Helicobacter pylori Mutations Detected by Next-Generation Sequencing in Formalin-Fixed, Paraffin-Embedded Gastric Biopsy Specimens Are Associated with Treatment Failure

Abstract

Helicobacter pylori antibiotic resistance is widespread and increasing worldwide. Routine detection of H. pylori mutations that invoke antimicrobial resistance may be a useful approach to guide antimicrobial therapy and possibly avert treatment failure. In this study, formalin-fixed, paraffin-embedded (FFPE) gastric biopsy specimens from a cohort of individuals from northern Ohio in the United States were examined using a next-generation sequencing (NGS) assay to detect H. pylori mutations that are known to confer resistance to clarithromycin, levofloxacin, and tetracycline. From January 2016 to January 2017, 133 H. pylori-infected gastric biopsy specimens were identified histologically and subsequently analyzed by NGS to detect mutations in gyrA, 23S rRNA, and 16S rRNA genes. The method successfully detected H. pylori in 126 of 133 cases (95% sensitivity). Mutations conferring resistance were present in 92 cases (73%), including 63 cases with one mutation (50%) and 29 cases with mutations in multiple genes (23%). Treatment outcomes were available in 58 cases. Sixteen of the 58 cases failed therapy (28%). Therapy failure correlated with the number of mutated genes: no failure in cases with no mutations (0/15), 19% (5/27) failure in cases with one gene mutation, and 69% (11/16) failure in cases with more than one mutated gene. Common 23S rRNA mutations (A2142G or A2413G) were present in 88% (14/16) of failed cases as opposed to in only 10% (4/42) of eradicated cases (P Ͻ 0.001). This NGS assay can be used on remnant specimens collected during standard-of-care testing to detect mutations that correlate with increased risk of treatment failure. A prospective study is needed to determine if the risk of treatment failure can be decreased by using this assay to guide antibiotic therapy.

Helicobacter pylori Infections in the Bronx, New York: Surveying Antibiotic Susceptibility and Strain Lineage by Whole-Genome Sequencing

“…In addition, we detected mutations in the 16S rRNA gene (A926G) in four tetracyclinesensitive isolates, but the significance of this finding is unclear, since this mutation has been associated with both low-level resistance and phenotypic susceptibility (16,47,48). In a recent study that examined the correlation between the detection of mutations directly from formalin-fixed, paraffin-embedded tissue and patient outcomes, only one treatment failure was noted in six patients with the A926G mutation, but studies with larger numbers of patients treated with tetracycline-containing regimens are needed to understand the clinical significance of this mutation (49).…”

What Is New in Helicobacter pylori Diagnosis. An Overview

“…Alternatively, molecular tests to detect H. pylori and its susceptibility to antibiotics can be performed on gastric juice [ 90 , 91 , 92 ]. A droplet-digital PCR may also be applied to formalin-fixed, paraffin-embedded gastric tissue to determine the presence of clarithromycin resistance [ 93 ], or by next generation sequencing to determine levofloxacin and tetracycline resistance [ 94 ] ( Figure 2 ).…”

Review of current diagnostic methods and advances inHelicobacter pyloridiagnostics in the era of next generation sequencing

“…In an effort to decrease turn-around-times and apply diagnostic workflows that do not require endoscopy, future studies should aim at detecting H. pylori and associated resistance mutations directly from clinical specimens (gastric biopsies or stool) using meta-genomic and/or meta-transcriptomic sequencing. Our literature search yielded primary research articles that have successfully applied WGS directly on gastric biopsies for the detection of H. pylori [116,132,144,145]. One major limitation for the cost-effectiveness and feasibility of clinical meta-genomic and meta-transcriptomic sequencing has always been the rather big amounts of RNA or DNA required for subsequent library preparation and high human DNA background requiring deep sequencing.…”