EP2685891A2 - Diagnostic method and breath testing device - Google Patents

Diagnostic method and breath testing device

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Publication number
EP2685891A2
EP2685891A2 EP12757976.1A EP12757976A EP2685891A2 EP 2685891 A2 EP2685891 A2 EP 2685891A2 EP 12757976 A EP12757976 A EP 12757976A EP 2685891 A2 EP2685891 A2 EP 2685891A2
Authority
EP
European Patent Office
Prior art keywords
sensor
hydrogen
detector
detector according
concentration
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP12757976.1A
Other languages
German (de)
French (fr)
Other versions
EP2685891A4 (en
Inventor
Anastasia Rigas
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Individual
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Individual
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Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP2685891A2 publication Critical patent/EP2685891A2/en
Publication of EP2685891A4 publication Critical patent/EP2685891A4/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/08—Detecting, measuring or recording devices for evaluating the respiratory organs
    • A61B5/082—Evaluation by breath analysis, e.g. determination of the chemical composition of exhaled breath
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/42—Detecting, measuring or recording for evaluating the gastrointestinal, the endocrine or the exocrine systems
    • A61B5/4216—Diagnosing or evaluating gastrointestinal ulcers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2562/00—Details of sensors; Constructional details of sensor housings or probes; Accessories for sensors
    • A61B2562/02—Details of sensors specially adapted for in-vivo measurements
    • A61B2562/0285—Nanoscale sensors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/08—Detecting, measuring or recording devices for evaluating the respiratory organs
    • A61B5/097—Devices for facilitating collection of breath or for directing breath into or through measuring devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/497—Physical analysis of biological material of gaseous biological material, e.g. breath
    • G01N2033/4975—Physical analysis of biological material of gaseous biological material, e.g. breath other than oxygen, carbon dioxide or alcohol, e.g. organic vapours
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
    • G01N27/04—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance
    • G01N27/12—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance of a solid body in dependence upon absorption of a fluid; of a solid body in dependence upon reaction with a fluid, for detecting components in the fluid
    • G01N27/125—Composition of the body, e.g. the composition of its sensitive layer
    • G01N27/127—Composition of the body, e.g. the composition of its sensitive layer comprising nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/0004—Gaseous mixtures, e.g. polluted air
    • G01N33/0009—General constructional details of gas analysers, e.g. portable test equipment
    • G01N33/0027—General constructional details of gas analysers, e.g. portable test equipment concerning the detector
    • G01N33/0036—Specially adapted to detect a particular component
    • G01N33/005—Specially adapted to detect a particular component for H2

Definitions

  • the present invention relates to a diagnostic method and breath-testing device for the diagnosis of celiac disease or other gastrointestinal malabsorption in adults and children.
  • Celiac disease is defined as a disease of the small bowel caused by a permanent intolerance to gluten. Most common symptoms of the disease are weight loss due to malabsorption, diarrhea, anemia, and growth failure. Celiac disease affects people of all ages and gender. Its incidence is on the rise worldwide due to environmental and genetic factors, currently affecting, on average, 1/300 people (children and adults) in the United States and in other developed countries. Up until recently celiac disease was considered a disease affecting predominantly people of Northern European origin this, however, is changing as cases are now reported in Asia and the Middle East, albeit at a much lower rate.
  • Definitive diagnosis of the disease is made on the basis of small bowel biopsies obtained while the patient is on gluten containing diet and after the patient is taken off gluten and free of symptoms. While small bowel biopsy is an invasive diagnostic modality there are other diagnostic methods currently available.
  • blood serum can be tested for the presence of D-xylose, a sugar detected in serum one hour after the ingestion of carbohydrates.
  • D-xylose test is performed less frequently due to low sensitivity and specificity for the diagnosis of celiac disease and due to the broad use of the more specific antibody diagnostic tests.
  • the present invention consists of a novel, inexpensive, non-invasive, point-of-care, handheld, device for the diagnosis of celiac disease using a selective hydrogen gas nanosensor.
  • a person, adult or child, suspected of having celiac disease or other type of gastrointestinal malabsorption will provide one fasting breath sample through the mouth piece of the device. No ingestion of carbohydrate substrate is required.
  • Figure 1 is a schematic block diagram of a breath analyzer device for the detection of hydrogen in breath according to the invention
  • Figure 2a is a scanning electron micrograph in cross-section of ZnO nanowires grown in deposition region at the entrance zone (ratio 0/Zn>l);
  • Figure 2b is a top view scanning micrograph of ZnO nanowires grown at the exit zone (ratio 0/Zn ⁇ 1);
  • Figure 3 is a graph showing the 3 ⁇ 4 gas response for a lOOnm diameter ⁇ ,, ⁇ NW-based sensor.
  • Figure 4 shows electronic circuitry of a device according to the invention.
  • the breath analyzer detects the presence of hydrogen in a human breath sample.
  • Figure 1 shows a sensor device which consists of a mouth piece, a trap for CO 2 , NH 3 and H 2 0, a hydrogen nanosensor, constant dry air flow and electronics.
  • the electronics include an acquisition module, a memory/computation module, and two displays.
  • the numerical display provides the concentration.
  • the YES/NO display will provide the person with an answer indicating high (above the established for this device threshold) or low (below the established for this device threshold) level of hydrogen in breath.
  • a high level will indicate celiac disease or another type of gastrointestinal malabsorption, and a low level will indicate normal gastrointestinal absorption i.e. less likely celiac disease.
  • the sensor may be a nanosensor, or may be a hydrogen-selective film available from Giner, Inc., Newton, MA, which it calls a trace gas sensor. (See, www. ginerinc.com.)
  • the characteristics of the sensor according to the invention are that 1) it is hydrogen selective; 2) can be ZnO nanowire-based (100 ⁇ in diameter) or other type of hydrogen- selective film or nanosensor; 3) can be a single ZnO nanowire sensor fabricated using focused ion beam (FIB/SEM) instrument (5); and 4) can be capable of operating at room temperature requiring a small pulse UV light to recover.
  • FIB/SEM focused ion beam
  • Figure 2a is a scanning electron micrograph in cross-section of ZnO nanowires grown in deposition region at the entrance zone (ratio 0/Zn>l).
  • Figure 2b is a top view scanning micrograph of ZnO nanowires grown at the exit zone (ratio 0/Zn ⁇ 1 ).
  • Figure 3 is a graph showing the IF gas response for a lOOnm diameter ⁇ ,, ⁇ NW-based sensor. The response curves of the individual nanowire-based sensor toward lOppm IF pulses. Before the test the nanowire was preconditioned in a constant dry air flow. An UV pulse is applied after 3 ⁇ 4 is turned off. Lupan O. el al. (5).
  • Figure 4 shows the electronic circuitry of the device. Figure 4 shows a sensor, interface circuitry and display.
  • a micro-controller ( ⁇ €) contains the Analog-to-Digital Converter memory (SRAM), and an Arithmetic Logic Unit (ALU).
  • the V test is a voltage proportional to the resistance of the sensor.
  • the sensor will detect hydrogen gas. If one wishes to detect gases other than hydrogen, other sensors could be used. More than one sensor could be incorporated, with a switch to select connection of the sensor to the circuit for the specific gas to be detected.

Abstract

The present invention relates to a diagnostic method and breath-testing device for the diagnosis of Celiac Disease or other gastrointestinal malabsorption in adults and children, and uses a hydrogen selective sensor in the form of a ZnO nanowire-based sensor fabricated using a focused ion beam (FIB/SEM) instrument or a thin film.

Description

TITLE: DIAGNOSTIC METHOD AND BREATH TESTING DEVICE CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority on U.S. Provisional Application 61/452,507 filed March 14, 201 1 which is incorporated herein by reference. This application also incorporates by reference U.S. Serial No. 61/379,963 filed September 3, 2010, U.S. Serial No. 13/226,082 filed September 6, 2011, and U.S. Serial No. 61/452,391 filed March 14, 2011.
TECHNICAL FIELD
The present invention relates to a diagnostic method and breath-testing device for the diagnosis of celiac disease or other gastrointestinal malabsorption in adults and children.
BACKGROUND OF THE INVENTION
The present application contains references to other publications. These publications are incorporated herein by reference.
Celiac disease is defined as a disease of the small bowel caused by a permanent intolerance to gluten. Most common symptoms of the disease are weight loss due to malabsorption, diarrhea, anemia, and growth failure. Celiac disease affects people of all ages and gender. Its incidence is on the rise worldwide due to environmental and genetic factors, currently affecting, on average, 1/300 people (children and adults) in the United States and in other developed nations. Up until recently celiac disease was considered a disease affecting predominantly people of Northern European origin this, however, is changing as cases are now reported in Asia and the Middle East, albeit at a much lower rate.
Definitive diagnosis of the disease is made on the basis of small bowel biopsies obtained while the patient is on gluten containing diet and after the patient is taken off gluten and free of symptoms. While small bowel biopsy is an invasive diagnostic modality there are other diagnostic methods currently available.
Currently available blood serum tests (Table 1) take place in centralized laboratories and measure antibodies to antigens produced in the patient's body due to gluten intolerance. Results are usually available in several days to a few weeks. Table 1. Diagnostic blood tests for the diagnosis of celiac disease
In addition to blood testing for the presence of specific antibodies, as described in Table 1, blood serum can be tested for the presence of D-xylose, a sugar detected in serum one hour after the ingestion of carbohydrates. The D-xylose test is performed less frequently due to low sensitivity and specificity for the diagnosis of celiac disease and due to the broad use of the more specific antibody diagnostic tests.
Breath testing for the diagnosis of celiac disease has been investigated but is not currently used as a standard diagnostic method due to lower sensitivity (88%) and specificity (84%) when compared to the serum antibodies (Table 1). Tveito K. et al. (1) claim that 13C-xylose and 14C-xylose breath tests are diagnostic of celiac disease patients and both tests demonstrate high sensitivity (88%) and specificity (84%). These investigators also claim that the 13C-sorbitol breath test (2) for the diagnosis of celiac disease is superior to H2- sorbitol test due to higher sensitivity and specificity, however well below the
specificity /sensitivity of 13C-xylose breath test. All of these breath tests require the patient to ingest a carbohydrate substrate, either xylose or sorbitol and undergo breath testing every 30 minutes starting one hour post ingestion up to four hours post ingestion of the substrate. Hryniuk A et al. (3) who investigated the presence of volatile alcohols in the breath of patients with celiac disease report that no differences were found in the breath levels of methanol, propanol, butanol, heptanol or hexanol investigated using chemical ionization of breath air with H30+ and/or NO+ precursor ions in patients with celiac disease when compared to health subjects. All of the above described breath tests are based on spectroscopic analysis of the human breath content and take place at centralized laboratories. Rana, S V et al. (4) report that, at baseline, patients with celiac disease have significantly higher hydrogen in their breath than patients with functional bowel disease and normal subjects. Hydrogen breath test is a standard diagnostic modality for the diagnosis of lactose intolerance and other carbohydrate malabsorption but not for the diagnosis of celiac disease. The existing methodology for the detection of hydrogen in human breath in patients with lactose intolerance and bacterial overgrowth is through gas chromatography (Quintron Instrument Company).
SUMMARY OF THE INVENTION
The present invention consists of a novel, inexpensive, non-invasive, point-of-care, handheld, device for the diagnosis of celiac disease using a selective hydrogen gas nanosensor.
Based on the present invention, a person, adult or child, suspected of having celiac disease or other type of gastrointestinal malabsorption, will provide one fasting breath sample through the mouth piece of the device. No ingestion of carbohydrate substrate is required.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic block diagram of a breath analyzer device for the detection of hydrogen in breath according to the invention;
Figure 2a is a scanning electron micrograph in cross-section of ZnO nanowires grown in deposition region at the entrance zone (ratio 0/Zn>l);
Figure 2b is a top view scanning micrograph of ZnO nanowires grown at the exit zone (ratio 0/Zn< 1);
Figure 3 is a graph showing the ¾ gas response for a lOOnm diameter Ζ,,Ο NW-based sensor; and
Figure 4 shows electronic circuitry of a device according to the invention.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
A preferred embodiment will be described, but the invention will not be limited to this embodiment.
The breath analyzer detects the presence of hydrogen in a human breath sample.
Figure 1 shows a sensor device which consists of a mouth piece, a trap for CO2, NH3 and H20, a hydrogen nanosensor, constant dry air flow and electronics. The electronics include an acquisition module, a memory/computation module, and two displays. The numerical display provides the concentration.
The YES/NO display will provide the person with an answer indicating high (above the established for this device threshold) or low (below the established for this device threshold) level of hydrogen in breath. A high level will indicate celiac disease or another type of gastrointestinal malabsorption, and a low level will indicate normal gastrointestinal absorption i.e. less likely celiac disease.
The sensor may be a nanosensor, or may be a hydrogen-selective film available from Giner, Inc., Newton, MA, which it calls a trace gas sensor. (See, www. ginerinc.com.)
The characteristics of the sensor according to the invention are that 1) it is hydrogen selective; 2) can be ZnO nanowire-based (100η in diameter) or other type of hydrogen- selective film or nanosensor; 3) can be a single ZnO nanowire sensor fabricated using focused ion beam (FIB/SEM) instrument (5); and 4) can be capable of operating at room temperature requiring a small pulse UV light to recover.
Figure 2a is a scanning electron micrograph in cross-section of ZnO nanowires grown in deposition region at the entrance zone (ratio 0/Zn>l). Lupan O. et. al. (5)
Figure 2b is a top view scanning micrograph of ZnO nanowires grown at the exit zone (ratio 0/Zn< 1 ). Lupan O, et. al. (5)
Figure 3 is a graph showing the IF gas response for a lOOnm diameter Ζ,,Ο NW-based sensor. The response curves of the individual nanowire-based sensor toward lOppm IF pulses. Before the test the nanowire was preconditioned in a constant dry air flow. An UV pulse is applied after ¾ is turned off. Lupan O. el al. (5). Figure 4 shows the electronic circuitry of the device. Figure 4 shows a sensor, interface circuitry and display. A micro-controller (μ€) contains the Analog-to-Digital Converter memory (SRAM), and an Arithmetic Logic Unit (ALU). The V test is a voltage proportional to the resistance of the sensor. The sensor will detect hydrogen gas. If one wishes to detect gases other than hydrogen, other sensors could be used. More than one sensor could be incorporated, with a switch to select connection of the sensor to the circuit for the specific gas to be detected.
Although one preferred embodiment has been shown and described, the invention is not limited to this embodiment, and the scope is defined by reference to the following claims.
REFERENCES
1. 13C-xylose and 14C-xylose breath test for the diagnosis of coeliac disease.
Scandinavian J Gastroenterol 2008; 43(2): 166-73; Tveito K. et al
2. A novel one-hour 13C-sorbitol breath test versus the H2-sorbitol breath test for assessment of coeliac disease. Scand J Gastroenterol. 2009;44(7):813-9; Tveito K. et al
3. A preliminary investigation of exhaled breath from patients with celiac disease using selected ion flow tube mass spectrometry. J Gastrointestin Liver Pis. 2010
Mar;19(l):15-20; Hrvniuk A Ross BM
. Influence of previously ingested wheat on fasting breath hydrogen in celiac patients.
Pis Pis Sci. 2009 Jun;54(6):1276-9. Rana SV et al.
. Selective hydrogen gas nanosensor using individual ZnO nanowire with fast response at room temperature. Sensors and Actuators B 144 (2010) 56-66; Lupan O. et al

Claims

WHAT IS CLAIMED IS:
1. A detector for detecting gastrointestinal malabsorption, comprising a hydrogen selective sensor which detects the presence of hydrogen in breath, which thereby detects the presence of gastrointestinal malabsorption.
2. The detector according to Claim 1, wherein the sensor is a nanosensor.
3. The detector according to Claim 1, wherein the sensor is a film sensor.
4. The detector according to Claim 3, wherein the sensor is a thin film sensor.
5. The detector according to Claim 1, wherein the detector is in the form of a hand-held, portable device.
6. The detector according to Claim 1, wherein the detector further comprises an acquisition module for receiving an output signal from the hydrogen selective sensor composite having a characteristic indicating the detected concentration of hydrogen and producing an output signal, a computation module connected to receive the output signal from the acquisition module and producing an output signal, and a display for displaying the results of the detector, based on the acquisition module output signal.
7. The detector according to Claim 6, wherein the display comprises a numerical display for displaying the concentration of detected hydrogen.
8. The detector according to Claim 6, wherein the display comprises a visual indicator which displays the results of a threshold detection indicating the presence of detected hydrogen above a threshold concentration.
9. The detector according to Claim 1, wherein the hydrogen selective sensor comprises a ZnO nanowire-based sensor.
10. The detector according to Claim 1, wherein the hydrogen selective sensor is fabricated using a focused ion beam (FIB/SEM) instrument.
11. The detector according to Claim 1, further comprising a UV light generator which emits a pulse of UV light, for recovering the detector for repeated uses.
12. The detector according to Claim 1 , wherein the detector detects the presence of celiac disease.
13. A method of detecting gastrointestinal malabsorption using a hydrogen selective sensor which detects the presence of hydrogen in breath, which thereby detects the presence of gastrointestinal malabsorption.
14. The method according to Claim 13, wherein the sensor is a nanosensor.
15. The method according to Claim 13, wherein the sensor is a film sensor.
16. The method according to Claim 15, wherein the sensor is a thin film sensor.
17. The method according to Claim 13, further comprising using a detector in the form of a hand-held, portable device.
18. The method according to Claim 13, further comprising acquiring an output signal from the hydrogen selective sensor which has a characteristic indicating the detected concentration of hydrogen, using a computation module to complete the detected hydrogen concentration, and displaying the results of the computation.
19. The method according to Claim 13, comprising displaying a numerical result indicating the concentration of detected hydrogen.
20. The method of Claim 13, comprising comparing the detected hydrogen concentration to a threshold amount, and displaying whether the amount has been exceeded, or is less than the threshold.
21. The method of Claim 13, comprising using a hydrogen selective sensor comprising a ZnO nanowire -based sensor.
22. The method of Claim 13, further comprising fabricating the hydrogen selective sensor using a focused ion beam (FI B/SEM) instrument.
23. The method of Claim 13, further comprising using a UV light pulse to recover the detector for repeated uses.
24. The method of Claim 13, wherein the gastrointestinal malabsorption is celiac disease.
25. A detection for detecting the presence of hydrogen in breath, comprising: a hand-held portable housing;
a sensor in the housing for detecting the presence hydrogen in a concentration sufficiently low to detect, from a user's breath, the presence of celiac disease;
an electronic circuit connected to the sensor for providing an output representative of the concentration of hydrogen detected; and
an output display for displaying a visual signal in response indicating the concentration of hydrogen detected.
26. The detector according to Claim 25, wherein the sensor is a nanosensor.
27. The detector according to Claim 25, wherein the sensor is a film sensor.
28. The detector according to Claim 27, wherein the sensor is a thin film sensor.
29. The detector according to Claim 25, wherein the detector further comprises an acquisition module for receiving an output signal from the hydrogen selective sensor composite having a characteristic indicating the detected concentration of hydrogen and producing an output signal, a computation module connected to receive the output signal from the acquisition module and producing an output signal, and a display for displaying the results of the detector, based on the acquisition module output signal.
30. The detector according to Claim 25, wherein the display comprises a numerical display for displaying the concentration of detected hydrogen.
31. The detector according to Claim 25, wherein the display comprises a visual indicator which displays the results of a threshold detection indicating the presence of detected hydrogen above a threshold concentration.
32. The detector according to Claim 25, wherein the hydrogen selective sensor comprises a ZnO nanowire -based sensor.
33. The detector according to Claim 25, wherein the hydrogen selective sensor is fabricated using a focused ion beam (F1B/SEM) instrument.
34. The detector according to Claim 25, further comprising a UV light generator which emits a pulse of UV light, for recovering the detector for repeated uses.
EP12757976.1A 2011-03-14 2012-03-14 Diagnostic method and breath testing device Withdrawn EP2685891A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201161452507P 2011-03-14 2011-03-14
PCT/US2012/029087 WO2012125734A2 (en) 2011-03-14 2012-03-14 Diagnostic method and breath testing device

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US10401318B2 (en) 2011-03-14 2019-09-03 Anastasia Rigas Breath analyzer and breath test methods

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EP2685891A4 (en) 2015-03-18
WO2012125734A2 (en) 2012-09-20

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