Methods of diagnosing or treating h. pylori infection

Abstract

Described herein is a method of diagnosing a H. pylori infection in a subject. The method comprises: determining a fatty acid profile in a stool sample of the subject; and comparing the fatty acid profile with a reference profile indicating H. pylori infection or a second reference profile indicating H. pylori infection. The subject is diagnosed to have H. pylori infection when a level of at least one fatty acid in the fatty acid profile is different than a level of the at least one fatty acid in the first reference profile; or a level of at least one fatty acid in the fatty acid profile matches a level of the at least one fatty acid in the second reference profile. Also described is a method of treating H. pylori infection based on the diagnostic method.

Description

[0001] Attorney Docket No. 370431-1054WO1 (00351)

[0002] METHODS OF DIAGNOSING

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 725,161, filed November 26, 2024, which is incorporated herein by reference in its entirety.

[0005] BACKGROUND

[0006] Helicobacter pylori infects approximately half of the world’s population and is associated with, among others, peptic ulcers, non-ulcer dyspepsia, nd gastric cancers. Pretreatment antimicrobial susceptibility testing is generally not available and treatment regimens are chosen empirically, which reduces the likelihood of successful eradication. Traditional first-line treatment regimens have been increasingly ineffective against / / , pylori due to emerging antibiotic resistance. As the burden of antibiotic resistance increases, the need for rapid diagnostic methods to indicate H. pylori infection and its eradication after treatment has become more pressing.

[0007] Accordingly, there is a need for methods for diagnosing and / or treating H. pylori infections. The present invention addresses this need.

[0008] SUMMARY

[0009] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0010] Method of diagnosing H. pylori infection

[0011] In some aspects, the present invention is directed to a method of diagnosing H. pylori infection in a subject.

[0012] In some embodiments, the method comprises: determining a fatty acid profile in a stool sample of the subject; and comparing the fatty acid profile with a first reference profile indicating H. pylori infection or a second reference profile indicating H. pylori infection.

[0013] In some embodiments, the subject is diagnosed to have H. pylori infection when a level of at least one fatty acid in the fatty acid profile is different than a level of the at least one fatty acid in the first reference profile.

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[0015] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351)

[0016] In some embodiments, the subject is diagnosed to have H. pylori infection when a level of at least one fatty acid in the fatty acid profile matches a level of the at least one fatty acid in the second reference profile.

[0017] In some embodiments, the method further comprises obtaining the stool sample from the subject.

[0018] In some embodiments, the subject is a human.

[0019] In some embodiments, at least one fatty acid comprises a metabolite of the omega-3 fatty acids pathway, a metabolite of the omega-6 fatty acids pathway, and / or a metabolite of the omega-9 fatty acids pathway.

[0020] In some embodiments, the at least one fatty acid comprises margaric acid (17:0), eicosapentaenoic acid (20:5n3), erucic acid (22: ln9), docosapentaenoic acid (22:5n3), eicosatetraenoic acid (20:4n3), docosahexaenoic acid (22:6n3), gamma-linolenic acid (18:3n6), and / or osbond acid (22:5n6).

[0021] In some embodiments, the subject is diagnosed to have H. pylori infection when the at least one fatty acid selected from the group consisting of margaric acid (17:0), eicosapentaenoic acid (20:5n3), erucic acid (22: ln9), docosapentaenoic acid (22:5n3), eicosatetraenoic acid (20:4n3), and docosahexaenoic acid (22:6n3) in the fatty acid profile is higher than in the first reference profile indicating free of H. pylori infection.

[0022] In some embodiments, the subject is diagnosed to have H. pylori infection when the at least one fatty acid selected from the group consisting of gamma-linolenic acid (18:3n6), and osbond acid (22:5n6) in the fatty acid profile is lower than in the first reference profile indicating free of H. pylori infection.

[0023] In some embodiments, the subject is diagnosed to have H. pylori infection when the diagnosis takes in to consideration whether the subject consumes alcohol, whether the subject smokes, and / or whether the subject has non-ulcer dyspepsia.

[0024] In some embodiments, the first reference profile comprises a fatty acid reference profile indicating a non-alcohol-consuming subject free from H. pylori infection.

[0025] In some embodiments, the first reference profile comprises a fatty acid reference profile indicating an alcohol-consuming subject free from H. pylori infection.

[0026] In some embodiments, the first reference profile comprises a fatty acid reference profile indicating a non-smoking subject free from H. pylori infection.

[0027] In some embodiments, the first reference profile comprises a fatty acid reference profile indicating a smoking subject free from H. pylori infection.

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[0030] In some embodiments, the first reference profile comprises a fatty acid reference profile indicating a non-ulcer dyspepsia-free subject free from H. pylori infection.

[0031] In some embodiments, the first reference profile comprises a fatty acid reference profile indicating a non-ulcer dyspepsia subject free from H. pylori infection.

[0032] In some embodiments, the second reference profile is a fatty acid reference profile indicating a non-alcohol-consuming subject having H. pylori infection.

[0033] In some embodiments, the second reference profile is a fatty acid reference profile indicating an alcohol-consuming subject having / / , pylori infection.

[0034] In some embodiments, the second reference profile is a fatty acid reference profile indicating a non-smoking subject having H. pylori infection.

[0035] In some embodiments, the second reference profile is a fatty acid reference profile indicating a smoking subject having / , pylori infection.

[0036] In some embodiments, the second reference profile is a fatty acid reference profile indicating a non-ulcer dyspepsia-free having from H. pylori infection.

[0037] In some embodiments, the second reference profile is a fatty acid reference profile indicating a non-ulcer dyspepsia subject having H. pylori infection.

[0038] In some embodiments, the first reference profile comprises a first reference profile-A indicating non-alcohol-consuming subject free from H. pylori infection, and a first reference profile-B indicating alcohol-consuming subject free from H. pylori infection, and wherein a level of at least one fatty acid selected from the group consisting of myristic acid (14:0), oleic acid (18:ln9), gamma-linolenic acid (18:3n6), docosapentaenoic acid (22:5n3), and osbond acid (22:5n6) is higher in the first reference profile-B than in the first reference profile-A.

[0039] In some embodiments, the second reference profile comprises a second reference profile- A indicating non-alcohol-consuming subject having / / , pylori infection, and a second reference profile-B indicating alcohol-consuming subject having H. pylori infection.

[0040] In some embodiments, a level of at least one fatty acid selected from the group consisting of behenic acid (22:0), oleic acid (18: ln9), and stearidonic acid (18:4n3) is lower in the second reference profile-B than in the second reference profile-A.

[0041] In some embodiments, a level of at least one fatty acid selected from the group consisting of eicosatetraenoic acid (20:4n3), eicosapentaenoic acid (20:5n3), docosahexaenoic acid (22:6n3), and gamma-linolenic acid (18:3n6) is higher in the second reference profile-B than in the second reference profile-A.

[0042] In some embodiments, the second reference profile comprises a second reference profile-C indicating non-smoking subject having / / , pylori infection, and a second reference

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[0044] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) profile-D indicating smoking subject having H. pylori infection, and wherein a level of at least one fatty acid selected from the group consisting of pentadecanoic acid (15:0), behenic acid (22:0), stearidonic acid (18:4n3), oleic acid (18: ln9), eicosapentaenoic acid (20:5n3), and osbond acid (22:5n6) is lower in the second reference profile-D than in the second reference profile-C.

[0045] In some embodiments, the first reference profile comprises a first reference profile-E indicating non-ulcer dyspepsia-free subject free from H. pylori infection, and a first reference profile-F indicating non-ulcer dyspepsia subject free from H. pylori infection, and wherein a level of at least one fatty acid selected from the group consisting of palmitoleic acid (16: ln7), cis-l l-eicosaenoic acid (20: ln9), gamma-linolenic acid (18:3n6), linoleic acid (18:2n6), cisl l, 14-eicosadienoic acid (20:2n6), and osbond acid (22:5n6) is higher in the first reference profile-F than in the first reference profile-E.

[0046] In some embodiments, the second reference profile comprises a second reference profile-E indicating non-ulcer dyspepsia-free subject having H. pylori infection, and a second reference profile-F indicating non-ulcer dyspepsia subject having H. pylori infection.

[0047] In some embodiments, a level of at least one fatty acid selected from the group consisting of docosapentaenoic acid (22:5n3) and docosahexaenoic acid (22:6n3) is higher in the second reference profile-F than in the second reference profile-E.

[0048] In some embodiments, a level of at least one fatty acid selected from the group consisting of stearidonic acid (18:4n3), oleic acid (18:ln9), gamma-linolenic acid (18:3n6), adrenic acid (22:4n6), and eicosapentaenoic acid (20:5n3) is lower in the second reference profile-F than in the second reference profile-E.

[0049] In some embodiments, determining the fatty acid profile in the stool sample comprises analyzing the stool sample with a chromatography method, a mass spectrometry method, a colorimetric method, a fluorometric method, or an assay analyzing interactions between fatty acids and proteins.

[0050] In some embodiments, the fatty acid profile is determined by the assay analyzing interactions between fatty acids and proteins, and wherein the type and / or amount of fatty acids in the stool sample are determined according to interactions between fatty acids in the stool sample and one or more proteins having different binding affinity toward different fatty acids.

[0051] In some embodiments, the one or more proteins comprise serum albumin and / or a fatty acid binding protein (FABP).

[0052] In some embodiments, the FABP comprises FABP1, FABP2, FABP3, and / or FABP4.

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[0055] In some embodiments, the assay analyzing interactions between fatty acids and proteins is a lateral flow assay, and the proteins are immobilized on a substrate and the stool sample is allowed to flow on the substrate.

[0056] In some embodiments, the method is a point-of-care test method or a home diagnostic test method.

[0057] Method of treating H. pylori infection

[0058] In some aspects, the present invention is directed to a method of treating H. pylori infection in a subject in need thereof.

[0059] In some embodiments, the method comprises determining that the subject has H. pylori infection. In some embodiments, the subject is determined to suffer from H. pylori infection according to the diagnosis herein.

[0060] In some embodiments, the method further comprises administering to the subject an effective amount of a treatment for H. pylori infection.

[0061] In some embodiments, the treatment for H. pylori infection comprises an antibiotic, a proton-pump inhibitor, bismuth subsalicylate, and / or a histamine (H-2) blocker.

[0062] In some embodiments, the treatment for H. pylori infection comprises a proton-pump inhibitor, clarithromycin and at least one selected from the group consisting of amoxicillin and metronidazole.

[0063] BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The following detailed description of exemplary embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating, non-limiting embodiments are shown in the drawings. It should be understood, however, that the instant specification is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0065] Fig. 1 illustrates certain aspects of the lipidome analyses of healthy controls versus Helicobacter pylori patients, in accordance with some embodiments. Concentrations of significantly differing fatty acids in two cohorts are presented. Fecal fatty acid profiles of H. pylori patients (n = 68) and healthy controls (n = 35) were compared. Metabolites of interest include: 18:3n6 = gamma-linolenic acid. 22:5n6 = osbond acid. 17:0 = margaric acid. 22:5n3 = docosapentaenoic acid. 22: ln9 = erucic acid. 20:5n3 = eicosapentaenoic acid. 20:4n3 = eicosatetraenoic acid. 22:6n3 = docosahexaenoic acid.

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[0068] Figs. 2A-2B illustrate certain aspects of the fatty acid composition of subjects who consumed alcohol as compared to subjects who did not consume alcohol, in accordance with some embodiments. Fig. 2A: Fatty acid levels in H. pylori patients were compared between those who consumed alcohol (n = 18) and those who did not (n = 50). Fig. 2B: Fatty acid levels in healthy controls were compared between those who consumed alcohol (n = 19) and those who did not (n = 16). Metabolites of interest include: 22: ln9 = erucic acid. 14:0 = myristic acid. 22:5n3 = docosapentaenoic acid. 18: ln9 = oleic acid. 22:5n6 = osbond acid. 18:3n6 = gamma-linolenic acid. 22:0 = behenic acid. 18:4n3 = stearidonic acid. 18:3n3 = alpha-linolenic acid. 18:2n6 = linoleic acid. 22:6n3 = docosahexaenoic acid. 20:4n3 = eicosatetraenoic acid. 20:5n3 = eicosapentaenoic acid.

[0069] Fig. 3 illustrates certain aspects of the fatty acid composition of Helicobacter pylori- positive samples who smoked as compared to Helicobacter pylori-^os iNQ patients who did not smoke, in accordance with some embodiments. Metabolites of interest include: 15:0 = pentadecanoic acid. 22:0 = behenic acid. 18:4n3 = stearidonic acid. 18:0 = stearic acid. 18: ln9 = oleic acid. 20:5n3 = eicosapentaenoic acid. 22:5n6 = osbond acid.

[0070] Figs. 4A-4B illustrate certain aspects of the fatty acid composition of subjects who have non-ulcer dyspepsia as compared to subjects who do not have non-ulcer dyspepsia, in accordance with some embodiments. Fig. 4A: Fatty acid levels in H. pylori patients were compared between those who have non-ulcer dyspepsia (n = 16) and those who do not (n = 52). Fig. 4B: Fatty acid levels in healthy controls were compared between those who have non-ulcer dyspepsia (n = 5) and those who do not (n = 30). Metabolites of interest include: 16: ln7 = palmitoleic acid. 20: ln9 = cis-11-eicosaenoic acid. 18:2n6 = linoleic acid. 20:2n6 = cis-l l,14-eicosadienoic acid. 18:3n6 = gamma-linolenic acid. 22:5n6 = osbond acid. 22:4n6 = adrenic acid. 18:4n3 = stearidonic acid. 18: ln9 = oleic acid. 20:5n3 = eicosapentaenoic acid. 22:5n3 = docosapentaenoic acid. 22:6n3 = docosahexaenoic acid.

[0071] Figs. 5A-5D illustrate the model of alterations in fatty acid metabolism in active Helicobacter pylori infection, in accordance with some embodiments. Fatty acid metabolites that are upregulated (green) or downregulated (red) in fecal samples from H. pylori patients are highlighted. Fig. 5A: Possible influence of H. pylori on omega-6 metabolism (blue lines) based on current literature (blue rectangles). Fig. 5B: Possible influence of H. pylori on omega-9 metabolism (blue lines) based on current literature (blue rectangles). Fig. 5C: Possible influence of host response to H. pylori infection on omega-3 metabolism (pink lines) based on current literature (pink rectangles). Fig. 5D: Possible influence of host response to H. pylori infection on omega-9 metabolism (pink lines) based on current literature (pink

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[0073] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) rectangles). 18:3n3 = alpha-linolenic acid. 18:4n3 = stearidonic acid. 20:4n3 = eicosatetraenoic acid. 20:5n3 = eicosapentaenoic acid. 22:5n3 = docosapentaenoic acid. 22:6n3 = docosahexaenoic acid. 18:0 = stearic acid. 17:0 = margaric acid. 18: ln9 = oleic acid. 20: ln9 = cis- 11 -eicosaenoic acid. 22: ln9 = erucic acid. 24: ln9 = nervonic acid. 18:2n6 = linoleic acid. 18:3n6 = gamma-linolenic acid. 20:3n6 = dihomo-gamma-linolenic acid. 20:4n6 = arachidonic acid. 22:4n6 = adrenic acid. 22:5n6 = osbond acid. 20:2n6 = cis- 11,14- eicosadienoic acid. A4 = delta-4-desaturase. A5 = delta-5-desaturase. A6 = delta-6-desaturase. A8 = delta-8-desaturase. A9 = delta-9-desaturase.

[0074] Figs. 6A-6C illustrate certain aspects of the effect of smoking, alcohol consumption, and non-ulcer dyspepsia on fatty acid metabolism in active Helicobacter pylori infection, in accordance with some embodiments. Fatty acid metabolites that are upregulated (green) or downregulated (red) in fecal samples of H. pylori patients are highlighted. Fig. 6A: Influence of smoking, alcohol consumption, and non-ulcer dyspepsia on omega-3 metabolites (neutral = dash; increased = up arrowhead; decreased = down arrowhead). Fig. 6B: Influence of smoking, alcohol consumption, and non-ulcer dyspepsia on omega-6 metabolites (neutral = dash; increased = up arrowhead; decreased = down arrowhead). Fig. 6C: Influence of smoking, alcohol consumption, and non-ulcer dyspepsia on omega-9 metabolites (neutral = dash; increased = up arrowhead; decreased = down arrowhead). 18:3n3 = alpha-linolenic acid. 18:4n3 = stearidonic acid. 20:4n3 = eicosatetraenoic acid. 20:5n3 = eicosapentaenoic acid. 22:5n3 = docosapentaenoic acid. 22:6n3 = docosahexaenoic acid. 18:0 = stearic acid. 17:0 = margaric acid. 18: ln9 = oleic acid. 20: ln9 = cis- 11 -eicosaenoic acid. 22: ln9 = erucic acid. 24: ln9 = nervonic acid. 18:2n6 = linoleic acid. 18:3n6 = gamma-linolenic acid. 20:3n6 = dihomo-gamma-linolenic acid. 20:4n6 = arachidonic acid. 22:4n6 = adrenic acid. 22:5n6 = osbond acid. 20:2n6 = cis-l l,14-eicosadienoic acid. A4 = delta-4-desaturase. A5 = delta-5- desaturase. A6 = delta-6-desaturase. A8 = delta-8-desaturase. A9 = delta-9-desaturase.

[0075] Figs. 7A-7C illustrate certain aspects of the quantification of fatty acid presence by the release of an indicator, in accordance with some embodiments. Fig. 7A: Characterization of serum albumin / indicator binding. Fig. 7B: Displacement of indicator by fatty acids. Fig. 7C: Quantification of indicator release.

[0076] Figs. 8A-8C are the overview of the proposed assay, in accordance with some embodiments. Fig. 8A: Fatty acids displace indicator molecules from serum albumin / fatty acid binding protein complex. Fig. 8B: Design of paper-based diagnostic. Fig. 8C: Paper based diagnostic device. The central channel in the printed paper diagnostic represents the

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[0078] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) diagnostic region expanded in Fig. 8B. BSA remains immobilized in this channel when subjected to flow while indicator solution migrates across paper device.

[0079] DETAILED DESCRIPTION

[0080] The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and / or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and / or configurations discussed.

[0081] The current clinically used diagnostic methods for H. pylori require laboratory setting and are not point-of-care tests. Two types of home detection tests are available on commercial websites, however are not validated clinically. These are either blood-based tests that detect anti- . pylori antibodies with limitations such as (i) less ease of sample collection or (ii) inability to distinguish active from past infection or stool-antigen-based tests, the limitation being heterogeneity of results caused by geographic-region-based antigen variability. Therefore, the major need in the field of H. pylori is identification of diagnostic indicators that will facilitate a point-of-care or home diagnostic test.

[0082] H. pylori is the most common gastrointestinal (GI) infection. The health, economic, and social costs of H. pylori infection are driven primarily by the rising antibiotic resistance of this pathogen, which in turn is caused by the lack of rapid diagnoses and prompt treatment. Eradication tests offer critical information reflecting local antibiotic resistance and cure rates but may be inconsistently completed. Current tests require laboratory setting. H. pylori patient compliance for reporting to the clinic either for the initial appearance of symptoms or for follow up visits after completion of antibiotic regimens is low. This significantly contributes to development of antibiotic resistance. In a study of 481 H. pylori patients to identify barriers to eradication test completion, -40% of patients did not complete eradication tests. Univariate analysis identified multiple factors including sex and socioeconomic factors

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[0084] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) associated with failure to complete eradication tests. Patient’s workload to engage with medical care including attending appointments, taking medications, and making appropriate lifestyle changes along with health literacy, finances, and access to transportation and care contributed to nonadherence. Therefore, the major need in the field of H. pylori is identification of diagnostic indicators that will facilitate a point-of-care / home diagnostic test. Point-of-care / home tests have become a norm after the CO VID-19 pandemic, which should positively impact patient’s reception of the H. pylori at-home diagnostic tests. A positive and rapid diagnosis of an H. pylori infection is crucial for its successful management as untreated H. pylori can cause chronic gastrointestinal symptoms and can advance to gastric cancer and is also associated with increased colorectal cancer risk.

[0085] H. pylori resides in the stomach and leads to changes in the microbial compositions of the gastric mucosa. The role of H. pylori spans beyond gastric microbiome, affects downstream gastrointestinal microbiome and can increase colorectal cancer risk. Gut bacteria can inhibit H. pylori by competing for nutrients, producing bactericidal substances (e.g. bactericidal SCFAs by Lactobacillus sp), competitive inhibition of adherence, and stimulating host functions and immunity. Dysbiosis is associated with an H. pylori infection. H. pylori impacts the gut microbiome composition, gastric H. pylori infection affects local and distant microbial populations and host responses, H. pylori infection is associated with fecal microbiota composition and diversity, and H. pylori can influence gut bacteria outside of its natural habitat, the stomach. It is suggested that H. pylori leads to changes in both gastric and gut microbiome, which in turn can be reflected in changes in metabolome and thus in fecal FAs. However, compared to microbiome studies, fewer metabolome studies have been reported for H. pylori .

[0086] H. pylori diagnostic methods include both invasive (endoscopy, culture, histopathology, rapid urease test, and molecular methods such as in situ hybridization, PCR etc) and noninvasive methods (respiratory tests, fecal antigens and serology). Limitations of invasive methods include (i) invasiveness, (ii) high cost, (iii) skill requirements, (iv) potential requirements for specific transport conditions, (v) false positives, e.g. for PCR due to DNA fragments from dead bacteria etc. Non-invasive methods have been increasingly explored in the last few years. All of these diagnostic methods are carried out in a laboratory setting and are not point-of-care diagnostic tests. There are two types of home detection tests available on popular commercial websites, but are not validated by physicians. One of these tests is blood-based and detects anti- / / , pylori antibodies, the limitations of which include (i) less ease involved in sample collection and (ii) inability to distinguish active from past infection.

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[0089] The second test is stool-antigen-based; its limitation includes heterogeneity of results caused by geographic-region-based antigen variability. According to the World Gastroenterology Organization Global Guidelines, despite the widespread validation of noninvasive diagnostic tests, there is a major unmet clinical need for a low cost, non-invasive test.

[0090] In the study described herein, it was discovered that the fatty acid profiles of stool samples from patients infected with H. pylori are different from those from healthy subjects. It was further discovered that H. pylori infections can be diagnosed (and thus treated) by determining the stool fatty acid profiles in the subject, and comparing the profiles with reference profiles, such as profiles indicating H. pylori infection or profiles indicating free from such infection. Notably, it is proposed that stool fatty acid profiles can be analyzed by point-of-care or home diagnostic test methods.

[0091] In addition, the present study further discovered that the fatty acid profiles of stool samples of H. pylori patients are affected by alcohol consumption, smoking, as well as nonulcer dyspepsia. In some embodiments, this discovery allows for accurate diagnosis of H. pylori infection by considering the subjects’ drinking, smoking, and / or non-ulcer dyspepsia status.

[0092] Accordingly, in some aspects, the present invention is directed to a method of diagnosing H. pylori infection in a subject.

[0093] In some aspects, the present invention is directed to a method of treating H. pylori infection in a subject.

[0094] Definitions

[0095] As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein are those well-known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

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[0098] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0099] As used herein, the terms “effective amount,” “pharmaceutically effective amount” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0100] The term “independently selected from” as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase “X1, X2, and X3are independently selected from noble gases” would include the scenario where, for example, X1, X2, and X3are all the same, where X1, X2, and X3are all different, where X1and X2are the same but X3is different, and other analogous permutations.

[0101] The terms “patient,” “subject,” or “individual” are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human.

[0102] A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

[0103] The terms “treat,” “treating” and “treatment,” as used herein, means reducing the frequency or severity with which symptoms of a disease or condition are experienced by a subject by virtue of administering an agent or compound to the subject.

[0104] Diagnostic of H. pylori Infection

[0105] In some aspects, the instant specification is directed to a method of diagnosing H. pylori infection in a subject.

[0106] In some embodiments, the subject is a human subject. In some embodiments, the subject is a non-human mammalian subject.

[0107] In some embodiments, the method comprises: determining a fatty acid profile in a stool sample of the subject; and comparing the fatty acid profile with a reference profile indicating H. pylori infection or a second reference profile indicating H. pylori infection.

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[0110] In some embodiments, the subject is diagnosed to have H. pylori infection when a level of at least one fatty acid in the fatty acid profile is different than a level of the at least one fatty acid in the first reference profile.

[0111] In some embodiments, the subject is diagnosed to have H. pylori infection when a level of at least one fatty acid in the fatty acid profile matches a level of the at least one fatty acid in the second reference profile.

[0112] In some embodiments, the first reference profile is a fatty acid reference profile indicating free from H. pylori infection.

[0113] In some embodiments, the second reference profile is a fatty acid reference profile indicating H. pylori infection.

[0114] In some embodiments, the method further comprises obtaining the stool sample from the subject.

[0115] In some embodiments, the at least one fatty acid comprises a metabolite of the omega- 3 fatty acids pathway, a metabolite of the omega-6 fatty acids pathway, and / or a metabolite of the omega-9 fatty acids pathway.

[0116] In some embodiments, the at least one fatty acid comprises margaric acid (17:0), eicosapentaenoic acid (20:5n3), erucic acid (22: ln9), docosapentaenoic acid (22:5n3), eicosatetraenoic acid (20:4n3), docosahexaenoic acid (22:6n3), gamma-linolenic acid (18:3n6), and / or osbond acid (22:5n6).

[0117] In some embodiments, the subject is diagnosed to have H. pylori infection when the at least one fatty acid selected from the group consisting of margaric acid (17:0), eicosapentaenoic acid (20:5n3), erucic acid (22: ln9), docosapentaenoic acid (22:5n3), eicosatetraenoic acid (20:4n3), and docosahexaenoic acid (22:6n3) in the fatty acid profile is higher than in the first reference profile indicating free of H. pylori infection.

[0118] In some embodiments, the subject is diagnosed to have H. pylori infection when the at least one fatty acid selected from the group consisting of gamma-linolenic acid (18:3n6), and osbond acid (22:5n6) in the fatty acid profile is lower than in the first reference profile indicating free of H. pylori infection.

[0119] In some embodiments, the at least one fatty acid comprises margaric acid (17:0), gamma-linoleic acid (18:3n6), dihomo-gamma-linolenic acid (20:3n6), arachidonic acid (20:4n6), docosapentaenoic acid (22:5n3), and / or nervonic acid (24: ln9).

[0120] In some embodiments, the subject is diagnosed with H. pylori infection when a level of margaric acid (17:0) in the fatty acid profile is higher than that in the first reference profile and / or matches that in the second reference level.

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[0123] In some embodiments, the subject is diagnosed with H. pylori infection when a level of eicosapentaenoic acid (EPA) / 20:5n3 in the fatty acid profile is higher than that in the first reference profile and / or matches that in the second reference level.

[0124] In some embodiments, the subject is diagnosed with H. pylori infection when a level of erucic acid / 22: ln9 in the fatty acid profile is higher than that in the first reference profile and / or matches that in the second reference level.

[0125] In some embodiments, the subject is diagnosed with H. pylori infection when a level of docosapentaenoic acid (DPA) / 22:5n3 in the fatty acid profile is higher than that in the first reference profile and / or matches that in the second reference level.

[0126] In some embodiments, the subject is diagnosed with H. pylori infection when a level of eicosatetraenoic acid / 20:4n3 in the fatty acid profile is higher than that in the first reference profile and / or matches that in the second reference level.

[0127] In some embodiments, the subject is diagnosed with H. pylori infection when a level of docosahexaenoic acid (DHA) / 22:6n3 in the fatty acid profile is higher than that in the first reference profile and / or matches that in the second reference level.

[0128] In some embodiments, the subject is diagnosed with H. pylori infection when a level of gamma-linoleic acid (18:3n6) in the fatty acid profile is lower than that in the first reference profile and / or matches that in the second reference level.

[0129] In some embodiments, the subject is diagnosed with H. pylori infection when a level of osbond acid / 22 :5n6 in the fatty acid profile is lower than that in the first reference profile and / or matches that in the second reference level.

[0130] In some embodiments, the diagnosis takes in to consideration whether the subject consumes alcohol, whether the subject smokes, and / or whether the subject has non-ulcer dyspepsia.

[0131] In some embodiments, the first reference profile comprises a fatty acid reference profile indicating a non-alcohol-consuming subject free from H. pylori infection.

[0132] In some embodiments, the first reference profile comprises a fatty acid reference profile indicating an alcohol-consuming subject free from H. pylori infection.

[0133] In some embodiments, the first reference profile comprises a fatty acid reference profile indicating a non-smoking subject free from H. pylori infection.

[0134] In some embodiments, the first reference profile comprises a fatty acid reference profile indicating a smoking subject free from H. pylori infection.

[0135] In some embodiments, the first reference profile comprises a fatty acid reference profile indicating a non-ulcer dyspepsia-free subject free from H. pylori infection.

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[0138] In some embodiments, the first reference profile comprises a fatty acid reference profile indicating a non-ulcer dyspepsia subject free from H. pylori infection.

[0139] In some embodiments, the second reference profile is a fatty acid reference profile indicating a non-alcohol-consuming subject having H. pylori infection.

[0140] In some embodiments, the second reference profile is a fatty acid reference profile indicating an alcohol-consuming subject having / / , pylori infection.

[0141] In some embodiments, the second reference profile is a fatty acid reference profile indicating a non-smoking subject having H. pylori infection.

[0142] In some embodiments, the second reference profile is a fatty acid reference profile indicating a smoking subject having / , pylori infection.

[0143] In some embodiments, the second reference profile is a fatty acid reference profile indicating a non-ulcer dyspepsia-free having from H. pylori infection.

[0144] In some embodiments, the second reference profile is a fatty acid reference profile indicating a non-ulcer dyspepsia subject having H. pylori infection.

[0145] In some embodiments, the first reference profile comprises a first reference profile-A indicating non-alcohol-consuming subject free from H. pylori infection, and a first reference profile-B indicating alcohol-consuming subject free from H. pylori infection, and wherein a level of at least one fatty acid selected from the group consisting of myristic acid (14:0), oleic acid (18:ln9), gamma-linolenic acid (18:3n6), docosapentaenoic acid (22:5n3), and osbond acid (22:5n6) is higher in the first reference profile-B than in the first reference profile-A.

[0146] In some embodiments, the second reference profile comprises a second reference profile- A indicating non-alcohol-consuming subject having / / , pylori infection, and a second reference profile-B indicating alcohol-consuming subject having H. pylori infection, and wherein at least one of the following applies:

[0147] (a) a level of at least one fatty acid selected from the group consisting of behenic acid (22:0), oleic acid (18: ln9), and stearidonic acid (18:4n3) is lower in the second reference profile-B than in the second reference profile- A; and / or

[0148] (b) a level of at least one fatty acid selected from the group consisting of eicosatetraenoic acid (20:4n3), eicosapentaenoic acid (20:5n3), docosahexaenoic acid (22:6n3), and gamma-linolenic acid (18:3n6) is higher in the second reference profile-B than in the second reference profile-A.

[0149] In some embodiments, the second reference profile comprises a second reference profile-C indicating non-smoking subject having / / , pylori infection, and a second reference profile-D indicating smoking subject having H. pylori infection, and wherein a level of at

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[0151] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) least one fatty acid selected from the group consisting of pentadecanoic acid (15:0), behenic acid (22:0), stearidonic acid (18:4n3), oleic acid (18: ln9), eicosapentaenoic acid (20:5n3), and osbond acid (22:5n6) is lower in the second reference profile-D than in the second reference profile-C.

[0152] In some embodiments, the first reference profile comprises a first reference profile-E indicating non-ulcer dyspepsia-free subject free from H. pylori infection, and a first reference profile-F indicating non-ulcer dyspepsia subject free from H. pylori infection, and wherein a level of at least one fatty acid selected from the group consisting of palmitoleic acid (16: ln7), cis-l l-eicosaenoic acid (20: ln9), gamma-linolenic acid (18:3n6), linoleic acid (18:2n6), cisl l, 14-eicosadienoic acid (20:2n6), and osbond acid (22:5n6) is higher in the first reference profile-F than in the first reference profile-E.

[0153] In some embodiments, the second reference profile comprises a second reference profile-E indicating non-ulcer dyspepsia-free subject having H. pylori infection, and a second reference profile-F indicating non-ulcer dyspepsia subject having H. pylori infection, and wherein at least one of the following applies:

[0154] (a) a level of at least one fatty acid selected from the group consisting of docosapentaenoic acid (22:5n3) and docosahexaenoic acid (22:6n3) is higher in the second reference profile-F than in the second reference profile-E; and / or

[0155] (b) a level of at least one fatty acid selected from the group consisting of stearidonic acid (18:4n3), oleic acid (18: ln9), gamma-linolenic acid (18:3n6), adrenic acid (22:4n6), and eicosapentaenoic acid (20:5n3) is lower in the second reference profile-F than in the second reference profile-E.

[0156] In some embodiments, determining a fatty acid profile in the stool sample comprises analyzing the stool sample with a chromatography method, a mass spectrometry method, a colorimetric method, a fluorometric method, or an assay analyzing interactions between fatty acids and proteins.

[0157] In some embodiments, the fatty acid profile is determined by the assay analyzing interactions between fatty acids and proteins, and wherein the type and / or amount of fatty acids in the stool sample are determined according to interactions between fatty acids in the stool sample and one or more proteins having different binding affinity toward different fatty acids.

[0158] In some embodiments, the one or more proteins comprise serum albumin and / or a fatty acid binding protein (FABP).

[0159] In some embodiments, the FABP comprises FABP1, FABP2, FABP3, and / or FABP4.

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[0162] In some embodiments, the assay analyzing interactions between fatty acids and proteins is a lateral flow assay. In some embodiments, the proteins are immobilized on a substrate and the stool sample is allowed to flow on the substrate.

[0163] In some embodiments, the diagnostic method herein is a point-of-care test method or a home diagnostic test method.

[0164] Treatment of H. pylori Infection

[0165] In some aspects, the present invention is directed to a method of treating H. pylori infection in a subject in need thereof.

[0166] In some embodiments, the method comprises performing the diagnostic method herein to determine that the subject has H. pylori infection, and administering to the subject an effective amount of a treatment for H. pylori infection.

[0167] In some embodiments, the treatment for H. pylori infection comprises an antibiotic, a proton-pump inhibitor, bismuth subsalicylate, and / or a histamine (H-2) blocker.

[0168] Non-limiting examples of antibiotics useful as H. pylori infection treatments include amoxicillin, clarithromycin, metronidazole, tetracycline, and the like.

[0169] Non-limiting examples of proton-pump inhibitors useful as H. pylori infection treatments include lansoprazole, omeprazole, pantoprazole, rabeprazole, esomeprazole, and the like.

[0170] Non-limiting examples of histamine (H-2) blockers useful as H. pylori infection treatments include cimetidine, nizatidine, and the like.

[0171] In some embodiments, the H. pylori infection is treated with the so-called triple therapy, in which the subject is administered with an effective amount of a proton-pump inhibitor, clarithromycin, and at least one selected from the group consisting of amoxicillin and metronidazole.

[0172] Examples

[0173] The instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the instant specification should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

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[0176] Example 1A: H. pylori infection is associated with change in fecal fatty acid profile

[0177] The present study proposes to evaluate fatty acids (FAs) as diagnostic biomarkers for an H. pylori infection and use the FA data to develop a pilot, point-of-care diagnostic test that can rapidly identify presence of H. pylori.

[0178] To address this need, a pilot study to decipher differences in the gut microbiome and lipidomic profiles was carried out using stool samples of healthy controls and H. pylori patients. It was observed that H. pylori infection is associated with changes in gut bacterial profiles with concomitant changes in the gut lipidomic profiles of the patients. These changes in lipidomic profiles were correlated with changes in the FAs excreted in the fecal samples.

[0179] In the pilot study (N=35; w=19 H. pylori patients; w=16 controls), a striking decrease in the concentration of the fecal PUFA (polyunsaturated fatty acid) C18:3n6 in H. pylori patients was observed. Interestingly, among the multiple liposomal fatty acids evaluated by Thamphiwatanam el al. (Proceedings of the National Academy of Sciences of the United States of America, 111, 17600-17605), only the linolenic acid formulation (C18:3) showed bactericidal activity by impacting the H. pylori membrane structure and stability. A second set of lipidome analysis with larger sample size (N=103; / / =68 H. pylori patients; n=35 controls) confirmed the decrease in C18:3n6 and exhibited additional fatty acids levels of which changed.

[0180] Example IB: Overview

[0181] Based on the observation that there are distinct differences in the fecal FAs of H. pylori patients and healthy controls, it is proposed that fecal fatty acids (FAs) can be used as biomarkers of H. pylori. It is further proposed that a study with higher statistical power will elucidate if margaric acid / 17:0, eicosapentaenoic acid (EPA) / 20:5n3, erucic acid / 22: ln9, docosapentaenoic acid (DPA) / 22:5n3, eicosatetraenoic acid / 20:4n3, docosahexaenoic acid (DHA) / 22:6n3, gamma-linolenic acid / 18:3n6 and osbond acid / 22:5n6 can serve as biomarkers of H. pylori and may reveal additional FAs with similar potential. To avoid the influence of diet on participants’ lipidome profiles, the subjects enrolled in the study are controlled for diet. Specifically, there are two aims:

[0182] Aim 1. Determine the lipidomic profiles of H. pylori patients and healthy controls. Participants follow a bland diet for a month, which helps remove variability and output biases due to diet, if any. The present study collects demographic and medical information and stool samples (N=300; n=150 each H. pylori patients and controls) and carries out blinded lipidome analyses. Samples are stratified for FA changes correlated to the

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[0184] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) presence / absence of H. pylori to address differential detection of (i) margaric acid / 17:0, eicosapentaenoic acid (EPA) / 20:5n3, erucic acid / 22: ln9, docosapentaenoic acid (DPA) / 22:5n3, eicosatetraenoic acid / 20:4n3, docosahexaenoic acid (DHA) / 22:6n3, gammalinolenic acid / 18:3n6 and osbond acid / 22:5n6 and (ii) additional FAs using gas chromatography-mass spectrometry (GC-MS).

[0185] Aim 2. Development of biosensor-array-based, pilot diagnostic assay for H. pylori. The present study develops a biosensor-array -based, point-of-care, pilot diagnostic assay. The assay is developed for margaric acid / 17:0, eicosapentaenoic acid (EPA) / 20:5n3, erucic acid / 22: ln9, docosapentaenoic acid (DPA) / 22:5n3, eicosatetraenoic acid / 20:4n3, docosahexaenoic acid (DHA) / 22:6n3, gamma-linolenic acid / 18:3n6 and osbond acid / 22:5n6 and for other H. pylori biomarker candidate FAs as they emerge from Aim 1. The present study creates, among others, a paper-based device for the assay which involves the binding of fecal FAs by serum albumin and fatty acid binding proteins (biosensors) resulting in displacement of indicators from association with fatty acid binding proteins and visualization of the liberated dye molecules. The present study establishes a FA-biosensor ensemble by generating a matrix of outputs for each pair of indicator / biosensor by determination of indicator response to specific FAs. The matrix of collected ensemble biosensor FA binding data is statistically analyzed using principal component analysis (PCA). Assay results are validated by (i) comparing results obtained with lipidome analysis of 300 patient stool samples by GC / MS in Aim 1 and (ii) by evaluating against the patient medical data to gauge the performance of the test in terms of its specificity, sensitivity, and false positive / negative rates. The biosensor ensemble described in Aim 2 is developed independent from Aim 1 using the proof-of-concept FAs, margaric acid / 17:0, eicosapentaenoic acid (EPA) / 20:5n3, erucic acid / 22: ln9, docosapentaenoic acid (DPA) / 22:5n3, eicosatetraenoic acid / 20:4n3, docosahexaenoic acid (DHA) / 22:6n3, gamma-linolenic acid / 18:3n6 and osbond acid / 22:5n6.

[0186] Example 1C: Pilot study

[0187] A biosensor-array -based, pilot diagnostic assay for H. pylori was performed. The work here also provides actionable information that can guide targeted therapies, readily assess treatment outcomes, and help mitigate the impact of increased antibiotic usage. As an infection with H. pylori is significantly associated with ethnic minority or underserved communities, findings from this study will also help address health disparities.

[0188] Lipidome profiles of H. pylori patients and healthy controls: Studies were carried out, where analyzed fecal FAs of H. pylori patients and healthy controls were analyzed by GC-

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[0191] MS using a 30-fatty acid (2-22 carbons) panel that included short-chain (SCFA), long-chain (LCFA), monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA). Concentrations of significantly differing FAs, separated by H. pylori status are presented in volcano plot analysis in Fig. 1. The results show an increase in margaric acid / 17:0, eicosapentaenoic acid (EPA) / 20:5n3, erucic acid / 22:ln9, and docosapentaenoic acid (DPA) / 22:5n3 as well as eicosatetraenoic acid / 20:4n3 and docosahexaenoic acid (DHA) / 22:6n3 in H. pylori patients relative to the healthy control subjects. In contrast, the PUFAs, gamma-linolenic acid / 18:3n6 and osbond acid / 22:5n6, were decreased in the H. pylori patients relative to healthy controls. The data demonstrates that these differences in FAs should be explored as diagnostic markers for H. pylori.

[0192] In the preliminary data, changes in gut bacterial profiles with concomitant changes in the gut lipidomic profiles of H. pylori patients were observed (Fig. 1), suggesting that metabolome profiling can yield new markers of H. pylori infection.

[0193] The pilot studies showed distinct differences in the levels of certain FAs in H. pylori patients compared to healthy controls (Fig. 1). Therefore, it is posited that these lipids can be used for the diagnosis of H. pylori. The present study evaluates if higher level of margaric acid / 17:0, eicosapentaenoic acid (EPA) / 20:5n3, erucic acid / 22: ln9, docosapentaenoic acid (DPA) / 22:5n3, eicosatetraenoic acid / 20:4n3, and docosahexaenoic acid (DHA) / 22:6n3 and lower level of gamma-linolenic acid / 18:3n6 and osbond acid / 22:5n6 can serve as diagnostic biomarkers of H. pylori. As mentioned above, rapid, point-of-care / home diagnosis of H. pylori remains a challenge.

[0194] Example ID: Lipidomic profile determination (prophetic)

[0195] The assay of the present study is non-invasive and uses excreted stool samples. To the best of the inventor’s knowledge, there are no existing methods that use metabolites in the stool samples for the detection of H. pylori, making it a novel and unique approach. In addition, the biosensor ensemble (Aim 2) is developed fully independent from Aim 1 and is anticipated to be readily extended for the identification of diverse changes in FA metabolic profile, a feature anticipated to be broadly relevant for diagnosis in many human diseases.

[0196] The present study includes a prospective, human subjects study of H. pylori patients and healthy control participants. The study recruits H. pylori patients with ongoing care at the Cooper University Hospital (CUH) in Camden, New Jersey. The subjects are identified by using the electronic medical record system (EPIC) by ICD-9 codes of 041.86, 531.90, 535.60, 795.79, 008.47, 531.00, V12.08. The study recruits patients whose H. pylori infection is confirmed by biopsy. Control subjects will be recruited from a healthy population, also seen

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[0198] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) at CUH for well visits. Healthy control subjects are included if they have (i) no history of GI symptoms, (i) no history of organic GI disease, (iii) not on any chronic opioid medications. Exclusion criteria will include: (i) vulnerable population such as people who are unable to consent including those who are <18 years age, pregnant women (pregnancy is a confounding factor) and prisoners, and (ii) patients with antibiotics history for the past three months.

[0199] Determine the lipidomic profiles of H. pylori patients and healthy controls.

[0200] The present study carries out blinded lipidome analyses of all samples stratified for changes in the FAs and correlate that to the presence / absence of H. pylori infection. This correlation addresses differential detection of (i) margaric acid / 17:0, eicosapentaenoic acid (EPA) / 20:5n3, erucic acid / 22:ln9, docosapentaenoic acid (DPA) / 22:5n3, eicosatetraenoic acid / 20:4n3, docosahexaenoic acid (DHA) / 22:6n3, gamma-linolenic acid / 18:3n6 and osbond acid / 22:5n6 and (ii) additional fatty acids (FAs) in the large, diet-controlled cohort using GC- MS. Certain FAs are differentially detected in the stool samples of H. pylori patients compared to the healthy subjects and thus can be used as diagnostic biomarkers of H. pylori.

[0201] Collection of samples and surveys: The present study collects stool samples, and demographic, medical and dietary information. Data pertinent to sex, ethnicity / race, height, weight, colonoscopy / endoscopy, medications, hypertension, diabetes, cholesterol, psychological comorbidities, smoking, and alcohol intake will be collected. Medical records are reviewed to confirm clinical details. The bland diet intervention is implemented to minimize influence of diet either directly on the fecal FA profiles or through its effect on gut microbiome. This standardizes the diet for all participants and minimize variability and output biases. Participants are asked to follow a bland diet (H., W.S.a.A. (2024) Bland Diet. SlalPearls: Vanhauwaert et al., AdvNutr, 6, 820-827; Lenhart et al., Clinical gastroenterology and hepatology: the official clinical practice journal of the American Gastroenterological Association, 20, e465-e483; Nguyen et al., Gastrointestinal endoscopy, 83, 499-507 e491; and Perler et al., Annual review of physiology, 85, 449-468) for 4 weeks prior to stool collection. The bland diet comprises of easily digestible foods that are soft- consistency, low-fiber, cooked, gentle to the gastrointestinal tract and non-spicy. Dietary compliance is ensured by asking all the participants to complete the Diet History Questionnaire III (DHQ III), a food frequency questionnaire (FFQ) developed by the National Cancer Institute (Barandouzi et al., Journal of personalized medicine, 11). They also complete three 24-hour diet recalls with a dietitian in the week prior to collecting stool samples. Cooper IRB (17-077EX) and appropriate Rowan lab safety approvals for the proposed experiments have been obtained. The present study applies for an IRB amendment

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[0203] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) to include bland diet intervention, however, as dietary surveys are included in the existing approval, this amendment will likely be approved before funding of this proposal. The present study optimizes a stool collection system that preserves the need for sterility and sample integrity, while maintaining a patient-centered, user-friendly approach (White et al., World journal of gastroenterology, 27, 5575-5594; Paripati et al., Microorganisms, 11; O'Neill et al., Obesity surgery, 32, 480-488; Awan et al., Pediatric gastroenterology, hepatology & nutrition, 26, 99-115). In brief, patients are given kits that include a handout with simple and concise picture-based instructions, a stool collection kit, gloves, and zip lock bags for discarding materials. Stool samples are collected using Norgen Biotek preservation tubes and also on cotton swabs and stored at -80°C (White et al., World journal of gastroenterology, 27, 5575-5594). The subject recruitment, collection of medical data and collection of samples are being overseen. Feedbacks, guidances and clinical insights throughout this project are provided.

[0204] Sample size computations: Study design has been reviewed for statistical validity; data analyses are performed. A two-sample independent means power analysis study using the pilot data indicated that a sample size of 150 in each of the 2 groups (H. pylori patients and controls) is sufficient to provide at least 80% power to detect a 30% change in the FAs of interest between the patient group and the control group at a significance level alpha=0.05.

[0205] Lipidomic profiling of patient samples: There is a major need for identification of diagnostic markers for H. pylori. H. pylori causes gut dysbiosis. Gut dysbiosis can lead to changes in metabolic profiles of affected individuals. Indeed, corresponding changes in the gut microbiome and various metabolites from H. pylori patients have been shown. Detection of several of these metabolites and products of enzymatic reactions is not readily adaptable for point-of-care diagnostics. The present study focuses on FA changes as colorimetric assays can be developed for detection of fecal FAs. The present study uses a 30-FA panel that includes SCFA, LCFA, MUFA and PUFA (2 to 22 carbons in length). These FAs can be readily and accurately measured in stool samples (White et al., World journal of gastroenterology, 27, 5575-5594; O'Neill et al., Obesity surgery, 32, 480-488; and Awan et al., Pediatric gastroenterology, hepatology & nutrition, 26, 99-115). The preliminary study identified a specific pattern of FA levels (Fig. 1) with respect to H. pylori infection. Aim 1 builds on these observations.

[0206] Statistical analysis: Statistical methods for lipidome profiles are used. Quantitation is performed using both linear and quadratic regression analysis generated from fortified calibration standards prepared immediately prior to each run. Raw data is collected and

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[0208] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) processed using Agilent MassHunter GC / MS Acquisition B.07. 04.2260 and Agilent MassHunter Workstation Software Quantitative Analysis for GC / MS B.09.00 / Build 9.0.647.0. Data reduction is performed using Microsoft Office 365 ProPlus Excel. Differences between groups for numerical variables are be assessed using ANOVA / independent t-test for normally distributed data, or Kruskal-Wallis ANOVA / Wilcoxon for non-normal data. Where appropriate, analyses are followed up with multiple pairwise comparisons adjusting for multiplicity using Tukey’s post hoc Honest Significant Difference test. Mann-Whitney U test is also used to differentiate between FAs. Volcano and boxplot analyses are presented. The bland diet intervention minimizes the influence of diet either directly on the fecal FA profiles or through its effect on gut microbiome. The goal for collection of dietary data is to ensure that bland diets are followed and diet is not an evaluable variable in the study.

[0209] Outcomes, challenges and alternative approaches: Quantification of FAs provides a chemical profile associated with an H. pylori infection. The blinded analysis of lipidomic profiles with the strong statistical power of 300 samples determines if margaric acid / 17:0, eicosapentaenoic acid (EPA) / 20:5n3, erucic acid / 22: ln9, docosapentaenoic acid (DPA) / 22:5n3, eicosatetraenoic acid / 20:4n3, docosahexaenoic acid (DHA) / 22:6n3, gammalinolenic acid / 18:3n6 and osbond acid / 22:5n6 can serve as H. pylori diagnostic biomarkers. Levels of all three FAs positively identifies H. pylori patients. The measured number of H. pylori office visits per year (>1500) at CUH will ensure sufficient cohort for recruitment. The present study have optimized patient recruitment (15-20 participants / month) by (i) researching patient chart reviews in advance to determine upcoming appointments, (ii) consenting and mailing kits prior to visits, (iii) streamlining sample collection, and (iv) inclusion of additional consent in Spanish to facilitate recruitment of Spanish-speaking patients, which form a large percentage of the population. Efficient procedures for sample and data transfer for metabolite analysis with Metabolon, LLC have already established (White et al., World journal of gastroenterology, 27, 5575-5594; Paripati et al., Microorganisms, 11; O'Neill et al., Obesity surgery, 32, 480-488; Awan et al., Pediatric gastroenterology, hepatology & nutrition, 26, 99-115). These methods have been standardized for reproducible results with samples stored for prolonged time at minus 80°C. Furthermore, the present study investigates if other FAs can serve as potential diagnostic markers. The present study observed other possible targets in the pilot study (White et al., World journal of gastroenterology, 27, 5575-5594). Other targets as proposed above are evaluated, as well. Another challenge is sampling and regional biases. The local community

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[0211] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) is considered one of the poorest and most economically distressed communities in the United States with the greatest socioeconomic disparities among ethnic minority groups. As H. pylori incidences are higher in similar communities, the observations may be useful for those communities as well. A chart review of more than 2,000 H. pylori patients showed that factors such as age, sex, ethnicity, previous H. pylori infection, previous antibiotics and proton pump inhibitor usage affect the H. pylori patient population in general. An international study carried out from Russia and UK reported that FA profiles in blood samples of participants show changes specific to H. pylori infection and suggested that these may potentially serve as a biomarker for this disease. This is consistent with the differences observed in fecal FAs of the H. pylori patients compared to controls. Thus, it is expected that the FAs identified in this study are of general relevance for H. pylori diagnosis. It is also reported that different GI diseases lead to different types of perturbations in the gut lipidome profiles, for example, there is an increase in palmitic acid and margaric acid in IBS patients, which is different from that observed in the H. pylori patients. The present study observed a striking decrease in the concentration of C18:3n6 in H. pylori patients. It is noted that the linolenic acid formulation (C18:3) showed bactericidal activity by impacting the H. pylori membrane structure and stability. Remarkable increases in the production of long chain fatty acid derivatives by H. pylori using C18:3n6 has been reported. This utilization of C18:3n6 may contribute to the diminished concentration of this metabolite in H. pylori patients. The present study makes an effort to keep inclusion of participants from major ethnic / racial groups comparable. Patient parameters collected via chart review and surveys are included in the statistical analyses. These include but are not limited to sex, ethnicity / race, age, alcohol intake, smoking and medications. Adherence of participants to bland diet for 4 weeks may be a possible challenge. To address this, the present study ensures their willingness to be compliant at the time of recruitment and follow up with the help of dietary intake records. The patients are offered appropriate compensation, which is already approved by CUH IRB.

[0212] Example IE: Development of biosensor-array-based, pilot diagnostic assay for / / . pylori (prophetic)

[0213] The lipidome analyses in Aim 1 are performed via a highly sensitive detection method, GS-MS. While GS-MS is robust, it does require multiple sample preparation steps, specialized instrumentation / facilities and is time consuming and costly. It is hypothesized that an ensemble biosensor array will enable selective identification of FA biomarkers in stool samples. The present study proposes to create a simple, biosensor-array -based, pilot point-of-

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[0215] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) care detection assay for fecal FAs that can be used to indicate presence / absence of an H. pylori infection. One of the goals for this Aim is to develop a pilot assay to show feasibility, which can be fine-tuned and field tested in a future study. This is cognizant of the budget and time allocated for this proposal.

[0216] The design is based on the recognition that only few examples of molecular recognition methods that allow structure-specific discrimination for FAs have been reported (Kubarych et al., Organic letters, 12, 4780-4783; Richieri et al., Biochemistry, 32, 7574- 7580; and Demant et al., Anal Biochem, 267, 366-372). Further, the preliminary lipidomics herein revealed that an H. pylori infection results in changes to several FAs. Accordingly, structure-specific discrimination would necessitate independent development of fully selective sensors for each FA of interest. Contrasting this against “lock and key” recognition model, the present study proposes employing proteins known to bind FA although not with exclusive binding selectivity. Each of the biosensors employed here do not provide structurespecific discrimination individually. However, by using an array of biosensors, each with distinct patterns of FA binding preference, statistically analyze the data via principal component analysis (PCA) that enables differentiation of FA can be performed. This approach is available for the analysis of chemical sensor arrays (Jurs et al., Chemical reviews, 100, 2649-2678).

[0217] Biosensor selection. Serum albumin (SA) and fatty acid binding protein (FABP) bind and transport FAs with preference for the binding of a range of specific FA (Table 1). Human SA has 7 high affinity (Ka~107-108M'1) and 20 low affinity binding sites. Notably, differences in binding affinity are on the basis of subtle changes in FA chain length and structure (Curry et al., Nature structural biology, 5, 827-835). Human FABPs belong to a family of proteins predominately expressed in the liver (FABP1), intestine (FABP2), heart (FABP3), and adipocyte (FABP4). These proteins display considerable sequence diversity based on their tissue-specificity and display some preference for binding to structurally specific FAs with potent binding affinities (Ka~106-109M'1) and up to 100-fold selectivity among FAs dependent on their structure. Based on this, the present study uses SA and FABP as biosensors. Importantly, binding constants support the hypothesis that these proteins display unique patterns of binding preference dependent on FA structure, therefore, robust sensitivity is anticipated. The present study is interested in, inter alia, selectivity, the relative binding of one FA in preference to structurally distinct FAs (structure selective FA binding) (Hanhoff et al., Molecular and cellular biochemistry, 239, 45-54; Richieri et al., The Journal

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[0219] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) of biological chemistry, 269, 23918-23930; Richieri et al., Biochemistry, 39, 7197-7204; and Kingma et al., Biochemistry, 37, 3250-3257).

[0220] Table 1: dissociation constants (Kd) for fatty acid protein binding, in accordance with some embodiments. All Kd values are given in nM. Selected patterns of fatty acid binding preference are underlined.

[0221] FA biosensor array. The present study collects a baseline matrix of data for PCA by characterizing the displacement of indicator molecules from FA binding proteins through the titration of the existing target FAs. The present study evaluates the displacement of indicator molecules (2AC, 1,8-ANS and fluorescein) from FABP1 through FABP4 and BSA by the titration of pure FA. Each biosensor is complexed to indicator molecule(s) prior to characterizing the release of these indicator molecules by the addition of specific free FA, e.g., BSA bound to the fluorescent indicator, 2-anthracene carboxylate (2AC) in which fluorescence of 2 AC at 422 nm decreased with addition of BSA supporting 1 : 1 complex formation (Fig. 7A). Titration of 2.5 pM 2AC / 3.75 pM BSA with steric acid (C18:0) (0 pM- 700 pM) resulted in decreased fluorescence (Fig. 7B). This data can be fit to enable quantification of the dose-dependent binding affinity of steric acid for BSA (Fig. 7C). Each biosensor-indicator pair will be evaluated by the titration of a library of 24 structurally distinct FAs ranging from C16 to C26. The FAs selected for this analysis will be based on the preliminary lipidomics analysis and will include: 1) the highest abundance FAs and 2) the FAs demonstrating the greatest Log2 fold change (Fig. 1). This analysis will be completed in 96-well format in triplicate. The matrix of FA binding data (response of each biosensor to different FAs and output of each pair) is analyzed using PCA (Jurs et al., Chemical reviews, 100, 2649-2678). PCA enables the transformation of a large and complex set of variables into a smaller data set that still maintains critical differentiating information contained in the parent data set. The primary PC represents the aspects of the ensemble biosensor matrix that

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[0223] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) provide the maximal levels of variance enabling the identification of target FAs. Loading plots are used to evaluate the biosensors contributing most significantly to each PC and for optimization of the composition of the biosensor ensemble. Selection of the biosensors in the ensemble that contribute most significantly to the data variance for discrimination of target FA is performed. Sensors which provide similar information are eliminated and selected for biosensors providing unique information.

[0224] Ensemble biosensor validation. To evaluate the fidelity and sensitivity of FA identification, an array of FA mixtures with broad range of concentrations is prepared and evaluated using the final optimized ensemble biosensor array in 96-well plate format. This includes known combinations of FAs identified in the H. pylori fecal FA profiles identified in the preliminary study. Assay results are additionally validated by (i) comparing results obtained with lipidome analysis of 300 stool samples by GC / MS in Aim 1 and (ii) by evaluating against the medical data to gauge the performance of the test in terms of its specificity, sensitivity, and false positive / negative rates.

[0225] Paper-based biosensor. Paper-based devices offer rapid, low-cost diagnostics in a non-lab oratory environment (e.g. point-of-care diagnostics) (Martinez et al. Analytical chemistry, 82, 3-10; Yuan et al., Biomicrofluidics, 15, 041303). The design of the biosensor assay is based on the binding of FAs by SA and / or FABP resulting in displacement of indicator molecules from protein association and the ultimate colorimetric or fluorometric readout of the liberated indicators (Fig. 8A). The present study immobilizes indicator loaded SA or FABP onto a paper device (Fig. 8B). The present study found that indicator-bound BSA is largely immobilized onto cellulose paper without requiring covalent surface attachment (Fig. 8C). Application of a solution containing fecal FAs leads to the displacement of the indicator from SA which elutes on the paper providing a quantifiable signal. The parallel analysis of a biosensor matrix of multiple SA / FABP and multiple indicators, representing the sum of the ensemble biosensor identified in the proposed studies, enables the identification of patterns of that would collectively provide a response profile for FAs of interest. This device therefore leverages the collective selectivity of an array of FA binding protein / indicator pairs for the identification of specific FAs from fecal samples. This biosensor-array -based approach builds on the concept of differential sensing of single or mixtures of FA by promiscuous biosensors and contrasts from the highly specific molecular recognition events required for detection following the traditional lock and key principle for substrate binding. Each SA / FABP displays differential binding affinity to the indicator molecules. Additionally, the selectivity for dye displacement by long chain fecal FAs are

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[0227] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) different for each SA / FABP-indicator pair. Accordingly, the presence and abundance of the collective FA profile lead to the displacement of a different pattern of colored dyes from the biosensor array (Fig. 8A: nl, n2 and n3) which can be individually displayed and quantified following in situ chromatographic separation on the paper device. In summary, the proposed paper-based implementation of the biosensor ensemble provides a readout of the relative proportions of diagnostic fecal FAs showing significant differences between controls and H pylori patients and thus enables point-of-care diagnosis for H. pylori infection

[0228] Statistical analysis. PCA is used for the analysis of the biosensor ensemble data as discussed above. PCA is widely employed in sensor arrays. This approach is appropriate for the system in which high linear dependence across the biosensor outputs is anticipated. After data have been transformed via their PCs, classification algorithms, such as logistic regression, neural networks, and machine learning are be employed to classify each sample to presen ce / absence of H. pylori infection. Each of the analyses is used to predict the H. pylori status of each of the 300 participants which then will be cross-validated with the actual diagnosis. This approach allows gauging the methods performance in terms of its estimated specificity, sensitivity, and false positive / negative rates.

[0229] Outcomes, challenges and alternative approaches: The biosensor is able to identify specific fecal FAs indicative of an H. pylori diagnosis. The proposed device requires analysis of only ~40pg of fecal material to enable quantification of key FAs. This sensitivity greatly enhances the potential utility and ease of the proposed assay for the characterization of fecal FAs. One challenge is that, while the characterized FA binding proteins do show binding preferences for FA on the basis of FA chemical structure, this selectivity is not exclusive. This means binding of FA to SA / FABP does not follow the simplistic convention of "lock and key" binding. It is noted that this feature is advantageous in enabling a small biosensor array to discriminate between a large number of FAs instead of requiring a unique biosensor for each FA. However, this feature could prove limiting in the ability to distinguish between certain combinations of FA or due to indicator displacement from other components (confounding compounds) present in fecal samples. Optimization of the biosensor / indicator molecule pairing provides a solution to these limitations. Alternatively, the specific factor(s) causing the limitation are determined and methods to remove the confounding component (sample “pre-processing”, such as desalting, filtration, etc.) or modify the sensor array are identified. Alternatively, the present study finds evaluation of components along an alternative PC that provides clustering of target FAs while reducing the contribution from

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[0231] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) interfering factors. Variations exist in the absolute concentrations of fecal FAs due to diet and / or physical characteristics of the samples used in the analysis. The present study aims to develop a device that is insensitive to these variations in the absolute concentrations of FAs from stool samples. The present study thus reports on the abundance of a target FA with comparison to a reference, universal FA as an internal control. The present study identified several such pairs of “diagnostic” versus “internal control” FAs. Thus, it is not necessary to follow a particular diet to use the proposed point-of-care test. Importantly, dietary intervention was not part of the two pilot studies, which included lipidome analyses of two independent cohorts of patients using two different methods of stool collection, but yielded same results

[0232] Example 2: Lipidome based pathophysiology of H. pylori Infection

[0233] H. pylori is a pathobiont that infects around two-thirds of the global population and has demonstrated a rise in antibiotic resistance, warranting a search for alternative treatments. As fatty acid biosynthesis is central to membrane structure and function, and H. pylori is correlated with the erosion of the mucosal barrier, lipidome analysis can elucidate the role of fatty acid metabolism in H. pylori infection and yield potential targets for intervention. Fecal samples from 68 H. pylori patients and 35 healthy control subjects were analyzed for fatty acid composition using gas chromatography-mass spectrometry. The present study observed an increase in margaric acid / 17:0, EPA / 20:5n3, erucic acid / 22:ln9, and docosapentaenoic acid (DPA) / 22:5n3 as well as eicosatetraenoic acid / 20:4n3 and DHA / 22:6n3 in H. pylori patients relative to the healthy control subjects. In contrast, the PUFAs gamma-linolenic acid / 18:3n6 and osbond acid / 22:5n6 were decreased in the H. pylori patients relative to healthy controls. Most of the fatty acids that differ in quantity between H. pyl ri-^os wQ samples and controls are metabolites of omega-3 and omega-6 fatty acid metabolism. Smoking, alcohol use, and non-ulcer dyspepsia further influenced fatty acid metabolism during H. pylori infection. Based on the results, the present study proposes a model for pathophysiology of H. pylori infection by exploring the gut lipid signatures of H. pylori patients and health control subjects. The results may provide insight on how H. pylori infection leads to changes in the fatty acid metabolism, how the host responds, and which metabolites may serve as potential candidates for future interventions.

[0234] The present study shows that H. pylori infection presents a defined pattern of chemical cross-talk between mammalian signaling circuits and bacterial regulatory circuits involving fatty acids. The present study proposes a model for the underlying pathophysiology

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[0236] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) of H. pylori infection, the first of this kind using human participants. The present study also demonstrates the effect of smoking, alcohol, and non-ulcer dyspepsia on fatty acid metabolism in H. pylori patients. The results may provide insight on fatty acids as potential intervention candidates for H. pylori infection.

[0237] Example 2A:

[0238] Helicobacter pylori (H. pylori) is a gram-negative spiral bacillus that infects approximately two-thirds of the global population. Despite its high infection rate, the majority of people with H. pylori do not develop symptoms. The mechanism by which H. pylori infection transitions from asymptomatic to symptomatic is unclear. In some patients, chronic inflammation due to H. pylori infection causes peptic ulcers, non-atrophic and atrophic gastritis, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. H. pylori resides in the stomach and leads to changes in the microbial compositions of the gastric mucosa. Recent work by several groups has also shown that the role of H. pylori spans beyond gastric microbiome, affects downstream gastrointestinal microbiome, and can increase colorectal cancer risk. Various groups have also shown that gut bacteria can inhibit H. pylori by competing for nutrients, producing bactericidal substances, competitive inhibition of adherence, and stimulating host functions and immunity. A number of studies support the notion that dysbiosis is associated with an H. pylori infection, describing the impact of H. pylori on the gut microbiome composition, how gastric H. pylori infection affects local and distant microbial populations and host responses, how H. pylori infection is associated with fecal microbiota composition and diversity, and how H. pylori can influence gut bacteria outside of its natural habitat, the stomach. These findings and suggest that H. pylori leads to changes in both gastric and gut microbiome, which in turn can be reflected in changes in gut lipidome.

[0239] In the mixed microbial environment of the GI tract, chemical signals such as fatty acids produced by bacteria and host influence bacterial gene regulation. Deviations in the chemicals in this environment will have an impact on the bacteria living within that environment. Fatty acid composition in the gut is impacted by mammalian biosynthetic pathways and microbial metabolism. Fatty acids play central roles in chemical signaling, and membrane structure and function for both the host and the microbes. Accordingly, characterization of the fatty acid profile provides insight into the availability of a class of molecules at the intersection of the human host and the gut microbial community. Studies suggest that fatty acid metabolism plays a role in the pathophysiology of various diseases

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[0241] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) such as gastric cancer and liver cirrhosis. Fatty acids are also implicated in having a direct signaling role in both bacteria and mammalian systems including inter-kingdom signaling. Further, many fatty acids are processed biosynthetically into a structurally diverse array of potent and essential chemical signaling molecules collectively referred to as oxylipins, which are prominent regulators of the mammalian inflammation response. As fatty acid biosynthesis is central to membrane structure and function, and H. pylori is correlated with the erosion of the mucosal barrier, the present study aims to capture this aspect of infection through differential profiling of fecal fatty acids.

[0242] Here the present study explores the pathophysiology of H. pylori infection by exploring the gut lipid signatures of H. pylori patients and health control subjects. Understanding lipid metabolism in the context of H. pylori infection may further elucidate bacterial mechanisms of infection, host defense, and possibly introduce new targets for intervention or as adjuvants to current therapy. Currently, the guideline-directed standard treatment for H. pylori infection is a quadruple therapy including bismuth, a PPI, a tetracycline, and metronidazole followed by rifampin therapy . Due to increased antibiotic resistance, there has been waning effectiveness of clarithromycin-based triple therapy. Resistance to at least one component of the standard treatment is often prevalent in patients who are being treated for H. pylori infection for the first time. Due to antibiotic resistance, there is currently no treatment that achieves over a 90% eradication rate in patients. This leads to multiple treatments. Increased use of antibiotics to target H. pylori has led to high antibiotic resistance observed in this pathogen. With high antibiotic resistance, there is a need for exploring alternative therapies against H. pylori. Understanding the alterations that occur in the gut lipidome during H. pylori infection will allow us to better understand H. pylori disease progression. Alternative therapeutic targets found through gut lipidome profiling can serve as potential points for intervention or as adjuvants to current therapies, which will allow for decreased reliance on antibiotics and provide different means for decreasing H. pylori disease progression. Additionally, the characterization of patterns of lipidome changes correlated to H. pylori infection may enable the development of clinically relevant non- invasive diagnostics to report on the identified disease biomarkers.

[0243] Example 2B: Materials and Methods

[0244] Patient recruitment and fecal sample collection

[0245] H. pylori patients and healthy controls were recruited for the present study from the Cooper Digestive Health Institute in Camden, New Jersey from September 1, 2022 to January

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[0248] 31, 2023. Patients who were 18 years of age or older who were diagnosed with H. pylori via esophagogastroduodenoscopy (EGD) biopsy met the inclusion criteria for this study. Patients and healthy controls were excluded from recruitment who were under 18 years of age, were pregnant, or were taking antibiotics for three months before sampling. The present study identified H. pylori patients through positive EGD biopsy sampling in the electronic medical record system (EPIC). H. pylori infection was determined by histological examination of biopsied samples. Healthy controls were also recruited from the Cooper Digestive Health Institute. Lack of previous H. pylori infection and lack of any gastrointestinal symptoms at the time of recruitment was verified for all control subjects. To minimize subjective bias, anonymous codes were given to each of the H. pylori patients and healthy controls. All patients and healthy controls who participated were included in subsequent analyses.

[0249] Institutional Review Board (IRB)-approved team members obtained written informed consent for every patient and healthy control. Information about demographic attributes and pertinent medical history was obtained through a survey and EPIC. H. pylori patients submitted a stool sample before starting eradication treatment. Patients and healthy control subjects were instructed on how to properly collect a stool sample, and a kit was provided containing all required materials for collection, including a Stool Nucleic Acid Collection and Preservation Tube (Cat. No. 45630, 45660, Norgen Biotek Corp., Thorold, Ontario, Canada). Samples were stored at -80°C until fatty acid analysis was carried out.

[0250] Non-targeted fatty acid analysis

[0251] Samples were prepared and fatty acid analysis was conducted through gas chromatography-mass spectrometry (Metabolon Inc., Durham, NC, United States). This method measures the concentrations of 30 fatty acid methyl esters (FAMEs) in stool samples. This corresponds to the concentration of these 30 fatty acids in the stool samples and are weight corrected in (pg / g) for each sample. Briefly, the stool samples were homogenized, and 100 mg of each of the suspensions was aliquoted into test tubes. To extract the fatty acids, Liquid / Liquid extraction was performed, which removed the nucleic acid preservative, leaving the fatty acid extract. 250 pL of each extract was added to a clean analysis tube and a stream of nitrogen was used to evaporate the solvent, which creates a dried sample extract. To the dried sample extracts, quality controls (QCs), and calibration standards, an internal standard solution was added. After the internal standard solution was added, another stream of nitrogen was again used to evaporate the solvent, creating dried samples. Methyl esters (FAMEs) of corresponding free fatty acids and conjugated fatty acids were created from the

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[0253] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) dried samples and QCs through methylation / transm ethylation with methanol / sulfuric acid. The reaction mixtures were extracted with hexanes after neutralization. An aliquot of the hexanes layer was injected onto an Agilent 7890 / 5975 GC / MS system (RRID:SCR_018695). Using single ion monitoring positive mode with electron ionization, mass spectrometric analysis was performed. Fortified calibration standards were prepared immediately before each run. Both linear and quadratic regression analyses were generated from these standards, and, through these regression analyses, fatty acids were quantified. The raw data was collected, and Agilent MassHunter GC-MS Acquisition B.07.04.2260 (Agilent Technologies (RRID:SCR_013575) and Agilent MassHunter Workstation - Qualitative Analysis for GC / MS (RRID:SCR_016657) was used to process the data. Data reduction was performed using Microsoft Excel (RRID:SCR_016137) from Microsoft 365 MSO.

[0254] Statistical Analysis for Fatty Acid Metabolism

[0255] Statistical analysis was carried out to compare lipidome profiles from H. pylori patients and healthy controls. Microsoft Excel (RRID:SCR_016137) from Microsoft 365 MSO was used for tabulating the data and Volcano plots were generated using GraphPad Prism (RRID:SCR_002798). For both groups, each of the fatty acids was individually averaged and standard deviation was determined for fatty acid composition for H. pylori patients and healthy controls. Fold change for each fatty acid was calculated through the ratio of the fatty acid average of H. pylori patient to the same fatty acid average of healthy control, - values were determined for statistical significance through Student’s t-test. The fold change and the p-value were logarithmically transformed by calculating the log2 of the fold change for each of the fatty acids and the negative log of the / >-value. Volcano plots were created using the logarithmic transformed data to determine the fatty acid changes between H. pylori patients and healthy controls. On volcano plots, fatty acid changes presented as greater than log2 (fold change) of 1 represent those that increased two-fold, and those that are presented as less than log2 (fold change) of U represent those that decreased two-fold.

[0256] To determine influence of alcohol on lipidome profile, if any, the fatty acid composition for each of the fatty acids was individually averaged and standard deviation was determined for fatty acid composition for H. pylori patients and healthy controls who consume alcohol and those did not. Volcano plots were generated as described above. Similar analyses were carried out to study the influence of smoking and non-ulcer dyspepsia in healthy controls and H. pylori patients. Corresponding Volcano plots were generated.

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[0259] Ethics approval

[0260] This study was approved by the Cooper Health System IRB (17-077EX) and all the steps were completed per the IRB standards.

[0261] Example 2C: Results

[0262] Demographic information from the 68 H. pylori-^osh NQ patients and 35 healthy controls is listed in Table 2. All patient and healthy control samples were included in the analysis. The long chain fatty acids (LCFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) included in our lipidome analysis, along with their fold changes and corresponding p-values, are listed in Table 3.

[0263] Concentrations of significantly differing fatty acids, separated by H. pylori status are presented in volcano plot analysis in Fig. 1. It shows an increase in the LCFA margaric acid / 17:0 and the PUFAs eicosapentaenoic acid (EPA) / 20:5n3, erucic acid / 22: ln9, docosapentaenoic acid (DPA) / 22:5n3, eicosatetraenoic acid / 20:4n3, and docosahexaenoic acid (DHA) / 22:6n3 in H. pylori patients relative to the healthy controls. The increase observed in margaric acid / 17:0, EPA20:5n3, erucic acid / 22: ln9 and DPA / 22:5n3 was of larger magnitude. In contrast, the PUFAs gamma-linolenic acid / 18:3n6 and osbond acid / 22:5n6 were found to be decreased in the H. pylori patients relative to healthy controls. Many of the fatty acids that show statistically significant differences between H. pylori patients and healthy controls are metabolites of omega-3 and omega-6 fatty acid metabolism.

[0264] Table 2. Patient Demographics and Clinical Characteristics

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[0267] Table 3: Fold change of fatty acids with corresponding p-values.

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[0270] To determine if alcohol consumption has an effect on the pathophysiology of H. pylori infection, the present study explored the lipidome profiles of the (i) H. pylori patients who consumed alcohol and those who did not as well as (ii) healthy control subjects who consumed alcohol and those who did not. Differences in the lipidome profiles with respect to alcohol consumption for H. pylori patients (Fig. 2 A) and control subjects (Fig. 2B) are shown. There were significant decreases in behenic acid / 22:0, oleic acid / 18: ln9, and stearidonic acid / 18:4n3 (Fig. 2Af m H. pylori positive patients who consumed alcohol. Other fatty acids such as linoleic acid / 18:2n6 and osbond acid / 22:5n6 also decreased but to a lesser extent. On the other hand, fatty acids such as eicosatetraenoic acid / 20:4n3, EPA / 20:5n3, DHA / 22:6n3, and gamma-linolenic acid / 18:3n6 increased in patients with alcohol consumption. In controls who consumed alcohol, myristic acid / 14:0, oleic acid / 18: ln9, gamma-linolenic acid / 18:3n6, DPA / 22:5n3, and osbond acid / 22:5n6 were increased (Fig. 2B).

[0271] To determine if smoking has an effect on the pathophysiology of H. pylori infection, the present study explored the lipidome profiles of the H. pylori patients who smoked and those who did not. Differences in the lipidome profiles with respect to smoking are shown for H. pylori patients (Fig. 3). A significant decrease was observed in pentadecanoic acid / 15:0, behenic acid / 22:0, stearidonic acid / 18:4n3, and oleic acid / 18: ln9 in H. pylori patients that smoked. Other fatty acids such as EPA / 20:5n3 and osbond acid / 22:5n6 were also decreased, but to a lesser extent. Similar analysis could not be carried out in the healthy control subjects due to the lack of a significant number of smokers in this cohort.

[0272] It has been reported in literature that the prevalence ofH. pylori infection may be higher in patients with non-ulcer dyspepsia compared to healthy controls (38). The present study thus analyzed the lipidome profiles of the H. pylori patients with and without non-ulcer dyspepsia. Differences in the lipidome profiles with respect to non-ulcer dyspepsia are shown for H. pylori patients (Fig. 4A). In patients who have non-ulcer dyspepsia, an increase was observed in DPA / 22:5n3 and DHA / 22:6n3, while significant decreases were observed in

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[0274] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) stearidonic acid / 18:4n3, oleic acid / 18: ln9, gamma-linolenic acid / 18:3n6, and adrenic acid / 22:4n6. Fatty acid EPA / 20:5n3 was also decreased in patients with non-ulcer dyspepsia. Similar analysis was carried out for healthy control subjects with and without nonulcer dyspepsia. Differences in the lipidome profiles with respect to non-ulcer dyspepsia are shown for H. pylori patients (Fig. 4A). Fatty acids such as palmitoleic acid / 16: ln7 and cis-11- eicosaenoic acid / 20: ln9 were significantly increased. An increase was also observed in gamma-linolenic acid / 18:3n6, linoleic acid / 18:2n6, cis-11,14-eicosadienoic acid / 20:2n6, and osbond acid / 22:5n6.

[0275] Example 2D:

[0276] The preliminary study showed that H. pylori infection appears to present a defined pattern of chemical cross-talk between mammalian signaling circuits and bacterial regulatory circuits involving fatty acids. The present study elucidated mechanisms underlying the pathophysiology of H. pylori infection with respect to the differential lipidome profiles observed. The study included patients and healthy controls that reside in the underprivileged community. The population in Camden, New Jersey is 53% Hispanic or Latino and 42% African American, with an estimated 30% of people living in poverty. Given that H. pylori infection is more prevalent in Hispanics, Latinos, African Americans, and those of lower socioeconomic status, the community and those with similar demographics are faced with a disproportionate rate of H. pylori infection.

[0277] Based on the results of the differential lipidome profiles overserved, the present study proposes a schematic model (Figs. 5A-5D) of fatty acid metabolism in response to H. pylori infection, influenced by both H. pylori and the host. To enhance the comprehensiveness of the model, the present study also included relevant information from literature, which is indicated in the figure as such (information presented in blue or pink rectangles).

[0278] Example 2E: Possible influence of H. pylori on omega-6 and omega-9 fatty acids pathways

[0279] The Figs. 5A-5B show possible effects of H. pylori infection on the omega-6 and omega-9 fatty acid pathways, respectively. In the omega-6 pathway (Fig. 5A), level of the fatty acid, gamma-linolenic acid (18:3n6), is decreased with H. pylori infection. Interestingly, the downstream fatty acids in the pathway, dihomo-gamma-linolenic acid (20:3n6), arachidonic acid (20:4n6), and adrenic acid (22:4n6P) however are not affected. It was reported that a decrease in gamma-linolenic acid / 18:3n6 in H. pylori patients (White et al.,

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[0282] World J Gastroenterol 2T. 5575-5594, 2021). The current observations were consistent with that. Interestingly, among the multiple liposomal fatty acids evaluated using in vivo mouse models by Thamphiwatana el al. (Proceedings of the National Academy of Sciences of the United States of America 111 : 17600-17605, 2014), only the linolenic acid formulation (C18:3) showed bactericidal activity by impacting the H. pylori membrane structure and stability. Linolenic acid / 18:3 has anti-bacterial activity against H. pylori, and gammalinolenic acid / 18:3n6 is an intermediate in the omega-6 fatty acid metabolism pathway. In H. pylori patients, the omega-6 pathway in gastric mucosal cells mostly bypasses the formation of gamma-linolenic acid (18:3n6) from linoleic acid / 18:2n6 and is favored towards formation of arachidonic acid / 20:4n6. It is interesting to note that the arachidonic acid / 20:4n6 is a PGE2 precursor involved in the breakdown of gastric mucosa, which may facilitate H. pylori colonization and survival in the stomach. Given the anti-bacterial capability of linolenic acid / 18:3 and the pro-inflammatory role that arachidonic acid / 20:4n6 plays in H. pylori infection, it is suggested that H. pylori may influence the production of gamma-linolenic acid / 18:3n6 by favoring the alternate delta-8-desaturase pathway of omega-6 metabolism, and / or rapidly metabolizing any gamma-linolenic acid / 18:3n6 that is present.

[0283] In the omega-9 fatty acid pathway (Fig. 5B), two fatty acids are upregulated in the H. pylori patients, margaric acid / 17:0 and erucic acid / 22: ln9. Both of these fatty acids share stearic acid / 18:0 as a precursor. Although its role in H. pylori infection is unknown, it is worth noting that margaric acid / 17:0 has been shown to reduce biofilm formation of other gram-negative bacteria by restricting bacterial quorum sensing activity. Upregulation of fecal margaric acid / 17:0 is also a characteristic finding in patients with chronic atrophic gastritis, which is a possible sequelae of H. pylori infection. It can be speculated that the anti-quorum sensing activity of margaric acid / 17:0 helps to control the growth of competing microbes, to the benefit of H. pylori colonization and growth. It is also possible that margaric acid / 17:0 is synthesized by host cells or the host microbiome in response to H. pylori infection.

[0284] Example 2F: Possible influence of host on omega-3 and omega-9 fatty acids pathways

[0285] The Figs. 5C and 5D show possible effects of host on the omega-3 and omega-9 fatty acid pathways, respectively. Most of the fatty acids in the omega-3 pathway (Fig. 5C) such as 20:4n3 = eicosatetraenoic acid / 20:4n3, eicosapentaenoic acid(EPA) / 20:5n3, docosapentaenoic acid / 22:5n3, docosahexaenoic acid / 22:6n3 are increased in the H. pylori patients. It is interesting to note that in in vitro and in vivo studies, EPA / 20:5n3 has been shown to inhibit and reduce the colonization of H. pylori. It is also known that EPA / 20:5n3

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[0287] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) has anti-inflammatory effects and is upregulated in chronic inflammation. In the context of ulcerative colitis in in vivo and human patients, EPA / 20:5n3 has been shown to protect intestinal mucosa, decrease inflammation and oxidative stress, reduce symptoms, and restore the gut microbiome. EPA / 20:5n3 was also shown to reduce the growth of colorectal cancer cells, decrease inflammation, and improve the efficacy of chemotherapy drugs in vitro and in vivo. The risk of developing colorectal cancer in humans is greater in patients when low levels of omega-3 fatty acids, particularly EPA / 20:5n3 and DHA / 22:6n3, are detected in various tissues. These finding are consistent with the observation of significant increase seen in the level of EPA / 20:5n3. This increase may be due to (i) the chronic nature of H. pylori infection, and (ii) the production by the host during an H. pylori infection to attenuate the bacterial load.

[0288] DPA / 22:5n3 is another omega-3 pathway metabolite that significantly increased in expression in patients with H. pylori. DPA / 22:5n3 is a precursor for some anti-inflammatory metabolites, including docosanoids derived from DHA / 22:6n3. In the context of ulcerative colitis, DPA / 22:5n3 was shown to restore the gut microbiome and metabolome from dysbiosis and relieve several colitis symptoms. Since DPA / 22:5n3 contributes to the formation of anti-inflammatory metabolites, it is possible that the observed increase in DPA / 22:5n3 is due to the host’s response to H. pylori infection. The downstream fatty acid in the omega-3 pathway, DHA / 22:6n3 that was elevated in H. pylori patients is a product of desaturation of DPA / 22:5n3, which was also significantly increased in H. pylori patients. In in vitro studies, DHA / 22:6n3 has been shown to decrease H. pylori growth and has decreased colonization of H. pylori in mouse models. DHA / 22:6n3 has also been added to nanoparticle encapsulation alone or combined with amoxicillin to treat H. pylori, and has been shown to decrease the growth of H. pylori. DHA / 22:6n3 also has general anti-inflammatory properties, as it is an omega-3 fatty acid. In gastric and esophageal tumors, DHA / 22:6n3 has been seen to decrease growth and progression. As DHA / 22:6n3 has anti-bacterial and anti-inflammatory properties, the observed increase in DHA / 22:6n3 in the results may be a possible host response to attenuate inflammation and inhibit the growth of H. pylori.

[0289] The omega-9 fatty acid pathway (Fig. 5D) is described above. It is also possible that margaric acid / 17:0 is synthesized by host cells or the host microbiome in response to H. pylori infection. Further studies are necessary to understand the role of margaric acid / 17:0 with respect to H. pylori.

[0290] The results suggest possible antibacterial activity of certain fatty acids against H. pylori. Other fatty acids with anti-bacterial activity against H. pylori include capric acid / 10:0,

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[0292] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) lauric acid / 12:0 and myristoleic acid / 14:ln5. The mechanism by which particular fatty acid inhibits H. pylori growth is not fully understood. Studies suggest that lipids that may have anti-bacterial activity against H. pylori share common features, such as a higher degree of unsaturation or longer carbon chain. A common mechanism of action of anti-bacterial fatty acids is disruption of the bacterial outer membrane, resulting in an unfavorable lipid composition, coccoid shape, and / or alteration of outer membrane proteins, all of which may negatively impact the pathogen’s ability for adhesion to and colonization of target mucosal cells. Ong et al. recently demonstrated that phosphatidylethanolamine composed of capric acid / 10:0 acyl chains is the preferred substrate for the enzyme cholesteryl alpha-glucoside 6’- acyltransferase, a key enzyme for H. pylori pathogenesis that alters lipid metabolism in gastric mucosal cell membranes. In Staphylococcus aureus, unsaturated fatty acids such as oleic acid / 18: ln9, linoleic acid / 18:2n6, linolenic acid / 18:3, and arachidonic acid / 20:4n6 exert anti-bacterial effect by inhibiting the FabI protein involved in bacterial fatty acid biosynthesis. H. pylori contains FabI, and thus may be a target for these fatty acids. Based on the results, margaric acid / 17:0, gamma-linolenic acid / 18:3n6, EPA / 20:5n3, DPA / 22:5n3, and DHA / 22:6n3 would be reasonable candidates for further study on their mechanism of action in H. pylori infection of human hosts.

[0293] Example 2G: Effect of smoking, alcohol consumption and presence of non-ulcer dyspepsia on lipidome profiles

[0294] Figs. 6A-6C summarize possible influence of smoking, alcohol consumption and presence of non-ulcer dyspepsia on omega-3 (Fig. 6A), omega-6 (Fig. 6B) and omega-9 (Fig. 6C) fatty acid pathways. Fatty acids that are described above (Figs. 6A-6D) as upregulated (green) or downregulated (red) in H. pylori patients are highlighted and up or down arrowheads indicate level of the respective fatty acid with respect to smoking, alcohol consumption or non-ulcer dyspepsia in these pathways. Smoking seems to nullify the overall upregulation of the omega-3 fatty acid pathway in H. pylori patients (Fig. 6A), while it does not have much effect on the omega-6 and omega-9 fatty acids pathways (Figs. 6B-6C).

[0295] Effect of alcohol consumption is synergistic to the effect caused by H. pylori infection on several fatty acids in the omega-3 pathway (Fig. 6A), while alcohol consumption does not have significant effect on the omega-6 and omega-9 fatty acids pathways (Figs. 6B-6C). The present study did observe an increase in pro-inflammatory omega-6 fatty acids gammalinolenic acid / 18:3n6 in healthy controls who consume alcohol.

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[0298] There has been a rise of antibiotic resistance of H. pylori, which is a limiting factor for its universal eradication. Antibiotic stewardship is a necessity in the treatment of H. pylori, and other avenues of treatment need to be explored. Targeting fatty acid metabolism or treating with particular fatty acids are possible alternatives. Various in vivo models demonstrate anti-bacterial activity, restoration of gut dysbiosis, or improvement of gastrointestinal disease symptoms after oral administration of fatty acids.

[0299] Enumerated Embodiments

[0300] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0301] Embodiment 1 : A method of diagnosing H. pylori infection in a subject, the method comprising: determining a fatty acid profile in a stool sample of the subject; and comparing the fatty acid profile with a first reference profile indicating H. pylori infection or a second reference profile indicating H. pylori infection, wherein the subject is diagnosed to have H. pylori infection when: a level of at least one fatty acid in the fatty acid profile is different than a level of the at least one fatty acid in the first reference profile; or a level of at least one fatty acid in the fatty acid profile matches a level of the at least one fatty acid in the second reference profile.

[0302] Embodiment 2: The method of Embodiment 1, further comprising: obtaining the stool sample from the subject.

[0303] Embodiment 3: The method of Embodiment 1 or 2, wherein the subject is a human.

[0304] Embodiment 4: The method of any one of Embodiments 1-3, wherein at least one fatty acid comprises a metabolite of the omega-3 fatty acids pathway, a metabolite of the omega-6 fatty acids pathway, and / or a metabolite of the omega-9 fatty acids pathway.

[0305] Embodiment 5: The method of any one of Embodiments 1-4, wherein the at least one fatty acid comprises margaric acid (17:0), eicosapentaenoic acid (20:5n3), erucic acid (22: ln9), docosapentaenoic acid (22:5n3), eicosatetraenoic acid (20:4n3), docosahexaenoic acid (22:6n3), gamma-linolenic acid (18:3n6), and / or osbond acid (22:5n6).

[0306] Embodiment 6: The method of any one of Embodiments 1-5, wherein the subject is diagnosed to have H. pylori infection when:

[0307] (a) the at least one fatty acid selected from the group consisting of margaric acid (17:0), eicosapentaenoic acid (20:5n3), erucic acid (22:ln9), docosapentaenoic acid (22:5n3),

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[0309] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) eicosatetraenoic acid (20:4n3), and docosahexaenoic acid (22:6n3) in the fatty acid profile is higher than in the first reference profile indicating free of H. pylori infection; and / or

[0310] (b) the at least one fatty acid selected from the group consisting of gamma-linolenic acid (18:3n6), and osbond acid (22:5n6) in the fatty acid profile is lower than in the first reference profile indicating free of H. pylori infection.

[0311] Embodiment 7: The method of any one of Embodiments 1-6, wherein the diagnosis takes in to consideration whether the subject consumes alcohol, whether the subject smokes, and / or whether the subject has non-ulcer dyspepsia.

[0312] Embodiment 8: The method of any one of Embodiments 1-7, wherein at least one of the following applies:

[0313] (a) the first reference profile comprises a fatty acid reference profile indicating a nonalcohol-consuming subject free from H. pylori infection;

[0314] (b) the first reference profile comprises a fatty acid reference profile indicating an alcohol-consuming subject free from H. pylori infection;

[0315] (c) the first reference profile comprises a fatty acid reference profile indicating a nonsmoking subject free from H. pylori infection;

[0316] (d) the first reference profile comprises a fatty acid reference profile indicating a smoking subject free from H. pylori infection;

[0317] (e) the first reference profile comprises a fatty acid reference profile indicating a nonulcer dyspepsia-free subject free from H. pylori infection;

[0318] (f) the first reference profile comprises a fatty acid reference profile indicating a nonulcer dyspepsia subject free from H. pylori infection;

[0319] (g) the second reference profile is a fatty acid reference profile indicating a nonalcohol-consuming subject having H. pylori infection;

[0320] (h) the second reference profile is a fatty acid reference profile indicating an alcohol- consuming subject having H. pylori infection;

[0321] (i) the second reference profile is a fatty acid reference profile indicating a nonsmoking subject having H. pylori infection;

[0322] (j) the second reference profile is a fatty acid reference profile indicating a smoking subject having H. pylori infection;

[0323] (k) the second reference profile is a fatty acid reference profile indicating a non-ulcer dyspepsia-free having from H. pylori infection; and / or

[0324] (l) the second reference profile is a fatty acid reference profile indicating a non-ulcer dyspepsia subject having H. pylori infection.

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[0327] Embodiment 9: The method of any one of Embodiments 1-8, wherein the first reference profile comprises a first reference profile-A indicating non-alcohol-consuming subject free from H. pylori infection, and a first reference profile-B indicating alcohol- consuming subject free from H. pylori infection, and wherein a level of at least one fatty acid selected from the group consisting of myristic acid (14:0), oleic acid (18: ln9), gammalinolenic acid (18:3n6), docosapentaenoic acid (22:5n3), and osbond acid (22:5n6) is higher in the first reference profile-B than in the first reference profile-A.

[0328] Embodiment 10: The method of any one of Embodiments 1-9, wherein the second reference profile comprises a second reference profile-A indicating non-alcohol-consuming subject having H. pylori infection, and a second reference profile-B indicating alcohol- consuming subject having H. pylori infection, and wherein at least one of the following applies:

[0329] (a) a level of at least one fatty acid selected from the group consisting of behenic acid (22:0), oleic acid (18: ln9), and stearidonic acid (18:4n3) is lower in the second reference profile-B than in the second reference profile- A; and / or

[0330] (b) a level of at least one fatty acid selected from the group consisting of eicosatetraenoic acid (20:4n3), eicosapentaenoic acid (20:5n3), docosahexaenoic acid (22:6n3), and gamma-linolenic acid (18:3n6) is higher in the second reference profile-B than in the second reference profile-A.

[0331] Embodiment 11 : The method of any one of Embodiments 1-10, wherein the second reference profile comprises a second reference profile-C indicating non-smoking subject having H. pylori infection, and a second reference profile-D indicating smoking subject having H. pylori infection, and wherein a level of at least one fatty acid selected from the group consisting of pentadecanoic acid (15:0), behenic acid (22:0), stearidonic acid (18:4n3), oleic acid (18: ln9), eicosapentaenoic acid (20:5n3), and osbond acid (22:5n6) is lower in the second reference profile-D than in the second reference profile-C.

[0332] Embodiment 12: The method of any one of Embodiments 1-11, wherein the first reference profile comprises a first reference profile-E indicating non-ulcer dyspepsia-free subject free from H. pylori infection, and a first reference profile-F indicating non-ulcer dyspepsia subject free from H. pylori infection, and wherein a level of at least one fatty acid selected from the group consisting of palmitoleic acid (16: ln7), cis- 11 -eicosaenoic acid (20: ln9), gamma-linolenic acid (18:3n6), linoleic acid (18:2n6), cis-11,14-eicosadienoic acid (20:2n6), and osbond acid (22:5n6) is higher in the first reference profile-F than in the first reference profile-E.

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[0335] Embodiment 13: The method of any one of Embodiments 1-12, wherein the second reference profile comprises a second reference profile-E indicating non-ulcer dyspepsia-free subject having H. pylori infection, and a second reference profile-F indicating non-ulcer dyspepsia subject having H. pylori infection, and wherein at least one of the following applies:

[0336] (a) a level of at least one fatty acid selected from the group consisting of docosapentaenoic acid (22:5n3) and docosahexaenoic acid (22:6n3) is higher in the second reference profile-F than in the second reference profile-E; and / or

[0337] (b) a level of at least one fatty acid selected from the group consisting of stearidonic acid (18:4n3), oleic acid (18: ln9), gamma-linolenic acid (18:3n6), adrenic acid (22:4n6), and eicosapentaenoic acid (20:5n3) is lower in the second reference profile-F than in the second reference profile-E.

[0338] Embodiment 14: The method of any one of Embodiments 1-13, wherein determining the fatty acid profile in the stool sample comprises analyzing the stool sample with a chromatography method, a mass spectrometry method, a colorimetric method, a fluorometric method, or an assay analyzing interactions between fatty acids and proteins.

[0339] Embodiment 15: The method of Embodiment 14, wherein the fatty acid profile is determined by the assay analyzing interactions between fatty acids and proteins, and wherein the type and / or amount of fatty acids in the stool sample are determined according to interactions between fatty acids in the stool sample and one or more proteins having different binding affinity toward different fatty acids.

[0340] Embodiment 16: The method of Embodiment 15, wherein the one or more proteins comprise serum albumin and / or a fatty acid binding protein (FABP).

[0341] Embodiment 17: The method of Embodiment 16, wherein the FABP comprises FABP1, FABP2, FABP3, and / or FABP4.

[0342] Embodiment 18: The method of any one of Embodiments 15-17, wherein the assay analyzing interactions between fatty acids and proteins is a lateral flow assay, and wherein the proteins are immobilized on a substrate and the stool sample is allowed to flow on the substrate.

[0343] Embodiment 19: The method of any one of Embodiments 1-18, which is a point-of- care test method or a home diagnostic test method.

[0344] Embodiment 20: A method of treating H. pylori infection in a subject in need thereof, comprising:

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[0346] 56736055.2 Attorney Docket No. 370431-1054WO1 (00351) performing the method of any one of Embodiments 1-19 to determine that the subject has H. pylori infection, and administering to the subject an effective amount of a treatment for H. pylori infection.

[0347] Embodiment 21: The method of Embodiment 20, wherein the treatment for H. pylori infection comprises an antibiotic, a proton-pump inhibitor, bismuth subsalicylate, and / or a histamine (H-2) blocker.

[0348] Embodiment 22: The method of any one of Embodiments 20-21, wherein the treatment for H. pylori infection comprises a proton-pump inhibitor, clarithromycin and at least one selected from the group consisting of amoxicillin and metronidazole.

[0349] The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

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[0351] 56736055.2

Claims

Attorney Docket No. 370431-1054WO1 (00351)CLAIMSWhat is claimed is:

1. A method of diagnosing H. pylori infection in a subject, the method comprising: determining a fatty acid profile in a stool sample of the subject; and comparing the fatty acid profile with a first reference profile indicating H. pylori infection or a second reference profile indicating H. pylori infection, wherein the subject is diagnosed to have H. pylori infection when: a level of at least one fatty acid in the fatty acid profile is different than a level of the at least one fatty acid in the first reference profile; or a level of at least one fatty acid in the fatty acid profile matches a level of the at least one fatty acid in the second reference profile.

2. The method of claim 1, further comprising: obtaining the stool sample from the subject.

3. The method of claim 1 or 2, wherein the subject is a human.

4. The method of any one of claims 1-3, wherein at least one fatty acid comprises a metabolite of the omega-3 fatty acids pathway, a metabolite of the omega-6 fatty acids pathway, and / or a metabolite of the omega-9 fatty acids pathway.

5. The method of any one of claims 1-4, wherein the at least one fatty acid comprises margaric acid (17:0), eicosapentaenoic acid (20:5n3), erucic acid (22: ln9), docosapentaenoic acid (22:5n3), eicosatetraenoic acid (20:4n3), docosahexaenoic acid (22:6n3), gammalinolenic acid (18:3n6), and / or osbond acid (22:5n6).

6. The method of any one of claims 1-5, wherein the subject is diagnosed to have H. pylori infection when:(a) the at least one fatty acid selected from the group consisting of margaric acid (17:0), eicosapentaenoic acid (20:5n3), erucic acid (22:ln9), docosapentaenoic acid (22:5n3), eicosatetraenoic acid (20:4n3), and docosahexaenoic acid (22:6n3) in the fatty acid profile is higher than in the first reference profile indicating free of H. pylori infection; and / or4556736055.2Attorney Docket No. 370431-1054WO1 (00351)(b) the at least one fatty acid selected from the group consisting of gamma-linolenic acid (18:3n6), and osbond acid (22:5n6) in the fatty acid profile is lower than in the first reference profile indicating free of H. pylori infection.

7. The method of any one of claims 1-6, wherein the diagnosis takes in to consideration whether the subject consumes alcohol, whether the subject smokes, and / or whether the subject has non-ulcer dyspepsia.

8. The method of any one of claims 1-7, wherein at least one of the following applies:(a) the first reference profile comprises a fatty acid reference profile indicating a nonalcohol-consuming subject free from H. pylori infection;(b) the first reference profile comprises a fatty acid reference profile indicating an alcohol-consuming subject free from H. pylori infection;(c) the first reference profile comprises a fatty acid reference profile indicating a nonsmoking subject free from H. pylori infection;(d) the first reference profile comprises a fatty acid reference profile indicating a smoking subject free from H. pylori infection;(e) the first reference profile comprises a fatty acid reference profile indicating a nonulcer dyspepsia-free subject free from H. pylori infection;(f) the first reference profile comprises a fatty acid reference profile indicating a nonulcer dyspepsia subject free from H. pylori infection;(g) the second reference profile is a fatty acid reference profile indicating a nonalcohol-consuming subject having H. pylori infection;(h) the second reference profile is a fatty acid reference profile indicating an alcohol- consuming subject having H. pylori infection;(i) the second reference profile is a fatty acid reference profile indicating a nonsmoking subject having H. pylori infection;(j) the second reference profile is a fatty acid reference profile indicating a smoking subject having H. pylori infection;(k) the second reference profile is a fatty acid reference profile indicating a non-ulcer dyspepsia-free having from H. pylori infection; and / or(l) the second reference profile is a fatty acid reference profile indicating a non-ulcer dyspepsia subject having H. pylori infection.4656736055.2Attorney Docket No. 370431-1054WO1 (00351)9. The method of any one of claims 1-8, wherein the first reference profile comprises a first reference profile-A indicating non-alcohol-consuming subject free from H. pylori infection, and a first reference profile-B indicating alcohol-consuming subject free from H. pylori infection, and wherein a level of at least one fatty acid selected from the group consisting of myristic acid (14:0), oleic acid (18: ln9), gamma-linolenic acid (18:3n6), docosapentaenoic acid (22:5n3), and osbond acid (22:5n6) is higher in the first reference profile-B than in the first reference profile-A.

10. The method of any one of claims 1-9, wherein the second reference profile comprises a second reference profile- A indicating non-alcohol-consuming subject having H. pylori infection, and a second reference profile-B indicating alcohol-consuming subject having H. pylori infection, and wherein at least one of the following applies:(a) a level of at least one fatty acid selected from the group consisting of behenic acid (22:0), oleic acid (18: ln9), and stearidonic acid (18:4n3) is lower in the second reference profile-B than in the second reference profile- A; and / or(b) a level of at least one fatty acid selected from the group consisting of eicosatetraenoic acid (20:4n3), eicosapentaenoic acid (20:5n3), docosahexaenoic acid (22:6n3), and gamma-linolenic acid (18:3n6) is higher in the second reference profile-B than in the second reference profile-A.

11. The method of any one of claims 1-10, wherein the second reference profile comprises a second reference profile-C indicating non-smoking subject having H. pylori infection, and a second reference profile-D indicating smoking subject having H. pylori infection, and wherein a level of at least one fatty acid selected from the group consisting of pentadecanoic acid (15:0), behenic acid (22:0), stearidonic acid (18:4n3), oleic acid (18: ln9), eicosapentaenoic acid (20:5n3), and osbond acid (22:5n6) is lower in the second reference profile-D than in the second reference profile-C.

12. The method of any one of claims 1-11, wherein the first reference profile comprises a first reference profile-E indicating non-ulcer dyspepsia-free subject free from H. pylori infection, and a first reference profile-F indicating non-ulcer dyspepsia subject free from H. pylori infection, and wherein a level of at least one fatty acid selected from the group consisting of palmitoleic acid (16: ln7), cis- 11 -eicosaenoic acid (20: ln9), gamma-linolenic4756736055.2Attorney Docket No. 370431-1054WO1 (00351) acid (18:3n6), linoleic acid (18:2n6), cis-11,14-eicosadienoic acid (20:2n6), and osbond acid (22:5n6) is higher in the first reference profile-F than in the first reference profile-E.

13. The method of any one of claims 1-12, wherein the second reference profile comprises a second reference profile-E indicating non-ulcer dyspepsia-free subject having H. pylori infection, and a second reference profile-F indicating non-ulcer dyspepsia subject having H. pylori infection, and wherein at least one of the following applies:(a) a level of at least one fatty acid selected from the group consisting of docosapentaenoic acid (22:5n3) and docosahexaenoic acid (22:6n3) is higher in the second reference profile-F than in the second reference profile-E; and / or(b) a level of at least one fatty acid selected from the group consisting of stearidonic acid (18:4n3), oleic acid (18: ln9), gamma-linolenic acid (18:3n6), adrenic acid (22:4n6), and eicosapentaenoic acid (20:5n3) is lower in the second reference profile-F than in the second reference profile-E.

14. The method of any one of claims 1-13, wherein determining the fatty acid profile in the stool sample comprises analyzing the stool sample with a chromatography method, a mass spectrometry method, a colorimetric method, a fluorometric method, or an assay analyzing interactions between fatty acids and proteins.

15. The method of claim 14, wherein the fatty acid profile is determined by the assay analyzing interactions between fatty acids and proteins, and wherein the type and / or amount of fatty acids in the stool sample are determined according to interactions between fatty acids in the stool sample and one or more proteins having different binding affinity toward different fatty acids.

16. The method of claim 15, wherein the one or more proteins comprise serum albumin and / or a fatty acid binding protein (FABP).

17. The method of claim 16, wherein the FABP comprises FABP1, FABP2, FABP3, and / or FABP4.4856736055.2Attorney Docket No. 370431-1054WO1 (00351)18. The method of any one of claims 15-17, wherein the assay analyzing interactions between fatty acids and proteins is a lateral flow assay, and wherein the proteins are immobilized on a substrate and the stool sample is allowed to flow on the substrate.

19. The method of any one of claims 1-18, which is a point-of-care test method or a home diagnostic test method.

20. A method of treating H. pylori infection in a subject in need thereof, comprising: performing the method of any one of claims 1-19 to determine that the subject has H. pylori infection, and administering to the subject an effective amount of a treatment for H. pylori infection.

21. The method of claim 20, wherein the treatment for H. pylori infection comprises an antibiotic, a proton-pump inhibitor, bismuth subsalicylate, and / or a histamine (H-2) blocker.

22. The method of any one of claims 20-21, wherein the treatment for H. pylori infection comprises a proton-pump inhibitor, clarithromycin and at least one selected from the group consisting of amoxicillin and metronidazole.4956736055.2

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