Molecular Characterization of Helicobacter pylori Virulence Genes and Antimicrobial Resistance Genes in Gastric Biopsies among Symptomatic Patients, Khartoum State-Sudan

Abstract

Helicobacter pylori is a gastrointestinal bacterium that causes peptic ulcer and stomach cancer in about half of the world's population. The outcome of H. pylori-related disorders is influenced by many virulence factors. Clarithromycin- resistant H. pylori strains represent a worldwide health problem. This is descriptive cross sectional study aimed to detect the frequency of H. pylori, the virulence factors of H. pylori, and clarithromycin resistant associated mutations in Sudanese patients with gastritis symptoms. Also to study molecular characterization of H. pylori virulence genes and antimicrobial resistance genes by PCR and sequencing. Gastric biopsies were obtained from 384 patients with gastritis symptoms from different hospitals in Khartoum State from December 2018 to January 2021, their ages ranged from 14 to 88 years and mean age of 43 years. Out of them, 209 (54.4%) were males, while 175 (45.6%) were females. All gastric biopsies were subjected to polymerase chain reaction (PCR) to detect H. pylori 16S rRNA gene, virulence genes (cagA, cagE, vacA, iceA1, and dupA), and Clarithromycin resistance genes. Allele-specific PCR and DNA sequencing were used to screen for the presence of A2142G and A2143G point mutations. From 384 patients, two hundred sixty-nine (70.0%) patients were diagnosed with gastritis which was found to be the most common endoscopic findings, thirty-eight (9.9%) as a gastric ulcer, twenty-eight (7.3%) as a duodenal ulcer, sixteen (4.2%) as esophagitis and thirty-three (8.6%) were of normal mucosa. H. pylori was detected in 28.4% (109/384) specimens by PCR using specific H. pylori 16S rRNA, there was no significant association between the presence of H. pylori, socio-demographic data (gender, age groups, and geographical distribution), and clinical outcome among study population. The positive specimens were genotyped using PCR targeting cagA, cagE, vacA, dupA, and iceA1 genes. All of strains were vacA positive 100% (109/109) followed by dupA 44.0% (48/109), cagA 38.5% (42/109), cagE gene 37.6% (41/109), and iceA1 gene was detected in only 18.3% (20/109). The vacA s1/m1 70% (77/109) was the most prevalent vacA subtype. There was no significant difference between the presence of H. pylori virulence genes in regards to gender with different age groups. Although there was no significant association of H. pylori cagA, cagE, iceA1, and vacA s/m status according to endoscopic findings, the dupA gene was significantly associated with the clinical outcome (p. value of 0.016). Allele-specific PCR detected the variant A2142G in the 9/53 (~ 17%) specimen, while A2143G mutation was not found in any specimen. The DNA sequencing revealed the presence of mutations associated with clarithromycin-resistance in 36% (9/25) of samples; the A2142G was present in one sample, A2143G in 5 samples, and T2182C in 4 samples. Additionally, another point mutation (C2195T) was detected in 3 samples. The study conclude H. pylori virulence genes were extremely prevalent and diverse among Sudanese gastritis patients. H. pylori dupA gene was associated with the clinical outcome. A high frequency of mutations associated with clarithromycin resistance using DNA sequencing of the 23S rRNA gene’s V domain. This information should be taken into consideration to avoid eradication therapy failure.