pylori. In fact, the 18-bp deletion type appeared to be a marker of Vietnamese H. pylori. Comparison of two geographically distant cities in Vietnam, Hanoi and Ho Chi Minh, showed that the vacA m1 genotype, thought to be more toxic than the vacA m2 type, is more prevalent in Hanoi, where the incidence of gastric cancer is higher than in Ho Chi Minh. Our data support the hypothesis that the vacA m1 type is closely associated with gastric carcinogenesis. Methods Patients and H. pylori H. pylori strains were obtained from the gastric mucosa of H. pylori-infected patients who underwent endoscopy at 108 Hospital,

Hanoi, and Cho Ray Hospital, Ho Chi Minh. The biopsy specimens learn more were immediately placed in Portagerm pylori (BioMérieux,

Nürtingen, Germany)[28] at 4°C and then sent to Oita University, Oita, Japan. H. pylori was cultured as described previously [14]. Informed consent was obtained from all participants and the protocol was approved by the local hospital ethics committees. Patients with a history of partial gastric resection, H. pylori eradication therapy or treatment MK-0457 with antibiotics, bismuth-containing compounds, H2-receptor blockers or proton pump inhibitors within 4 weeks prior to the study were excluded. H. pylori genotyping For DNA extraction, multiple colonies on blood agar plates were harvested together, and bacterial genomic DNA was extracted according to the CTAB (hexadecyltrimethylammonium bromide) method [29] and subsequently suspended in TE buffer (10 mM Tris HCl and 1 mM EDTA). A DNA fragment covering approximately 300 bp upstream from the first EPIYA motif in the cagA 3′ repeat region, which we designated the pre-EPIYA region in this study, was amplified by PCR using the following primer sets: T5: 5′-AAG CGT TAG CCG ATC TCA AA-3′ (forward), and 1-AS: 5′-CAT Dolutegravir cell line TAC CGA CTA GGG TTC C-3′ (reverse) [27]. The amplified DNA fragments were separated by electrophoresis on 2% agarose gel, stained with ethidium bromide, and finally visualized under ultraviolet light. For sequencing of the pre-repeat region

of the cagA gene, a DNA fragment of approximately 1,100 bp covering both the pre- EPIYA region and repeat region was initially amplified by PCR using the following primer sets: 2059f: 5′-GAA TTG TCT GAT AAA CTT G-3′ (forward), and 3156r: 5′-GCG TAT GTG GCT GTT AGT AGC G-3′ (reverse), then the amplified DNA fragments were sequenced with an ABI Prism 310 Genetic Analyzer [27] (Applied Biosystems, CA) in accordance with the manufacturer’s instructions. Multiple sequence alignments of the cagA pre-EPIYA sequences were generated using the ClustalX programs (downloaded from ftp://​ftp.​ebi.​ac.​uk/​pub/​software/​clustalw2). The vacA genotyping (signal regions s1 and s2, and middle regions m1 and m2) and cag BVD-523 right-junction motif genotyping (type I to V) were performed as described previously [11, 18, 21].