Evaluation of a new test, genotype HelicoDR, for molecular detection of antibiotic resistance in Helicobacter pylori

Abstract

The eradication rate of Helicobacter pylori by standard therapy is decreasing due to antibiotic resistance, mainly to clarithromycin. Our aim was to provide a new molecular test to guide the treatment of new and relapsed cases. We first studied 126 H. pylori strains for phenotypic (MIC) and genotypic resistance to clarithromycin (rrl mutation) and levofloxacin (gyrA mutation) and then developed a DNA strip genotyping test on the basis of the correlation results and literature data. Clinical strains (n = 92) and gastric biopsy specimens containing H. pylori (n = 105) were tested blindly with the new molecular test GenoType HelicoDR. The presence of mutations or the absence of hybridization with wild-type sequences was predictive, in rrl for clarithromycin resistance in 91 cases (mostly the A2147G mutation) and in gyrA for levofloxacin resistance in 58 cases (mutations at codon 87 or 91). Genotyping revealed a mix of genotypes in 33% of the cases, reflecting a coinfection or selection for resistant mutants. The sensitivity and specificity of detecting resistance were 94% and 99% for clarithromycin and 87% and 98.5% for levofloxacin, respectively. The concordance scores were 0.96 for clarithromycin and 0.94 for levofloxacin. With global resistance rates of 46% for clarithromycin and 25% for levofloxacin, which were observed for consecutive positive biopsy specimens from 2007 and 2008, the positive and negative predictive values for detecting resistance were 99% and 94% for clarithromycin and 96% and 96% for fluoroquinolone. GenoType HelicoDR is efficient at detecting mutations predictive of antibiotic resistance in H. pylori when applied to strains or directly to gastric biopsy specimens.

FIG. 1.

Prototype of the strip of the GenoType HelicoDR test. Lane 1, gyrA pattern with a wt1 codon at position 87, a wt codon at codon 91, and a wild-type rrl pattern; lane 2, gyrA pattern with a MUT codon at position 87, a wt codon at codon 91, and a wild-type rrl pattern; lane 3, gyrA pattern with a wt2 codon at position 87, a MUT2 codon at position 91, and a wild-type rrl pattern; lane 4, gyrA pattern with a wt3 codon at position 87, a wt codon at position 91, and a MUT1 rrl mutation; lane 5, double mutation in gyrA with mutation at both 87 (MUT) and 91 (MUT3) and a wild-type rrl pattern.

FIG. 2.

Representative GenoType HelicoDR DNA strip results obtained with H. pylori strains and biopsy specimens showing (i) rrl alleles that are wild type (lanes 1 to 11) or mutated (12 to 15), (ii) gyrA alleles that are wild type (lanes 1, 3 to 11, and 15) or mutated (a mutation at codon 87 in lane 12 and no hybridization with probes of codon 87 in lane 2), and (iii) heterogeneity of the H. pylori population (lanes 7, 11, 12, and 15). NC, negative control; CC, conjugate control; AC, amplification control.