided with a plurality of optionally removable collection surfaces positioned in the path of exhaled breath, each of the plurality of collection surfaces characterized by a plurality of binding regions, each of the binding regions in the form of binding molecules and / or surface features configured to reversibly bind at least one material indicative of the presence of the pathogen.

56. 54. A system for carrying out the method of any one of claims 1 to 53, said system comprising one or more optionally removable sampling units positioned in a path of the exhaled breath, each of said sampling units comprising binding regions in the form of binding molecules and / or surface features configured to reversibly bind to at least one marker present in the exhaled breath.

57. 1. A method for determining the onset of a bacterial condition, disease, or disorder in a subject, the method comprising: collecting a volume of exhaled air from the breath of the subject; and detecting (1) the presence of at least one substance indicative of bacterial disease, and (2) a change in concentration of the substance over time, wherein the change in the concentration of the substance over time is indicative of the bacterial condition, disease, or disorder.

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