DESC:
F O R M 2

THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10 and rule 13]

1. TITLE OF THE INVENTION: INTESTINAL MICROBIAL HEALTH MARKER BASED ON INTESTINAL MICROBIAL PROFILING

2. APPLICANT (A) NAME: LEUCINE RICH BIO PVT LTD

(B) ADDRESS: # 283, 3RD FLOOR, PUTTALINGIAH RD, GURURAJA LAYOUT, PADMANABHANAGAR, BENGALURU, KARNATAKA 560070

3. NATIONALITY (C) INDIA

THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE NATURE OF THIS INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED
[001] TECHNICAL FIELD OF THE INVENTION
[002] The present invention is in the technical field of gut microbiome, more particularly a eubiosis/dysbiosis index, for establishing the state of eubiosis or degree of dysbiosis of an individual’s gut microbiome and for monitoring intestinal microbial health.
[003] BACKGROUND OF THE INVENTION
[004] ‘Microbiome’ refers to the entire habitat of the microorganisms (bacteria, archaea, lower eukaryotes, and viruses), their genomes, and the surrounding environmental conditions [Ex. human gut]1. This definition is based on that of biome, the biotic and abiotic factors of given environments. Further, the term ‘Microbiota’ is defined as the entire community of microorganisms present in a defined environment [such as human gut or skin], while the term ‘Metagenome’ is the collection of genomes and genes from the members of a microbiota1,2. In the recent past, the distinct physio-chemical properties, including but not limited to microbial structures, metabolites, mobile genetic elements of the microbiome, has been reported to play a significant role in human health and long-term wellbeing.
[005] Due to their symbiotic/parasitic nature, there is a significant interplay between individual species of the microbiome, leading to either eubiosis [if more symbiotic species colonize/transit] or dysbiosis [if more parasitic/opportunistic species colonize/transit]3.
[006] Further, because of its ability to establish an axis with several extraintestinal organs, such as kidneys, liver, cardiovascular, the bone system and the brain, the symbiosis or dysbiosis of the gut microbiome has gained a lot of traction in the recent past due to its axial effect4.
[007] Furthermore, it has also emerged as a virtual metabolic and endocrine organ, given the impact of the eubiosis or dysbiosis on a large spectrum of digestion and metabolism of the food that we consume5,6,7.
[008] Most importantly, the dysbiosis of the gut microbiome has been shown to be linked with various chronic diseases such as IBD, type 2 diabetes, non-alcoholic fatty acid liver disease (NAFLD)8–12 etc.
[009] Because of such significant impact, establishing the state of eubiosis or dysbiosis of the gut microbiome has been deemed critical.
[010] The need to perform microbiome analysis, focused on the health and wellness domain of the public sector, has exponentially increased to determine the effect of the gut microbial community on the host [humans].
[011] Specifically, the characterization of identity, abundance, and/or ratio of gut microflora (Bacteria, Archaea, Fungi, Virus, Metazoa and Protozoa) in a host is critical to recommend one or more dietary, pre-, and pro-biotic centred approach to improve overall health and wellbeing.
[012] Significant improvements have been made in sequencing and bioinformatics tools for identification and quantification of individual species, to characterize even those microorganisms that cannot be cultured in a laboratory.
[013] However, there is a significant lag in establishing the cumulative state of eubiosis or dysbiosis from this data.
[014] Few indices that are available13–15 are limited in their accuracy and effectiveness, either because they are based only on bacterial component of the gut microbiome [16s sequencing based], or because they focus only a single aspect [composition or abundance or diversity] of a comprehensive characterization of all microbial [Bacterial, Archaeal, Fungal, Viral, Metazoal and Protozoal] components.
[015] Both cases do not provide an effective index, let alone provide a single score, for representing the state of ‘eubiosis/dysbiosis’.
[016] Furthermore, most of the indices focus on establishing if the sample represents a healthy gut, while its being increasingly proven that there is nothing such as a healthy gut.
[017] Hence, there is an urgent need to develop an index/score that can accurately and effectively indicate the eubiotic/dysbiotic state of one’s gut microbiome to be used as a simple marker for monitoring intestinal microbial health.
[018] SUMMARY OF THE INVENTION
[019] The primary objective of the present invention is to generate a gut microbiome eubiosis/dysbiosis marker score for establishing the state of eubiosis or degree of dysbiosis of an individual’s gut microbiome and using the marker for monitoring intestinal microbial health.
[020] In an embodiment, a method for determining gut microbiome eubiosis/dysbiosis marker in a subject, the method comprising the steps of:
a) receiving a biological sample from a subject;
b) extracting DNA;
c) sequencing DNA through next generation sequencing;
d) analyzing the microbial quality, quantity and diversity;
e) determining microbiome marker score;
f) gathering information from microbiome profile;
g) compiling the effective score; and
h) interpreting eubiosis/dysbiosis marker, The Rych Index Marker of the subject.
[021] In further embodiment, the Rych Index correlates with various disease conditions is provided based on individual’s gut microbiota.
[022] In further embodiment, the said disease conditions are Irritable Bowel Syndrome, Colorectal Neoplasm, Constipation, Diabetes Mellitus Type 2, Inflammatory Bowel Disease, Non-Alcoholic Fatty Liver Disease, Ulcerative Colitis, Aerobic Endurance, Crohn’s Disease, Physical Endurance, Clostridium Difficile Infection, Power, Depression, Prone to Fatigue, Anxiety, Gluten intolerance, Rheumatoid Arthritis, Asthma, Chronic Kidney Disease, Eczema, Atherosclerosis, Atopic dermatitis, Obesity, Acne, Hypertension, Effect of Smoking, Parkinson’s Disease, Autism spectrum disorder, Alzheimer’s disease, Preeclampsia or any combination thereof.
[023] In an embodiment, a method for monitoring the progress of microbiome-based disease condition comprising the steps of:
[024] obtaining sample comprising the gut microbiome from a human patient to be diagnosed
[025] extracting DNA;
[026] sequencing DNA;
[027] analyzing the data;
[028] concluding on microbiota in said patient, wherein an increase of the abundance of a said species as detected in said sample compared to a sample from a control patient;
[029] monitoring the disease progress; and
[030] recommending a personalized nutritional requirement.
[031] In an embodiment, a system comprising:
[032] a portable device to capture microbial quality, quantity and diversity data from biological sample of a subject;
[033] a server system to:
[034] compute a plurality of input characteristics of dysbiosis to eubiosis, as a function of time or dietary or prebiotic or probiotic based on said input characteristics; and
[035] send for display a comparison of said dysbiosis to eubiosis score to a user;
[036] wherein said score enables recommending a personalized nutritional regimen based on gut microbiota of the subject.
[037] In an embodiment, the said microbiome marker score utilizes the information gathered from the microbiome profile from the stool of the patient using next generation sequencing. The microbial identity, abundance and diversity is characterized, and an effective score is compiled.
[038] In an embodiment, the information pertaining to microbial association with that of eubiosis or dysbiosis, and in extension to various diseases, is curated from known medica/scientific literature and compiled into a database called MicroByte.
[039] In exemplary embodiment, all said data is incorporated in defining the projected microbiome eubiosis/dysbiosis marker score which gives a threshold value called - The Rych Index Marker.
[040] According to the exemplary aspect of the present invention, the Rych Index marker can be effectively utilized as a decision maker in medical and or wellness domains to describe intestinal gut microbial health which will enable clinicians, nutritionists and other service providers to take effective measures and monitor the health.
[041] In an embodiment, physicians and clinicians, addressing disease(s) involving or influenced by gut microbiome, do not have any single and simple representation of the extent of dysbiosis associated with that disease, which is necessary for taking quick measures.
[042] In further embodiment, nutritionists, to rely on gut microbiome for customizing the dietary recommendations, various microbial and metagenomic indices must be understood.
[043] In an embodiment, the Rych Index can also aid the physicians, clinicians, Ayurveda practitioners, naturopaths and nutritionists to monitor the impact of their therapeutic or nutritional intervention, since the Rych Index scores shifts from a dysbiosis score [pre-intervention] to an eubiosis score [post-intervention] depending on the kind and impact of the intervention.
[044] In further embodiment, the current metagenomic approaches have failed to conceptualize a universal measure that can indicate the state of eubiosis or degree of dysbiosis and largely rely on technical indices and jargon involving diversity indices and other ratios (Ex. Bacteroidetes to Firmicutes ratio). This is the key issue we are trying to address with this invention.
[045] Single score-based marker, simple and effective way of representation of gut eubiosis / dysbiosis for Direct to Consumer (DTC) solutions, involving characterizing the microbial composition of individuals using metagenomics approaches.
[046] According to the exemplary aspect of the present invention, The Rych Index marker can be effectively utilized as a good representative of the intestinal gut microbial health which will enable the clinicians and nutritionists to take effective interventional measures and monitor the effectiveness of such steps.
[047] Several aspects of the invention are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the invention can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well-known structures, materials, or operations are not shown in detail to avoid obscuring the features of the invention. Furthermore, the features/aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.
[048] BRIEF DESCRIPTION OF THE DRAWINGS
[049] Example embodiments of the present invention will be described with reference to the accompanying drawings briefly described below.
[050] FIG. 1 illustrates the Illustrating the Rych Index, according to the aspects of the invention.
[051] FIG. 2 Illustrates a block diagram illustrating the details of a digital processing system in which various aspects of the present invention are operative by execution of appropriate execution modules.
[052] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.
[053] DETAILED DESCRIPTION OF THE INVENTION
[054] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.
[055] The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.
[056] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a dosage” refers to one or more than one dosage.
[057] The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements, or method steps.
[058] A microbiota profile in accordance with the methods of the invention is a numerical representation of such levels obtained from an analysis of a GI tract sample from the subject. The individual values for such levels-individual profile, may be qualitative, quantitative or semiquantitative, preferably quantitative.
[059] The term "amount" could be used in place of "levels" if required.
[060] The profiling of the GI tract sample may involve any convenient means by which the levels of microorganism or group of microorganisms may be measured, preferably quantified.
[061] The term, “Disease" refers to a state of pathological disturbance relative to normal which may result, for example, from infection or an acquired or congenital genetic imperfection.
[062] The term "condition" refers to a state of the mind or body of an organism which has not occurred through a recognised disease, e.g., the presence of an agent in the body such as a toxin, drug or pollutant, or pregnancy.
[063] The subject may be any human or non-human animal subject, but more particularly may be a vertebrate, e.g., a mammal, including livestock and companion animals.
[064] A "normal" or "healthy" subject is a subject that is not known to have the illness or disease or other medical condition, the diagnosis of which is the object of the method in question, or a disease, illness or other medical condition that is similar thereto or shares common features and symptoms.
[065] All documents cited in the present specification are hereby incorporated by reference in their totality. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.
[066] Example embodiments of the present invention are described with reference to the accompanying figures.
[067] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.
[068] 1. EMBODIMENTS OF THE INVENTION:
[069] In an embodiment, a method for determining gut microbiome eubiosis/dysbiosis marker in a subject, the method comprising the steps of:
i) receiving a biological sample from a subject;
j) extracting DNA;
k) sequencing DNA through next generation sequencing;
l) analyzing the microbial quality, quantity and diversity;
m) determining microbiome marker score;
n) gathering information from microbiome profile;
o) compiling the effective score; and
p) interpreting eubiosis/dysbiosis marker, The Rych Index Marker of the subject.
[070] In further embodiment, the Rych Index correlates with various disease conditions is provided based on individual’s gut microbiota.
[071] In an embodiment, the method estimates the extent of dysbiosis.
[072] In an embodiment, the method monitors the extent of shift from dysbiosis to eubiosis, as a function of time or dietary, prebiotic, probiotic, or any therapeutic intervention.
[073] In further embodiment, the said disease conditions are Irritable Bowel Syndrome, Colorectal Neoplasm, Constipation, Diabetes Mellitus Type 2, Inflammatory Bowel Disease, Non-Alcoholic Fatty Liver Disease, Ulcerative Colitis, Aerobic Endurance, Crohn’s Disease, Physical Endurance, Clostridium Difficile Infection, Power, Depression, Prone to Fatigue, Anxiety, Gluten intolerance, Rheumatoid Arthritis, Asthma, Chronic Kidney Disease, Eczema, Atherosclerosis, Atopic dermatitis, Obesity, Acne, Hypertension, Effect of Smoking, Parkinson’s Disease, Autism spectrum disorder, Alzheimer’s disease, Preeclampsia or any combination thereof.
[074] In an embodiment, the method for estimating the intestinal health of the subject based on the gut microbiota profile.
[075] In further embodiment, the said analyzing comprises a method, such as a quantitative method, selected from the group of PCR, rtPCR, qPCR, multiplex PCR, high-throughput sequencing, metatranscriptomic sequencing, identification of strain-specific markers, such as genes and/or proteins, and 16S rDNA analysis.
[076] In an embodiment, a method for monitoring the progress of microbiome-based disease condition comprising the steps of:
[077] obtaining sample comprising the gut microbiome from a human patient to be diagnosed
[078] extracting DNA;
[079] sequencing DNA;
[080] analyzing the data;
[081] concluding on microbiota in said patient, wherein an increase of the abundance of a said species as detected in said sample compared to a sample from a control patient;
[082] monitoring the disease progress; and
[083] recommending a personalized nutritional requirement.
[084] In an embodiment, a method for assessing the status of a disease, condition, pathology, or cell state, wherein the disease, condition, pathology, or cell state is associated with the presence or level of a cellular component, the method comprising the steps of detecting and/or isolating or removing the cellular component associated with the disease, condition, pathology, or cell state, and correlating the presence or level of the cellular component with disease, condition, pathology, or cell state.
[085] In an embodiment, a system comprising:
[086] a portable device to capture microbial quality, quantity and diversity data from biological sample of a subject;
[087] a server system to:
[088] compute a plurality of input characteristics of dysbiosis to eubiosis, as a function of time or dietary or prebiotic or probiotic based on said input characteristics; and
[089] send for display a comparison of said dysbiosis to eubiosis score to a user;
[090] wherein said score enables recommending a personalized nutritional regimen based on gut microbiota of the subject.
[091] In an embodiment, a computer-readable medium comprising a plurality of digitally-encoded profiles wherein each profile of the plurality has a plurality of values, each value representing the abundance of a biomolecule in a subject host microbiome.
[092] In further embodiment, the computer readable medium, wherein each profile of the plurality of digitally-encoded expression profiles are associated with a compound related disorder.
[093] Inventors developed and compiled a system and method for generating the Rych Index marker – A gut microbiome eubiosis/dysbiosis marker for establishing the state of eubiosis or degree of dysbiosis of an individual’s gut microbiome and using the marker for monitoring intestinal microbial health.
[094] The Rych Index marker can be effectively utilized as a decision maker in medical and or wellness domains to describe intestinal gut microbial health which will enable clinicians, service provider and nutritionists to take effective measures and monitor the progress of health.
[095] The invention is relevant to the fields of,
[096] Microbiome, Gut Microbiome
[097] Metagenomics, High-throughput sequencing technology and its applications
[098] Precision medicine
[099] Health and Wellness, Direct to Consumer (DTC) omics
[0100] For, physicians and clinicians addressing issues using gut microbiome and metagenomics approach (Ex. Gastroenterologists).
[0101] For nutritionists – using gut microbiome and metagenomics approach for their nutritional recommendations.
[0102] In a nutshell, the current metagenomic approaches have failed to conceptualize a universal measure that can indicate the state of eubiosis or degree of dysbiosis and largely rely on technical indices and jargon involving diversity indices and other ratios (Ex. Bacteroidetes to Firmicutes ratio). This is the key issue we are trying to address with this invention.
[0103] The Rych Index marker can be effectively utilized as an accurate representative of the intestinal gut microbial health which will enable the clinicians and nutritionists to take effective interventional measures and monitor the effectiveness of such steps.
[0104] Following diseases are known to be influenced by dysbiosis in the intestine. Rych index marker can be used effectively in monitoring the intestinal health of these diseases.
Irritable Bowel Syndrome Colorectal Neoplasm
Constipation Diabetes Mellitus Type 2
Inflammatory Bowel Disease Non-Alcoholic Fatty Liver Disease
Ulcerative Colitis Aerobic Endurance
Crohn’s Disease Physical Endurance
Clostridium Difficile Infection Power
Depression Prone to Fatigue
Anxiety Gluten intolerance
Rheumatoid Arthritis Asthma
Chronic Kidney Disease Eczema
Atherosclerosis Atopic dermatitis
Obesity Acne
Hypertension Effect of Smoking

Parkinson’s Disease Autism spectrum disorder
Alzheimer’s disease Preeclampsia
TABLE A: 30 diseases based on gut microflora profile
[0105] Furthermore, the Rych Index can also aid the physicians, clinicians, and nutritionists to monitor the impact of their therapeutic or nutritional intervention, since the Rych Index scores shifts from a dysbiosis score [pre-intervention] to an eubiosis score [post-intervention].
[0106] 2. PROCESS FLOW:
[0107] The Rych Index marker estimates “the state of eubiosis or the degree of dysbiosis” of the gut microbiome of an individual, as a single score on a simple yet effective gradient.
[0108] The Index Score is established by cumulatively considering the gut microbiota composition, abundance, diversity, and other key microbial indicators.
[0109] The established index is represented on a gradient of +5 to -5, with a single score representing the “current status” of the individual’s gut microbiome.
[0110] A score leaning towards +5 represents an ideal state of eubiosis and hence largely beneficial, while a score leaning towards -5 represents a non-ideal state of dysbiosis and hence potentially detrimental.
[0111] FIG 1 shows The Rych Index in one of the example embodiments. It shows one such gradient.
[0112] The process of arriving at this Rych index largely includes the following steps,
[0113] Any gut microbiome metagenomics sample characterized by any metagenomics analysis pipeline can be used as the input to arrive at The Rych Index.
[0114] The index utilizes,
[0115] The composition – different types of microorganisms identified.
[0116] The abundance – the normalized quantity of the identified microorganism
[0117] The diversity measure – how diverse the microorganisms are.
[0118] Other critical markers of eubiosis/dysbiosis curated within MicroByte.
[0119] The method cumulatively estimates a value applying statistical measures on all these values and assigns a final score on a scale of -5 to +5.
[0120] As mentioned above,
[0121] a score leaning towards +5 represents an ideal state of eubiosis and hence largely beneficial and have a healthy gut microbiome. Also, represents a state where the individual is at a relatively lower risk of developing diseases that might be influenced by the intestinal microbial health.
[0122] a score leaning towards -5 represents a non-ideal state of dysbiosis and hence potentially detrimental and can be associated with many diseases. Also, represents a state where the individual is at a relatively higher risk of developing diseases. Examples as listed above.
[0123] Furthermore, the Rych Index can also aid the physicians, clinicians, and nutritionists to monitor the impact of their therapeutic or nutritional intervention, since the Rych Index scores shifts from a dysbiosis score leaning towards -5 [pre-intervention] to an eubiosis score leaning towards +5 [post-intervention].
[0124] Overall, The Rych Index score can easily be estimated using any metagenomic data and represented as simple and effective was of representing gut eubiosis or dysbiosis.
[0125] 3. EXAMPLE EMBODIMENTS:
[0126] EXAMPLE EMBODIMENT 1
[0127] The index (marker) has been successfully utilized to represent the overall eubiotic or dysbiotic state of hundreds of stool samples from individuals, who have undergone gut microbiome test. In this section, inventors have exclusively tested the accuracy and the finer aspects of the Rych Index with data generated from these microbiomes, especially pertaining to its extended utility of indicating diseased states.
[0128] To begin with, the Rych Index scoring could characterize the microbial sequencing data into accurate gut microbiome profiles of individuals, bunning them into eubiotic or dysbiotic state. The key validation from these experimental data confirmed our algorithm’s ability to assign accurate Rych Index Scores for each individual being tested. The process flow [discussed above] could accurately assign Rych Index scores, which correlated with any clinical diagnosis of the individuals [as consented and disclosed by them].
[0129] As described under process flow, the Rych Index score was estimated after considering the composition, abundance, ratio, diversity, and other factors of the microbiome, overlay that with evidence curated in MicroByte. Later, the score was estimated on a scale of -5 to +5.
[0130] The Rych Index scoring could not only indicate the overall dysbiotic state of the gut microbiome but also aligned within a specific ranges of Rych Index score [detailed below] on a continuous scale of -5 to +5, strongly indicating the correlation between a specific Rych Index Score and specificity to various diseased states.
[0131] The shift in Rych Index score, post prebiotic nutritional intervention, from a negative score to a positive score, validated the extended utility of the scoring method to monitoring prognosis of any intervention.
[0132] Overall, the study validated the utility of The Rych Index and its utility to represent the state of eubiosis or degree of dysbiosis, and the extent of change in this eubiosis/dysbiosis index.
[0133] The experimental data provided below pertains to few specific diseases, representing its corresponding broader class. We have correlated the accuracy of our Rych Index Score with that of the dysbiosis of one’s gut microbiome, translated as a specific disease, for the following diseases:
[0134] Functional Gastrointestinal Disorders [FGIDs] – representing the ability to estimate the risk of most FGIDs [IBS, IBD, UC, CD, Constipation etc.]
[0135] Hypertension and Atherosclerosis – representing the ability to estimate the risk of other cardiovascular diseases [CVDs].
[0136] Type-2 Diabetes – representing the ability to estimate the risk of other lifestyle diseases or diseases due to systemic/chronic inflammation.
[0137] Depression - representing the ability to estimate the risk of other mental disorders.
[0138] We have also validated the utility of Rych Index as a prognostic marker of any therapeutic/nutritional interventions, estimating the Rych Index scores before and after a prebiotic intervention.
[0139] EXAMPLE EMBODIMENT 2
[0140] Functional Gastrointestinal Disorders [FGIDs]
[0141] Data included a total of 30 gut microbiome profiles from 30 individuals who consented for providing the stool samples for the purpose of this study, of which 15 were diagnosed with at least one of the FGIDs [IBD, IBS or Constipation] [as consented and disclosed by individuals] and 15 were Non-FGID controls. All data were received from patients with their informed consent.
[0142] The following were the key observations:
[0143] There was a strong correlation between the Rych Index score and dysbiotic state of the microbiome. The individuals with already diagnosed FGIDs had moderate to severe dysbiosis as represented by Rych Index, mostly leaning towards -3 of the indices.
[0144] The Index score had a Mean ± S.D of -2.78 ± 0.86.
[0145] Further, the lower the score (toward -5) the higher the number of diseases (FGIDs or otherwise) that the individual was susceptible for.
[0146] Individuals with a Rych Index score in the range of -1.2 to -2.5 were either diagnosed with one of FGIDs with no other comorbidities such as diabetes or hypertension.
[0147] On the other hand, individuals with a severe dysbiosis (close to -4 or -5) were not only diagnosed with one key FGID but were also recorded to be diagnosed with other comorbidities such as hypertension, rheumatoid arthritis, anxiety, and other diseases [as consented and disclosed by individuals].
Non-FGIDs Rych Index Score FGIDs Rych Index Score
Sample_1 2.12 Sample_1 -4.33
Sample_2 2.25 Sample_2 -4.31
Sample_3 2.67 Sample_3 -3.24
Sample_4 2.89 Sample_4 -3.23
Sample_5 3.10 Sample_5 -3.19
Sample_6 3.45 Sample_6 -3
Sample_7 3.51 Sample_7 -2.93
Sample_8 3.57 Sample_8 -2.91
Sample_9 3.6 Sample_9 -2.58
Sample_10 3.63 Sample_10 -2.56
Sample_11 3.66 Sample_11 -2.45
Sample_12 3.78 Sample_12 -2.12
Sample_13 4.29 Sample_13 -1.94
Sample_14 4.5 Sample_14 -1.63
Sample_15 4.95 Sample_15 -1.27

[0148] EXAMPLE EMBODIMENT 3
[0149] Cardiovascular Diseases [CVDs].
[0150] Data included a total of 29 gut microbiome profiles from 29 individuals who consented for providing the stool samples for the purpose of this study, of which 14 were diagnosed with at least one of the CVDs [Hypertension, Atherosclerosis] [as consented and disclosed by individuals] and 15 were Non-CVD controls. All data were received from patients with their informed consent.
[0151] The individuals with already diagnosed CVDs had moderate to severe dysbiosis as represented by Rych Index, mostly leaning towards -1.6 of the indices.
[0152] The Index score for individuals with CVDs had a Mean ± S.D of -1.61 ± 2.51.

Non-CVDs Rych Index Score CVDs Rych Index Score
Sample_1 2.12 Sample_1 -3.55
Sample_2 2.25 Sample_2 -3.5
Sample_3 2.67 Sample_3 -3.24
Sample_4 2.89 Sample_4 -3.2
Sample_5 3.10 Sample_5 -3.07
Sample_6 3.45 Sample_6 -2.14
Sample_7 3.51 Sample_7 -1.92
Sample_8 3.57 Sample_8 2.5
Sample_9 3.6 Sample_9 -3.42
Sample_10 3.63 Sample_10 -3.07
Sample_11 3.66 Sample_11 -2.14
Sample_12 3.78 Sample_12 -1.92
Sample_13 4.29 Sample_13 2.79
Sample_14 4.5 Sample_14 3.37
Sample_15 4.95

[0153] EXAMPLE EMBODIMENT 4
[0154] Depression
[0155] Data included a total of 20 gut microbiome profiles from 20 individuals who consented for providing the stool samples for the purpose of this study, of which 5 were diagnosed depression [as consented and disclosed by individuals] and 15 were Non-Depressed controls. All data were received from patients with their informed consent.
[0156] The individuals with already diagnosed depression had moderate to severe dysbiosis as represented by Rych Index, mostly leaning towards -1.9 of the indices.
[0157] The Index score for individuals with depression had a Mean ± S.D of -1.93 ± 3.08.

Non-Depressed Rych Index Score Depressed Rych Index Score
Sample_1 2.12 Sample_1 -3.93
Sample_2 2.25 Sample_2 -3.42
Sample_3 2.67 Sample_3 -3.07
Sample_4 2.89 Sample_4 -2.76
Sample_5 3.10 Sample_5 3.52
Sample_6 3.45
Sample_7 3.51
Sample_8 3.57
Sample_9 3.6
Sample_10 3.63
Sample_11 3.66
Sample_12 3.78
Sample_13 4.29
Sample_14 4.5
Sample_15 4.95

[0158] EXAMPLE EMBODIMENT 5
[0159] Type 2 Diabetes Mellitus [T2DM]
[0160] Data included a total of 24 gut microbiome profiles from 24 individuals who consented for providing the stool samples for the purpose of this study, of which 9 were diagnosed T2DM [as consented and disclosed by individuals] and 15 were Non-Depressed controls. All data were received from patients with their informed consent.
[0161] The individuals with already diagnosed depression had moderate to severe dysbiosis as represented by Rych Index, mostly leaning towards -0.83 of the indices.
[0162] The Index score for individuals with depression had a Mean ± S.D of -0.83 ± 3.08.

Non-T2DM Rych Index Score T2DM Rych Index Score
Sample_1 2.12 Sample_1 -3.73
Sample_2 2.25 Sample_2 -3.24
Sample_3 2.67 Sample_3 -3.07
Sample_4 2.89 Sample_4 -2.98
Sample_5 3.10 Sample_5 -2.14
Sample_6 3.45 Sample_6 -1.92
Sample_7 3.51 Sample_7 2.79
Sample_8 3.57 Sample_8 3.37
Sample_9 3.6 Sample_9 3.43
Sample_10 3.63
Sample_11 3.66
Sample_12 3.78
Sample_13 4.29
Sample_14 4.5
Sample_15 4.95

[0163] EXAMPLE EMBODIMENT 6
[0164] Rych Index as a Prognostic Marker to track the effect of therapeutic/nutritional intervention in disease
[0165] The Rych Index could also capture the changes in gut microbiome due to intervention with prebiotic. The pre and post samples showed increasing index score and shift from dysbiosis to eubiosis.
[0166] Data included a total of 12 gut microbiome profiles from 12 individuals who consented for providing the stool samples for the purpose of this study. 12 samples were collected, processed and Rych index was estimated before and after the prebiotic intervention. All data were received from patients with their informed consent.
[0167] The Index score for individuals pre-intervention had a Mean ± S.D of -1.24 ± 1.86, which shifted to a Mean ± S.D of +2.31 ± 0.48 post-intervention.

Pre-Intervention Rych Index Score Post-Intervention Rych Index Score
Sample_1 1.99 Sample_1 2.42
Sample_2 1.73 Sample_2 2.26
Sample_3 1.61 Sample_3 2.36
Sample_4 -1.27 Sample_4 1.88
Sample_5 -2.27 Sample_5 1.02
Sample_6 -1.94 Sample_6 2.12
Sample_7 -2.26 Sample_7 2.38
Sample_8 -2.41 Sample_8 2.62
Sample_9 -2.48 Sample_9 2.62
Sample_10 -2.49 Sample_10 2.65
Sample_11 -2.49 Sample_11 2.74
Sample_12 -2.62 Sample_12 2.67
[0168] Further, the risk for FGID the individual was susceptible for also reduced along with other ancillary risk factor estimated, all based on associations between the individual’s change in microbiome profile.
[0169] Furthermore, these change in index score, indicating a shift towards eubiosis post prebiotic intervention, was also corroborated with several clinical parameters supporting the efficiency of the index even further.
[0170] Please note: the extent of shift on the index range (of -5 to +5) is function of the efficacy of the prebiotic. The Rych Index does not claim the efficacy of the prebiotic and is restricted to capture only the extent of change in the overall gut microbiota. However, the utility of the index in such trials is conceivable.
[0171] 4. USES OF THE INVENTION:
[0172] Overall, the innovation enables.
[0173] The use of a universal indexing system as a marker for intestinal microbial health, with a single, simple to represent and comprehend score, that can be implemented in Direct-to-Consumer products, utilizable by nutritionists, researchers, and clinicians.
[0174] Direct consumers can understand the status of their gut health and focus on improving this single score, rather than addressing multiple modalities of gut microbiome.
[0175] Physicians and clinicians, addressing disease(s) involving or influenced by gut microbiome, can utilize this representation of the extent of dysbiosis associated with that disease, and take quick measures for its correction.
[0176] The intestinal microbial health of the following diseases can be effectively monitored, and intervention planned:-
Irritable Bowel Syndrome Colorectal Neoplasm
Constipation Diabetes Mellitus Type 2
Inflammatory Bowel Disease Non-Alcoholic Fatty Liver Disease
Ulcerative Colitis Aerobic Endurance
Crohn’s Disease Physical Endurance
Clostridium Difficile Infection Power
Depression Prone to Fatigue
Anxiety Gluten intolerance
Rheumatoid Arthritis Asthma
Chronic Kidney Disease Eczema
Atherosclerosis Atopic dermatitis
Obesity Acne
Hypertension Effect of Smoking

Parkinson’s Disease Autism spectrum disorder
Alzheimer’s disease Preeclampsia
[0177] Nutritionists, who wants to rely on gut microbiome for customizing the dietary recommendations, can utilize this index easily. Further, they can monitor this index periodically, and customize the recommendations over a period.
[0178] Clinical trials studying the associations of gut microbiome and disease, effect of dietary, prebiotic, probiotic or any therapeutic intervention on disease can utilize the index to,
[0179] Estimate the extent of dysbiosis.
[0180] Monitor the extent of shift from dysbiosis to eubiosis, as a function of time or dietary, prebiotic, probiotic, or any therapeutic intervention.
[0181] Based on the extent of shift, fine tune their intervention
[0182] Overall, has great implications in representing any gut microbiome related functionality to wide array of reciprocates.
[0183] It has a universal appeal, from Direct to Consumer to nutritionists to researchers to clinicians and clinical trials.
[0184] 5. HARDWARE
[0185] Digital Processing System
[0186] Fig 2 is a block diagram illustrating the details of a digital processing system in which various aspects of the present invention are operative by execution of appropriate execution modules, firmware or hardware components. Digital processing system 500 may correspond to any of monitoring devices 120a-120c or server system 150.
[0187] Digital processing system 500 may contain one or more processors (such as a central processing unit (CPU) 501), random access memory (RAM) 502, secondary memory 503, graphics controller 506, display unit 507, network interface 508, and input interface 509. All the components except display unit 507 may communicate with each other over communication path 505 which may contain several buses as is well known in the relevant arts. The components of FIG. 9 are described below in further detail.
[0188] CPU 501 may execute instructions stored in RAM 502 to provide several features of the present invention. CPU 501 may contain multiple processing units, with each processing unit potentially being designed for a specific task. Alternatively, CPU 501 may contain only a single general-purpose processing unit. RAM 502 may receive instructions from secondary memory 503 using communication path 505.
[0189] Graphics controller 506 generates display signals (e.g., in RGB format) to display unit 507 based on data/instructions received from CPU 501. Display unit 507 contains a display screen to display the images defined by the display signals. Input interface 509 may correspond to a keyboard and a pointing device (e.g., touch-pad, mouse), which enable the various inputs to be provided.
[0190] Network interface 508 provides connectivity to a network (e.g., using Internet Protocol), and may be used to communicate with other connected systems. Network interface 508 may provide such connectivity over a wire (in the case of TCP/IP based communication) or wirelessly (in the case of WIFI, Bluetooth based communication).
[0191] Secondary memory 503 may contain hard drive 503a, flash memory 503b, and removable storage drive 503c. Secondary memory 503 may store the data and, which enable digital processing system 500 to provide several features in accordance with the present invention.
[0192] Some or all of the data and instructions may be provided on removable storage unit 504, and the data and instructions may be read and provided by removable storage drive 503c to CPU 501. Floppy drive, magnetic tape drive, CD-ROM drive, DVD Drive, Flash memory, removable memory chip (PCMCIA Card, EPROM) are examples of such removable storage drive 503c.
[0193] Removable storage unit 64 may be implemented using storage format compatible with removable storage drive 503c such that removable storage drive 63c can read the data and instructions. Thus, removable storage unit 504 includes a computer readable storage medium having stored therein computer software (in the form of execution modules) and/or data.
[0194] However, the computer (or machine, in general) readable storage medium can be in other forms (e.g., non-removable, random access, etc.). These “computer program products” are means for providing execution modules to digital processing system 500. CPU 501 may retrieve the software instructions (forming the execution modules) and execute the instructions to provide various features of the present invention described above.
[0195] Digital processing system may correspond to each of user system: local system or remote and server noted above. Digital processing system may contain one or more processors (such as a central processing unit (CPU)), random access memory (RAM), secondary memory, graphics controller (GPU), primary display unit, network interfaces like (LAN, Wi-Fi), and input interfaces (not shown).
[0196] CPU executes instructions stored in RAM to provide several features of the present invention. CPU may contain multiple processing units, with each processing unit potentially being designed for a specific task. Alternatively, CPU may contain only a single general purpose processing unit. RAM may receive instructions from secondary/system memory.
[0197] Graphics controller (GPU) generates display signals (e.g., in RGB format) to primary display unit based on data/instructions received from CPU. Primary display unit contains a display screen (e.g. monitor, touchscreen enabled monitor) to display the images defined by the display signals. Input interfaces may correspond to a keyboard, a pointing device (e.g., touch-pad, mouse), a touchscreen, etc. which enable the various inputs to be provided. Network interface provides connectivity to a network (e.g., using Internet Protocol), and may be used to communicate with other connected systems. Network interface may provide such connectivity over a wire (in the case of TCP/IP based communication) or wirelessly (in the case of Wi-Fi, Bluetooth based communication).
[0198] Secondary memory may contain hard drive (mass storage), flash memory, and removable storage drive. Secondary memory may store the data (e.g., the specific requests sent, the responses received, etc.) and executable modules, which enable the digital processing system to provide several features in accordance with the present invention.
[0199] Some or all of the data and instructions may be provided on a removable storage unit (SD card), and the data and instructions may be read and provided by removable storage drive to CPU. Floppy drive, magnetic tape drive, CD-ROM drive, DVD Drive, Flash memory, removable memory chip (PCMCIA Card, EPROM) are other examples of such removable storage drive.
[0200] Software Architecture:
[0201] Removable storage unit may be implemented using storage format compatible with removable storage drive such that removable storage drive can read the data and instructions. Thus, removable storage unit includes a computer readable storage medium having stored therein executable modules and/or data. However, the computer (or machine, in general) readable storage medium can be in other forms (e.g., non-removable, random access, etc.). CPU may retrieve the executable modules and execute them to provide various features of the present invention described above.
[0202] 6. ADVANTAGES OF THE INVENTION:
[0203] The Rych Index rely on comprehensive analysis of all [Bacterial, Archaeal, Fungal, Viral, Metazoal and Protozoal] composition, abundance, diversity and many more ‘eubiosis/dysbiosis’ factors into consideration.
[0204] Simple yet effective index with and single score representing ‘eubiosis/dysbiosis’.
[0205] The Rych Index indicates, utilizing all those key factors, achieves exactly this and hence is more robust.
[0206] It can be applicable in all areas from DTC products to health and wellness representations, nutritional, research and clinical practices.
[0207] The present disclosure provides for an understanding the association between gut microbiota, health, and disease states, and provides for potential diagnostic and therapeutic targets.
[0208] 7. BEST MODE TO PRACTICE
[0209] The best mode to practice the invention is through commercialization of this invention through the distributors, e-commerce sites, and marketing to reach private clinics and hospitals.
[0210] According to aspects of the present invention, the invention also provides kits that are useful for the practice of the methods of the invention.
[0211] 8. USES, APPLICATIONS, AND BENEFITS OF THE INVENTION
[0212] Direct to Consumer.
[0213] Helping in decision making of doctors and dieticians.
[0214] End to end solution from registration to report.
[0215] Merely for illustration, only representative number/type of graph, chart, block, and sub-block diagrams were shown. Many environments often contain many more block and sub-block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed.
[0216] While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.
[0217] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
[0218] It should be understood that the figures and/or screenshots illustrated in the attachments highlighting the functionality and advantages of the present invention are presented for example purposes only. The present invention is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.
[0219] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

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,CLAIMS:CLAIMS

I/ We claim,
1) A method for determining gut microbiome eubiosis/dysbiosis marker in a subject, the method comprising the steps of:
a) receiving a biological sample from a subject;
b) extracting DNA;
c) sequencing DNA through next generation sequencing;
d) analyzing the microbial quality, quantity and diversity;
e) determining microbiome marker score;
f) gathering information from microbiome profile;
g) compiling the effective score; and
h) interpreting eubiosis/dysbiosis marker, The Rych Index Marker of the subject.
2) The method as claimed in claim 1, wherein the Rych Index correlates with various disease conditions is provided based on individual’s gut microbiota.
3) The method as claimed in claim 1, wherein the method estimates the extent of dysbiosis.
4) The method as claimed in claim 1, wherein the method monitors the extent of shift from dysbiosis to eubiosis, as a function of time or dietary, prebiotic, probiotic, or any therapeutic intervention.
5) The method as claimed in claim 1 or 2, wherein said disease conditions are Irritable Bowel Syndrome, Colorectal Neoplasm, Constipation, Diabetes Mellitus Type 2, Inflammatory Bowel Disease, Non-Alcoholic Fatty Liver Disease, Ulcerative Colitis, Aerobic Endurance, Crohn’s Disease, Physical Endurance, Clostridium Difficile Infection, Power, Depression, Prone to Fatigue, Anxiety, Gluten intolerance, Rheumatoid Arthritis, Asthma, Chronic Kidney Disease, Eczema, Atherosclerosis, Atopic dermatitis, Obesity, Acne, Hypertension, Effect of Smoking, Parkinson’s Disease, Autism spectrum disorder, Alzheimer’s disease, Preeclampsia or any combination thereof.
6) The method as claimed in claim 5, wherein the method for estimating the intestinal health of the subject based on the gut microbiota profile.
7) The method as claimed in claim 5, wherein the said analyzing comprises a method, such as a quantitative method, selected from the group of PCR, rtPCR, qPCR, multiplex PCR, high-throughput sequencing, metatranscriptomic sequencing, identification of strain-specific markers, such as genes and/or proteins, and 16S rDNA analysis.
8) A method for monitoring the progress of microbiome-based disease condition comprising the steps of:
a) obtaining sample comprising the gut microbiome from a human patient to be diagnosed
b) extracting DNA;
c) sequencing DNA;
d) analyzing the data;
e) concluding on microbiota in said patient, wherein an increase of the abundance of a said species as detected in said sample compared to a sample from a control patient;
f) monitoring the disease progress; and
g) recommending a personalized nutritional requirement.
9) A method for assessing the status of a disease, condition, pathology, or cell state, wherein the disease, condition, pathology, or cell state is associated with the presence or level of a cellular component, the method comprising the steps of detecting and/or isolating or removing the cellular component associated with the disease, condition, pathology, or cell state by a method according to any of Claims 1 or claim 8, and correlating the presence or level of the cellular component with disease, condition, pathology, or cell state.
10) A system comprising:
a portable device to capture microbial quality, quantity and diversity data from biological sample of a subject;
a server system to:
compute a plurality of input characteristics of dysbiosis to eubiosis, as a function of time or dietary or prebiotic or probiotic based on said input characteristics; and
send for display a comparison of said dysbiosis to eubiosis score to a user;
wherein said score enables recommending a personalized nutritional regimen based on gut microbiota of the subject.
11) A computer-readable medium comprising a plurality of digitally-encoded profiles wherein each profile of the plurality has a plurality of values, each value representing the abundance of a biomolecule in a subject host microbiome.
12)The computer readable medium of claim 11, wherein each profile of the plurality of digitally-encoded expression profiles are associated with a compound related disorder.

Dated this 17th day of April
Signature:
Name: Dr Lipika Sahoo
Registration Number: IN/PA 2467
Agent for Applicant