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.
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.