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We read with interest the study by Simrén et al 1 addressing the correlation between GI symptoms and functional GI disorders (eg, irritable bowel syndrome (IBS)) and the comment by Hadizadeh et al 2 reporting abdominal pain sensation to be associated with an altered faecal microbiota composition. Hadizadeh et al 2 propose from their study on 159 individuals that their results may allow to develop novel tools for diagnosis and management of IBS and dyspepsia. We tried to replicate their findings in the population-based Study of Health in Pomerania (SHIP-Trend)3 using stool samples from 906 volunteers to analyse faecal microbiota composition and diversity by 16S rRNA gene sequencing as previously described.4 Twenty participants were excluded for incomplete phenotypic data or antibiotic treatment at sample collection. Of the remaining 886 individuals (age: 51 years (40–61), median (first to third quartiles); female: 56.4%), 172 (19.4%) reported abdominal pain or discomfort for at least 3 days per month during the last 3 months (cases), whereas 714 did not (controls). To estimate the association of abdominal pain with beta-diversity, we performed permutational multivariate analysis of variance (PERMANOVA, vegan function ‘adonis’)5 using Bray-Curtis dissimilarity and Jaccard distance, finding significant associations at the genus level (p=0.029 and p=0.010, respectively). At the operational taxonomic unit level (OTU, species proxy), only Jaccard distance was significantly associated with abdominal pain (p=0.005), suggesting a first confirmation of the observation by Hadizadeh et al.2
In a next step, we excluded all individuals with a history of known GI disorders (lactose intolerance, IBD, colorectal cancer, liver cirrhosis, pancreatitis, peptic ulcer disease, recent gallstone disease, cholecystitis or gastritis). Of the remaining 727 study subjects, 113 participants (15.5%) reported GI symptoms, whereas 614 did not. Again we performed PERMANOVA to estimate the association of abdominal pain with beta-diversity. Additionally we also adjusted for the putative confounders age and sex. This analysis did not reveal any significant differences between cases and controls—neither at the genus nor the OTU level. Figure 1 shows the result of a principal coordinate analysis corrected for age and sex (vegan function ‘capscale’) based on Bray-Curtis dissimilarity. There was no substantial shift of cases compared with controls. To further evaluate variations in taxon abundance between cases and controls, we compared all the genera present in at least 20% of all samples using a log-transformed linear model including age and sex as covariates. Again, no significant differences were found after adjusting for multiple testing (Benjamini-Hochberg procedure, BH). Notably, also a direct comparison of unadjusted abundance values between cases and controls with respect to the four genera associated with abdominal pain reported by Hadizadeh et al (Prevotella, Blautia, Streptococcus and Lactobacillus) did not yield any significant differences (BH-corrected).
We see two possible—not mutually exclusive—reasons for the different results between Hadizadeh et al and our study. First, due to a different study design, Hadizadeh et al had selected participants for the epidemiological analysis of IBS.6 Therefore their sample population is likely biased towards a history of abdominal pain with possibly more severe manifestations. This explanation would be supported by the higher proportion of cases (32.7%)2 than in our data set. Second, differences in phenotypic variables between cases and controls, such as age and sex, or known disorders associated with GI symptoms such as gallstone disease or inflammatory disorders, may have confounded putative associations. When we corrected for these factors, we no longer confirmed any association between GI pain and discomfort and intestinal microbiota composition in a truly population-based cohort.
At this point the role of intestinal microbiota in functional GI disorders remains unclear. To what extent they contribute to this group of, probably multifactorial, disease entities clearly requires further investigation in ethnically and phenotypically more precisely defined and larger study populations.
We thank Anja Wiechert, Susanne Wiche and Doris Jordan for technical assistance.
Contributors Planning and concept of study: MML, AF, HV, AAA, FUW, JM. Acquisition of data: FF, TK, FUW, MCR, F-AH. Statistical analysis: FF, TK, MCR. Data interpretation and manuscript revision: FF, TK, MCR, AF, F-AH, UV, HV, AAA, JM, FUW, GH, MML. Writing committee: FF, MML.
Funding SHIP is part of the Research Network Community Medicine of the University Medicine Greifswald, which is supported by the German Federal State of Mecklenburg-West Pomerania.
Competing interests None declared.
Patient consent Not required.
Ethics approval Local institutional review board, University Medicine Greifswald, Germany, registration numbers BB122/13 and BB174/15.
Provenance and peer review Not commissioned; internally peer reviewed.
Data sharing statement All samples and corresponding phenotypic data were obtained from the Study of Health in Pomerania (SHIP) and can be accessed through a data access application form (https://www.fvcm.med.uni-greifswald.de/dd_service/data_use_intro.php).
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