WO2017123582A1

WO2017123582A1 - Methods for detecting bacterial lung infections

Info

Publication number: WO2017123582A1
Application number: PCT/US2017/012928
Authority: WO
Inventors: David Karshmer; John Maynard
Original assignee: Avisa Pharma Inc.
Priority date: 2016-01-11
Filing date: 2017-01-11
Publication date: 2017-07-20

Abstract

The present disclosure is directed to methods of diagnosing bacterial lung infections by administering an effective amount of carbon-13 isotopically labeled urea to a subject and analyzing exhaled breath samples for the presence of ¹³CO₂ originating from the lungs of the subject.

Description

METHODS FOR DETECTING BACTERIAL LUNG INFECTIONS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No.

62/277,121, filed January 11, 2016, the entirety of which is incorporated herein.

BACKGROUND

[0002] Certain bacteria have enzymes that can metabolize particular compounds into cleavage or fermentation products. For example, Franscisells tularensis has an enzyme that can convert citrulline to carbon dioxide. Mycobacterium tuberculosis and Pseudomonas aeruginosa have enzymes ("ureases") that can convert urea to carbon dioxide. These enzymatic processes can be exploited to diagnose the presence, and in some cases, the severity or location, of a particular bacterial lung infection. See, e.g., U.S. 9,074,237; U.S. 7,897,400; RE 44,544; U.S. Published Application Nos. 2015/0275265; 2015/0125898; 2014/0179809; and 2014/0114206.

[0003] In these methods, a ¹ C-labeled compound, for example ¹ C-urea, is

administered to the lungs of the patient. If a bacteria is present in the patient's lungs that can metabolize the ¹ C-labeled compound to carbon dioxide, that bacteria will convert the ^Relabeled compound to ¹ C0₂, which will be expelled in the patient's breath. Samples of the exhaled breath of the patient can then be analyzed for the ratio of ¹ C0₂ to ¹²C0₂. If the patient's lungs are infected, the ratio of ¹ C0₂ to ¹²C0₂ in the patient's breath after the ¹ C-labeled compound administration will typically be higher, as compared to the ratio of ¹ C0₂ to ¹²C0₂ in a control sample.

[0004] While these methods can be used to diagnose a bacterial lung infection, in some instances, a "mouth signal" can interfere with the sample analysis. In these instances, the patient's mouth will include pathogens that can also metabolize the ¹ C-labeled compound to carbon dioxide, for example, planktonic pathogens and pathogens that are resident in mouth biofilms, Streptococci, and the like. When the patient's exhaled breath samples are collected, one is unable to ascertain whether the amount of ¹ C0₂ in those samples has come from a bacterial infection in the lungs and/or from a pathogen in the mouth. Methods of minimizing or eliminating interfering mouth signal are needed.

SUMMARY [0005] The disclosure is directed to methods for diagnosing a urease-positive bacterial infection in the lungs of a subject comprising administering to the lungs of the subject, via nebulization or dry powder inhalation, an effective amount of carbon-13 isotopically labeled urea; collecting one or more samples of exhaled breath from the subject; and analyzing said samples to determine a concentration of ¹ CC>2 in said samples; said concentration indicating the presence or absence of the urease-positive bacterial infection in the lungs of the subject; the methods further comprising rinsing the mouth of the subject at least once with a solution at a time that is before the administration, after the administration, or both before and after the administration.

[0006] The disclosure is also directed to methods for diagnosing a urease-positive bacterial infection in the lungs of a subject comprising administering to the lungs of the subject, via nebulization, an effective amount of carbon-13 isotopically labeled urea; collecting one or more samples of exhaled breath from the subject, wherein the collection minimizes the amount of mouth sample; and analyzing said samples to determine a concentration of ¹ CC>2 in said samples; said concentration indicating the presence or absence of the urease-positive bacterial infection in the lungs of the subject.

[0007] The disclosure is also directed to methods for diagnosing a urease-positive bacterial infection in the lungs of a subject comprising administering to the lungs of the subject, via nebulization, an effective amount of carbon-13 isotopically labeled urea, wherein at least a portion of the interior mouth surfaces are covered during the administration; collecting one or more samples of exhaled breath from the subject; and analyzing said samples to determine a concentration of ¹ CC>2 in said samples; said concentration indicating the presence or absence of the urease-positive bacterial infection in the lungs of the subject

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0008] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. [0009] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0010] As used in the specification and in the claims, the term "comprising" may include the embodiments "consisting of and "consisting essentially of. " The terms

"comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients/steps and permit the presence of other ingredients/steps. However, such description should be construed as also describing compositions or processes as "consisting of and "consisting essentially of the enumerated ingredients/steps, which allows the presence of only the named ingredients/steps, along with any impurities that might result therefrom, and excludes other ingredients/steps.

[0011] Numerical values in the specification and claims of this application, particularly as they relate to polymers or polymer compositions, reflect average values for a composition that may contain individual polymers of different characteristics. Furthermore, unless indicated to the contrary, the numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0012] All ranges disclosed herein are inclusive of the recited endpoint and

independently combinable (for example, the range of "from 2 grams to 10 grams" is inclusive of the endpoints, 2 grams and 10 grams, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and/or values.

[0013] As used herein, approximating language may be applied to modify any quantitative representation that may vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as "about" and "substantially," may not be limited to the precise value specified, in some cases. In at least some instances, the approximating language may correspond to the precision of an instrument for measuring the value. The modifier "about" should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression "from about 2 to about 4" also discloses the range "from 2 to 4." The term "about" may refer to plus or minus 10% of the indicated number. For example, "about 10%" may indicate a range of 9% to 1 1%, and "about 1 " may mean from 0.9-1.1. Other meanings of "about" may be apparent from the context, such as rounding off, so, for example "about 1" may also mean from 0.5 to 1.4.

[0014] The present disclosure is directed to methods of minimizing or eliminating the amount of interfering mouth signal when analyzing exhaled breath samples for the presence of ¹ C0₂ originating from the lower airway gas of a subject.

[0015] As used herein, "mouth signal" refers to an amount of ¹ C0₂ originating from the metabolism of carbon-13 labeled urea by a pathogen residing in the mouth of the subject.

[0016] As used herein, an "interfering mouth signal" refers to an amount of ¹ C0₂ originating from the metabolism of carbon-13 labeled urea by a pathogen residing in the mouth of the subject wherein the pathogen is not diagnostic of a bacterial lung infection. When interfering mouth signals are present in the exhaled breath samples to be analyzed, the ratio of ¹ C02 to ¹²C02 in the lower airway gas of the subject cannot be accurately measured.

[0017] As used herein, "mouth breath" refers to exhaled breath from the subject that substantially includes breath from the subject's mouth with only a minimal concentration of breath from the lower airway gas of the subject.

[0018] As used herein, "subject" is used to describe an individual subject or patient, a mammal, generally a human, who is at risk for developing a urease-positive bacterial lung infection, who is suspected of being infected with a urease-positive bacterial lung infection, or who has been previously diagnosed with a urease-positive bacterial lung infection.

[0019] Preferred aspects of the disclosure are directed to methods of diagnosing a bacterial infection, preferably a urease-positive bacterial infection, in the lungs of a subject. An exemplary bacterial lung infection that can be diagnosed using the methods described herein is a Mycobacterium tuberculosis infection. Other aspects of the disclosure are directed to methods of diagnosing a bacterial infection, preferably a urease-positive bacterial infection, in the gastrointestinal tract of a subject, e.g., a Helicobacter pylori infection. The methods described herein are performed so as to minimize or eliminate the amount of interfering mouth signal present in the exhaled breath sample to be analyzed.

[0020] According to the methods of the disclosure, an effective amount of carbon-13 isotopically labeled urea (NH₂-¹ C(0)-NH₂) is administered to the subject's lungs. The administration can be via nebulization, using methods known in the art. Alternatively, the administration can be via inhalation of a dry powder using methods known in the art.

[0021] After the carbon-13 isotopically labeled urea is administered, and after a period of time that is sufficient for any urease-positive bacteria to metabolize urea to carbon dioxide, one or more samples of the subject's exhaled breath is collected. The collection can be into any suitable vessel, for example a breath collection bag, bottle, or vial. The samples are then analyzed to determine a concentration of ¹ CC>2 in the samples, wherein the concentration is indicative of the presence or absence of the urease-positive bacterial infection in the lungs of the subject. For example, the analysis can comprise comparing the ratio of ¹ CC>2 to ¹²CC>2 in the post-administration, exhaled breath samples to the ratio of ¹ CC>2 to ¹²CC>2 in a control sample. In some aspects, an increase in the ratio of ¹ CC>2 to ¹²CC>2 in the post-administration, exhaled breath samples to the ratio of ¹ CC>2 to ¹²CC>2 in the control sample indicates the presence of the urease-positive bacterial infection in the lungs of the subject. The control sample(s) can be taken from the subject prior to administration of the carbon- 13 isotopically labeled urea. Alternatively, the control sample can be obtained from a collection of known, non-infected individuals.

Exemplary methods for performing the analysis are described in, for example, U.S. 9,074,237; U.S. 7,897,400; RE 44,544; U.S. Published Application Nos. 2015/0275265; 2015/0125898; 2014/0179809; and 2014/0114206, the entireties of which are incorporated herein. In embodiments wherein the control sample(s) is taken from the subject prior to administration of the carbon- 13 isotopically labeled urea, the methods described herein for minimizing interfering mouth signal can also be applied to that pre-administration, exhaled breath sample collection.

[0022] In order to minimize or eliminate the amount of interfering mouth signal occurring in the methods of the disclosure, in some aspects, the mouth of the subject is rinsed at least once with a solution. The solutions useful in the present disclosure are preferably aqueous solutions that do not include any components that may negatively influence a spectrometric analysis. A preferred solution is water, for example, spring water, distilled water, mineral water, well water, or municipal water. Alcohol-based antiseptic mouthwashes that may also include menthol, thymol, methyl salicylate, and/or eucalyptol are not useful in the described methods.

[0023] The rinsing can occur at a time that is before the administration of the carbon- 13 isotopically labeled urea, e.g., before the nebulized administration or dry powder inhalation administration of the carbon- 13 isotopically labeled urea. Alternatively, the rinsing can occur at a time that is after the administration of the carbon- 13 isotopically labeled urea, e.g., after the nebulized administration or dry powder inhalation administration of the carbon-13 isotopically labeled urea. In other aspects, the rinsing can occur at a time that is both before and after the administration of the carbon-13 isotopically labeled urea e.g., both before and after the nebulized administration or dry powder inhalation administration of the carbon-13 isotopically labeled urea. [0024] In some aspects of the disclosure, wherein the rinsing occurs before the administration of the carbon- 13 isotopically labeled urea, the solution can comprise unlabeled urea. In these embodiments, it is envisioned that the unlabeled urea will be absorbed by any mouth pathogens that might be present in the subject's mouth that can metabolize urea to carbon dioxide. In these methods, the mouth pathogens would then be unable to absorb any clinically significant amount of the administered carbon- 13 isotopically labeled urea, thus minimizing or eliminating interfering mouth signal.

[0025] In some aspects, the mouth is rinsed with a pre-determined volume of the solution. This volume may be used to rinse the mouth in a single rinse. Alternatively, the predetermined volume can be divided over more than one rinse of the mouth, for example, the predetermined volume can be divided over two, three, or four mouth rinses.

[0026] The pre-determined volume will be the amount of solution that results in the minimization or elimination of interfering mouth signal. This pre-determined volume can be ascertained by one skilled in the art without undue experimentation. In some embodiments, the pre-determined volume of the solution used to rinse the subject's mouth is between about 10 mL and about 100 mL. In some aspects, the pre-determined volume of the solution used to rinse the subject's mouth is between about 20 mL and about 60 mL. For example, the pre-determined volume of the solution used to rinse the subject's mouth is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100 mL.

[0027] In those aspects wherein the pre-determined volume is divided over more than one rinse of the mouth, the pre-determined volume can be divided into doses that are between about 10 mL and about 50 mL. For example, each rinsing dose can be about 10, 15, 20, 25, 30, 35, 40, 45, or about 50 mL.

[0028] In some aspects, the mouth is rinsed with the solution for a pre-determined amount of time. This pre-determined amount of time can be the time for a single mouth rinse. Alternatively, the pre-determined amount of time can be distributed over more than one rinse of the mouth, for example, the pre-determined amount of time can be divided over two, three, or four mouth rinses.

[0029] The pre-determined time for rinsing will be the amount of rinsing time that results in the minimization or elimination of interfering mouth signal. This pre-determined time for mouth rinsing can be ascertained by one skilled in the art without undue experimentation. In some embodiments, the pre-determined amount of mouth rinsing time is about 20 seconds to about 120 seconds. In some aspects, the pre-determined amount of mouth rinsing time is about 40 seconds. For example, the pre-determined amount of mouth rinsing time is about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or about 120 seconds.

[0030] In some aspects, the pre-determined about of mouth rinsing time with the solution is the time for a single rinse. In other aspects, the pre-determined about of rinsing time with the solution is divided over two or more rinses, for example, divided over two, three, or four rinses. For example, each rinse can be about 10 to about 40 seconds, preferably about 20 seconds for each rinse. The amount of time for each rinse can be substantially the same, i.e., each rinse can be for about 20 seconds. Alternatively, the times for each rinse can be different, i.e. , one rinse can be for about 20 seconds and another rinse can be for about 30 seconds.

[0031] The use of mouth rinses to minimize the interfering mouth signal can be combined with other methods for reducing interfering mouth signal. In some aspects, the use of mouth rinses can be combined with collection methods and/or administration methods, as described herein, that minimize or eliminate interfering mouth signal. For example, the collection of the breath samples can begin after a pre-determined period of time that results in the minimization or elimination of the interfering mouth signal. In some embodiments, the predetermined period after which the collection begins is between about 2 minutes and about 5 minutes.

[0032] In those aspects wherein the control sample(s) is obtained from the subject prior to administration of the carbon-13 isotopically labeled urea, the pre-administration exhaled breath collection can begin after a pre-determined period of time that results in the minimization or elimination of the interfering mouth signal in the analysis of the pre-administration, exhaled breath sample. In some embodiments, the pre-determined period after which the collection begins is between about 2 minutes and about 5 minutes.

[0033] In other aspects, the use of mouth rinses can be combined with collection methods that result in the concentration of lower airway gas in the exhaled breath samples being analyzed being greater than the amount of mouth breath in the samples. In still other aspects, the use of mouth rinses can be combined with collection methods that result in the amount of lower airway gas in the exhaled breath samples being analyzed being greater than the amount of mouth breath and greater than the amount of upper respiratory breath in the samples. Such methods are described in more detail herein.

[0034] In other aspects, the use of mouth rinses can be combined with administration methods that minimize the exposure of the carbon-13 labeled urea to surfaces of the subject's mouth. For example, the administration can occur through the nares of the subject. In other aspects, at least a portion of the interior surfaces of the subject's mouth are covered during administration of the carbon-13 labeled urea. Such methods are described in more detail herein.

[0035] The disclosure is also directed to methods of minimize interfering mouth signal without the use of mouth rinses. In these aspects, a urease-positive bacterial infection is diagnosed in the lungs of a subject by administering to the lungs of the subject, via nebulization or via dry powder inhalation, an effective amount of carbon-13 isotopically labeled urea.

[0036] After the carbon-13 isotopically labeled urea is administered, and after a period of time that is sufficient for any urease-positive bacteria to metabolize urea to carbon dioxide, one or more samples of the subject's exhaled breath is collected. The collection can be into any suitable vessel, for example a breath collection bag, bottle, or vial. The samples are then analyzed to determine a concentration of ¹ CC>2 in the samples, wherein the concentration is indicative of the presence or absence of the urease-positive bacterial infection in the lungs of the subject. Exemplary methods for performing the analysis are described in, for example, U.S. 9,074,237; U.S. 7,897,400; RE 44,544; U.S. Published Application Nos. 2015/0275265;

2015/0125898; 2014/0179809; and 2014/0114206, the entireties of which are incorporated herein. In these methods, the collection of the exhaled breath samples is performed so as to minimize the amount of interfering mouth signal.

[0037] In one aspect, the collection is performed such that the amount of lower airway gas in the exhaled breath sample to be analyzed is greater than the amount of mouth breath present in the exhaled breath sample to be analyzed. In another aspect, the collection is performed such that the amount of lower airway gas in the sample is greater than the amount of mouth breath and upper respiratory breath in the exhaled breath sample to be analyzed.

[0038] In those aspects wherein the control sample(s) is obtained from the subject prior to administration of the carbon-13 isotopically labeled urea, the pre-administration exhaled breath collection can be performed such that the amount of lower airway gas in the pre- administration, exhaled breath sample to be analyzed is greater than the amount of mouth breath present in the pre-administration, exhaled breath sample to be analyzed. In aspects wherein the control sample(s) is obtained from the subject prior to administration of the carbon-13 isotopically labeled urea, the pre-administration exhaled breath collection can be performed such that the amount of lower airway gas in the pre-administration, exhaled breath sample to be analyzed is greater than the amount of mouth breath and upper respiratory breath present in the pre-administration, exhaled breath sample to be analyzed. [0039] In one aspect, the breath samples - either pre- or post-carbon- 13 labeled urea administration - are collected so as to minimize exposure of the exhaled breath samples to the subject's mouth cavity. For example, one method to minimize exposure of the exhaled breath samples to the subject's mouth cavity is to collect the breath samples from the nares of the subject.

[0040] In other embodiment, the breath samples - either pre- or post-carbon- 13 labeled urea administration - are collected while restricting the volume of the subject's mouth cavity. This restriction can be accomplished by mechanical means, i.e., using a device adapted to prevent the subject from expanding the volume of the subject's mouth cavity. Alternatively, the subject can exhale while voluntarily restricting the volume of the subject's mouth cavity.

[0041] One example of a collection method that minimizes or eliminates interfering mouth signal - either pre- or post-carbon- 13 labeled urea administration - is the use of a collection vessel that has a volume greater than the tidal volume of the subject. Subject tidal volumes can be determined or estimated using techniques known in the art.

[0042] In some aspects, the collection vessel has a volume that is greater than 300 mL. In other aspects, the vessel has a volume of between about 400 mL and 3000 mL. In yet other aspects, the vessel has a volume of between about 400 mL and 1500 mL. In still other aspects, the vessel has a volume of between about 450 mL and 1000 mL. For example, the collection vessel can have a maximum volume of 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or about 3000 mL. While not wishing to be held to any particular theory, it is believed that by collecting a larger exhaled breath sample volume, i.e., a breath sample volume greater than 300 mL or approaching the subject's tidal volume, the exhaled breath sample will include a higher percentage of gas from the lower airways of the subject. If the sample includes a higher percentage of gas from the lower airways, as compared to the percentage of breath from the mouth, any interfering signal that would have originated from the mouth will be minimized.

[0043] In another aspect, the amount of mouth sample in the exhaled breath sample - either pre- or post-carbon- 13 labeled urea administration - is minimized by fractionating the exhaled breath sample. This fractionation can be via mechanical means using methods known in the art. This fractionation can alternatively be via capnography fractionation using methods known in the art. The earlier portions of an exhaled breath sample typically include a greater percentage of mouth breath. Collection and subsequent analysis of the later portions of an exhaled breath sample, while discarding the earlier portions of the exhaled breath sample (e.g., discarding the first 100, 125, 150, 175, or 200 mL of exhaled breath), will minimize the amount of interfering mouth signal present in the exhaled samples being analyzed.

[0044] In other aspects of these methods wherein the collection methods minimize or eliminate the amount of interfering mouth signal, the carbon-13 isotopically labeled urea is administered to the subject's lungs using methods that minimize exposure of the carbon-13 isotopically labeled urea to the surfaces of the subject's mouth. For example, the carbon-13 isotopically labeled urea can be administered to the subject's lungs via the subject's nares. In other aspects, the carbon-13 isotopically labeled urea is administered to the lungs of the subject wherein at least a portion of the interior mouth surfaces are covered during the administration using methods as described herein.

[0045] Other aspects of the disclosure are directed to methods of minimizing or eliminating interfering mouth signal without the use of mouth rinses and without the use of collection methods that minimize or eliminate interfering mouth signal. In these methods, a urease-positive bacterial lung infection in a subject is diagnosed by administering to the lungs of subject, via nebulization or via dry powder inhalation, an effective amount of carbon-13 isotopically labeled urea. In these aspects, at least a portion of the interior mouth surfaces are covered during the administration so as to minimize the exposure of the interior mouth surfaces, and any pathogens residing therein, to the carbon-13 isotopically labeled urea. By minimizing the exposure of the mouth surfaces to the carbon-13 isotopically labeled urea, the amount of interfering mouth signal will be reduced. The interior mouth surfaces can be covered using any known method. For example, the interior mouth surfaces can be covered with a "dam"-type device or a "scuba"-type mouth device.

[0046] After the carbon-13 isotopically labeled urea is administered, and after a period of time that is sufficient for any urease-positive bacteria to metabolize urea to carbon dioxide, one or more samples of the subject's exhaled breath is collected. The collection can be into any suitable vessel, for example a breath collection bag, bottle, or vial. The samples are then analyzed to determine a concentration of ¹ CC>2 in the samples, wherein the concentration is indicative of the presence or absence of the urease-positive bacterial infection in the lungs of the subject. Exemplary methods for performing the analysis are described in, for example, U.S. 9,074,237; U.S. 7,897,400; RE 44,544; U.S. Published Application Nos. 2015/0275265; 2015/0125898; 2014/0179809; and 2014/0114206, the entireties of which are incorporated herein.

Claims

What is Claimed:

1. A method for diagnosing a urease-positive bacterial infection in the lungs of a subject comprising:

administering to the lungs of the subject, via nebulization or dry powder inhalation, an effective amount of carbon-13 isotopically labeled urea;

collecting one or more samples of exhaled breath from the subject; and

analyzing said samples to determine a concentration of ¹ CC>2 in said samples;

said concentration indicating the presence or absence of the urease-positive bacterial infection in the lungs of the subject;

the method further comprising rinsing the mouth of the subject at least once with a solution at a time that is before the administration, after the administration, or both before and after the administration.

2. The method of claim 1, wherein the rinsing occurs before the administration.

3. The method of claim 2, wherein the solution comprises unlabeled urea.

4. The method of any one of the preceding claims, wherein the solution is an aqueous

solution.

5. The method of any one of the preceding claims, wherein the mouth is rinsed with a predetermined volume of the solution.

6. The method of claim 5, wherein the pre-determined volume of the solution is divided over two, three, or four mouth rinses.

7. The method of any one of the preceding claims, wherein the mouth is rinsed with 20 mL to 60 mL of the solution.

8. The method of any one of the preceding claims, wherein the rising occurs for a predetermined amount of time.

9. The method of claim 8, wherein the pre-determined amount of time is about 40 seconds to about 120 seconds.

10. The method of any one of the preceding claims, wherein the rising occurs for a total time of at least 40 seconds.

11. The method of any one of the preceding claims, wherein the mouth is rinsed two, three, or four times.

12. The method of any one of claims 1 and 4 to 11, wherein the rising occurs after the

nebulized administration the effective amount of the carbon- 13 isotopically labeled urea.

13. The method of any one of the preceding claims, wherein the collection minimizes the amount of mouth sample.

14. The method of any one of the preceding claims, wherein the collection begins after a predetermined period of time that results in minimization of the contribution of mouth signal.

15. The method of claim 14, wherein the pre-determined period after which the collection begins is 2 minutes to 5 minutes.

16. The method of any one of the preceding claims, wherein the collection results in the amount of lower airway gas in the sample being greater than the amount of mouth breath in the sample.

17. The method of claim 16, wherein the collection results in the amount of lower airway gas in the sample being greater than the amount of mouth breath and upper respiratory breath in the sample.

18. A method for diagnosing a urease-positive bacterial infection in the lungs of a subject comprising:

collecting one or more samples of exhaled breath from the subject, wherein the collection

minimizes the amount of mouth sample; and

analyzing said samples to determine a concentration of ¹ CC>2 in said samples;

said concentration indicating the presence or absence of the urease-positive bacterial infection in the lungs of the subject.

19. The method of claim 18, wherein the breath samples are collected in a vessel having a volume greater than the tidal volume of the subject.

20. The method of claim 19, wherein the vessel is configured to accept two or more samples of exhaled breath.

21. The method of any one of claims 18 to 20, wherein the breath samples are collected in a vessel having a volume greater than 300 mL.

22. The method of claim 21, wherein the vessel has a volume of between 400 mL and 3000 mL, preferably between 400 mL and 1500 mL, and more preferably between 450 mL and 1000 mL.

23. The method of claim 18, wherein the exhaled breath samples are fractionated.

24. The method of claim 23, wherein the fractionation is mechanical fractionation.

25. The method of claim 23, wherein the fractionation is capnography fractionation.

26. The method of claim 18, wherein the administration of the carbon- 13 isotopically labeled urea and/or the samples of exhaled breath are collected from the nares of the subject.

27. The method of claim 18, wherein the breath samples are collected while restricting the volume of the mouth cavity.

28. The method of claim 18, wherein the administration minimizes exposure of the carbon- 13 isotopically labeled urea with the surfaces of the subject's mouth.

29. The method of claim 18, wherein the collection minimizes exposure of the breath

samples with the surfaces of the subject's mouth.

30. The method of claim 18, wherein the collection results in the amount of lower airway gas in the sample being greater than the amount of mouth breath in the sample.

31. The method of claim 30, wherein the collection results in the amount of lower airway gas in the sample being greater than the amount of mouth breath and upper respiratory breath in the sample.

32. A method for diagnosing a urease-positive bacterial infection in the lungs of a subject comprising:

administering to the lungs of the subject, via nebulization or dry powder inhalation, an effective amount of carbon- 13 isotopically labeled urea, wherein at least a portion of the interior mouth surfaces are covered during the administration;

collecting one or more samples of exhaled breath from the subject; and

analyzing said samples to determine a concentration of ¹ CC>2 in said samples;

said concentration indicating the presence or absence of the urease-positive bacterial infection in the lungs of the subject

33. The method of any one of the preceding claims, wherein the analysis comprises

comparing the ratio of CO2 to CO2 in said samples to the ratio of CO2 to CO2 in a control sample.

34. The method of claim 33, wherein an increase in the ratio of ¹ CC>2 to ¹²CC>2 in said

samples to the ratio of ¹ CC>2 to ¹²CC>2 in the control sample indicates the presence of the urease-positive bacterial infection in the lungs of the subject.

35. The method of any one of the preceding claims, wherein the urease positive bacterial infection is a Mycobacterium tuberculosis infection.