MATERIALS AND METHODS: Specific primers and probes were designed to amplify ureA and mutations in 23S rRNA and gyrA genes. Singleplex and triplex qPCR assays were optimized and the assay's sensitivities and specificities were determined. The optimized multiplex qPCR assay was performed on 571 gastric biopsies.
RESULTS: In this study, 14.7% (84/571) of the gastric biopsies were positive for H. pylori by conventional methods and 23.8% (136/571) were positive by the ureA-qPCR with 96.4% sensitivity and 88.5% specificity, while the +LR and -LR were 8.72 and 0.04, respectively. The ureA-positive samples (n=136) were subjected to multiplex qPCR which detected A2142G and A2143G mutations in the 23S rRNA gene (20.6%, 28/136) conferring clarithromycin resistance and gyrA mutations N87K, N87I, D91N, and D91Y (11.8%, 16/136) leading to levofloxacin resistance. The sensitivity and specificity of qPCR of 23S rRNA gene were 100% and 98.7%, respectively, while 100% and 99.8% for qPCR of gyrA, respectively.
CONCLUSION: The effectiveness of this qPCR is that it is sensitive in detecting low bacterial load and will help in timely detection of clarithromycin- and levofloxacin-resistant strains, especially in case of mixed infections. Since it is culture independent, it can inform clinicians about antibiotics to be included in the first-line therapy, thereby improving the management of H. pylori infection at a much greater pace.
Materials and methods: Gastric biopsies from antrum (n=288) and corpus (n=283) were obtained from 288 patients who underwent endoscopy at Universiti Kebangsaan Malaysia Medical Center (UKMMC), Kuala Lumpur, Malaysia. Antibiotic susceptibility to six classes of antibiotics was determined by the E-test. Mutations conferring in resistance in functional genes were identified by PCR and sequencing.
Results: Overall resistance rates to metronidazole, clarithromycin and levofloxacin were 59.3% (35/59), 35.6% (21/59) and 25.4% (15/59), respectively. Secondary isolates showed significantly higher resistance rates to clarithromycin compared to the primary isolates. Mixed infection with susceptible and resistant isolates was observed in 16.2% (6/37) of cases, of which 83.3% (n=5) had infection with the same strain. 41% (18/44) of isolates were resistant to more than one class of antibiotics of which 50% (9/18) were multidrug-resistant, two being primary and seven being secondary isolates. Mutations in rdxA, 23S rRNA and gyrA genes were associated with resistance to metronidazole, clarithromycin and levofloxacin, respectively.
Conclusion: The high level of resistance to metronidazole, clarithromycin and levofloxacin seen in H. pylori isolates in our setting warrants the need for continuous surveillance and highlights caution in use of antibiotics generally used as first-line therapy in H. pylori eradication regimen.