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

a 2022

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

SLÁMOVÁ, Terezie, Natálie MLČŮCHOVÁ, Zdeněk KALA, Zdeněk PAVLOVSKÝ, Jan BÖHM et. al.

Basic information

Original name

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

Authors

SLÁMOVÁ, Terezie (203 Czech Republic), Natálie MLČŮCHOVÁ (203 Czech Republic), Zdeněk KALA (203 Czech Republic), Zdeněk PAVLOVSKÝ (203 Czech Republic), Jan BÖHM (203 Czech Republic), Radek KROUPA (203 Czech Republic), Jiří DOLINA (203 Czech Republic), Vladimír PROCHÁZKA (203 Czech Republic), Tomáš GROLICH (203 Czech Republic), Jitka VACULOVÁ (203 Czech Republic), Lumír KUNOVSKÝ (203 Czech Republic), Petra BRENEROVÁ (203 Czech Republic), Petr ANDRLA (203 Czech Republic), Ondřej URBAN (203 Czech Republic), Vít NAVRÁTIL, Tomáš HARUSTIAK, Robert LISCHKE (203 Czech Republic), Tereza DEISSOVÁ (203 Czech Republic), Jan LOCHMAN (203 Czech Republic), Lydie IZAKOVIČOVÁ HOLLÁ (203 Czech Republic), Ondřej SLABÝ (203 Czech Republic), Eva BUDINSKÁ (703 Slovakia) and Petra BOŘILOVÁ LINHARTOVÁ (203 Czech Republic, guarantor, belonging to the institution)

Edition

Setkání biochemiků a molekulárních biologů, 2022, 2022

Other information

Language

English

Type of outcome

Konferenční abstrakt

Field of Study

10608 Biochemistry and molecular biology

Country of publisher

Czech Republic

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

poster

RIV identification code

RIV/00216224:14310/22:00127181

Organization unit

Faculty of Science

Keywords in English

Helicobacter pylori; immunohistochemistry; adenocarcinoma; Barrett's esophagus; 16S rRNA sequencing

Změněno: 18/11/2022 11:47, Mgr. Terezie Slámová

Abstract

V originále

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.

Links

LM2018121, research and development project

Name: Výzkumná infrastruktura RECETOX (Acronym: RECETOX RI)

Investor: Ministry of Education, Youth and Sports of the CR, RECETOX RI

NU20-03-00126, research and development project

Name: Hostitelský mikrobiom ve vztahu k rozvoji Barrettova jícnu a adenokarcinomu jícnu

Investor: Ministry of Health of the CR, Host microbiome in relation to Barrett ́s esophagus and esophageal adenocarcinoma development, Subprogram 1 - standard

Citovat

SLÁMOVÁ, Terezie, Natálie MLČŮCHOVÁ, Zdeněk KALA, Zdeněk PAVLOVSKÝ, Jan BÖHM, Radek KROUPA, Jiří DOLINA, Vladimír PROCHÁZKA, Tomáš GROLICH, Jitka VACULOVÁ, Lumír KUNOVSKÝ, Petra BRENEROVÁ, Petr ANDRLA, Ondřej URBAN, Vít NAVRÁTIL, Tomáš HARUSTIAK, Robert LISCHKE, Tereza DEISSOVÁ, Jan LOCHMAN, Lydie IZAKOVIČOVÁ HOLLÁ, Ondřej SLABÝ, Eva BUDINSKÁ and Petra BOŘILOVÁ LINHARTOVÁ. Helicobacter analysis in gastric mucosa samples – comparison of two methodical approaches. In Setkání biochemiků a molekulárních biologů, 2022. 2022.

@proceedings{2232463,
   author = {Slámová, Terezie and Mlčůchová, Natálie and Kala, Zdeněk and Pavlovský, Zdeněk and Böhm, Jan and Kroupa, Radek and Dolina, Jiří and Procházka, Vladimír and Grolich, Tomáš and Vaculová, Jitka and Kunovský, Lumír and Brenerová, Petra and Andrla, Petr and Urban, Ondřej and Navrátil, Vít and Harustiak, Tomáš and Lischke, Robert and Deissová, Tereza and Lochman, Jan and Izakovičová Hollá, Lydie and Slabý, Ondřej and Budinská, Eva and Bořilová Linhartová, Petra},
   booktitle = {Setkání biochemiků a molekulárních biologů, 2022},
   keywords = {Helicobacter pylori; immunohistochemistry; adenocarcinoma; Barrett's esophagus; 16S rRNA sequencing},
   language = {eng},
   title = {Helicobacter analysis in gastric mucosa samples – comparison of two methodical approaches},
   url = {https://ucnmuni-my.sharepoint.com/:b:/g/personal/106107_muni_cz/EWVXKsMQzohOnbY0aXD1X6EBoDm9GVT5ad4qJ5mTJNtQ9Q?e=HkOb81},
   year = {2022}
}

TY  - CONF
ID  - 2232463
AU  - 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
PY  - 2022
TI  - Helicobacter analysis in gastric mucosa samples – comparison of two methodical approaches
KW  - Helicobacter pylori
KW  - immunohistochemistry
KW  - adenocarcinoma
KW  - Barrett's esophagus
KW  - 16S rRNA sequencing
UR  - https://ucnmuni-my.sharepoint.com/:b:/g/personal/106107_muni_cz/EWVXKsMQzohOnbY0aXD1X6EBoDm9GVT5ad4qJ5mTJNtQ9Q?e=HkOb81
N2  - 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.
ER  -

SLÁMOVÁ, Terezie, Natálie MLČŮCHOVÁ, Zdeněk KALA, Zdeněk PAVLOVSKÝ, Jan BÖHM, Radek KROUPA, Jiří DOLINA, Vladimír PROCHÁZKA, Tomáš GROLICH, Jitka VACULOVÁ, Lumír KUNOVSKÝ, Petra BRENEROVÁ, Petr ANDRLA, Ondřej URBAN, Vít NAVRÁTIL, Tomáš HARUSTIAK, Robert LISCHKE, Tereza DEISSOVÁ, Jan LOCHMAN, Lydie IZAKOVIČOVÁ HOLLÁ, Ondřej SLABÝ, Eva BUDINSKÁ and Petra BOŘILOVÁ LINHARTOVÁ. Helicobacter analysis in gastric mucosa samples – comparison of two methodical approaches. In \textit{Setkání biochemiků a molekulárních biologů, 2022}. 2022.

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