Helicobacter Pylori and Vonoprazan Dual Therapy

Upper gastrointestinal endoscopy examinations were performed for all participants andbiopsies from gastric antrum and body were obtained. All the collected gastric biopsyspecimens were sent to Jiangxi Provincial Key Laboratory of Digestive Diseases, FirstAffiliated Hospital of Nanchang University for susceptibility testing of antibiotics. Theminimum inhibitory concentrations of antibiotics (amoxicillin, metronidazole,clarithromycin, levofloxacin and tetracycline) were determined by E‐test. The inhibitionzone for furazolidone was determined by the Kirby‐Bauer disc diffusion method. Detailedprocedure for detecting antibiotics resistance were consistent with our previous study. Astrain was considered as resistant if minimum inhibitory concentration >0.125 μg/mL foramoxicillin, >8 μg/mL for metronidazole, ≥1 μg/mL for clarithromycin, >2 μg/mL forlevofloxacin, ≥2 μg/mL for tetracycline, the inhibition zone was ≤7mm for furazolidone.The detailed demographics and characteristics (sex, age, nationality, height, weight,education status, dwelling area, history of smoking and alcohol etc.) were recorded. Thetreatment‐related adverse events (TEAEs) were recorded. We defined adherence as good ifthe participants took ≥80% drugs of the regimen during the consecutive 14 days. The H.pylori status after therapy was evaluated by ¹³C‐UBT at least 6 weeks after completion oftreatment. Proton pump inhibitors and antibiotics were stopped at least 2 and 4 weeksbefore ¹³C‐UBT, respectively.The stool samples were collected at baseline (before treatment), week 2 (aftereradication) and week 8‐10 (confirmation of H. pylori status). We sent the stool samplesfrom subjects with successful eradication for metagenome DNA extraction and shotgunsequencing to avoid the influence of H. pylori eradication failure on gut microbiota.Briefly, OMEGA Mag‐Bind Soil DNA Kit (Omega Bio‐Tek, Norcross, GA, USA) was used toextract total microbial genomic DNA samples. Metagenome shotgun sequencing libraries fromextracted microbial DNA was constructed by Illumina TruSeq Nano DNA LT LibraryPreparation Kit, which was then sequenced by Illumina NovaSeq platform (Illumina, USA)with PE150 strategy at Personal Biotechnology Co., Ltd. (Shanghai, China).Raw sequencing reads were subjected to processing to yield quality‐filtered readssuitable for further analysis, including removal of adapter and low‐quality reads.Subsequently, minimap2 was utilized to align reads to the host genome of human andeliminate host contamination. Gene prediction was carried out on the generated contigsfrom each sample, whose translated protein sequences were subsequently pooled andclustered using mmseqs2. The lowest common ancestor taxonomy of the non‐redundant geneswas ascertained using mmseqs2 in "taxonomy" mode, by aligning them against a customizeddatabase comprising protein sequences of bacteria from GTDB (release 207:https://data.ace.uq.edu.au/public/gtdb/data/releases/), fungi from NCBI‐nr(https://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/), and viruses from RVDB (version 24.1:https://rvdb.dbi.udel.edu/download/). In order to assess the abundance of genes,high‐quality reads from each sample were mapped onto the contigs using minimap2 and readcounts were computed using htseq. Abundance values in metagenomes were normalized usingcopies per kilobase per million mapped reads. Clean high‐quality reads were processed andprofiled with ARGs‐OAP (version 2.0) by querying against the SARG (version 3.0‐F)database, which is a structural antimicrobial resistance genes database containing 32types and 2842 subtypes of antibiotic resistance genes. In order to perform thequantification and downstream analysis of diversity indices, resistome profiling, andprevalence ranking, the abundance of antibiotic resistance genes at type and subtypelevel were normalised to the number of 16S rRNA genes.