WO2019125299A1

WO2019125299A1 - Method for analysis of health status

Info

Publication number: WO2019125299A1
Application number: PCT/SE2018/051362
Authority: WO
Inventors: Hans Fischer; Walter Fischer
Original assignee: Gutfeeling Labs Ab
Priority date: 2017-12-22
Filing date: 2018-12-21
Publication date: 2019-06-27
Also published as: EP3728645A4; EP3728645A1

Abstract

The present invention relates to a method for determining gut microbe profile in a sample earlier obtained, comprising the steps: a) providing a sample; b) analyzing the sample of step a) for telomere length.

Description

METHOD FOR ANALYSIS OF HEALTH STATUS

Field of the invention

The present invention relates to a method for determining gut microbe profile in a sample of a human. More particular, the gut microbe profile is to be defined by at least the telomere length, but also in combination with microbe composition including the composition of bacteria, parasites, and/or fungus.

Technical Background

People are becoming more and more healthy and more and more aware about one’s health. Preventive measures such as healthy food and regular exercise is being accompanied by various devices to measure parameters such as pulse, number of hours of sleep and steps taken during the day. People want to know more and have it measured. It is also known that much of the human immune system is regulated in cooperation with the bacteria of our intestines and several probiotics have reached the market. Still people visit physicians with discomfort and some cases cannot be clearly diagnosed. It is known that the microbiome health status is of importance for the general condition of the individual. However, even if the awareness of the importance of microbiomes for the general health it is still connected with great complexity and lack of knowledge. There is still a need for more knowledge of components and parameters and their interrelation with each other and their implications on other parts of the human body.

Summary of the invention

The present invention disclose a method for determining gut microbe profile in a sample earlier obtained, comprising the steps: a) providing a sample; b) analyzing the sample of step a) for telomere length.

In one embodiment of the invention, the step b) further includes analyzing the sample of step a) for microbiome composition.

In one embodiment the present invention is a method for determining gut microbe profile in a sample earlier obtained, comprising the steps: a) providing a sample;

b) analyzing the sample of step a) for microbiome composition, optionally the analysis of step b) comprises measurement of telomere length.

In one embodiment the present invention is a method, wherein the sample of step a) may be selected from the group consisting of fecal, blood, saliva, urine, vaginal epithelial cells, and any combination thereof.

In one embodiment the present invention is a method, wherein the analysis of step b) comprise measurement of telomere length.

In one embodiment the present invention is a method, wherein the analysis of step b) comprise measurement of presence of bacterial, and presence of fungal, parasite or any combination thereof.

In one embodiment the present invention is a method, wherein the analysis of step b) is a DNA analysis, preferably using the human lactase gene to determine levels of lactase enzyme activity.

In one embodiment the present invention is a method, wherein the DNA analysis further comprise the steps separate PCR amplification of telomere DNA sequences and/or length determination.

In one embodiment the present invention is a method, further comprising; c. separate parasite/fungal PCR amplification, preferably using 18S and ITS specific primers; and/or

d. separate bacterial PCR amplification, preferably using 16S specific primers;

e. optionally pooling of samples of step c and d, preferably after genetic bar-coding (DNA-indexes);

f. DNA sequencing of samples of step e, preferably using NGS.

In one embodiment the present method may be used to discover, monitor, and/or facilitate treatment of conditions, preferably selected from the group consisting of inflammatory diseases, metabolic disorders, cardiovascular diseases, autoimmune diseases, neurological diseases, psychiatric disorders / neuropsychiatric disabilities, respiratory diseases, and skin disorders. And in one embodiment the conditions may be selected from the group consisting of fibromyalgia, intestinal inflammation, especially diabetes and obesity, rheumatic diseases, MS, Alzheimer, Parkinson, ALS, autism, ADHD, asthma, COPD, eczema, and psoriasis.

In one embodiment the present invention comprises measurement and analysis of bacteria, fungi, and parasites. This measurement may be performed stepwise, simultaneously or separately.

In one embodiment the present invention comprises measurement and analysis of bacteria, fungi, parasites, and telomeres. This measurement may be performed stepwise, simultaneously or separately.

By the method it is possible to provide a description of present status of the gut microbe profile, as well as develop an individual guidance of how to improve the conditions, for example by food, by specified diet, or by food supplement(s). When describing the gut profile, it is with focus on the content of the microbiome composition with respect of content of bacteria, parasites, and fungus. Also the condition of human telomeres is included in the gut profile. The length of the telomeres reflects the conditions and health of the individual.

Figure

Figure 1 - result of the measurement of telomere length and content of GAPDH in sample taken is presented.

Detailed description of the invention

By the method herein described the telomere length is analyzed. It is a general desire to have and keep the telomeres as long as possible.

For example, the method includes analyzing human intestinal status by using fecal samples for the analysis of telomere length in chromosomes in shredded cells from the gut epithelium of the human host in the presence or absence of human disease, along microbial profile, as well as presence of parasites and/or fungal cells.

It has been found that awareness of the telomere length is important. It is known that the length of telomeres is affected by stress, inflammatory conditions, etc. Therefore, it is of importance to get the knowledge of telomere length of the individual. In particular, by the invention it is possible to provide a guidance of how the individual can lengthen the telomeres. By selecting suitable food, by diet, or by specific food supplement the environment of the gastrointestinal area can be changed, for example the amount of bacteria may increase, the amount of parasites and fungus may decrease, and the length of the telomeres may increase.

Further, it is an aim to increase the amount of Akkermansia ( Akkermansia muciniphilra) in the intestinals The amount of Akkermansia bacteria can be increased by adding the microbe. However, a more suitable way would be to improve the environment and conditions for the Akkermansia microbes already existing in the intestinal flora. For example, supplement with inulin and/or oligofructose creates favourable conditions for growth of the microbes.

By the method herein defined it is possible to provide a guidance for an individual of how to increase the amount of beneficial bacteria in the intestinal system. The guidance may imply than an increase of intake of food rich in inulin and oligofructose is recommended to the individual. Such food is for example asparagus, banana, endives, garlic, onion, etc. Akkermansia is also favoured by increased amounts of polyphenols. Food rich in polyphenols are for example fruit, vegetables, cranberries, tea, coffe, red wine, chocolate, olives, etc.

The guidance can also focus on increasing the amount of “anti-inflammatory bacteria”, thus bacteria producing anti-inflammatory substances. Example of anti-inflammatory substances are butyric acid and propionic acid, but is shall be noted that this list is not exhaustive. For example, Akkermansia and Anaerostipes are bacteria producing butyric acid and propionic acid. Therefore, by increasing the amount of the beneficial bacteria, the amount of the anti inflammatory substances increases.

With the method of the invention it is also possible to indicate the level of parasites present in the individual. Following the result of the analysis of the sample, also a guidance for handling the levels of parasites present in the body may be provided. If a person tests positive for a parasite, for example Dientamoeba fragilis and also has gastrointestinal problems, such as bloating, abdominal pain, and/or loose stool/diarrhea, our guidance is: seek advise from a medical doctor (regarding possible treatment with, for example, metronidazole, and/or follow a guidance on food regimes, remedies, diets and related treatments in order to eradicate the parasite in question.

Shortening of telomeres can result in telomere fusions and increased levels of chromosomal instability, which in turn is a key initiatory event in many cancers including lung, breast, colon and prostate cancer as well as in certain leukemia’s.

Birds with chronic infection by Haemoproteus and Plasmodium parasites (bird malaria) suffered long-term adverse effects which correlated with the shortening of the telomeres with consequences for survival, reproductive function and quality of the offspring.

Telomeres are nucleoprotein structures that cover the ends of the chromosomes. The integrity of the telomere structure and the repetition sequence of its DNA (TTAGGG) is critical for the protection of chromosomes from degradation and for maintaining overall genomic stability.

The number of repeats of the DNA (TTAGGG) is reduced during each cell division in differentiated cells, and as a result, telomere length (TL) often decreases in most differentiated cells throughout the life of the organism.

It has also been shown that telomere shortening is associated with lung, breast, colon, prostate cancer and certain leukemia’s, dementia and reduced cognition.

Further, in a remarkable study from Lund, it has been shown that birds with chronic infection of Haemoproteus and Plasmodium parasites (bird malaria) suffered long-term adverse effects, which correlated with the shortening of the telomeres with consequences for survival, reproductive function and quality of the offspring. The mechanism by which telomeres are shortened in birds with malaria is suggested to be through oxidative stress, which supposedly inhibits the telomerase enzyme activity, an enzyme that extends the telomeres after each cell division.

Physically, our body is a union of human cells and our microbes, where the incomparably greatest bacterial diversity is concentrated to the lower part of the intestinal tract, namely in the colon. Previously, the only possibility to identify microbes in the intestine was by microscopy or by culture, but since most of the microbes in the intestinal flora do not live isolated but in coexistence with other microbes, it was not possible to culture all the intestinal microbes in the laboratory. In fact, only about 80% of the bacterial flora of the intestine has been identified by in vitro culture. Recent studies have shown a connection between the composition of microbial communities in the intestine and various aspects of the host's physiology, including the host's metabolism and immunological functions. Particularly large resources are concentrated to investigate the potential causal link between probable disease-inducing mechanisms from a disrupted bacterial flora in the development of ill health / diseases. And vast resources are invested in finding for example new probiotics.

Therefore, it is of particular interest that, for example, it has been shown that the human microbiome (genes from all microbes in an ecosystem) plays an important role in the maturity of the immune system, neurological signaling, synthesis of vitamins, nerve transmitters, elimination of ingested toxins, as well as an identified production of a variety of substances with unknown function. Given the varying functional repertoire of the intestinal flora, it is therefore natural that this is the focus of research in a wide range of chronic diseases such as:

Inflammatory diseases (e.g. fibromyalgia, intestinal inflammation).

Metabolic disorders (especially diabetes and obesity).

Cardiovascular disease

Autoimmune diseases (e.g. rheumatic diseases, MS)

Neurological diseases (e.g. Alzheimer, Parkinson, ALS)

Psychiatric disorders / neuropsychiatric disabilities (e.g. autism, ADHD) Respiratory diseases (especially asthma and COPD),

Skin disorders (e.g. eczema and psoriasis).

There are two major projects for the analysis of public intestinal flora, namely uBiome (USA) and American Gut (USA) who map the bacterial flora of the gut, but not the presence of parasites such as Blastocystis and fungi such as Candida. Since the intestinal flora consists not only of bacteria, but also of fungi and parasites, there exists a "blind spot" in the above-mentioned projects. It is impossible to make assumptions about any correlation between the gut flora and diseases/health problems without taking into account the potential overgrowth of parasites and/or fungi.

The present invention discloses a method, measuring parasitic and/or fungal presence in the human intestine. The present invention further correlates these measures with the shortening of the host telomeres. In order to correlate telomere length with the presence or absence of parasites, fungus and with bacterial profiles, the present invention use next generation sequencing (NGS) to identify the above mentioned microbes in the same fecal sample.

The method herein described includes a step of providing a sample. The sample is taken from a suitable place depending on the desired guidance to be provided. The method includes samples to be selected from the group consisting of fecal, blood, saliva, vaginal epithelial, urine, and any

combination thereof.

With sample from fecal, urine, and vaginal epithelial cells information both of microbiome composition and telomere length may be obtained.

With samples from blood and saliva information of telomere length may be obtained. Therefore, it might be advantageously to collect samples from different sources, for providing a more accurate result.

The purpose of the present invention is to provide a method comprising innovative steps of using, for example, fecal samples to investigate both chromosomal stability of the host, which indicates the level of stress affecting telomerase activity (an enzyme crucial for the extension of the telomeres in the human host) and the microbial profile of the gut.

The fecal sample is processed in such a way that the human DNA from the naturally shredded gut epithelium and the gut parasite-, fungal and bacterial DNA is preserved during the extraction procedure.

In one embodiment the present method comprise a sample obtained from the mouth cavity.

Specifically, this is achieved by the following: The extraction buffer in the sample tube contain inhibitors of DNA- and RNA degrading enzymes. This is especially important since the time it takes for the sample to arrive at the lab from client is 2-5 days. Other extraction buffers in the sample tube containing inhibitors of DNA- and RNA degrading enzymes can possibly be used instead of the transportation buffer disclosed. The present invention is not to be seen as limited by a specific extraction buffer.

Silica sand and glass beads in the sample tubes are used to shred cells in order to expose, but not disrupt the DNA. Other substances apart from silica sand and glass beads can be introduced into the sample tubes to expose the DNA without disrupting it and the present invention is not to be seen as limited by means for disrupting the DNA.

In order to mechanically disrupt the cell membranes of the human cells and microbes without damaging the long DNA strands, especially the telomeres, a shorter shaking process is performed. Usually shaking cycles of minutes are used. In one embodiment the shaking is performed in an interval of between 1 - 50 seconds. In one embodiment the shaking is performed in an interval of between 1 -30 seconds. In one embodiment the shaking is performed in an interval of between 1 -20 seconds. In one embodiment the shaking is performed in an interval of between 1 -10 seconds. In one embodiment the shaking is performed in an interval of between 1 -5 seconds.

Magnetic rods are used in the purification procedure to decrease risk of DNA shearing of chromosomal DNA (this is especially important for the measurement of telomere length).

Measurement of telomere length and analysis of DNA from parasites, fungus and bacteria can be performed on the same fecal sample at the same time point. This means that the correlation analysis between telomere length and parasite/fungal load can be performed.

Same sample procedure:

Separate PCR amplification of telomere DNA sequences and length determination. Separate PCR amplification of housekeeping gene GAPD for total DNA measurement. Separate parasite/fungal PCR amplification using 18S and ITS specific primers.

Separate bacterial PCR amplification using 16S specific primers.

Pooling of samples (b and c) after bar-coding.

DNA sequencing (of d) using NGS.

For the PCR amplification of telomere DNA sequences and length determination, this is possible to perform on samples from other sources of the body such as the blood, saliva and urine.

There are technical advantages with the present invention since it makes it possible to analyze telomere length in fecal samples as a marker for the local and systemic chromosomal stability in an individual.

Further, there are commercial advantages with the present invention since there is a growing interest in the community concerning the possible involvement of the gut flora in health.

Flowever, the presently commercially available companies of significance performing gut flora analysis offer only analysis of bacteria but not identification of parasites or fungus.

With the analysis of telomere length, there is presented an opportunity to provide a highly relevant measure of chromosome stability. Since the shortened telomere length is partly reversible, motivation for anti-parasite and anti-fungal treatment as well as lifestyle changes to balance the gut bacterial flora may be expected in the wake of analysis according to the present invention.

Providing that bacterial status is known, the oxidative stress provided by an overgrowth of parasites and/or fungi, being related to a telomere length shortening, may disclose presence of parasite/fungi.

Further, providing the parasite/fungi content is known, along with telomere length, i.e. shortening of telomeres, presence of bacteria and/or bacteria populations that stress the body may be identified.

Examples of such bacteria being present may be:

1 . Clostridia or predatory bacteria that give "ADFID". 2. Absence of mucin-growing bacteria that protect and stimulate intestinal mucosa production, thereby protecting the gut epithelial cell barrier or intestinal lymph nodes from direct contact with feces proteins, which may cause inflammation.

3. Cyanobacter that may cause Alzheimer's disease.

4. Bacteria that folds the alpha-synuclein peptide so that after transport via the vagus nerve from the intestine to the brain it catalyzes harmless alpha-synuclein to plaque-forming substances that kill substantia nigra.

By measuring the composition of bacteria, fungi and parasites along with teleomer length a more complete picture of intestinal status may be obtained and thereby success of various treatments may increase along improved health. Through individual guidance on how to balance gut flora, reduce parasite and fungus content it is possible to increase content of fatty acids having an antiinflammatory effect, and to increase telomer length.

EXAMPLES

Eight (8) individuals have been included in a study (denoted FT, ET, MR, MM, MME, L, B, and NN in figure 1 ).

Samples (feces) were taken and tested according to the method herein described.

For analysis and comparison also the content of GAPDFI (glyceraldehyde 3- phosphate dehydrogenase) a housekeeping gene present in human chromosomes was measured. The content of GAPDFI functions as a positive control, indicating the presence of human cells in the sample (feces).

In figure 1 the result of the measurement of telomere length and content of GAPDFI in the sample have been presented (the number indicates PCR cycles, the higher the lower amount of DNA in the sample).

Total DNA (GAPDFI) and telomere signals vary between individuals where e.g ”B” (fungal overgrowth in the gut and low levels of Akkermancia muciniphila) had lower telomere signal than GAPDFI indicating shorter telomeres. On the other hand, MR , MME and L ( no significant amount parasites or fungus, and high levels of Akkermancia muciniphila) showed higher telomere signals compared to GAPDH indicating longer telomers. MM had leukemia and had received chemotherapy before submitting a fecal sample. F showed no human DNA signals illustrating the importance of securing an additional sample from e.g. saliva/mouth epithelium

Claims

1. Method for determining gut microbe profile in a sample earlier

obtained, comprising the steps:

a) providing a sample;

b) analyzing the sample of step a) for telomere length.

2. Method according to claim 1 , wherein the analysis of step b) comprise analysis for microbiome composition.

3. Method according to claims 1 or 2, wherein the sample of step a) may be selected from the group consisting of fecal, blood, saliva, urine, vaginal epithelial cells, and any combination thereof.

Method according to any of claims 1 to 3, wherein the analysis of step b) comprise measurement of presence of bacterial, and presence of fungal, parasite or any combination thereof.

5. Method according to any of claims 1 to 4, wherein the analysis of step b) is a DNA analysis, preferably using the human lactase gene to determine levels of lactase enzyme activity.

Method according to claim 5, wherein the DNA analysis further comprise the steps separate PCR amplification of telomere DNA sequences and/or length determination.

7. Method according to claim 6, further comprising;

c. separate parasite/fungal PCR amplification, preferably using 18S and ITS specific primers; and/or

d. separate bacterial PCR amplification, preferably using 16S specific primers; e. optionally pooling of samples of step c and d, preferably after genetic bar-coding;

f. DNA sequencing of samples of step e, preferably using NGS.

8. Method according to any one of the preceding claims, wherein the present method may be used to discover, monitor, and/or facilitate treatment of conditions, preferably selected from the group consisting of inflammatory diseases, metabolic disorders, cardiovascular diseases, autoimmune diseases, neurological diseases, psychiatric disorders / neuropsychiatric disabilities, respiratory diseases, and skin disorders.

9. Method according to claim 8, wherein the conditions may be selected from the group consisting of fibromyalgia, intestinal inflammation, especially diabetes and obesity, rheumatic diseases, MS, Alzheimer,

Parkinson, ALS, autism, ADHD, asthma, COPD, eczema, and psoriasis.