Droplet Digital PCR-Based Detection of Clarithromycin Resistance in Helicobacter pylori Isolates Reveals Frequent Heteroresistance

Abstract

Chronic infection with Helicobacter pylori causes peptic ulcers and stomach cancer in a subset of infected individuals. While standard eradication therapy includes multiple antibiotics, treatment failure due to resistance is an increasing clinical problem. Accurate assessment of H. pylori antimicrobial resistance has been limited by slow growth and sampling of few isolates per subject. We established a method to simultaneously quantify H. pylori clarithromycin-resistant (mutant) and -susceptible (wild-type) 23S rRNA gene alleles in both stomach and stool samples using droplet digital PCR (ddPCR). In 49 subjects, we assessed the performance of these assays alongside clarithromycin MIC testing of up to 16 H. pylori isolates per subject and included both cancer (25 subjects) and noncancer (24 subjects) cases. Gastric ddPCR and H. pylori culture showed agreement with urea breath test (UBT) detection of infection in 94% and 88% of subjects, respectively, while stool ddPCR showed agreement with UBT in 92% of subjects. Based on MIC testing of 43 culture-positive cases, 20 subjects had only susceptible isolates, 14 had a mix of susceptible and resistant isolates, and 9 had only resistant isolates. ddPCR of gastric samples indicated that 21 subjects had only wild-type alleles, 13 had a mixed genotype, and 9 had only mutant alleles. Stool ddPCR detected mutant alleles in four subjects for which mutant alleles were not detected by stomach ddPCR, and no resistant isolates were cultured. Our results indicate that ddPCR detects H. pylori clarithromycin resistance-associated genotypes, especially in the context of heteroresistance.

Keywords: Helicobacter pylori; clarithromycin resistance; droplet digital PCR; heteroresistance; noninvasive detection.

FIG 1

MIC distribution of H. pylori isolates in culture-positive gastric samples. Each column represents the MIC distribution of a gastric antrum or corpus sample (W008, W014, W019, and W029 are from corpus samples, and the rest are from antrum samples). The diameter of each dot represents the number of isolates that show a corresponding MIC value (y axis) in each sample (x axis). Horizontal dotted lines separate three zones of clarithromycin resistance: resistant (≥0.75 mg/liter), intermediate (0.38 and 0.5 mg/liter), and sensitive (≤0.25 mg/liter). Vertical dotted lines separate subjects with only susceptible and intermediate isolates (left), with a mixture of susceptible, intermediate, and resistant isolates (middle), and with only resistant isolates (right).

FIG 2

Fractional abundance analysis of clarithromycin resistance alleles measured by ddPCR. Three kinds of clarithromycin resistance strain DNAs (A2143G, A2142G, and A2142C) were mixed with the wild-type strain at ratio of 0:1, 1:100, 1:10, 1:1, 10:1, 100:1, and 1:0 before being spiked into stool DNA. Two ddPCRs were run for each sample, and the results were combined. Bars indicate Poisson 95% confidence intervals.

FIG 3

Comparison of phenotypic (Etest) and genotypic (ddPCR) clarithromycin resistance detected in the stomach and stool among heteroresistant subjects. Each point represents a single subject. The fractional abundance in culture of clarithromycin-resistant (Cla^r) isolates is defined as the number of isolates with clarithromycin MICs of ≥1 mg/liter divided by the total number of isolates and multiplied by 100. For stomach ddPCR, the percentage of 23S mutant alleles (number of mutant alleles divided by the sum of wild-type and mutant alleles and multiplied by 100) is reported for the same stomach region used in the MIC testing for that subject (A) or reported as the average of the antrum and corpus measurements (B). Spearman correlation coefficients are 0.69 for panel A (P = 0.004) 0.74 for panel B (P = 0.003).