Why are international guidelines for gut microbiome testing in clinical practice important?

On December 5, 2024 an international group of microbiology scientists published their international consensus statement on microbiome testing in clinical practice in the Lancet (see link here). This paper was meant to give international guidelines on good gut microbiome testing practices for use in clinical practice. While scientific evidence continues to increase about the strong importance of the micro organisms in the gut microbiome and how they relate to many aspects of human health, experts believe that gut microbiome technology will be used more and more in clinical practice. Hence, it is important to setup international guidelines for the use of gut microbiome testing in clinical practice.

How does the InnerBuddies test compare to these international guidelines?

InnerBuddies is a science driven company and we are a spin-off from the University of Maastricht. That means we are always focused on making sure our products are up-to-date with the latest scientific knowledge and developments. So we obviously were eager to check our existing product against the international guidelines that were established by an international group of experts.

The international guidelines consist of 35 different statements divided into five different working groups. Below we will go through all 35 different statements and explain how the InnerBuddies technology matches with all of these statements.

As you can see below InnerBuddies fully meets 32 of the 35 guidelines and is partly compliant with the remaining 3 guidelines.

Working group 1: general principles and minimum requirements for providing microbiome testing

This working group focused on general principes and some minimum requirements for microbiome testing.

Statement 1: Providers of microbiome testing should communicate a reasonable, reliable, transparent, and scientific representation of the test, making customers clearly aware of the scarce evidence for its applicability in clinical practice

InnerBuddies is focused on:

• Only including functionality in its dashboard and reports for which we feel there is sufficient scientific evidence. All functionality that is launched in our platform needs to be signed off by Koen Venema, a well known international microbiology expert with more than 30 years of experience with background from Wageningen University, Maastricht University and TNO.
• Translating the scientific language into language that is understandable for regular consumers. This is exactly the most difficult part of our job, to translate all this scientific material into language that customers can understand.
• For proper use in clinical practice, gut microbiome testing should get closer to specific medical indications. However, at the moment there is still limited evidence (and accuracy) to be able to do this. That is why the current InnerBuddies product is focused on the healthy population and general health topics (and not specific medical indications).

Statement 2: The provision of a microbiome test involves a complex framework, from the collection of biological samples to the sequencing of the microbial genome and computational analyses, to the release of an interpretable report. Therefore,
providers of microbiome testing should include experts with multidisciplinary competences

This is actually where the InnerBuddies team has a strong Unique Selling Point (USP) since our team combines three different disciplines:

• Microbiology which is all about the knowledge and expertise around lab testing
• Dieticians which is all about the knowledge and expertise around Food & Nutrition & Health but also about how to present everything to consumers so that they can understand what they are doing and that nutritional advice is presented in an actionable way to consumers
• Artificial Intelligence (A.I.) which in this case is all about utilizing the Big Data from microbiology DNA testing to uncover all of the information included in the DNA sequences.

Statement 3: Any change in the clinical management of the patients based on microbiome testing should be made only by their referring physicians or health-care professionals

For InnerBuddies clinical use of gut microbiome testing refers to the use of gut microbiome testing related to specific medical conditions. We have translated this in the following ways:

• We separate "consumer-use" of gut microbiome testing from "health care professional-use" of gut microbiome testing. We serve both types of customers. But our experience has also learned that consumers require the information in a very simple and actionable way while health care professionals want to see more technical information. That is why we will create a separate "expert information" module for health care professionals which includes more detailed technical information on the gut microbiome test.
• In our current InnerBuddies product we do not provide any medical condition related assessment of the gut microbiome or advice related to any specific medical conditions. However, the InnerBuddies team is in the process of organizing independent clinical trials (with universities) on specific medical conditions where the InnerBuddies personalized nutrition advice will be tailored to specific medical conditions. After positive clinical trials, this functionality is targeted to become available in separate products which will be available through reimbursement under medical device regulation for these specific medical conditions.

Statement 4: Laboratories that provide gut microbiome testing should guarantee high quality standards and protection of patient data, and be accredited, registered, and regulated

When InnerBuddies initially started we used the laboratory of the University of Maastricht. At the moment we use two different external laboratories for our gut microbiome testing. Both of these laboratories have high quality standards and are accredited and regulated. With both laboratories we have GDPR processing agreements in place.

 

Statement 5: Validated and up-to-date computational software pipelines and databases aimed at delineating microbial taxonomy are required to provide microbiome testing

This is actually one of the strongest points of the InnerBuddies team since we have both a deep expertise in microbiology and ICT and Artificial Intelligence in our team. We have the following things in place:

• With our two existing external laboratories, we use two different DNA sequencing technologies for gut microbiome testing: Illumina MiSeq and Oxford Nanopore. Both DNA sequencers are among the state-of-the-art in gut microbiome testing. Illumina MiSeq allows accurate microbial taxonomy up to Genus level while Oxford Nanopore Full 16S sequencing allows accurate microbial taxonomy up to Species level.
• We tested multiple different calculation pipelines for each of these DNA sequencers with mock samples to find the best calculation pipeline (highest accuracy in terms of finding the bacteria but also finding the correct abundance of each bacteria). Based on these comparisons, we can state that we have the most accurate microbial taxonomy calculations that are currently available on the market.
• We use the SILVA database for microbial taxonomy. This database is also used by scientists around the world since this is currently the best available validated database for microbial taxonomy.
• We tested both of our external laboratories with mock samples to move to the most accurate lab process for our samples. For example, it is very important to apply specific lab processes to make sure both gram-positive and gram-negative bacteria are found correctly.

Working group 2: procedural steps before testing

 This working group focuses on best practices to apply before the actual gut microbiome testing takes place.

Statement 6: As there is little evidence for the applicability of gut microbiome testing in clinical practice, the direct request by patients for microbiome testing without a clinical recommendation is discouraged

InnerBuddies recognizes that there are two markets for gut microbiome testing:

• Consumers that are curious and want to perform gut microbiome testing to see how their gut microbiome looks like. These consumers also want personalized nutritional advice, personalized food diary advice and personalized probiotics & prebiotics advice. In general, these consumers form part of the healthy population and are eager to work on their preventive general health.
• Testing through health care professionals (clinical practice). These are typically customers with specific medical conditions that seek a solution to their issues. That is also why they talk to healthcare professionals (HCP's). HCP's can then decide whether a gut microbiome test makes sense for that particular patient.

InnerBuddies is not marketing its gut microbiome test based on medical conditions. Because at the moment there is indeed still limited scientific evidence to use gut microbiome testing for specific medical conditions. This decision is left to healthcare professionals who can provide patients with clinical recommendations.

Statement 7: Before testing, key clinical data of the patient, including that which might influence gut microbiome characteristics, should be collected; essential information to be captured should at least include age, gender, BMI, dietary habits, smoking and alcohol status, gut transit time, comorbidities and medications, and past medical history

All InnerBuddies customers need to fill-in a survey which indeed includes questions about all of these topics. We include the answers to all of these questions also in the generation of the personalized advice in terms of nutrition, food diary or probiobitcs & prebiotics.

Statement 8: Patients should not suspend their therapy or change their usual diet before testing, unless recommended by the referring physician

This is correct, InnerBuddies does not provide any recommendation on suspending existing therapies. InnerBuddies does also not recommend to change diets before testing. We do provide diet & nutrition recommendations after performing the test. We do that in a number of ways:

• InnerBuddies provides a personalized food diary advice. This is based on the 3-day food diary that customers fill in plus the answers to the survey questions (this advice is not based on the gut microbiome test). This product generates a row-by-row advice on the 3-day food diary, similar to the normal process of dieticians. Since this advice is based on the existing diet followed by the customer, it makes it very practical and actionable for the customer to follow-up on.
• The personalized nutrition advice from InnerBuddies is based on the gut microbiome test and the answers to the questions from the survey. In this product we recommend specific food items that should be added to the diet and specific food items that should be avoided.

Statement 9: Collection of stool samples should avoid any environmental contamination and ensure genome preservation

InnerBuddies focuses on collecting samples with a good quality and stability. For this purpose we do two specific things:

• In our physical testkit we have included all types of materials that help the consumer easily collecting the stool sample in the toilet. This includes collection tape that you can attach to your toilet and with which you can easily catch the poop so that it does not fall into the toilet water.
• We also have a tube that contains a buffer fluid so that the collected material remains stable and shelf-stable for approximately 2 years. InnerBuddies is actually working together with suppliers to develop buffer liquids that keep the sample stable for a longer period of time.

Statement 10: Collected samples should be shipped to testing laboratories with assurance standards for microbiome sequencing within recommended timeframes and conditions described in the instructions of the collection kits. Once arrived, samples should be stored at –80°C until further processing

Both of our external labs store all received samples free-dried (at - 80 Degrees Celsius) when they arrive. Both labs have assurance standards and are accredited. Both laboratories use different DNA sequencing machines and their processing timelines are according to their Service Level Agreements (SLA's).

Statement 11: The analysis of the microbiome from biological samples other than from faeces, including vaginal, skin, and oral swabs, saliva, and breastmilk samples, should be processed according to existing scientific evidence and clinical indications

InnerBuddies currently only processes stool samples (from faeces) although we are working on multi-omics offerings where we also process other types of samples. In all of these cases, off course we will work according to existing scientific evidence and clinical indications.

Working group 3: microbiome analysis

This working group focused on the actual laboratory process and techniques and also the analysis part of the gut microbiome.

Statement 12: Appropriate methods for gut microbiome community profiling include amplicon sequencing and whole genome sequencing

InnerBuddies uses Next Generation Sequencing (NGS) technologies and specifically amplicon sequencing of the 16S region. For Illumina MiSeq we focus on the 16S V3/4 region while for Oxford Nanopore we focus on the Full16S region. At the moment we are also running some experiments with Shotgun Sequencing (whole genome sequencing).

While competitors sometimes use techniques like qPCR, PCR or flow-cytometry (these techniques are generally cheaper than NGS techniques), they also are less accurate and do not provide a full picture of the gut microbiome community. 

Statement 13: Multiplex PCR and bacterial cultures, although potentially useful, neither can be considered microbiome testing nor can be used as a proxy for microbiome profiling

See above with statement 12. InnerBuddies does not use techniques like Multiplex PCR and bacterial cultures. We use the more expensive (and more accurate) amplicon sequencing techniques.

Statement 14: The pre-processing of raw sequenced data should be detailed before analysis

Indeed InnerBuddies performs all types of pre-processing and filtering before performing the analysis:

1. Removing the primers
2. Filtering out low-quality sequences
3. Filtering out too short or too long sequences
4. Performing chimera filtering
5. Etcetera

Statement 15: The microbiome analysis should include alpha diversity metrics, including richness and evenness

InnerBuddies indeed provides alpha diversity metrics like Shannon's Index, Pielou Eveness (evenness) and number of OTU's (richness).

Alpha diversity metrics are the most important metrics of gut microbiome quality since they have been linked to many health indications in scientific research.

Statement 16: Beta diversity measures should be included in the microbiome analysis

InnerBuddies uses a healthy cohort which is basically a form of beta diversity. We compare the abundance of the top 35 bacteria (our keystone bacteria) to the healthy ranges of the healthy cohort. This compares the gut microbiome quality compared to a cohort of known healthy people and provides guidance to people where their gut microbiome quality could potentially be improved.

Statement 17: A complete taxonomic profiling of gut microbial communities is an essential component of microbiome testing

InnerBuddies performs a complete taxonomic profiling of the gut microbial communities. It is even possible for customers or healthcare professionals to download this data from our portal in CSV fornat. This provides full transparancy into the complete taxonomic profile of the stool sample.

Statement 18: Appropriate comparison to a matched healthy control group should be included in microbiome testing to aid the interpretation of patient taxonomic and diversity profile

InnerBuddies matches the taxonomic and diversity profile to a healthy cohort. Initially InnerBuddies started with a healthy cohort that we inherited from the University of Maastricht. However, after some time InnerBuddies collected a lot more customer data (with structured meta data) so now InnerBuddies is able to periodically update its healthy cohort based on its extensive database.

InnerBuddies is now also moving to a healthy cohort for bacterial pathways. Bacterial pathways are more stable across regions and continents and will even provide a better basis for healthy cohort comparison. This is even more advanced and up-to-date than what the microbiology experts, involving in the international guidelines, are suggesting.

Statement 19: A longitudinal assessment of the patient microbiome at different timepoints might be useful in specific clinical scenarios

InnerBuddies allows its customers (and healthcare practitioners) to take multiple tests, done at different points in time, and compare them against each other. InnerBuddies also offers annual subscriptions which include performing multiple tests during the year. This allows customers to follow the progress that they made by following-up on our different types of personalized advice.

The InnerBuddies platform contains extensive functionality that allows an easy comparison in detail on these different tests.

Statement 20: Metabolomic analysis of biofluids is not recommended in clinical practice. Inference of the patient microbiome “metabolic potential” by its taxonomic profile is discouraged

Most scientific research in the last two years on the human gut microbiome is focused on metabolomic functions of the bacteria versus looking at the bacteria population alone. Different bacteria can have similar biological functions. This is called "metabolic potential" of the gut microbiome since by looking at the metabolic pathways, you can basically look at the biological functions that are performed by the bacteria in the gut microbiome.

There are different technology libraries to be used for calculating metabolic pathways. InnerBuddies uses PictRust2 which is the most respected library and used in most scientific research papers. There are two types of DNA sequencing on which these libraries can be used:

• 16S sequencing (also called amplicon sequencing)
• Shotgun sequencing

Below you can find an overview of these two types of sequencing.

At the moment InnerBuddies utilizes 16S amplicon sequencing in its product. The reason is that this is the most accurate method for bacteria profiling. For functional profiling (predicting metabolic pathway potential) shotgun sequencing is more accurate, however this method is more than 2 times more expensive than 16S amplicon sequencing (which would increase the price of the InnerBuddies product with a factor 2).

InnerBuddies always strives for the best quality - cost ratio for its products. Based on scientific research we know that the functional profiling from 16S amplicon sequencing achieves roughly 90% of the accuracy obtained with functional profiling from shotgun sequencing. While for clinical practice, the actual price of the testing product might be less relevant, for consumer use of the InnerBuddies product price is very relevant (since customers need to pay for it themselves).

So from a scientific perspective, the most accurate would be to use 16S amplicon sequencing for the bacteria profiling while using shotgun sequencing for the functional profiling. In total, this would increase the price of the InnerBuddies product with a factor 3. InnerBuddies has therefore chosen to use the 16S amplicon sequencing for both purposes to keep the product pricing at a low level. Since the accuracy of functional profiling is roughly 90% of the accuracy from shotgun sequencing, this is for InnerBuddies an acceptable compromise.

When the InnerBuddies product will be used in clinical practice (for specific medical indications), this might lead us to also include shotgun sequencing inside the product. Since the clinical practice product would be likely covered under reimbursement from healthcare insurances, the balance in terms of quality versus cost would be different. For clinical practice purposes, we would more likely favour shotgun sequencing. InnerBuddies by the way is currently already using shotgun sequencing for its InnerSoils soil microbiome business.

Working group 4: characteristics of reports

This working group focused on what is good practice in the area of displaying the results and advice from microbiome analysis.

Statement 21: Data concerning the patient medical history should appear in the final report

InnerBuddies displays the answers to the survey questionaire as part of its customer dashboard. 

Statement 22: The report should briefly detail the test protocol, including methods of stool collection and storage, DNA extraction, amplification, sequencing, and post-sequencing analyses

We consider this type of information to belong to the Intellectual Property (IP) of InnerBuddies. It took a lot of time, cost and effort to arrive at a gut microbiome analysis workflow of a high & stable quality.

However, for clinical practice purposes we understand that this type of information is relevant for healthcare professionals. Once we launch our clinical practice products (with focus on specific medical conditions), we will obviously include this type of information.

Statement 23: Alpha and beta diversity measures assessed in the testing phase should be included in the final report

InnerBuddies includes both types of data both in its customer dashboard but also in its PDF reports. 

Statement 24: Microbiome composition should be described with the deepest possible taxonomic resolution

InnerBuddies provides genus level taxonomic resolution for the Illumina MiSeq 16S V3/4 region testing. We do not provide species level resolution for this analysis, since we know it is not accurate enough (although we do see competitors providing this with this type of technology).

InnerBuddies provides species level taxonomic resolution for the Oxford Nanopore Full16S testing. We do not provide strain level resolution for this analysis, since we know it is not accurate enough.

Our customers are able to download the full taxonomic profile in CSV and Excel formats from our customer dashboard. This is quite unique functionality since most gut microbiome testing companies do not provide this option.

Statement 25: The report should include all taxa that shift significantly from healthy matched controls and known microbial pathogens. The report of specific health-relevant taxa and clusters, regardless of their abundance, might be of interest, despite the scarce evidence for a causal connection with human diseases

InnerBuddies includes both aspects into its reporting. InnerBuddies is using its top 35 of important bacteria (keystone bacteria). This top 35 has been established as those bacteria for which science has most knowledge available. InnerBuddies divided these bacteria into positive bacteria, negative bacteria and other relevant bacteria (science knows these other relevant bacteria are important but science is not 100% clear yet on whether these bacteria are good or bad).

All top 35 bacteria are compared in terms of abundance with our healthy cohort. For each bacteria in the top 35 we include information on the role and function of that bacteria.

Statement 26: The reporting of Firmicutes-to-Bacteroidetes ratio in the microbiome testing is discouraged

While many other gut microbiome testing companies indeed provide this ratio in their report, InnerBuddies has decided not to do this. It is quite an old custom to include this specific ratio and science is currently aware that this ratio is not really important and conclusive.

Statement 27: There is insufficient evidence to include any dysbiosis index in the report of microbiome testing, but these metrics warrant further research

We have indeed seen other gut microbiome testing companies provide things like "Dysbiosis Index" or "General Gut Health Index", basically trying to display the gut health into a simple number. While many scientists are working on trying to find such a simple index, there is currently no generally accepted and correct method of displaying gut health into a simple number. Therefore, InnerBuddies has decided not to include such a number into its customer dashboard or PDF reports yet. Although we are working with universities on new innovations that could make such a simple gut health number possible.

Statement 28: Generally, there is not enough information to report strict healthy reference ranges of species relative abundance

One of the best practices mentioned above is utilizing a healthy cohort (beta diversity) to compare the gut microbiome composition from the stool sample with the healthy cohort. InnerBuddies utilizes a healthy cohort which is defined on "genus" level, not on "species" level. And this is exactly because of this guideline since there is not enough information to report min-max levels from a healthy cohort based on species relative abundance levels.

Statement 29: The use of a user-friendly infographic—eg, barplots or boxplots displaying the relative abundances of key taxa—is recommended to make the report easily interpretable, while simple ordinations of taxa should be avoided

Since its foundation in 2021, InnerBuddies has spent considerable time thinking about how best to display the gut microbiome testing information to its customers using visualization techniques. We are using infographic visualizations indeed to show how the abundance of a particular bacteria compares to the healthy ranges for that bacteria.

Statement 30: The panel discourages the reporting of any post-testing therapeutic advice by the testing provider

The expert panel has indicated that there is insufficient evidence yet to be able to provide post-testing therapeutic advice based on the gut microbiome testing. InnerBuddies however does provide three types of personalized advice:

1. Personalized nutrition advice
2. Personalized food diary advice
3. Personalized probiotics & prebiotics advice

However, InnerBuddies provides these advice products not only on the results from the gut microbiome test, but also on the answers to the survey questionaire and the 3-day food diary that customers fill in. Actually, the personalized food diary advice is primarily based on the 3-day food diary and it not based on the gut microbiome test. Also, the personalized probiotics & prebiotics advice is more based on the survey questionaire than on the gut microbiome test. There is a simple reason for this:

• Most probiotics have been clinically validated on specific functions and target groups. This information comes from the survey questionaire and not from the gut microbiome test.
• A lot of the bacterial strains that have been clinically validated are so-called "synthetic bacterial strains" which means they have been manufactured in a laboratory and do not naturally occur in the gut microbiome. Therefore, a gut microbiome test will typically not be able to measure these bacterial strains unless the customer has recently taken this specific probiotic strain as part of a food supplement.

The InnerBuddies personalized nutrition advice is based on a combination of all 3 data sources (questionaire, 3-day food diary and the gut microbiome test) but for the gut microbiome part, it is highly based on specific datasets and algorithms that are proprietary to InnerBuddies. These are not available to the scientific domain. InnerBuddies is working together with universities though to validate these proprietary datasets and algorithms in clinical trials. Once the research becomes available in terms of scientific publications, you will be able to find them on this website. But converting gut microbiome testing is personalized nutrition advice is definitely state-of-the-art and InnerBuddies is quite unique in this field.

Statement 31: Raw data can be provided to the patient upon request (eg, for a second-opinion analysis) in form of amplicon or metagenomic reads (based on the sequencing method)

Yes, InnerBuddies allows its customers to download the full taxonomic profile from its customer dashboard. 

Working group 5: relevance of microbiome testing in clinical practice: present and future

This working group primarily focused on the future use of gut microbiome testing in clinical practice.

Statement 32: There is insufficient evidence to widely recommend the routine use of microbiome testing in clinical practice, which should be supported by dedicated studies

Indeed, at the moment gut microbiome testing is not widely used in clinical practice. However, given the accumulating evidence of the importance of gut microbiome health for human health, experts expect gut microbiome testing to become just as normal as a blood test in clinical practice in the next 5 to 10 years. InnerBuddies wants to be at the forefront of this development. Therefore, InnerBuddies is actively working together with universities to test its personalization algorithm in clinical trials, targeted on specific medical conditions.

Statement 33: Qualitative or quantitative data retrievable from microbiome reports might be helpful in the management of several disorders, although there is still insufficient evidence to apply them in clinical practice

That is correct. However, multiple research projects are exploring the clinical practice use of gut microbiome testing for specific medical conditions. InnerBuddies for example is involved in studies on Obesity, IBS, Stress and other conditions. These products obviously will only be launched after positive clinical trial data becomes available.

Statement 34: Studies aimed at evaluating the value of microbiome profiling in different disorders are needed to enable testing to enter clinical practice

Yes, many studies are being done evaluating the accuracy of using gut microbiome testing to "test" for specific diseases with the hope that tests can be developed that can give an early warning for these diseases. InnerBuddies for example is involved in studies on Obesity, IBS, Stress and other conditions. These products obviously will only be launched after positive clinical trial data becomes available.

Statement 35: Disclosure of the potential benefits and pitfalls of microbiome testing, and training on the basics of microbiome science and on the interpretation of microbiome reports, are advocated to foster and disseminate their use in clinical practice

 Yes, InnerBuddies is working together with healthcare professionals to update them on the benefits of gut microbiome testing and what is scientifically possible today. InnerBuddies will continue to do this once more science becomes available and these types of tests will be used more widely in clinical practice.