Measurement of hydrogen sulfide during the breath test

Abstract

This document describes methods for detecting hydrogen sulfide (H2S) levels and diagnosing diseases or conditions that cause H2S to be present. Examples of diseases and conditions that cause H2S include small intestinal bacterial overgrowth (SIBO), diarrhea, fatigue, bowel urgency, and abdominal pain. The H2S level can guide treatment in individuals with elevated levels of this compound.

Description

FIELD OF INVENTION

[0001] This invention relates to small intestinal bacterial overgrowth, irritable bowel syndrome and clinical symptoms such as diarrhea and fatigue.BACKGROUND

[0002] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] The breath test (BT) is an important, noninvasive diagnostic test for small intestinal bacterial overgrowth (SIBO). BTs remain the simplest and most widely available evaluation in clinical practice. Presently, 3 gases are measured by this diagnostic modality including hydrogen (H 2 ), methane (CH 4 ) and carbon dioxide (CO 2 ), wherein the presence of detectable methane is notably correlated with constipation and the presence of detectable hydrogen is not demonstrated to correlate with any specific symptoms. Hydrogen sulfide (H 2 S) gas is another microbial byproduct but is not included in the current measurement in BTs. Described herein, we examine the levels of detectable H 2 S from breath testing. In various embodiments, the levels of detectable H 2 S are used to determine associations with specific patient symptoms, and for treatment of H 2 S related conditions such as H 2 S positive SIBO, diarrhea, and fatigue. US 2009 / 23388A1 discloses the use of a breath test to assess the severity of SIBO. In the Pediatric Critical Care Medicine (2017: Vol 18 No. 8 pages e327-e332)the authors report that Inflammatory Bowel Disease (IBD) patients who exhibit overlapping Irritable Bowel Syndrome (IBS)-like symptoms may have gut microbial dysbiosis and, as such, breath testing (BT) may be a useful diagnostic tool in such patients. However, this prior art does not contemplate diagnosing a condition selected from the group consisting of diarrhea, fatigue, bowel urgency, abdominal pain and combinations thereof, by measuring H 2 S as defined in the claims.SUMMARY OF THE INVENTION

[0004] The invention is set out in the appended set of claims.

[0005] Disclosed herein are methods of treating a hydrogen sulfide ("H 2 S") positive condition, comprising: administering a treatment for the H 2 S positive condition to a subject who has been diagnosed as having the H 2 S positive condition, wherein the H 2 S positive condition is selected from the group consisting of H 2 S positive small intestinal bacterial overgrowth ("SIBO"), H 2 S positive diarrhea, H 2 S positive fatigue, H 2 S positive bowel urgency, H 2 S positive abdominal pain and combinations thereof.

[0006] H 2 S positive conditions can be diagnosed by a method comprising: obtaining a biological sample from the subject; measuring the H 2 S level in the biological sample; and diagnosing the H 2 S positive condition if the H 2 S level is higher than a reference level.

[0007] Subjects having the H 2 S positive condition may be identified before administering the treatment for the H 2 S positive condition.

[0008] A test result regarding the H 2 S positive condition may be requested before administering the treatment for the H 2 S positive condition, wherein the test result can be obtained from a method comprising: obtaining a biological sample from the subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample, and diagnosing the H 2 S positive condition if the H 2 S level is higher than a reference level, wherein the H 2 S positive condition can be selected from the group consisting of H 2 S positive diarrhea, H 2 S positive fatigue, H 2 S positive bowel urgency, H 2 S positive abdominal pain and combinations thereof.

[0009] Disclosed herein are methods of detecting hydrogen sulfide ("H 2 S") in a subject, comprising: obtaining a biological sample from the subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and comparing the H 2 S level to a reference level, wherein the subject is suspected to have a H 2 S positive condition selected from the group consisting of, H 2 S positive diarrhea, H 2 S positive fatigue, H 2 S positive bowel urgency, H 2 S positive abdominal pain and combinations thereof.

[0010] Disclosed herein are methods of diagnosing a hydrogen sulfide ("H 2 S") positive condition, comprising: obtaining a biological sample from a subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and diagnosing H 2 S positive condition if the H 2 S level is higher than a reference level, wherein the H 2 S positive condition is selected from the group consisting of, H 2 S positive diarrhea, H 2 S positive fatigue, H 2 S positive bowel urgency, H 2 S positive abdominal pain and combinations thereof.

[0011] Various embodiments of the present invention provide for a method for selecting a therapy for a subject, comprising: obtaining a biological sample from a subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and selecting a therapy for an H 2 S positive condition if the H 2 S level is higher than a reference level, wherein the H 2 S positive condition is selected from the group consisting of H 2 S positive diarrhea, H 2 S positive fatigue, H 2 S positive bowel urgency, H 2 S positive abdominal pain and combinations thereof.

[0012] Various embodiments of the present invention provide for a method for selecting a subject for a clinical trial, comprising: obtaining a biological sample from a subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and selecting the subject for a clinical trial for the treatment of an H 2 S positive condition if the H 2 S level is higher than a reference level, wherein the H 2 S positive condition is selected from the group consisting of H 2 S positive diarrhea, H 2 S positive fatigue, H 2 S positive bowel urgency, H 2 S positive abdominal pain and combinations thereof.

[0013] Rifaximin may be used for treating subjects according to the invention.

[0014] The biological sample is a breath sample.

[0015] The subject may have or be suspected of having irritable bowel syndrome.

[0016] In various embodiments, H 2 S can be measured in these methods using a four gas detection device or system and the four gases can be H 2 , CH 4 , H 2 S, and CO 2 .

[0017] H 2 S may be measured after the subject ingests a controlled quantity of a substrate selected from the group consisting of lactulose, xylose, lactose, glucose, fructose and combinations thereof.

[0018] The reference level may be 6 parts per million (ppm).

[0019] The reference level may be 1.2 parts per million (ppm).

[0020] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 depicts diarrhea severity in relation to hydrogen sulfide concentration. When the area under the curve (AUC) for H 2 S is ≥8ppm there is a greater severity of diarrhea based on a 0-100mm visual analogue scale. Figure 2 depicts urgency severity in relation to hydrogen sulfide concentration. Similar to diarrhea, when the area under the curve (AUC) for H 2 S is ≥8ppm there is a greater severity of urgency based on a 0-100mm visual analogue scale. This provides internal consistency since urgency and diarrhea are related. Figure 3 depicts symptom severity (constipation) in relation to hydrogen sulfide concentration. Unlike diarrhea, H 2 S is not related to constipation. Figure 4 depicts average hydrogen sulfide concentration in patients with or without breath methane. As H 2 S and CH 4 compete for hydrogen as a fuel source for production, it is evident that when there is higher H 2 S, CH 4 is less and vice versa. Figure 5 depicts diarrhea severity in relation to hydrogen sulfide concentration cut-off set at 1.2 ppm. Another and perhaps better way to evaluate H 2 S is by peak value. As shown herein, peak H 2 S of ≥1.2ppm was associated with diarrhea severity. Figure 6 depicts urgency severity in relation to hydrogen sulfide concentration cut-off set at 1.2 ppm. Another and perhaps better way to evaluate H 2 S is by peak value. As shown herein, peak H 2 S of ≥1.2ppm was also associated with urgency severity. Figure 7 depicts H 2 S and H 2 interaction in non-CH 4 Producers. In another way of examining the competition for hydrogen as a fuel source, this graph shows that when a breath test is normal, both H 2 S and H 2 are low. However, as H 2 S increases, the utilization of H 2 increases resulting in consumption represented by ever lower H 2 levels. Figure 8 depicts Hydrogen distribution by Hydrogen Sulfide. As the consumption of H 2 increases to create more H 2 S at the expense of H 2 , there is ever increasing diarrhea. Figure 9 depicts body weight in relation to hydrogen sulfide concentration. It also appears that higher production of H 2 S is associated with greater BMI using an 8ppm cutoff for AUC. Figure 10 depicts H 2 S only on breath test versus none positive. In this graph, the presence of only H 2 S on the breath of ≥1.2ppm peak, was associated with diarrhea again. This time in a larger cohort of subjects. The H 2 S only subjects were compared to normal breath test subjects. Figure 11 depicts H 2 S only on breath test versus all others. Here the H 2 S only subjects were compared to all other subjects and again, diarrhea is statistically greater. Figure 12 shows that higher methane lower hydrogen. This figure demonstrates that at higher CH 4 levels from breath, there is lower hydrogen in line with the CH 4 consuming hydrogen to produce CH 4 . Figure 13 shows that hydrogen depends on methane. The dependence of CH 4 on hydrogen is shown more explicitly in strata of CH 4 levels detected in breath. The greater the peak CH 4 the more H 2 is suppressed. Figure 14 depicts methane and constipation. In this figure, when methane is the sole gas produced in high peak amounts (≥10ppm), subjects are more constipated than methane when other gases are competing and present. Figure 15 depicts diarrhea (Effect of hydrogen sulfide). In the analysis of the larger cohort of patients, once again H 2 S is seen to cause diarrhea. However, in the panel on the right, CH 4 overpowers H 2 S to neutralize the effect (CH 4 is constipating and H 2 S is diarrhea producing and they cancel). Figure 16 depicts constipation-diarrhea vs gas type. This is depiction based on determining the push and pull effect of diarrhea and constipation. In this figure, the diarrhea severity on a scale of 0-100mm is subtracted from the constipation severity on the same scale. A positive number implies constipation is winning. A negative number that diarrhea is winning. Clearly, CH 4 is associated with constipation and H 2 S leans in favor of diarrhea unless CH 4 is there which is almost 0 (balanced). Figure 17 depicts Proof of Hydrogen Consumption Total group. Compared methane and hydrogen sulfide pooled (n=118) and all other non-consumers. In this figure, the H 2 level is assessed based on whether is one (H 2 S or CH 4 ) or two (H 2 S and CH 4 ) present on the breath test. The greater number of competing gases means lower H 2 as it is being used. Figure 18 depicts the effects of rifaximin on H 2 S. In this figure, fresh stool was used to assess H 2 S over 1.5 and 4 hours of fermentation with or without the addition of the antibiotic rifaximin. In this graph, it is clear that rifaximin reduces H 2 S production. DESCRIPTION OF THE INVENTION

[0022] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application.

[0023] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

[0024] "Beneficial results" may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition and prolonging a patient's life or life expectancy.

[0025] "Mammal" as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus adult, child and newborn subjects, whether male or female, are intended to be including within the scope of this term.

[0026] Selecting a therapy as used herein, includes but is not limited to selecting, choosing, prescribing, advising, recommending, instructing, or counseling the subject with respect to the treatment.

[0027] "Therapeutically effective amount" as used herein refers to that amount which is capable of achieving beneficial results in a patient with a disease or condition. A therapeutically effective amount can be determined on an individual basis and will be based, at least in part, on consideration of the physiological characteristics of the mammal, the type of delivery system or therapeutic technique used and the time of administration relative to the progression of the disease.

[0028] "Treatment" and "treating," as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, slow down and / or lessen the disease even if the treatment is ultimately unsuccessful.

[0029] Described herein is the first clinical examination of detectable H 2 S in human BTs in North America. Without wishing to be bound by any particular theory, the inventors believe that H 2 S is an important missing link to SIBO, as H 2 S is herein found to be bioactive and like methane is important in predicting clinical symptoms such as diarrhea and fatigue. Embodiments of present invention are based, at least in part, on these findings.Diarrhea

[0030] Various embodiments of the present invention provide for a method of diagnosing diarrhea, comprising: obtaining a biological sample from a subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and diagnosing diarrhea if the H 2 S level is higher than a reference level.

[0031] Various embodiments of the present invention provide for a method for selecting a therapy for a subject, comprising: obtaining a biological sample from a subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and selecting a therapy for diarrhea if the H 2 S level is higher than a reference level.

[0032] Various embodiments of the present invention provide a method for selecting a subject for a clinical trial, comprising: obtaining a biological sample from a subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and selecting the subject for a clinical trial for the treatment of diarrhea if the H 2 S level is higher than a reference level.

[0033] Various embodiments of the present invention provide for a method of treating diarrhea, comprising: administering a treatment for diarrhea to a subject who has been diagnosed with diarrhea by a method comprising: obtaining a biological sample from the subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and diagnosing diarrhea if the H 2 S level is higher than a reference level.

[0034] Various embodiments of the present invention provide for a method of treating H 2 S positive diarrhea, comprising: administering a treatment to a subject diagnosed with H 2 S positive diarrhea. In various embodiments, the method comprises identifying the subject with H 2 S positive diarrhea before administering treatment.

[0035] Various embodiments of the present invention provide for a method of treating H 2 S positive diarrhea, comprising: administering a treatment for H 2 S positive diarrhea to a subject who has been diagnosed with H 2 S positive diarrhea by a method comprising: obtaining a biological sample from the subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and diagnosing diarrhea if the H 2 S level is higher than a reference level.

[0036] Various embodiments of the present invention provide for a method of treating diarrhea, comprising: requesting a test result obtained from a method comprising: obtaining a biological sample from the subject, measuring the hydrogen sulfide (H 2 S) level in the biological sample, and diagnosing diarrhea if the H 2 S level is higher than a reference level; and administering a treatment for diarrhea to the subject who has been diagnosed with diarrhea.

[0037] Various embodiments of the present invention provide for a method of treating H 2 S positive diarrhea, comprising: requesting a test result obtained from a method comprising: obtaining a biological sample from the subject, measuring the hydrogen sulfide (H 2 S) level in the biological sample, and diagnosing H 2 S positive diarrhea if the H 2 S level is higher than a reference level; and administering a treatment for H 2 S positive diarrhea to the subject who has been diagnosed with diarrhea.

[0038] In various embodiments, the treatment is rifaximin.

[0039] The reference level used in various embodiments of the present invention are described in more detail below.Abdominal Pain

[0040] Various embodiments of the present invention provide for a method of diagnosing H 2 S positive abdominal pain, comprising: obtaining a biological sample from a subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and diagnosing H 2 S positive abdominal pain if the H 2 S level is higher than a reference level.

[0041] Various embodiments of the present invention provide for a method for selecting a therapy for a subject, comprising: obtaining a biological sample from a subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and selecting a therapy for H 2 S positive abdominal pain if the H 2 S level is higher than a reference level.

[0042] Various embodiments of the present invention provide a method for selecting a subject for a clinical trial, comprising: obtaining a biological sample from a subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and selecting the subject for a clinical trial for the treatment of H 2 S positive abdominal pain if the H 2 S level is higher than a reference level.

[0043] Various embodiments of the present invention provide for a method of treating H 2 S positive abdominal pain, comprising: administering a treatment for H 2 S positive abdominal pain to a subject who has been diagnosed with H 2 S positive abdominal pain by a method comprising: obtaining a biological sample from the subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and diagnosing H 2 S positive abdominal pain if the H 2 S level is higher than a reference level.

[0044] Various embodiments of the present invention provide for a method of treating H 2 S positive abdominal pain, comprising: administering a treatment to a subject diagnosed with H 2 S positive abdominal pain. In various embodiments, the method comprises identifying the subject with H 2 S positive abdominal pain before administering treatment.

[0045] Various embodiments of the present invention provide for a method of treating H 2 S positive abdominal pain, comprising: administering a treatment for H 2 S positive abdominal pain to a subject who has been diagnosed with H 2 S positive abdominal pain by a method comprising: obtaining a biological sample from the subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and diagnosing H 2 S positive abdominal pain if the H 2 S level is higher than a reference level.

[0046] Various embodiments of the present invention provide for a method of treating H 2 S positive abdominal pain, comprising: requesting a test result obtained from a method comprising: obtaining a biological sample from the subject, measuring the hydrogen sulfide (H 2 S) level in the biological sample, and diagnosing H 2 S positive abdominal pain if the H 2 S level is higher than a reference level; and administering a treatment for H 2 S positive abdominal pain to the subject who has been diagnosed with H 2 S positive abdominal pain.

[0047] In various embodiments, the treatment is rifaximin.

[0048] The reference level used in various embodiments of the present invention are described in more detail below.Urgency

[0049] Various embodiments of the present invention provide for a method of diagnosing likelihood of having bowel urgency, comprising: obtaining a biological sample from a subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and diagnosing the subject as having bowel urgency if the H 2 S level is higher than a reference level.

[0050] Various embodiments of the present invention provide for a method for selecting a therapy for a subject, comprising: obtaining a biological sample from a subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and selecting a therapy for having bowel urgency if the H 2 S level is higher than a reference level.

[0051] Various embodiments of the present invention provide a method for selecting a subject for a clinical trial, comprising: obtaining a biological sample from a subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and selecting the subject for a clinical trial for the treatment bowel urgency if the H 2 S level is higher than a reference level.

[0052] Various embodiments of the present invention provide for a method of treating bowel urgency, comprising: administering a treatment for bowel urgency to a subject who has been diagnosed with having H 2 S positive bowel urgency. In various embodiments, the method comprises first identifying the subject who has H 2 S positive bowel urgency before administering treatment.

[0053] Various embodiments of the present invention provide for a method of treating bowel urgency, comprising: administering a treatment for bowel urgency to a subject who has been diagnosed with having bowel urgency by a method comprising: obtaining a biological sample from the subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and diagnosing bowel urgency if the H 2 S level is higher than a reference level.

[0054] Various embodiments of the present invention provide for a method of treating bowel urgency, comprising: requesting a test result obtained from a method comprising: obtaining a biological sample from the subject, measuring the hydrogen sulfide (H 2 S) level in the biological sample, and diagnosing bowel urgency if the H 2 S level is higher than a reference level; and administering a treatment for bowel urgency to the subject who has been diagnosed with bowel urgency.

[0055] In various embodiments, the treatment is rifaximin.

[0056] The reference level used in various embodiments of the present invention are described in more detail below.Fatigue

[0057] Various embodiments of the present invention provide for a method of diagnosing fatigue, comprising obtaining a biological sample from a subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and diagnosing fatigue if the H 2 S level is higher than a reference level.

[0058] Various embodiments of the present invention provide for a method for selecting a therapy for a subject, comprising: obtaining a biological sample from a subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and selecting a therapy for fatigue if the H 2 S level is higher than a reference level.

[0059] Various embodiments of the present invention provide for a method for selecting a subject for a clinical trial, comprising: obtaining a biological sample from a subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and selecting the subject for a clinical trial for the treatment of fatigue if the H 2 S level is higher than a reference level.

[0060] Various embodiments of the present invention provide for a method of H 2 S positive treating fatigue, comprising: administering a treatment for H 2 S positive fatigue to a subject who has been diagnosed with H 2 S positive fatigue. In various embodiments, the method comprises identifying the subject with H 2 S positive fatigue before administering treatment.

[0061] Various embodiments of the present invention provide for a method of treating fatigue, comprising: administering a treatment for fatigue to a subject who has been diagnosed with fatigue by a method comprising: obtaining a biological sample from the subject; measuring the hydrogen sulfide (H 2 S) level in the biological sample; and diagnosing fatigue if the H 2 S level is higher than a reference level.

[0062] Various embodiments of the present invention provide for a method of treating fatigue, comprising: requesting a test result obtained from a method comprising: obtaining a biological sample from the subject, measuring the hydrogen sulfide (H 2 S) level in the biological sample, and diagnosing fatigue if the H 2 S level is higher than a reference level; administering a treatment for fatigue to the subject who has been diagnosed with fatigue.

[0063] In various embodiments, the treatment is rifaximin.

[0064] The reference level used in various embodiments of the present invention are described in more detail below.Detection of H 2 S

[0065] Various embodiments of the present invention provide for a method of detecting H 2 S in a subject having or suspected of having a condition selected from the group consisting of diarrhea, bowel urgency, fatigue, irritable bowel syndrome (IBS), diarrhea predominant IBS (D-IBS), constipation predominant IBS (C-IBS), and combinations thereof, comprising: obtaining a biological sample from the subject having or suspected of having the condition; and measuring the hydrogen sulfide (H 2 S) level in the biological sample. Prior to the present invention, it was not known that H 2 S can be bioactive and cause diarrhea, bowel urgency, or fatigue and thus, patients having or suspected of having these conditions did not have a reason to test for H 2 S to see if their condition was caused by H 2 S.Subjects

[0066] The subject from whom a biological sample is obtained can be a subject who has or is suspected to have a disease or condition caused, at least in part, by having high H 2 S levels. Examples of these subjects are those who are or who are suspected to have irritable bowel syndrome (IBS), diarrhea predominant IBS (D-IBS), constipation predominant IBS (C-IBS), diarrhea, bowel urgency, fatigue, constipation, bloating, diabetes, or obesity. In various embodiments, the subject is a human subject.

[0067] In certain embodiments, the subject from whom a biological sample is obtained can be a subject who desires to know whether he or she is susceptible to a disease or condition caused, at least in part, by having high H 2 S levels. Examples of these subjects are those who desire to know whether he or she is susceptible to having irritable bowel syndrome (IBS), D-IBS, C-IBS, diarrhea, bowel urgency, fatigue, constipation, bloating, diabetes, or obesity.Reference levels

[0068] In embodiments wherein the biological sample is a breath sample, the reference level for H2S is 6 parts per million (ppm) or 1.2 parts per million (ppm). In other embodiments wherein the biological sample is a breath sample, the reference level for H 2 S is 1 ppm.

[0069] The reference level can depend on the type of disease or condition that will be determined, as well as the sensitivity of the detection method or system. Different reference levels can be set for different diseases or conditions and for different detection methods and systems.

[0070] In some embodiments, the reference level can be established from biological samples from a healthy subject. For example, if the biological sample is breath, then the reference level can be obtained from the breath of a healthy subject. In other embodiments, the reference level is the average H 2 S level for the same type of biological sample from a population of healthy subjects. In other embodiments, the reference level is the average plus one or two standard deviations of average H 2 S level for the same type of biological sample from a population of healthy subjects. In some embodiments, the population of healthy subjects can range from at least three healthy individuals to 25 healthy individuals, to 50 healthy subject, 75 healthy subject, and even more than 100 healthy individuals. In various embodiments, a heathy subject is a subject who on questionnaire, report no altered bowel function, no bloating and no abdominal pain (each less than 10mm on a 100mm VAS scale for the specific symptom).

[0071] In certain embodiments, wherein breath samples are taken, and methane and / or hydrogen are also measured, the methane reference value can be about 3 ppm or about 5ppm and the hydrogen reference value can be about 20 ppm.Biological Samples

[0072] The biological sample according to the invention is a breath sample. Examples of other biological samples include but are not limited to body fluids, whole blood, plasma, serum, stool, intestinal fluids or aspirate, and stomach fluids or aspirate, serum, breath, cerebral spinal fluid (CSF), urine, sweat, saliva, tears, pulmonary secretions, breast aspirate, prostate fluid, seminal fluid, cervical scraping, amniotic fluid, intraocular fluid, mucous, and moisture in breath. Other biological samples may be whole blood, blood plasma, blood serum, stool, intestinal fluid or aspirate or stomach fluid or aspirate, or stool.Breath Test

[0073] In various embodiments, H 2 S can be detected and measured via breath testing. In various embodiments the breath test can utilize (1) sorbent based technology; and / or (2) membrane based technology to measure the levels of gases exhaled by a subject.

[0074] One potential technology to measure the gas levels includes certain sorbent technologies. In general, these are substances that absorb gases. In order to measure a gas concentration using sorbents, the sorbents can be first weighed, then exposed to the gas. After exposure, the sorbents can be weighed again to determine the increase from the added mass of the absorbed gas. Alternatively, the change in mass may be measured by other means such as luminescence, color, transparency, conductivity, or resonance.

[0075] The following formula may be utilized to determine the concentration of H 2 in exhaled breath gases based on weighing the sorbents: V Lung V per mol of air ∗ C H 2 = N H 2 5 L 24 L mol ∗ 1 ppm = 2 × 10 − 7 mol N H 2 ∗ M Hydrogen = Total Weigh t Hydrogen per breath 2 x 10 − 7 mol ∗ 1 g mol ∗ 2 = 4 x 10 − 7 g = .4 μg per breath Where: V - Volume (litres) C - Concentration N - Number of mols (per breath) M - Molecular mass of hydrogen atoms (grams)

[0076] CO 2 (40000ppm), H 2 S (1 ppm) and methane (1 ppm) can be calculated in a similar manner and require scales sensitive to 0.6g, 6pg, and 3pg respectively. Technology such as a quartz crystal microbalance may be utilized to detect weights to that level of precision.

[0077] In some embodiments, sorbent materials may require a resetting process after each use to expel all of the absorbed gases. For instance, some sorbents require a heating cycle to force the sorbent to release the stored gas, or some similar process. In other embodiments, a sorbent material may be selected which rapidly releases the absorbed gases. Accordingly, devices utilizing sorbents may include a heating element or other processing technology that would be triggered after each use to expel the gases. In other embodiments, the sorbents may be disposed of and replaced instead of being reset, but would need to be mounted to the weight measurement device.

[0078] Types of sorbents that may be utilized include immobilized amine, aminosilane, and organoclay sorbents. The amine sorbents, for example, may be regenerable. Some examples of suitable sorbents include high performance hollow microspheres, hollow fibers and supported liquid membranes. Specifically, the high performance hollow microspheres include amine microspheres. In one example, the hollow microspheres may be made of biocompatible materials for use in medical applications. The hollow microspheres have geometries that allow for detection of several gases. Further, the organoclay sorbents may be used for CO 2 and H 2 S detection. In one example, the organoclay sorbent may be an amine based sorbent. Some sorbents are designed to be regenerable such that the modified amine is regenerated in the presence of water vapor.

[0079] Examples of sorbent based technology and methods used for detecting gases are described in, for example, US Patent 8,500,854, issued on Aug. 6, 2013, titled Regenerable Sorbent Technique for Capturing CO 2 using Immobilized Amine Sorbents, and U.S. Patent No. 7,288,126, issued on Oct. 30, 2007, titled High Capacity Immobilized Amine Sorbents,

[0080] In some embodiments, membrane based technology may be utilized to determine the concentrations of breath gases. For instance, membranes could first be utilized to selectively filter gases of interest. Then, another sensor technology may determine the concentration of the isolated gas that has permeated through the other side of the membrane. For example, pressure sensors (to detect partial pressure changes), gas chromatography, or a simple counter could be utilized. In some embodiments, the combination of membranes with other sensor technologies may enhance the selectivity of the device.

[0081] Some examples of membranes to be incorporated into the devices and methods disclosed herein include flat sheet membranes, hollow microspheres and mixed matrix membranes. Mixed matrix membranes, in particular, may be advantageous as they have different levels of bulk and surface porosity as well as customizable inner and outer diameter dimensions. The geometries of the membranes allow for maximum detection of several gases. Specifically, mixed matrix membranes with metal organic frameworks may be used for detection of CO 2 and CH 4 .

[0082] Breath H 2 S test is based fermentative bacteria found in the gastrointestinal tract producing detectable quantities of H 2 S as fermentation products from a substrate consumed by the host, under certain circumstances. Substrates include sugars such as lactulose, xylose, lactose, glucose, or fructose. The H 2 S produced in the small intestine then enters the blood stream of the host and are gradually exhaled.

[0083] Typically, after an ovemight fast, the patient swallows a controlled quantity of a sugar, such as lactulose, xylose, lactose, glucose, or fructose and breath samples are taken at frequent time intervals, typically every 10 to 15 minutes for a two- to four-hour period. In certain embodiments, samples are analyzed by gas chromatography or by other suitable techniques, singly or in combination. In other embodiments, samples are analyzed using a sorbent based technology and / or membrane based technology as described herein.

[0084] In various embodiments, the breath test can be performed utilizing a device or method as described in Intemational Application Publication No. WO 2017 / 040546.Therapies

[0085] In various embodiments, once a high H 2 S level is detected a therapy aimed at lowering the H 2 S level can be selected or administered to the subject.

[0086] In various embodiments, a therapy for the treatment of diarrhea can be selected or administered to the subject.

[0087] In various embodiments, a therapy for the treatment of fatigue can be selected or administered to the subject.

[0088] An antibiotic or a combination of two or more antibiotics can be selected and / or administered to subjects who have a H 2 S level higher than the reference level. Examples of antibiotics include but are not limited to aminoglycosides (e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, paromomycin), ansamycins (e.g., geldanamycin, herbimycin), carbacephems (e.g., loracarbef), carbapenems (e.g., ertapenem, doripenem, imipenem, cilastatin, meropenem), cephalosporins (e.g., first generation: cefadroxil, cefazolin, cefalotin or cefalothin, cefalexin; second generation: cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime; third generation: cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone; fourth generation: cefepime; fifth generation: ceftobiprole), glycopeptides (e.g., teicoplanin, vancomycin), macrolides (e.g., azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spectinomycin), monobactams (e.g., aztreonam), penicillins (e.g., amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, meticillin, nafcillin, oxacillin, penicillin, piperacillin, ticarcillin), antibiotic polypeptides (e.g., bacitracin, colistin, polymyxin b), quinolones (e.g., ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin), rifamycins (e.g., rifampicin or rifampin, rifabutin, rifapentine, rifaximin), sulfonamides (e.g., mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole (co-trimoxazole, "tmp-smx"), and tetracyclines (e.g., demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline) as well as arsphenamine, chloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin, pyrazinamide, quinupristin / dalfopristin combination, and tinidazole.

[0089] In various embodiments, the antibiotic selected, or directed and / or administered is rifaximin. The rifaximin therapy selected, directed and / or administered can be 200-2400 mg / dose, administered two or three times per day. In various embodiments the dosage can be about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 mg / dose. In particular embodiments, the dosage can be about 550 mg / dose. In various embodiments, the rifaximin therapy can be administered one, two, three, four or five times a day. In various embodiments, the therapy can be administered for 5, 7, 10, 14, 15, 20, 21, or 28 days. In various embodiments, the therapy can be re-administered after a period of no therapy.

[0090] The antibiotic selected and / or administered may be neomycin. The neomycin therapy selected, directed and / or administered can be 500-1000 mg / dose, administered two times per day. In various embodiments the dosage can be about 100, 200, 300, 400, 500, 600, 700, 750, 1000, 1100, 1200, 1300, 1400, or 1500 mg / dose. In various embodiments, the neomycin therapy can be administered one, two, three, four or five times a day. In various embodiments, the therapy can be administered for 5, 7, 10, 14, 15, 20, 21, or 28 days. In various embodiments, the therapy can be re-administered after a period of no therapy.

[0091] The antibiotic selected and / or administered may be vancomycin. The vancomycin therapy selected, directed, and / or administered can be about 125 mg / dose, administered four times per day. In various embodiments the dosage can be about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 mg / dose. In various embodiments, the vancomycin can be administered one, two, three, four or five times a day. In various embodiments, the therapy can be administered for 5, 7, 10, 14, 15, 20, 21, or 28 days. In various embodiments, the therapy can be re-administered after a period of no therapy.

[0092] The antibiotic selected and / or administered may be metronidazole. The metronidazole therapy selected, directed and / or administered can be 250-500 mg / dose, administered three times per day. In various embodiments the dosage can be about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, or 1000 mg / dose. In various embodiments, the metronidazole therapy can be administered one, two, three, four or five times a day. In various embodiments, the therapy can be administered for 5, 7, 10, 14, 15, 20, 21, or 28 days. In various embodiments, the therapy can be re-administered after a period of no therapy.

[0093] Particularly effective antibiotics may be non-absorbable antibiotics. Examples of non-absorbable antibiotics include but are not limited to rifaximin, neomycin, Bacitracin, vancomycin, teicoplanin, ramoplanin, and paramomycin.

[0094] A probiotic agent that inhibits the growth of H 2 S producing microorganisms can be selected or administered, for example, Bifidobacterium sp. or Lactobacillus species or strains, e.g., L. acidophilus, L. rhamnosus, L. plantarum, L. reuteri, L. paracasei subsp. paracasei, or L. casei Shirota, or probiotic Saccharomyces species, e.g., S. cerevisiae, is selected and / or administered. In some embodiments, the agent that inhibits the growth of H 2 S producing microorganisms can be methanogens or acetogens. These microorganisms can compete with the growth of H 2 S producing microorganisms and thus inhibit the growth and proliferation of H 2 S producing microorganisms. The methanogen can be from the genus Methanobrevibacter. Examples of Methanobrevibacter include but are not limited to M. acididurans, M. arboriphilus, M. curvatus, M. cuticularis, M. filiformis, M. gottschalkii, M. millerae, M. olleyae, M. oralis, M. ruminantium, M. smithii, M. thaueri, M. woesei, and M. wolinii. In certain embodiments, the Methanobrevibacter is Methanobrevibacter smithii (M. Smithii). The agent that inhibits the growth of H 2 S producing microorganisms can be typically administered in a pharmaceutically acceptable ingestible formulation, such as in a capsule, or for some subjects, consuming a food supplemented with the inoculum is effective, for example a milk, yoghurt, cheese, meat or other fermentable food preparation. These probiotic agents can inhibit the growth of H 2 S producing microorganisms, for example, by competing against H 2 S producing microorganisms for growth and thus reduce or inhibit the growth of H 2 S producing microorganisms.EXAMPLES

[0095] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.Example 1

[0096] Consecutive patients undergoing BTs at a tertiary care motility program were consented. For the BT, subjects presented after a 24 hour-preparation, beginning with 12-hour diet restrictions and a subsequent 12-hour fast. After a baseline breath sample, 10g of lactulose or 25g of fructose was administered, followed by collection of breath samples every 15 minutes for 120 minutes. Breath samples were then analyzed on a Quintron Breathtracker ™< gas chromatograph (Quintron Diagnostics, Milwaukee, WI) to measure H 2 and CH 4 after correction for CO 2 . Remaining breath samples in the collection bag were analyzed for detectable H 2 S (Alphasense, Essex, UK). Patients completed a questionnaire evaluating gastrointestinal symptoms, medical history, and demographics. The AUC of H 2 S over 120 minutes was used to determine the relevant clinical cutoffs. This was then compared to symptom severity by non-parametric testing and multivariate analysis to rule out effect of age and gender.

[0097] A total of 101 subjects were recruited for this study with 99 studies (62% female, mean age=46.2±16.3 years, 98 of these were lactulose test) having complete data on H 2 S measurements. For the entire group the mean AUC for H 2 S was 5.37±2.29 ppm (range=2.57-16.44 ppm). Using a sensitivity analysis, a significant relationship was seen between the level of H 2 S and specific symptoms. For diarrhea, AUC levels of H 2 S ≥ 6.0 were associated with greater severity of diarrhea (P=0.028, see table). The same cutoff was notable for a greater degree of fatigue in the high H 2 S category (P=0.035). Interestingly, the H 2 AUC was higher in the high H 2 S group (367±232 ppm) compared to the low H 2 S group (244±176 ppm) (P=0.02) consistent with H 2 consumption to produce H 2 S by sulfate-reducing bacteria. Age and gender were not confounding variables in this analysis. Table 1: Comparison of AUC 120 for hydrogen sulfide to symptoms during breath testing SymptomLow H 2 S (<6.0 ppm)High H 2 S (>6.0 ppm)P-valueDiarrhea48.1±33.163.5±24.50.028Constipation53.1±31.263.5±24.40.20Abdominal Pain59.9±27.869.1±19.40.16Fatigue61.6±31.279.2±14.70.04 Example 1B

[0098] 127 patients were recruited from GI Motility Program of Cedars-Sinai Medical Center. Subjects ≥ 18 years undergoing lactulose breath testing were eligible to participate in this study. Subjects prepared with standard-of-care diet restriction and fast. Baseline breath sample was taken; then ingestion of 10g lactulose was done; then collection of breath samples were performed every 15 mins for 120 mins.

[0099] Patients completed questionnaire during 120-minute breath test collection: Gastrointestinal symptoms, Medical / surgical history, Demographics.

[0100] Statistical Analyses: H 2 S levels were assessed by examining the AUC 120 or maximum H 2 S during the first 120 minutes of testing; Chi-squared (χ2) test was used for comparisons of qualitative data; Nonparametric analyses were used for non-normal data Comparisons; Spearman's rank correlation was applied in the comparison of interquartile assessment of H 2 S levels. Table 2: DemographicsH 2 S Cutoff<8 ppm (n=77)≥8 ppm (n=50)P-valueAge (years)46.2 ± 16.853.6 ± 16.7P=0.02Gender (%female)64.980.3P=0.07

[0101] H 2 S is important in breath testing and correlates with patient symptoms ≥8 ppm AUC or ≥1.2 ppm max appears to be initial candidate cut-offs for patient symptoms. H 2 S and CH 4 appear competitive. H 2 S can be an important gas to be assessed in relation to the gut microbiome.Example 2

[0102] The effects of rifaximin on H 2 S was examined. Fresh stool was used to assess H 2 S over 1.5 and 4 hours of fermentation with or without the addition of the antibiotic rifaximin. As shown in figure 18, it is clear that rifaximin reduces H 2 S production.Example 3 4-gas device for breath testing shows exhaled H 2 S is associated with diarrhea and abdominal pain in a large scale prospective trial

[0103] Adult subjects undergoing routine lactulose breath testing at a tertiary outpatient clinic were consented for the study. Subjects underwent a 24-hour pre-test preparation, following a specific diet for the first 12 hours and fasting for the remaining 12. Subjects provided a baseline breath sample and, after consuming 10g lactulose dissolved in 250 mL water, successive breath samples were collected every 15 minutes over the next 120 minutes. Each breath sample was analyzed using a 4-gas device, simultaneously measuring H 2 , CH 4 , CO 2 , and H 2 S. Subjects completed a questionnaire evaluating medical history, demographics, and severity of gastrointestinal symptoms (0- 100mm VAS scale). The gas profiles were then related to the presence and severity of symptoms.

[0104] Of the 300 subjects who consented for the study, 298 had complete data for analysis. Based on H 2 alone, breath testing was positive for SIBO in 182 (61%) subjects. Diarrhea severity was not different between those with (44.2±2.4) and without H 2 (43.2±3.0) (P=0.79). The range of H 2 S levels in these subjects was 0.43 to 1.99ppm. Using a sensitivity analysis, H 2 S was associated with more diarrhea and the cutoff for this discrimination was a maximum level of 1.2ppm at any point during the test. Using this cutoff, 72 subjects (24.2%) were positive for H 2 S. Diarrhea severity in H 2 S subjects was 52.1±3.8 compared to 41.2±2.1 in non-H 2 S subjects (P=0.01). In some cases, H 2 S was positive in addition to H 2 and / or CH 4 . However, the greatest severity of diarrhea was seen in subjects with H 2 S only (≥1.2ppm). Similarly, subjects with excess H 2 S but normal CH 4 and H 2 had greater abdominal pain (71.1±5.1) than normal breath test patients (52.9±3.8). In fact, the greatest abdominal pain was seen in subjects positive for H 2 S only (P<0.05).

[0105] In this large-scale trial, the 4-gas device for breath testing demonstrates the importance of H 2 S in diarrhea and abdominal pain. Despite the lack of correlation between H 2 and these two symptoms, subjects positive for H 2 S only (≥1.2ppm) had the greatest diarrhea and abdominal pain among subjects referred for testing. This finding is important as it complements the breath test panel for SIBO testing and can be a predictor for treatment.Example 4 Competitive hydrogen gas utilization by methane- and hydrogen sulfide-producing microorganisms and associated symptoms: results of a 4-gas breath test machine

[0106] Breath testing subjects at a tertiary care center were consented. After fasting, subjects provided a baseline breath sample, consumed 10g lactulose, and gave breath samples every 15 min for 120 min. H 2 , CH 4 , H 2 S, and CO 2 levels were measured using the 4-gas detection device. Subjects completed a medical history, demographics, and symptoms questionnaire. Diarrhea, bloating, constipation, and abdominal pain severity were measured using 0-100mm VAS scales. Positive breath testing was based on the North American consensus for H 2 (≥20ppm at or before 90 min) and CH 4 (≥10ppm during 120 min). Sensitivity analysis found H 2 S ≥1.2ppm at any point was clinically important. Competition for H 2 utilization was assessed by CH 4 and H 2 S. CH 4 and H 2 S levels were also correlated with symptoms.

[0107] Of 300 subjects enrolled, 298 had complete data (mean age=49.0±1.0yrs; 66% female). Among these, 8 distinct patterns were noted with 238 (80%) subjects positive for at least one gas. Of these, 25 subjects who would have been categorized as non-CH 4 / non-H 2 (i.e. flat-liners) were positive for H 2 S. Excess CH 4 production was associated with lower H 2 levels (32.2±4.3ppm) compared to normal CH 4 (60.1±2.7ppm; P<0.0001). After excluding CH 4 -positive subjects, H 2 levels were lower in subjects with excess H 2 S (34.8±3.7ppm) compared to those with normal H 2 S (60.1±2.7ppm; P<0.0001). H 2 was also lower in CH 4 -positive subjects (32.0±8.1ppm) compared to excess H 2 S producers (34.8±3.7ppm; P=0.81). H 2 levels were successively lower when comparing no consuming gases, one consuming gas (CH 4 or H 2 S), and two consuming gases (CH 4 and H 2 S) (figure 17) (ANOVA, p<0.05). CH 4 -positive subjects, irrespective of other gas present, had a constipation-predominant pattern. H 2 S-positive subjects exhibited a diarrhea-predominant pattern.

[0108] This 4-gas device can assess a more complete interaction of fermented gases in breath testing. This study shows that CH 4 and H 2 S are independently associated with lower H 2 , consistent with the known physiology of CH 4 and H 2 S production via H 2 consumption. For the first time, we demonstrate the dynamics between competing gases in terms of dominant symptom manifestation (diarrhea vs. constipation).

[0109] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). Although the openended term "comprising," as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as "consisting of" or "consisting essentially of."

Claims

1. A method of detecting a hydrogen sulfide ("H2S") positive condition in a subject, comprising: measuring the hydrogen sulfide (H2S) level in a breath sample obtained from the subject; and comparing the H2S level to a reference level, wherein the subject is suspected to have a H2S positive condition selected from the group consisting of H2S positive diarrhea, H2S positive fatigue, H2S positive bowel urgency, H2S positive abdominal pain and combinations thereof; and wherein the reference level is 1, 1.2 or 6 parts per million (ppm).

2. The method according to claim 1 wherein the H2S is detected for the purposes of diagnosing a H2S positive condition selected from the group consisting of H2S positive diarrhea, H2S positive fatigue, H2S positive bowel urgency, H2S positive abdominal pain and combinations thereof.

3. The method according to claim 1 wherein the H2S is detected for the purposes of selecting a therapy for the subject and wherein the therapy is for treating a H2S positive condition selected from the group consisting of H2S positive diarrhea, H2S positive fatigue, H2S positive bowel urgency, H2S positive abdominal pain and combinations thereof.

4. The method according to claim 1 wherein the H2S is detected for the purposes of selecting the subject for a clinical trial for the treatment of an H2S positive condition if the H2S level is higher than a reference level; and wherein the H2S positive condition is selected from the group consisting of H2S positive fatigue, H2S positive bowel urgency, H2S positive abdominal pain and combinations thereof.

5. A use of an antibiotic for treating a subject with a hydrogen sulfide ("H2S") positive condition, wherein the subject has been diagnosed as having the H2S positive condition according to the method of claim 2.

6. The use according to 5 wherein the subject has been identified as having the H2S positive condition before treatment of the H2S positive condition.

7. The use according to any of claims 5 or 6 wherein the antibiotic is rifaximin.

8. The method or use according to any preceding claim wherein the subject has or is suspected of having irritable bowel syndrome.

9. The method or use according to any preceding claim wherein H2S is measured using a four gas detection device or system and the four gases are H2, CH4, H2S, and CO2.

10. The method or use according to any preceding claim wherein H2S is measured after the subject ingests a controlled quantity of a substrate selected from the group consisting of lactulose, xylose, lactose, glucose, fructose and combinations thereof.

11. The method or use according to any preceding claim wherein the reference level is 6 parts per million (ppm).

12. The method or use according to any preceding claim wherein the reference level is 1.2 parts per million (ppm).

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