Laboratory study finds gut microbiota profiling feasible using leftover FIT samples

BackgroundNumerous countries use the EXTEL HEMO-AUTO MC Quantitative Faecal Immunochemical Test (qFIT) to screen for faecal haemoglobin. We aimed to determine if bacterial 16S rRNA gene sequencing results from the leftover qFIT cassettes would be stable over time and comparable with larger volume faecal collection protocols. MethodsFour qFIT probe samples were taken from each of sixteen fresh healthy volunteer stool samples and sequencing results were compared after 0, 4, 7 and 14 days to provide a baseline control and mimic postage and sample processing conditions in cancer screening programmes. qFIT results were then compared to those of standard laboratory processing of larger whole-stool samples. DNA was extracted from 100 NHS surplus qFIT samples from symptomatic patients reporting rectal bleeding and quantified to assess suitability for 16S rRNA gene sequencing. ResultsBacterial composition and diversity from healthy volunteer qFITs remained stable over 14 days with only minor differences compared to baseline (day 0) and larger stool control samples; at least 75% of the symptomatic qFITs yielded sufficient DNA for 16S rRNA gene sequencing. ConclusionqFIT samples were almost identical to control samples and stable over 14 days, allowing them to be used for large-scale low-cost population-based intestinal microbiota studies. Clinical Trial RegistrationThe study was registered on clinicaltrials.gov (NCT06100549). Data summaryThe datasets generated from human samples supporting the conclusions of this article are available in the Figshare repository: https://figshare.com/s/3e75fcba093f13750402. The raw sequencing datasets used for this study have been deposited in the NCBI Short Read Archive, under accession number PRJNA1268008 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1268008. Impact statementThe quantitative Faecal Immunochemical Test (qFIT) is now widely used in colorectal cancer screening and symptomatic testing to detect faecal haemoglobin, with millions of samples tested annually worldwide. As only microlitres of the sample-containing buffer are required for haemoglobin testing, this provides a potentially very valuable, otherwise wasted resource of surplus samples, which could be used to analyse the gut microbiota composition by 16S rRNA gene sequencing or metagenomic methods. We have demonstrated that the EXTEL HEMO-AUTO MC collection picker, widely used for screening and symptomatic testing, and which uses only 2 mg of faeces, yields sufficient DNA for microbiota sequencing (with stability over 14 days) and 16S rRNA gene sequencing results were generally comparable to those from larger, conventionally collected faecal samples. An ability to reliably measure gut microbiota composition in surplus qFIT samples, with associated data linkage, could lead to large-scale, low-cost, population-based studies, including longitudinal studies from ages 50 to 74 years. Such an approach would provide unparalleled statistical power to assess links between the gut microbiota and some of the most prevalent causes of poor health, including cardiovascular disease, chronic inflammatory disease and diabetes, as well as providing vital insights into the gut microbiota during cancer development and treatment response.