Stool Testing for the Microbiome - Ready for Primetime?

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2 Stool Testing for the Microbiome - Ready for Primetime? Gillian M. Barlow, PhD Project Scientist, Medically Associated Science and Technology (MAST) Program Cedars-Sinai Medical Center

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4 Take Charge of Your Gut Health Unlock Insights From Your Gut Microbiome Discover the Bacterial Populations that make Us and our Planet Unique Provide Targeted Recommendations for Your Unique Microbial Biology Translate your Unique Microbiome into Personalized Actionable Nutritional Insights Reveal the Deep Interconnections between Microbes and the Processes that Make Life on Earth Possible

5 One-time testing or subscribe and save. Test before and after a lifestyle change Test multiple microbiomes gut, mouth, nose, genitals, and skin Test at least 4 times a year to stay up-to-date on your ever-changing microbiome Provide dietary & supplement interventions Provide you ranking of all known [probiotic] strains and which ones will be the most beneficial for you Personalized nutrition app based on your unique microbiome track and maintain blood sugar levels

6 So Are We Ready for This? Have we reached the point where everyone who comes into GI should have their microbiome tested? Can we use the resulting data to diagnose or predict disease? Can we recommend personalized interventions?

7 Fecal Microbial Transplant (FMT) Promising treatment for Clostridium difficile Also being considered as a treatment for ulcerative colitis, obesity and IBS, amongst others Gut Apr; 66(4):

8 Fecal Microbial Transplant (FMT) FMT practice is booming, ranging from: highly organized stool banking programs individual treatments with patient-identified donors individual and harmful do-it-yourself practices

9 FMT Case 1 32-year-old female underwent FMT for Clostridium difficile infection Chose FMT using 16-year-old daughter as donor Donor screened for human immunodeficiency virus 1 and 2, syphilis, viral hepatitis A, B, and C, C. difficile, Giardia lamblia; routine stool culture for enteric pathogens FMT successful C. difficile resolved Reported by Alang N, Kelly CR. Open Forum Infectious Diseases

10 FMT - Case 1 At FMT Post FMT Donor Recipient Donor Recipient (16 months) 140 lbs 136 lbs BMI lbs 170 lbs BMI 33 Recipient (36 months) 177 lbs BMI 34.5 Alang and Kelly, Open Forum Infectious Diseases 2015

11 FMT - Case 1 Authors comments: Need for Note of caution when considering the use of non-ideal donors for FMT Recommend selecting non-overweight donors for FMT Professional stool donors for FMT are selected on the basis of good health, including a normal BMI Alang and Kelly, Open Forum Infectious Diseases 2015

12 FMT - Case 2 22-year-old female underwent FMT for Clostridium difficile infection FMT donor stepbrother FMT successful C. difficile resolved Chang and Rezaie, Am J Gastroenterol 2017

13 FMT - Case 2 Following FMT, patient developed severe post-prandial bloating, distension, fatigue, and constipation Antidepressants, low FODMAPs diet, peppermint, over-thecounter laxatives, linaclotide, lubiprostone, and prucalopride proved minimally effective Post FMT Recipient Methane 51 ppm Donor Methane 35 ppm

14 FMT - Case 2 First report of IBS-C contracted from an FMT donor Should we screen FMT donors for breath methane (and/or hydrogen)?

15 Lessons from FMT These cases are just two examples of AEs and SAEs resulting from FMT a systematic review in 2016 identified: 78 AEs in 50 papers reviewed Total incidence rate of AEs was 28.5% FMT-attributable AEs included: Cytomegalovirus colitis Norovirus gastroenteritis Relapse of severe C. difficile infection Bacteriemia Urinary tract infections Post infectious IBS symptoms Death Wang et al. PLoS One. 2016; 11(8): e

16 Lessons from FMT Donors were screened to the best of current knowledge BUT we didn t know what we didn t know We don t know enough yet about the gut microbiome and what constitutes a healthy microbiome AND we know much less about other gut colonizers fungi, archaea, viruses

17 What DOES Stool Microbiome Testing Test for? Testing for specific microbial panels Most companies offer full stool microbiome testing Some offer additional tests, such as: Pancreatic Elastase-1 - marker of exocrine pancreatic function Calprotectin, as a marker of neutrophil-driven inflammation Eosinophil Protein X - marker of eosinophil-driven inflammation and allergic response Fecal Secretory IgA - marker of gut secretory immunity and barrier function Beta-glucuronidase - inducible enzyme involved in the metabolism and bioavailability of food and drug compounds Short-Chain Fatty Acids - metabolomic indicator of GI microbiome health And many more

18 Caveat Sequencing Techniques Vary Some companies (not all) describe the sequencing technologies they are using. Descriptions include: Illumina Next-Generation sequencing Use Illumina MiSeq Machines to conduct 16s rrna microbial sequencing on the v4 region Ribosomal sequencing Most advanced sequencing technology available, including full shotgun metagenomic sequencing

19 Frequency So Results Can Vary Also Reporter with Science News sent two swabs of the same fecal sample to two different groups: American Gut Project Commercial company Your Sample Average Similar Diet Same Gender Similar BMI Similar Age Michael Pollan Firmicutes Bacteroidetes Proteobacteria Actinobacteria Verrucomicrobia Tenericutes Cyanobacteria Fusobacteria Other You Healthy Omnivores Vegetarians All You vs. Other Groups Firmicutes Bacteroidetes Proteobacteria Verrucomicrobia Actinobacteria

20 Genus Percent of Sample Genus Percent of Sample Bacteroides Bacteroides 20.3 Ruminococcus 4.78 Faecalibacterium 12.2 Parabacteroides 2.51 Blautia 9.28 Faecalibacterium 2.37 Ruminococcus 4.81 Blautia 2.21 Streptococcus 3.67 Sutterella 1.72 Roseburia 2.05 Lachnospira 1.29 Dorea 1.48 Akkermansia 1.23 Akkermansia 1.42 Ruminococcus* 0.94 Alistipes 1.39 Streptococcus 0.92 Parabacteroides 1.21 Possible explanations: Differences in DNA isolation techniques Differences in sequencing methods Differences in biostatistical analyses used

21 Not unique to commercial microbiome sampling Example from diabetes literature: Qin et al, Nature 2012;490:55-60 & Karlsson et al, Nature 2013;498: Metagenomics studies Qin et al Chinese subjects Karlsson et al - female European subjects in their 70s Qin - decreased levels of butyrate-producing bacteria, including Faecalibacterium prausnitzii, Roseburia intestinalis, R. inulinivorans and Eubacterium rectae Karlsson - decreases in Clostridium and increases in Lactobacillus species Discrepancies in potential metagenomic markers identified in both populations Due to dissimilarities in age, ethnicity and environment between the two cohorts, also sequencing approaches and reference gene catalogs used Qin et al. Nature. 2012;490:55-60; Karlsson et al. Nature. 2013;498:

22 Microbiome Quality Control Project Initiated by Rashmi Singha (National Cancer Institute epidemiologist), Rob Knight and other leaders in the field In order to transition from a basic research environment to the clinic, technologies and computational methods for assessing human-associated microbial communities must be standardized and quality controlled Collaborative effort to comprehensively evaluate methods for measuring the human microbiome, including Sampling human-associated microbes at different body sites Techniques and protocols for handling human microbiome samples Computational pipelines for microbiome data processing Voluntary basis!!!

23 How Do Commercial Companies Base Their Recommendations? How do they translate your unique microbiome into personalized actionable nutritional insights, and maintain normalized blood sugar levels? For the company that markets the app: Samples are carefully analyzed through our patent pending algorithm, producing a unique nutrition profile just for you

24 Zeevi et al Cell (5):

25 Cohort is representative of the adult nondiabetic Israeli population Also characteristic of the Western adult nondiabetic population Dietary intervention worked in 26 people What about different ethnicities etc?

26 What About Outputs? You vs. Other Groups Firmicutes Bacteroidetes Proteobacteria Verrucomicrobia Actinobacteria You Healthy Omnivores Vegetarians Phylum-level comparison to the rest of the population Some will also provide comparisons to other groups, e.g. other vegetarians, healthy omnivores, etc Genus and even species levels All

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29 Do We Need This Are We Ready? HUGE level of information for a lay person and possibly even for many physicians We don t know what many of these microbes do Links to conditions or disease states (e.g. T2D or obesity) are often only associative Sometimes performed only in specific populations may not be generalizable A lot more work is needed on different ethnic populations Some studies have been done in mice only Low diversity bad but what is low? Is there a diagnostic threshold? We are NOT ready yet

30 The Positives: Our understanding of the microbiome is growing daily There is growing evidence for some associations that may be causative - Even if some are still in mouse models, many have very strong evidence, e.g. brain-gut, autism - Some are progressing to human trials This is educating and engaging the public in science - and that is always good!