Assessment of target-enrichment library preparation and next-generation sequencing of paraffin-embedded gastric biopsies for H. pylori diagnosis and evaluation of virulome and resistome

The diagnosis of Helicobacter pylori infection from gastric biopsies requires polymerase chain reaction or culture. However, culture is often unsuccessful due to the bacterial fragility and complex growth requirements. Next-generation sequencing (NGS), performed on primary clinical samples, offers a promising alternative for analysis of the bacterial resistome. In this study, we describe the adaptation of a target-enrichment library preparation and NGS workflow for use on formalin-fixed, paraffin-embedded (FFPE) gastric biopsies to investigate both the H. pylori resistome and virulome. In total, 30 FFPE gastric biopsy samples were analyzed, all derived from patients infected with H. pylori, the majority of whom presented with gastritis or epigastric pain. The Agilent SureSelect XT protocol was modified for implementation on the Magnis automated system, and sequencing was performed using the iSeq 100 platform. RNA probes targeting key genes associated with virulence (e.g., cagA and vacA), antibiotic resistance (e.g., 23S rRNA, 16S rRNA, gyrA, and rpoB), and multilocus sequence typing (MLST) were employed. The resulting sequence data were compared to those obtained from cultured H. pylori strains isolated from the same patients. Mutations in 23S rRNA linked to macrolide resistance, those in the quinolone resistance-determining region of gyrA associated with levofloxacin resistance, and those conferring rifamycin resistance were accurately detected. The MLST profiles generated through this method were consistent with those obtained via Sanger sequencing. Furthermore, the cagA gene, including EPIYA motifs, and vacA genotypes were reliably identified. This target-enrichment technique provides accurate access to the H. pylori resistome and virulome directly from FFPE biopsy specimens, representing a significant technological advancement.