Accurate prediction of macrolide resistance in Helicobacter pylori by a PCR line probe assay for detection of mutations in the 23S rRNA gene: Multicenter validation study

Helicobacter pylori strains from 299 patients were tested in six laboratori es in different countries. Macrolide susceptibility of the strains was dete rmined by agar dilution (17.4%) or the epsilometer test (82.6%). Mutations in the 23S ribosomal DNA (rDNA) that are associated with macrolide resistan ce were analyzed by PCR and reverse hybridization (PCR-line probe assay [Li PA]). This method identifies A2115G, G2141A, A2142G, A2142C, A2142T, A2143G , and A2143C mutations in the 238 rDNA, vacA s-region (s1a, s1b, s1c, and s 2) and m-region (m1, m2a, and m2b) genotypes and cagA status were also dete rmined using another PCR-LiPA system. Of the 299 strains investigated by MI C testing, 130 (43.5%) were resistant and 169 (56.5%) were susceptible to c larithromycin, Of the 130 resistant strains, 127 (97.7%) contained 23S rDNA mutations, whereas 167 (98.8%) of the 169 susceptible strains contained wi ld-type sequences. The predominant mutations were A2143G (45.2%) and A2142G (33.3%). Twenty-eight (19.8%) strains contained multiple 23S rDNA mutation s. Only five resistant strains contained the A2142C mutation (three of thes e in combination with the A2142G mutation), and the A2115G, G2141A, A2142T, and A2143C mutations were not found, MICs of clarithromycin for the A2142G mutant strains were significantly higher than MICs for the A2143G strains. Although there was no significant association between 23S rDNA mutations a nd the vacA and cagA status, clarithromycin-susceptible strains more often contained mixed vacA genotypes, indicating the presence of multiple H. pylo ri strains. In conclusion, our data confirmed the very strong association b etween 238 rDNA mutations and macrolide resistance and showed that the PCR- LiPA permits accurate and reliable diagnosis of macrolide resistance in H. pylori.