Real-time PCR for Helicobacter pylori quantification and detection of clarithromycin resistance in gastric tissue from patients with gastrointestinal disorders

Helicobacter pylori gram-negative bacteria commonly infect the human gastrointestinal (GI) tract and are readily diagnosed by endoscopy. H. pylori infection causes a broad range of host symptoms from discomfort to significant GI disorders (GIDs). Severity of the clinical manifestations depends mainly upon bacterial load. In this cross-sectional study, we investigated the affects of 23S rRNA point mutations on H. pylori count in naturally infected human GI tissues. Two-hundred H. pylori patients with suspected GIDs were evaluated to determine bacteria concentration and presence of four known 23S rRNA point mutations, causing clarithromycin resistance. Gastric biopsy specimens were examined by rapid urease test and 16S rRNA-targeted PCR to identify H. pylori; then bacterial load was quantified by real-time PCR targeting wild type and known 23S rRNA mutations. Eighty-two percent of the samples were confirmed as H. pylori-positive, having 10(4)-10(12) colony-forming units (CFU)/ml. The 10(6) load was most strongly associated with peptidyltransferase point mutations of the 23S rRNA gene A2144G (p = 0.033), A2143G (p = 0.005), A2143C (p = 0.005), and A2142G (p = 0.015). Thus, our findings indicated that dominant 23S rRNA mutated H. pylori strains have the same growth rate as the wild type in a gastric environment.