Direct detection of Helicobacter pylori in biopsy specimens using a high-throughput multiple genetic detection system

Abstract

Aim: We evaluated the direct high-throughput multiple genetic detection system (dHMGS) for Helicobacter pylori in gastric biopsies.

Materials & methods: One hundred and thirty-three specimens were concurrently analyzed by dHMGS, rapid urease test, culture and sequencing.

Results: dHMGS was highly sensitive and specific for H. pylori identification compared with culture and rapid urease test. The correlation coefficient of the quantitative standard curve was R² = 0.983. A significant difference in the relative H. pylori DNA abundance was found in different gastroduodenal diseases. Concordance rates between dHMGS and sequencing for resistance mutations were 97.1, 100.0, 85.3 and 97.1%, respectively. Finally, dHMGS could efficiently distinguish mixed infection in biopsy specimens.

Conclusion: The dHMGS could efficiently diagnose and quantify H. pylori burden in biopsies, simultaneously screening for virulence, antibiotic resistance and presence of the multistrain infections.

Keywords: Helicobacter pylori; direct high-throughput multiple genetic detection system; gastric biopsy specimens; identification; mixed infection; quantitative analysis; resistance; virulence.

Figure 1. The position and peak area of ureC and β-globin gene in direct high-throughput multiple genetic detection system

The peak area of ureC and β-globin were 59146 and 111834, respectively. The ratio of the peak area between ureC and β-globin was 0.5289.

Figure 2. The quantitative standard curve of direct high-throughput multiple genetic detection system for biopsy specimens

The quantitative standard curve obtained by doubling dilutions from 0.004 to 0.032 ng (x-axis) and their corresponding ratio of direct high-throughput multiple genetic detection system peak area (y-axis), 0.21, 0.59, 1.26, 2.05, respectively.

Figure 3. Correlation of Helicobacter pylori DNA load and clinical characteristics

(A) Helicobacter pylori DNA load in gender groups; p = 0.5896 (p > 0.05). (B) Helicobacter pylori DNA load in different gastroduodenal diseases including CSG, CAG, PUD, p = 0.0371 (p < 0.05). (C) Helicobacter pylori DNA load among different age groups; p = 0.9721 (p > 0.05). y-axis represents the relative H. pylori DNA level while x-axis represents different parameters. CAG: Chronic atrophic gastritis; CSG: Chronic superficial gastritis; PUD: Peptic ulcer disease.

Figure 4. Concordance of direct high-throughput multiple genetic detection system and resistance mutation for antibiotics in biopsy specimens (see facing page)

(A) Resistance genes gyrA_C had the wild-type peak at 306 bp, were detected by direct high-throughput multiple genetic detection system, as well as showed the peak of base C in the sequencing result. (B) The peaks of wild-type and mutant resistance genes pbp1A_A/G were appeared in direct high-throughput multiple genetic detection system at 175 and 180 bp, respectively, as well as showed both the peaks of base A and G in the sequencing result.

Figure 5. Direct high-throughput multiple genetic detection system can efficiently distinguish multistrain infection in gastric biopsy specimens

(A) The peaks of wild-type pbp1A_A and mutant resistance genes pbp1A_A/G were appeared at 154 and 159 bp in biopsy specimen. (B) The peak of wild-type pbp1A_A appeared at 154 bp in the corresponding isolated strains. 23S rRNA_G, rdxA_C and gyrA_C had the single peak in biopsy specimen and corresponding isolated strains, appeared at 180, 253 and 306 bp, respectively. Human internal control β-globin had the specific peak appeared at 199 bp in biopsy specimens.