- Study design and participants
This study conducted a sub-analysis of H. pylori infection screening as part of the junior high school health screening system among third-year students in Saga Prefecture . Using local governmental grants, a screening and treatment program for eradicating H. pylori infection among third-grade junior high students was started in Saga Prefecture in 2016. The students underwent urinary anti-H. pylori antibody tests (RAPIRAN; Otsuka Pharmaceutical Co., Ltd. Tokyo, Japan), followed by H. pylori stool antigen tests (TESTMATE RAPID RYLORI ANTIGEN; Wakamoto Pharmaceutical Co., Ltd. Tokyo, Japan). To eradicate H. pylori infection, those who tested positive on both tests received triple therapy comprising 20 mg vonoprazan (Takeda Pharmaceutical Co., Ltd. Tokyo, Japan), 750 mg amoxicillin, and 200 mg clarithromycin twice a day for 7 days. Then, 8–12 weeks after the eradication therapy, the 13C-urea breath test was conducted at one of the cooperating medical institutions to assess for treatment efficacy. Breath samples were obtained 4 h after a meal and 20 min after the ingestion of 100 mg 13C-urea (UBIT® tablet, 100 mg). An infrared spectrometer (POC One®; Otsuka Electronics Co., Ltd., Hirakata, Japan) was used for the test.
Children with two positive test results were enrolled in the study between June 2017 and March 2019. Stool samples were collected prior to treatment (Before), 1–2 days after treatment (After), and after 8–12 weeks of eradication therapy (Judgment). At the time of judgment of eradication therapy, students with unsuccessful treatment were excluded to rule out the effect of the presence of H. pylori on the gut microbiota . The outcome of H. pylori eradication therapy was confirmed using the 13C-UBT. The stools were immediately stored at −20ºC until DNA extraction.
This study was approved by the ethics committee of Saga University (institutional review board no.: 2017-03-01) and was registered in the clinical trials registry (UMIN000028726) of the University Hospital Medical Information Network. All patients and their parents provided a written informed consent for participating in the study and for collecting fecal samples.
- DNA extraction
The specimens were stored at −20°C until DNA extraction. Bacterial DNA was extracted using the NucleoSpin Microbial DNA kit (MACHERY-NAGEL, Düren, Germany) according to the manufacturer’s instructions. The total DNA was eluted in 50 μL of elution buffer and was stored at −20°C. The V3–V4 hypervariable regions of 16S ribosomal DNA (rDNA) were amplified using the 16S (V3–V4) Metagenomic Library Construction Kit for NGS (Takara Bio Inc., Kusatsu, Japan) with primer pairs (341F 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3', 806R 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT-3'). The amplicon was purified using AMPure XP magnetic beads (Beckman Coulter, Brea, CA, the USA). The index PCR was performed using the Nextera XT Index Kit (Illumina, San Diego, CA, the USA). After purification with AMPure XP beads, sequencing was conducted on a MiSeq platform with the MiSeq Reagent Kit v3 and Phix Control Kit v3 (Illumina) from Takara Bio Inc.
- 16s rDNA sequence analysis
The 16s rDNA sequencing results were analyzed as follows: low-quality sequences were removed, chimeras were checked, operational taxonomic units (OTUs) were constructed, and taxonomy was assigned using CD-HIT-OTU, Quantitative Insights Into Microbial Ecology pipeline (http://qiime.org/). OTUs were established by clustering with a 97% identity threshold and were completed using an RDP classifier with the Green genes database. The observed OTUs, Chao1 (microbial species richness), and Shannon indices (microbial evenness) and PD whole tree (phylogenetic richness) were calculated to determine the alpha (α)-diversity of the microbiota in the samples. The beta (β)-diversity of our samples was calculated using the default beta-diversity metrics of the weighted and unweighted UniFrac distance. To compare the differences in the overall bacterial gut microbiota structure, a principal coordinates analysis was performed to reduce the dimensionality of the resulting distance.
- Statistical analysis
The raw data were expressed as percentage, mean, and standard deviation whenever applicable. For skewed data, the Mann–Whitney U test and Wilcoxon signed-rank test were used to compare variables between and within groups, respectively. The comparison of α-diversity and group distance for β-diversity were performed using the Monte Carlo two-sample test, followed by the false discovery rate and Bonferroni correction. A P value <0.05 was considered statistically significant. Statistical analyses of the microbiota were performed using Python. Other statistical analyses were performed using R (R Core Team 2018]. R: A language and environment for statistical computing; R Foundation for Statistical Computing, Vienna, Austria; URL: https://www.R-project.org/.).
- Study endpoint
The study endpoint was the change in the intestinal microbiota during H. pylori eradication with vonoprazan-containing triple therapy. In particular, the relative abundance, α-diversity, and β-diversity of the gut microbiota were compared before eradication, immediately after eradication, and during assessment of treatment efficacy. In addition, the adverse effects of H. pylori eradication with vonoprazan-containing triple therapy in children were evaluated.