A rapid and high-throughput multiplex genetic detection assay for detection, semi-quantification and virulence genotyping of Helicobacter pylori in non-invasive oral samples.

Aim: This study established a high-throughput multiplex genetic detection assay (HMGA) for rapid identification, semi-quantification and virulence analysis of Helicobacter pylori directly from the clinical non-invasive oral samples. Methods: The gastric mucosa and oral samples were collected from 242 patients in Shanghai from 2021 to 2022. All the samples were detected by routine clinical tests for H. pylori and Sanger sequenced for inconsistent results. A new multiplex PCR assay providing results within 4 hours was designed and optimized involving fluorescent dye-labeled specific primers targeted 16S rRNA gene, semi-quantitative gene ureC and 10 virulence genes of H. pylori. Semi-quantification was carried out by simulating the serial 10-fold dilutions of positive oral samples, and the H. pylori loads in different clinical samples were further compared. The mixed plasmids of virulence genes vacA s1, vacA m1 and vacA m2 were used to evaluate the performance on different genotypes. The consistency of 10 virulence genes in gastric mucosa, saliva, mouthwash and dental plaque of H. pylori-positive patients was compared. Results: The non-invasive HMGA was highly specific for detection of all 12 targets of H. pylori and human internal reference gene ß-globin, and the sensitivity to all target genes could reach 10 copies/µL. Compared with routine clinical tests and sequencing, non-invasive HMGA has a high level (>0.98) of sensitivity, specificity, accuracy, PPV, NPV and kappa coefficient for direct detection of H. pylori in oral samples. Moreover, by detecting peak area levels of ureC, it was confirmed that the H. pylori loads in gastric mucosa were significantly higher than those of the three kinds of oral samples (p<0.05). We also found that 45.0% (91/202) of patients had different H. pylori virulence genes in different oral samples. The concordance of positive detection rates of each virulence gene between saliva and gastric mucosa was more than 78% (p<0.05). Conclusion: The non-invasive HMGA proved to be a reliable method for the rapid H. pylori identification, semi-quantification and detection of 10 virulence genes directly in oral samples, providing a new idea for non-invasive detection of H. pylori.

Effect of posture on (13)C-urea breath test in partial gastrectomy patients.

AIM: To investigate whether posture affects the accuracy of (13)C-urea breath test ((13)C-UBT) for Helicobacter pylori (H. pylori) detection in partial gastrectomy patients. METHODS: We studied 156 consecutive residual stomach patients, including 76 with H. pylori infection (infection group) and 80 without H. pylori infection (control group). H. pylori infection was confirmed if both the rapid urease test and histology were positive during gastroscopy. The two groups were divided into four subgroups according to patients' posture during the (13)C-UBT: subgroup A, sitting position; subgroup B, supine position; subgroup C, right lateral recumbent position; and subgroup D, left lateral recumbent position. Each subject underwent the following modified (13)C-UBT: 75 mg of (13)C-urea (powder) in 100 mL of citric acid solution was administered, and a mouth wash was performed immediately; breath samples were then collected at baseline and at 5-min intervals up to 30 min while the position was maintained. Seven breath samples were collected for each subject. The cutoff value was 2.0‰. RESULTS: The mean delta over baseline (DOB) values in the subgroups of the infection group were similar at 5 min (P > 0.05) and significantly higher than those in the corresponding control subgroups at all time points (P < 0.01). In the infection group, the mean DOB values in subgroup A were higher than those in other subgroups within 10 min and peaked at the 10-min point (12.4‰ ± 2.4‰). The values in subgroups B and C both reached their peaks at 15 min (B, 13.9‰ ± 1.5‰; C, 12.2‰ ± 1.7‰) and then decreased gradually until the 30-min point. In subgroup D, the value peaked at 20 min (14.7‰ ± 1.7‰). Significant differences were found between the values in subgroups D and B at both 25 min (t = 2.093, P = 0.043) and 30 min (t = 2.141, P = 0.039). At 30 min, the value in subgroup D was also significantly different from those in subgroups A and C (D vs C: t = 6.325, P = 0.000; D vs A: t = 5.912, P = 0.000). The mean DOB values of subjects with Billroth I anastomosis were higher than those of subjects with Billroth II anastomosis irrespectively of the detection time and posture (P > 0.05). CONCLUSION: Utilization of the left lateral recumbent position during the procedure and when collecting the last breath sample may improve the diagnostic accuracy of the (13)C-UBT in partial gastrectomy patients.