PhD Scientific Days 2024

Pathological and Oncological Sciences I.

Detection of Helicobacter pylori and its clarithromycin resistance from formalin-fixed, paraffin-embedded gastric biopsy samples by quantitative real-time PCR.

Text of the abstract

Introduction: The diagnostic accuracy of detecting Helicobacter pylori (HP) and its clarithromycin susceptibility (Cla-susc) from gastric biopsies by immunohistochemistry (IHC) and fluorescence-in-situ-hybridization (FISH) compared to isolated DNA-based polymerase chain reaction (PCR) methods is still controversial and needs to be investigated.
Methods: Formalin-fixed paraffin-embedded (FFPE) gastric tissue samples from 209 patients were collected. Tissue sections were stained by Helicobacter-IHC and Cla-susc FISH. DNA was isolated for quantitative-PCR (qPCR), and a 122 bp sequence of the bacterial 23SrRNA gene was amplified using a custom designed primer pair.
Results: 108/209 specimens was HP-positive by IHC and FISH and 101 was HP-negative. Quantitative-PCR detected the bacterium from 102/108 IHC/FISH-HP-positive and 25/101 IHC/FISH-HP-negative cases. From the 42 samples Cla-susceptible by FISH, qPCR-melting point analysis identified 34/5/1 as Cla-susceptible/-heteroresistant/-homoresistant (and 2 HP-negative). From the 33 samples Cla-heteroresistant by FISH, 4/19/8 Cla-susceptible/-heteroresistant/-homoresistant (and 2 HP-negative) was found. From the 33 samples Cla-homoresistant by FISH, 0/2/29 Cla-susceptible/-heteroresistant/-homoresistant (and 2 HP-negative) was detected by qPCR.
Conclusion: QPCR performed adequately in detecting both HP and its clarithromycin-susceptibility status in IHC/FISH-positive cases. However, among IHC/FISH-negative cases, a substantial proportion (25%) was found to be HP-positive by qPCR. Owing to the discontinuous mucosal appearance of Helicobacter, it cannot be detected histopathologically in certain biopsy specimens, only by soluble DNA-based PCR, which is therefore recommended in IHC-negative cases.
Funding: The study was funded by the K_22 142604 grant of the National Research, Development and Innovation Office (Hungary), as well as the New National Excellence Program (ÚNKP-23-4-II-SE-24) and KDP-2023 Funding Scheme (2023-2.1.2-KDP-2023-00016) of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund, Hungary.