Next Generation Sequencing of Stool Samples Accurately Identified Antibiotic Resistance for Helicobacter Pylori

American College of Gastroenterology (ACG 2021): Sequencing of Stool Samples technology eliminates need for endoscopic gastric biopsy

Helicobacter pylori is a major human pathogen for which increasing antibiotic resistance constitutes a serious threat to human health. Antimicrobial resistance is an ever-growing problem for successful eradication of H. pylori, which is the most common chronic bacterial infection in humans and associated with 89% of gastric cancers.

Eradicating H. pylori using combination antibiotics prevents most ulcers and reduces gastric cancer risk by 50%, but increasing antibiotic resistance among H. pylori strains has become a serious problem worldwide, limiting the success of eradication therapy.

Profiling to determine antibiotic resistance to Helicobacter pylori (H. pylori) can be conducted as accurately using next generation sequencing of stool samples as it can using gastric biopsy, according to research presented at the Annual Scientific Meeting of the American College of Gastroenterology.

Key points

Using next generation sequencing through stool samples is a promising, noninvasive step toward identifying mutations associated with resistance and offers more targeted therapy for patients, especially in those with previous treatment failure.

Bacterial infection of the stomach by H. pylori is the most important cause of peptic ulcers and gastric cancer worldwide. Tailoring treatment based upon the results of antibiotic resistance measurement has become increasingly important but is inconvenient and underused in practice.

This study report shows that antibiotic sensitivity to six different antibiotics can now be evaluated accurately and easily via a novel molecular method from a sample of stool, obviating the need for endoscopy and gastric biopsy, which are invasive and expensive. This should lead to more widespread use of resistance testing and improved rates of success for H. pylori eradication.

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Abstract

Aim

H. pylori eradication rates have declined in concert with rising antimicrobial resistance worldwide. There is a need for rapid accurate, reliable antibiotic resistance testing, especially in refractory cases. Culture-based susceptibility testing requires endoscopic gastric biopsy, with resultant inconvenience and costs. Molecular testing using next generation sequencing (NGS) of stool potentially allows rapid prediction of resistance to all 6 commonly used antimicrobials. Study aimed to compare the results of H. pylori antibiotic resistance testing by NGS using stool and gastric biopsy specimens in the same patients.

Methods:

Patients scheduled for upper endoscopy from 4 clinical practices were recruited after informed consent. 2 gastric biopsies were taken for NGS (American Molecular Labs). A spontaneously passed stool specimen was also obtained within 2 weeks of endoscopy but before starting anti-H. pylori therapy. H. pylori was confirmed in biopsies by PCR followed by NGS designed to identify mutations associated with H. pylori antibiotic resistance. H. pylori in stools was confirmed by fecal antigen test and PCR. Stool samples positive by at least 2 stool tests were also examined by NGS to predict resistance to amoxicillin, clarithromycin, metronidazole, tetracycline, levofloxacin, and rifabutin. Agreement between the tests was expressed as a kappa coefficient.

Results:

262 patients were recruited; overall, 29% of patients were H. pylori positive by both biopsy and stool testing, and 2 had insufficient gastric DNA for analysis. Among the 71 evaluable positive cases, identical results for stool and biopsy samples were obtained for all six antibiotics in 91.5%. Among the remaining 6 cases where there was a mismatch between stool and biopsy samples, 4 cases were due to just one antibiotic-associated mutation difference.

Both gastric and stool samples revealed high rates of resistance-associated mutations for clarithromycin (54.9% for both), levofloxacin (32.4% and 28.2%, respectively), and metronidazole (32.4% and 29.6%, respectively). Resistance-associated mutations were much less likely to be found for tetracycline (9.9% for both), amoxicillin (6.0% for both), and rifabutin (0% for both).

Among the positive cases, 70.4% of gastric biopsies had at least one resistance-associated mutation, and 29.6% had no mutations. Results were similar with stool samples, where 68.5% had at least one resistance-associated mutation, and 31.5% had no mutations. The concordance between stool and gastric biopsies for individual antibiotics ranged from 89% (metronidazole) to 100% (tetracycline, amoxicillin, and rifabutin).

Conclusion:

Profiling H. pylori antibiotic resistance by NGS from stool samples provides rapid results highly comparable to those obtained from gastric biopsies. Using NGS to determine H. pylori antibiotic resistance using stool obviates the cost, inconvenience and risks of endoscopy for patients in whom resistance profiling is needed.