DOI: 10.5281/zenodo.18368165
title: Raw Data from Clinical Trial NCT03582826

description: Sample sets and linkage to symptom data

Gut microbiome samples were organized into several analytic sets based on the testing for TMAU (GUT1T1: positive for TMAU1 or genetic testing, GUT1T1: positive for TMAU2, GUT1TN: negative, GUT1NT, not tested, results from 2018) and time of testing (2018 vs 2019). Correct reporting of kit numbers in the online survey enabled reliable linkage between taxonomic profiles and symptom/quality-of-life (QoL) measures at the time of sampling. Remaining data with taxonomic information but incomplete or inconsistent kit identifier reporting was used for microbiome-only analyses and sensitivity checks.

Recruitment, consent, and privacy protections

Participants were recruited from prior related studies, social media, and ClinicalTrials.gov (NCT03582826). All participants provided electronic informed consent after receiving a complete description of the study, under a protocol approved by the MEBO Research Institutional Review Board (May 11, 2018). Study materials were available in English and Spanish, and the study followed ethical principles described in the Declaration of Helsinki and international biomedical research guidelines. Participants did not receive incentives.

An enrollment interview was administered remotely by the study coordinator to collect shipping information for test kits and to document relevant medical history and prior testing (e.g., TMAU urine tests, genetic testing, gastrointestinal, dermatologic, dental, urologic/endocrine evaluations), detailed descriptions of odor-related conditions, participants’ ability to self-detect malodor, and the availability of a trusted person (“buddy”) who could provide external feedback. Personal information was kept confidential. Each participant was assigned a de-identified unique identifier used for survey submission and kit reporting; research personnel accessed de-identified data only, while linkage between identifiers, kit IDs, and medical histories was maintained by the study coordinator under restricted access.

Surveys and symptom characterization

Participants completed an online survey at baseline and after sampling to capture symptoms and QoL indicators. QoL was assessed using a 24-item instrument adapted from the Halitosis Associated Life-quality Test (HALT) and WHOQOL-100 frameworks, primarily using 0–5 Likert scales; negatively framed items were transformed so higher scores reflected better QoL. The survey produced a total QoL score (range 20–150) spanning four domains: physical health, psychological health, social support, and environment. The survey did not collect personally identifiable information and relied on participant-entered identifiers for linkage to enrollment history.

Symptoms consistent with MEBO and PATM were assessed by participant report and, when available, trusted-buddy feedback. Measures included perceived disease state (active vs regression/remission), symptom frequency (5-point Likert scale referencing the previous 24 hours/few days), symptom detection distance (10-point scale), and symptom duration. Additional questions captured severity via behavioral impacts, psychological symptoms (e.g., anxiety, stress, depression), social interactions, physical health, and comorbidities. The survey was iteratively refined with community feedback and reviewed for usability in a small pilot.

Sample collection and sequencing

Consenting participants were mailed three gut microbiome sampling kits containing sterile polyester swabs, sterile water for pre-wetting, and collection tubes with zirconia beads plus lysis/stabilization buffer, along with written instructions. Participants collected samples following flare-ups or symptom improvements and returned samples by mail.

Microbial DNA was extracted using mechanical disruption with beads followed by guanidine thiocyanate silica column-based purification. The V4 region of the 16S rRNA gene was amplified using barcoded primers (515F/806R) and sequenced on an Illumina NextSeq 500 platform (2×150 bp paired-end). Reads were demultiplexed (Illumina BCL2FASTQ), quality filtered (average Q score >30), trimmed to remove primers and leading bases, and paired reads were appended. Sequences were clustered using Swarm (v2.1.5; distance=1) and taxonomically annotated against the SILVA v132 database, with species-level assignment applied when both forward and reverse reads matched the same SILVA reference at >97% identity.

Outcomes

The primary outcome was relative abundance of bacterial classes derived from operational taxonomic units (OTUs), with analyses comparing microbiome profiles across symptom states (active vs regression/remission). Secondary outcomes examined changes in fecal microbiota associated with symptom improvement.