Deviations in human gut microbiota: a novel diagnostic test for determining dysbiosis in patients with IBS or IBD

Abstract

Background: Dysbiosis is associated with many diseases, including irritable bowel syndrome (IBS), inflammatory bowel diseases (IBD), obesity and diabetes. Potential clinical impact of imbalance in the intestinal microbiota suggests need for new standardised diagnostic methods to facilitate microbiome profiling.

Aim: To develop and validate a novel diagnostic test using faecal samples to profile the intestinal microbiota and identify and characterise dysbiosis.

Methods: Fifty-four DNA probes targeting ≥300 bacteria on different taxonomic levels were selected based on ability to distinguish between healthy controls and IBS patients in faecal samples. Overall, 165 healthy controls (normobiotic reference collection) were used to develop a dysbiosis model with a bacterial profile and Dysbiosis Index score output. The model algorithmically assesses faecal bacterial abundance and profile, and potential clinically relevant deviation in the microbiome from normobiosis. This model was tested in different samples from healthy volunteers and IBS and IBD patients (n = 330) to determine the ability to detect dysbiosis.

Results: Validation confirms dysbiosis was detected in 73% of IBS patients, 70% of treatment-naïve IBD patients and 80% of IBD patients in remission, vs. 16% of healthy individuals. Comparison of deep sequencing and the GA-map Dysbiosis Test, (Genetic Analysis AS, Oslo, Norway) illustrated good agreement in bacterial capture; the latter showing higher resolution by targeting pre-determined highly relevant bacteria.

Conclusions: The GA-map Dysbiosis Test identifies and characterises dysbiosis in IBS and IBD patients, and provides insight into a patient's intestinal microbiota. Evaluating microbiota as a diagnostic strategy may allow monitoring of prescribed treatment regimens and improvement in new therapeutic approaches.

Figure 1

Target regions for the GA primer (1180 bp) and the Illumina primer (459 bp) showing variable (orange V1‐V9) and conserved (grey) regions in the bacterial 16S rRNA gene (1400 bp) utilised by the two methods. The numbers in V3 to V7 denote the number of GA probes targeting each variable region, in total 54 probes. *Illumina application note; http://res.illumina.com/documents/products/appnotes/appnote_16s_sequencing.pdf. ^†Position in E. coli (number of base pairs)40

Figure 2

Flow chart illustrating the GA‐map Dysbiosis Test development, starting with in silico development of bacterial probe set, standardisation of laboratory analysis process, model calibration and verification in healthy individuals (normobiotic reference collection), and validation in healthy, IBS and IBD individuals. Derivation of a DI based on bacterial 16S rRNA DNA analysis in faecal samples demonstrates that a DI score >2 confirms microbiota profile deviations from the normobiotic reference collection.

Figure 3

Distribution of DI scores 1–5 for the validation cohort as determined by GA‐map Dysbiosis Test, showing the increase in DI from healthy individuals through IBS patients and finally in IBD patients.

Figure 4

PCA scores for the first two principal components for validation cohort (n = 287) based on 54 probes. The two PCs account for 48% of the variation, and points are coloured according to (a) cohort: yellow – healthy, blue – IBS, and red – IBD; and (B) DI: grey = 1–2, orange = 3, red = 4, dark red = 5.

Figure 5

Mean normalised signal for top five probes sorted by absolute relative difference between dysbiotic (red) and nondysbiotic (grey) as determined by the GA‐map Dysbiosis Test for (a) IBS patients (n = 109), and (b) IBD patients (n = 135). Act, Actinobacteria; B/Prev, Bacteroides/Prevotella; Firm(b), Firmicutes (Bacilli); Firm (c), Firmicutes (Clostridia); F. prau, Faecalibacterium prausnitzii; Pb, Proteobacteria; Rum.g, Ruminococcus gnavus; Sh/Es, Shigella/Escherichia.

Figure 6

Mean normalised signal for probes sorted by absolute relative difference between dysbiotic (red) and nondysbiotic (grey) as determined by the GA‐map Dysbiosis Test for Spanish cohort (n = 24). Bf; Bifidobacterium, B. ster; Bacteroides stercoris, Parab; Parabacteroides, Pb; Proteobacteria, Sh/Es; Shigella/Escherichia.

Figure 7

Scores for the first three principal components from PCA of normalised data from five healthy subjects collected weekly for up to 14 weeks (n = 64). One point is one sample for donor x taken at time point y. The first three PCs account for 65% of the variation, and points are coloured according to donor.