High frequency of metronidazole and clarithromycin-resistant Helicobacter pylori in formalin-fixed, paraffin-embedded gastric biopsies

Background: Clarithromycin and metronidazole resistance of Helicobacter pylori is increasing worldwide and has resulted in a loss in the effectiveness of therapeutic regimens. We aimed to evaluate common mutations of resistance genes to clarithromycin (A2143G, A2142G and A2142C) and metronidazole (rdxA and frxA) in H. pylori strains in formalin-fixed, paraffin-embedded gastric biopsies. Methods: A total of 110 tissue blocks from children suspected of H. pylori infection were included. After DNA extraction, UreC PCR was performed. Specific primers and restriction enzymes by PCR-RFLP were used for analysis of A2143G and A2142G mutations. To detect A2142C and assess frequent mutations of metronidazole resistance, specific primers and PCR method were used. Results: One hundred cases of H. pylori (91%) were by PCR. Of 34 (34%) clarithromycin-resistant isolates 17 (50%), 10 (29%) and 7 (21%) had A2143G, A2142G, A2142C, respectively. Resistance rate to metronidazole was 60% (N = 60). In sequencing rdxA and frxA in the mutated strains, missense mutations were most frequent (60 and 57%, respectively), and there were differences in frameshift and non-sense mutations (p < 0.001). Conclusion: Resistance rate to clarithromycin was high and the highest percentage of mutation was of A2143G. PCR-RFLP was used directly with formalin-fixed gastric biopsies, thus, avoiding the requirement for time-consuming culture-based methods. The isolates that developed resistance were mainly associated with mutations of both rdxA and frxA genes.