PCR-Based Diagnosis of Helicobacter pylori Infection and Real-Time Determination of Clarithromycin Resistance Directly from Human Gastric Biopsy Samples

Abstract

A novel PCR detection assay that amplifies the Helicobacter pylori-specific vacuolating cytotoxin gene (vacA) and thus enables rapid diagnosis of infection is described. Additionally, a real-time probe hybridization melting point analysis assay to detect all three mutations in the 23S rRNA gene associated with clarithromycin resistance was applied directly to antral gastric biopsy samples. Comparison with culture and an alternative PCR assay targeting the 16S rrn gene showed that the vacA assay was sensitive and specific when tested on biopsy samples from 121 patients. Clarithromycin susceptibilities could be determined in the majority (92.3%) of culture-positive gastric biopsy samples analyzed, four of which generated melting peaks indicative of clarithromycin resistance by either an A3G or A3C mutation. The presence of the mutations correlated with the clarithromycin disk diffusion sensitivities of matched cultures. This PCR-based system was simple to perform and could be completed in 3 to 4 h, thereby overcoming the delays associated with conventional culture methods for H. pylori identification and susceptibility testing.Helicobacter pylori, a major cause of chronic gastritis, is strongly associated with the development of gastric and duodenal ulcers and has been linked with gastric adenocarcinoma and B-cell mucosa-associated lymphoid tissue lymphoma (15,17,18). Infection can be eradicated in up to 90% of patients using current combination triple therapies, of which the macrolide antibiotic clarithromycin is a key component (5). Rates of resistance to clarithromycin of 1 to 9% have been reported in several European countries and the United States, with even higher rates in some countries, such as France and Belgium (24). The development of clarithromycin resistance in H. pylori is recognized as a significant contributing factor in treatment failure (8,14), and the mechanism is attributed to single point mutations in the peptidyltransferase region of the 23S rRNA gene (25). Adenine residues at either position 2143 or 2144 can mutate. Transition to guanine (A2143G and A2144G) is the most common mutation type, with the transversion mutation to cytosine (A2143C) less common (16,21,22,25). Although culture of gastric biopsy samples allows further H. pylori strain analysis, including determination of antibiotic susceptibility, tests can take up to 2 weeks to complete. The aim of the present study was to develop a PCR-based system using conventional and real-time techniques enabling same-day diagnosis of H. pylori infection and determination of clarithromycin resistance. MATERIALS AND METHODSGastric biopsy samples and strain isolation. Two sets of gastric biopsy samples were used in this study. First, we examined a series of preserved (Ϫ20°C) gastric biopsy samples from 39 dyspeptic patients attending an open-access endoscopy clinic in Chelmsford during 1995 and 1996. These biopsy samples were confirmed positive for H. pylori by both culture and histology, and the patients were also confirmed seropositive for H. py...

Polymerase chain reaction versus culture in the diagnosis of Helicobacter pylori infection

“…The vacA gene, though also present in all strains, shows considerable polymorphism, 25 which could explain it to be less sensitive than ureC as a molecular diagnostic marker for H. pylori. 10,14 We found cagA to be present in 37% of the H. pylori strains in our study, which is in agreement with the low prevalences reported in Western populations. 26,27 There was a tendency towards better sensitivity for real-time PCR than conventional PCR, but the difference did not reach statistical significance.…”

“…Conventional PCR targeting the ureC, vacA and cagA genes was performed using PCR conditions as described elsewhere. 10,14,15 Realtime PCR for ureC was performed using the same primers as for conventional PCR and by constructing a TaqMan probe. Details of DNA primer and probe sequences are given in Table 1.…”

“…7,8 Besides accurately detecting H. pylori encompassing also non-culturable coccoid forms, 9 polymerase chain reaction (PCR) can be employed for strain characterization by detecting genes encoding pathogenic factors and identifying certain resistance traits by detecting microbial gene mutations. 10 PCR entails no special requirements in processing and transport, enabling a rapid and probably a cost-effective diagnosing of H. pylori, overcoming the delay associated with conventional culture methods. [10][11][12] About ten years ago, after having used a conventional PCR method as a supplement to our standard H. pylori culture in gastric mucosal biopsy specimens, a preliminary investigation in our hospital disclosed that culture often seemed to fail compared to PCR.…”

“…10 PCR entails no special requirements in processing and transport, enabling a rapid and probably a cost-effective diagnosing of H. pylori, overcoming the delay associated with conventional culture methods. [10][11][12] About ten years ago, after having used a conventional PCR method as a supplement to our standard H. pylori culture in gastric mucosal biopsy specimens, a preliminary investigation in our hospital disclosed that culture often seemed to fail compared to PCR. Culture gave a negative result in as much as 27% of PCR-positive biopsies, whereas 5% of positive cultures were found to have a negative PCR-result, necessitating a more thorough evaluation of the biopsy-based diagnostic methods in use.…”

Prevalence, diversity and disease association of Helicobacter pylori in dyspeptic patients from Pakistan

“…PCR amplifications were performed according to previously described protocols [35,36]. cagA and vacA genotyping cagA status was determined for H. pylori positive samples by polymerase chain reaction (PCR) using two different primers pairs as described previously [12,37] (Table 1).…”