H. pylori mixed infections were defined as having more than one allele of either the s-region or m-region of vacA or both iceA1 and iceA2 genotypes [22–24]. The rates of mixed infections were varied from 0% to 85% [14, 22, 26–28]. However, most studies used pooled cultures or biopsies for PCR, which may have yielded misleading results. Additionally, the sampling methods, including bacterial cultures from the sites of biopsies and analysis of the antimicrobial-resistant fractions or unselected strains, might have been attributed to the discrepant results . In this study, we used 6 distinct colonies from each culture plate for genotyping of the cagA, cagE, cagT, cagM, and vacA s- or m-regions. We evaluated a large number of colonies (n = 420) isolated from 70 patients. Moreover, 11 pairs of primers, including those for the genes cag-PAI and vacA s- or m-regions, were used. Therefore, we accurately and sensitively analyzed H. pylori mixed infections.