Pathology & Data Analytics, Leeds Institute of Medical Research at St James's University Hospital, University of Leeds, Level 4 Wellcome Trust Brenner Building, Leeds, LS9 7TF, UK.
Cancer Institute (WIA), Chennai, India.
Genome Med. 2021 Feb 16;13(1):27. doi: 10.1186/s13073-021-00844-8.
BACKGROUND: The incidence of colorectal cancer (CRC) is increasing in developing countries, yet limited research on the CRC- associated microbiota has been conducted in these areas, in part due to scarce resources, facilities, and the difficulty of fresh or frozen stool storage/transport. Here, we aimed (1) to establish a broad representation of diverse developing countries (Argentina, Chile, India, and Vietnam); (2) to validate a 'resource-light' sample-collection protocol translatable in these settings using guaiac faecal occult blood test (gFOBT) cards stored and, importantly, shipped internationally at room temperature; (3) to perform initial profiling of the collective CRC-associated microbiome of these developing countries; and (4) to compare this quantitatively with established CRC biomarkers from developed countries. METHODS: We assessed the effect of international storage and transport at room temperature by replicating gFOBT from five UK volunteers, storing two in the UK, and sending replicates to institutes in the four countries. Next, to determine the effect of prolonged UK storage, DNA extraction replicates for a subset of samples were performed up to 252 days apart. To profile the CRC-associated microbiome of developing countries, gFOBT were collected from 41 treatment-naïve CRC patients and 40 non-CRC controls from across the four institutes, and V4 16S rRNA gene sequencing was performed. Finally, we constructed a random forest (RF) model that was trained and tested against existing datasets from developed countries. RESULTS: The microbiome was stably assayed when samples were stored/transported at room temperature and after prolonged UK storage. Large-scale microbiome structure was separated by country and continent, with a smaller effect from CRC. Importantly, the RF model performed similarly to models trained using external datasets and identified similar taxa of importance (Parvimonas, Peptostreptococcus, Fusobacterium, Alistipes, and Escherichia). CONCLUSIONS: This study demonstrates that gFOBT, stored and transported at room temperature, represents a suitable method of faecal sample collection for amplicon-based microbiome biomarkers in developing countries and suggests a CRC-faecal microbiome association that is consistent between developed and developing countries.
背景：结直肠癌（CRC）在发展中国家的发病率正在上升，但由于资源匮乏、设施不足以及新鲜或冷冻粪便储存/运输困难，这些地区对 CRC 相关微生物组的研究有限。在这里，我们旨在：（1）建立一个广泛的代表不同发展中国家（阿根廷、智利、印度和越南）的代表性样本；（2）验证一种“资源节约型”的样本采集方案，该方案可在这些环境中使用愈创木脂粪便潜血试验（gFOBT）卡进行，这些卡片可在国际上室温下储存和运输；（3）对这些发展中国家的 CRC 相关微生物组进行初步分析；（4）并与发达国家已建立的 CRC 生物标志物进行定量比较。
方法：我们通过对来自五个英国志愿者的 gFOBT 进行重复评估，来评估国际储存和室温运输的效果，其中两个在英国储存，并将重复样本寄送到四个国家的研究所。接下来，为了确定英国储存时间延长的影响，对部分样本的 DNA 提取重复进行了长达 252 天的实验。为了分析发展中国家的 CRC 相关微生物组，我们从四个研究所的 41 名未经治疗的 CRC 患者和 40 名非 CRC 对照中收集了 gFOBT，并进行了 V4 16S rRNA 基因测序。最后，我们构建了一个随机森林（RF）模型，该模型经过训练并与来自发达国家的现有数据集进行了测试。
结果：当样品在室温下储存/运输以及在英国长期储存后，微生物组都能稳定地进行检测。大规模的微生物组结构按国家和大陆划分，CRC 的影响较小。重要的是，RF 模型的表现与使用外部数据集训练的模型相似，并确定了相似的重要分类群（Parvimonas、Peptostreptococcus、Fusobacterium、Alistipes 和 Escherichia）。
结论：这项研究表明，gFOBT 经室温储存和运输，是发展中国家基于扩增子的微生物组生物标志物粪便样本采集的一种合适方法，并表明 CRC 粪便微生物组与发达国家和发展中国家之间存在关联。
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