Detection of mixed clarithromycin-resistant and -susceptible Helicobacter pylori using nested PCR and direct sequencing of DNA extracted from faeces

Abstract

The major cause of chemotherapy failure in patients with chronic gastritis and peptic ulcers caused by Helicobacter pylori is clarithromycin (CAM) resistance due to a mutation in the 23S rRNA gene. This study describes a non-invasive and accurate method for the detection of mixed CAM-resistant and -susceptible H. pylori by sequencing of the H. pylori 23S rRNA gene. Faeces were crushed with beads and the 23S rRNA gene was amplified using a nested PCR on the extracted DNA. Mutation analysis of this gene using this method showed that 20.4 % of patients carried mixed CAM-susceptible (wild type) and -resistant (A2142G or A2143G mutant) H. pylori. Furthermore, it was found that 66.6 % of patients who had been treated unsuccessfully carried one of these mutations in the 23S rRNA gene (including the mixed type), whilst standard culture detected CAM-resistant isolates in only 22.2 % of patients with unsuccessful treatment. These data suggest that, for successful therapy, the diagnosis method described here would more accurately detect CAM-resistant H. pylori, including mixed infections. INTRODUCTIONHelicobacter pylori is a Gram-negative, spiral bacterium found in the human stomach. This micro-organism causes chronic gastritis and peptic ulcers (Kuipers, 1997) and is linked to gastric cancer and other non-gastrointestinal diseases (Kusters et al., 2006;Leong & Sung, 2002). To treat H. pylori infections, a triple therapy is used comprising a combination of a proton pump inhibitor and two antimicrobial agents. This is reported to be the most successful method for eradication of infection (Malfertheiner et al., 2002). In Japan, a combination of lansoprazole, amoxicillin and clarithromycin (CAM) (the LAC regimen) is commonly used to eliminate H. pylori (Asaka et al., 2001). Although most patients are treated successfully with this triple therapy, some failure has necessitated the use of additional drugs such as levofloxacin (Asaka et al., 2001;Kato et al., 2000). The major cause of therapy failure in patients is resistance to CAM (Kato et al., 2000; Rimbara et al., 2005a). Therefore, a reliable diagnostic test to detect macrolide resistance would be useful in planning a therapeutic regimen. Standard culture is generally used to determine the susceptibility of H. pylori to antimicrobial agents; however, acquiring a test sample is invasive for the patient, as endoscopy is required to obtain gastric specimens. Faecal culture is not possible, as H. pylori coccoid forms are unculturable; however, there are some reliable non-invasive tests for the diagnosis of H. pylori infections (Graham et al., 1987;Leodolter et al., 2003;Shuber et al., 2002;Vaira et al., 1999).Resistance to CAM in H. pylori is due to a mutation in the 23S rRNA subunit in the 50S ribosome. The two most common mutations are an adenine-to-guanine transition at position 2142 or 2143, the latter leading to an adenineto-cytosine transversion at position 2142 (Alarcon et al., 2003;van Doorn et al., 2001). Resistance due to mutations at other positions has also been rep...

Evaluation of a New Test, GenoType HelicoDR, for Molecular Detection of Antibiotic Resistance inHelicobacter pylori

“…The presence of more than one genotype in a single biopsy specimen has been shown to be the consequence of either different strains coexisting in the gastric mucosa or the presence of mutated and wild-type alleles in the same strain (26,32). Using culture, a similar proportion of 20 to 25% of the H. pylori cases was found.…”

“…Using culture, a similar proportion of 20 to 25% of the H. pylori cases was found. When a mixture of susceptible and resistant genotypes has been found, it has been related to clinical failure so far (26). In some cases, the results of genotyping by our test showed a mixture of wild-type alleles.…”

Clarithromycin-resistant Helicobacter pylori in Recife, Brazil, directly identified from gastric biopsies by polymerase chain reaction

“…In such situations, only an appropriate molecular method can discern a few H. pylori cells carrying resistant alleles (11,13) . Gastric samples showing H. pylori with resistant genotypes, 21 (77.8%) were also infected with strains carrying the wildtype allele, a high number of biopsies with mixed populations in comparison with other studies (11,12,13,17,20) . This was probably due to the design of the qPCR assay.…”

Phenotypic and genotypic correlation as expressed in Helicobacter pylori resistance to clarithromycin and fluoroquinolones

“…In cases of specimens demonstrating a genotypic mutant a susceptible phenotypic profile, a possible explanation could be the presence of a mixed bacterial population residing in the gastric specimens that may consist of susceptible and resistant strains [26]. In fact, this correlates with appearance of w.t.…”