Table 3.

List of recommendations suggested by microbiome experts

		Non-IVD testing kits	IVD testing kits
Intended use		Curiosity only	Clinical use, diagnostic use
Commercialization channel		Commercialized directly by the companies, publicly available	Commercialized by medical laboratories, medical prescription should be required
Recommendations related to the scientific validity and reliability of the results	Avoid bias in sample collection	•The companies should provide standard operating procedures (SOP) or instructions to the consumer for the sample collection, based on available international standards in order to improve the reproducibility and reliability of their analysis •The kits components for sample collection must be adapted to the intended analytical method •Use of validated sampling devices as well as compliance with international standards (such as CEN/TS 17626:2021) should be highly recommended	•The medical laboratories must provide standard operating procedures (SOP) to the patients for the sample collection, based on available international standards in order to ensure the reproducibility and reliability of their analysis •The kit components for sample collection must be validated and suitable for the intended analytical method •Use of validated sampling devices as well as compliance with international standards (such as CEN/TS 17626:2021) is mandatory
	Harmonize shipping and storage conditions	•Guidelines/consensus should be developed by the scientific community in order to ensure appropriate storage and shipping conditions (stabilizing buffer, temperature, time…) by the different companies •In the case where the consumer lives in another country and it is not possible to ensure good conditions and a reasonable delivery time for the preservation of the sample, the company should not offer its service	•Medical laboratories should provide a standardized sampling and clear instructions regarding the delay when shipping the sample. They then should ensure appropriate shipping and storage conditions for intended analysis, even if the sample is processed and analysed in another location
	Report methodology and its validation	•The company should provide at least a general description of the analytical method used and their potential validation. This should be adapted to the level of understanding of the consumer •The company must avoid over-interpretation of the data. The consumer must not be misled •As the reproducibility and comparability of the results can be limited, the company should not advise the consumer for repeated testing over time. In case of repeated testing, the company should propose to the consumer to re-analyse the previous sample(s) with a new sample in exchange for additional costs. This will ensure the comparison between different samples	•Medical laboratories must provide clear and complete information on the methods used and their validation (“more information leads to more confidence”). This should be adapted to the level of understanding of the end user (different information should be provided to the patient versus healthcare professional) •Medical laboratories must be clear about all of the limitations of the current analytical methods and not provide over-interpretation of the results (for example mentioning SCFA production based only on taxonomic data) •Interpretation of the results and medical consequences should be restricted to healthcare professionals
	Describe the reference cohort	•Transparency is mandatory for reference cohorts, and the main participants characteristics must be provided prior to analysis as well as in the final report •Reference cohorts should be appropriate to the claims •If a proprietary reference cohort is used, the update of the reference cohort (addition of new user/participants in the cohort) must be mentioned as well as the date of the latest update. If a publicly available reference cohort is used, the reference number of the registered cohort must be indicated prior to the analysis and in the report •The authorities should provide a list of main criteria for the cohort constitution, based on scientific evidence	•In order to support the development of IVD, collaborative efforts should be engaged to establish consensus on some important microbiome-related concepts such as “healthy microbiome”, “dysbiosis”,…. A regular update/revision of these concepts is greatly needed •In the future, if microbiome-based biomarkers were to be qualified, this would foster the development of IVD. All stakeholders should engage in collaborative efforts to support the qualification of microbiome-based biomarkers in order to support the development of useful tests for clinical routine
	Harmonize and clarify the status of metadata	•Metadata are necessary for the interpretation of the results. The authorities should provide and/or engage collaborative efforts to establish a validating questionnaire as a “gold metadata standard” to support company and medical laboratories in their activity and to ensure the application of the General Data Protection Regulation •A worldwide coverage concerning the metadata sets is claimed by the experts
	Ensure reliability of analytical methods and interpretation	•Collaborative efforts must be engaged and supported to lead to the standardization of all the steps required for microbiome testing (from sample collection up to downstream analysis of sequencing data)
Recommendations related to the interpretation of the results	Avoid over-promising interpretation of data	•Species claims should be made with caution, when analysis was performed using 16S rRNA gene sequencing. Additional evidence concerning species or strain level claims need to be provided regarding the identification (e.g. coverage, average nucleotide identity to type strain) •The different measures, scores and indices must be explained and supported by scientific validation •The results must not be over-interpreted since it dramatically affects the reliability of the results and the credibility of the field •The report for the consumer must be simple, easy to read and to understand without any mention of health and medical claims or any association to particular diseases or risk of disease •Nutritional and lifestyle advice based on microbiome characteristics are too premature, the scientific evidence does not support such recommendations at this time and the messages are often confusing. Specific nutritional recommendations linked to microbiome profiling should be removed from the reports	•Species claims should be made with caution, when analysis was performed using 16S rRNA gene sequencing. Additional evidence concerning species or strain level claims need to be provided regarding the identification (e.g. coverage, average nucleotide identity to type strain) •An indication of whether the species level is sufficient to assign clinical relevance to any organism mentioned in the report should be clearly stated •The different measures, scores and indices must be explained and supported by scientific validation (gut health index, dysbiosis index, …) •Only a trained healthcare professional should interpret the results of this test and make medical recommendations or a diagnostic •The report sent to the healthcare professional should include details of the analysis and analytical methods used. Another, simpler report could also be made available to the patient
Recommendations related to the regulatory status in Europe	Clarify the regulatory status	•A reinforced oversight from the European authorities is requested •Clarification of the regulatory status and framework are highly needed in Europe
	Avoid over-promising messages	•Health claims must be removed from commercial marketing messages •It is critical to be clear and honest on the goals achieved by this analysis, so as to avoid over-promising expectations •There is a clear need for educational information for the consumer	•No promotional messages, the analysis must be done under a medical prescription for a specific health condition •Health/medical claims are supported with scientific evidence •Regular training for the healthcare professionals to ensure an appropriate interpretation of microbiome data
Recommendations related to the legal notice, privacy policies and ethical aspects	Protect the consumer/patient data (microbiome data and personal data)	•The adoption of the best practices covering transparency related to data and samples use, retention and sharing with different entities should be mandatory •The privacy policy should be easily accessible on the company’s website (upon purchase) and should be included in the kit with a consent form to sign and to return to the company with the sample •Data policy for the samples’ retention time must be clear •For ensuring that consumer rights are upheld, data protections must extend to scenarios involving the sale or liquidation of a company	•For ensuring that consumer rights are upheld, data protections must extend to scenarios involving the closure or transfer of a medical laboratory
	Clarify the status of microbiome data (sensitive personal data or not)	•The authorities should help to define whether microbiome sequencing data are personal sensitive data or not (based on the GDPR)
	Respect ethical principles	•No reference to pathologies/diseases should appear on the report as the consumer can suffer from psychological consequences of receiving alarming messages •The price of these kits is a key concern, especially regarding the limited interpretation and recommendation that can be done today based on microbiome data. Repeated testing should not be recommended due to the price and limited added value of repeated testing (see above, lack of comparison of the data obtained during consecutive tests) •The business model of these companies is not clearly described, and the consumer has to be fully aware of the use of personal data to create private databases • “Name-dropping” should be avoided in the reports provided by the companies	•The sequencing raw data should be sent to the healthcare professional

Based on the analysis of the different services offered and the reports received after the analysis, and on the discussion with experts in the microbiome field during a workshop held on 7 March 2023, a list of recommendations has been established. Common and specific recommendations to support and encourage the development of microbiome testing services have been listed for non-IVD kits (intended for curiosity only) and for the in vitro diagnostic medical device (IVD, intended for diagnostic or clinical use)