Helicobacter analysis in gastric mucosa samples – comparison of two methodical approaches

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Authors

SLÁMOVÁ Terezie MLČŮCHOVÁ Natálie KALA Zdeněk PAVLOVSKÝ Zdeněk BÖHM Jan KROUPA Radek DOLINA Jiří PROCHÁZKA Vladimír GROLICH Tomáš VACULOVÁ Jitka KUNOVSKÝ Lumír BRENEROVÁ Petra ANDRLA Petr URBAN Ondřej NAVRÁTIL Vít HARUSTIAK Tomáš LISCHKE Robert DEISSOVÁ Tereza LOCHMAN Jan IZAKOVIČOVÁ HOLLÁ Lydie SLABÝ Ondřej BUDINSKÁ Eva BOŘILOVÁ LINHARTOVÁ Petra

Year of publication 2022
Type Conference abstract
MU Faculty or unit

Faculty of Science

Citation
Description Introduction: The presence of Helicobacter pylori of the Helicobacter genus, in the gastric mucosa was associated with both Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). The conventional diagnostic method for the detection of this bacteria in the tissue involves endoscopic biopsy, subsequent histological examination, and immunohistochemistry (IHC). Successful detection using this method, however, depends on the amount and location of H. pylori colonization in the gastric mucosa. 16S rRNA sequencing may provide an efficient culture-independent method of Helicobacter analysis. This study aims to compare results from the standard analysis of H. pylori and 16S rRNA sequencing analysis of gastric mucosal samples from patients with BE or EAC. Methods: Tissue samples were collected from the gastric antrum and the gastric body. In total, 52 patients were included, of which 26 suffered from BE and 26 from EAC. Samples for histological examination were fixed in neutral formalin and embedded in paraffin, and the presence or absence of H. pylori was determined by immunohistochemistry (IHC). Bacterial DNA from esophageal tissues was isolated using the AllPrep DNA/RNA Universal Kit (QIAGEN, Germany). The V1-V2 hypervariable region of the 16S rRNA was amplified using PCR and modified primers 68Fmod and 338R. The whole metagenomic library was prepared using the MiniSeq High Output Kit (2 X 150 paired-end sequencing) and was deeply sequenced on an Illumina MiniSeq 150 bp Instrument. Results: IHC analysis confirmed the presence of H. pylori solely in the gastric antrum in one BE patient, solely in the gastric body in one, and in both gastric antrum and body in 5 EAC patients. The high (? 90%) relative abundance of Helicobacter in both gastric antrum and body was more frequently observed in patients with EAC than in those with BE. In patients positive for H. pylori (in both samples) according to the IHC analysis, 16S rRNA sequencing showed relative abundances of Helicobacter higher than 90%. However, this high relative abundance of Helicobacter was also found in 3 patients in which IHC found no H. pylori. There is a strong co-occurrence relationship between results obtained by IHC and 16S rRNA sequencing, which are represented by the high H. pylori positivity and relative abundance of Helicobacter, respectively (Cramer's V = 0.7174). Hypothesis of independence of results between these two methodical approaches was rejected (Fisher’s exact test, p < 10-6). Conclusion: We found that 16S rRNA sequencing may provide sensitive identification of Helicobacter; the used NGS method is, however, unsuitable for taxonomic resolution at the strain/species level, and thus H. pylori detection. However, in all patients positive for H. pylori in gastric antrum and body on IHC, Helicobacter always dominated as results of 16S rRNA sequencing showed.
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