Development and Validation of Multiplex Quantitative PCR Assay for Detection of and Mutations Conferring Resistance to Clarithromycin and Levofloxacin in Gastric Biopsy.

AIMS AND OBJECTIVES

More than half of the world's population is infected with , which can cause chronic gastritis. WHO has regarded clarithromycin-resistant as a high priority pathogen. Hence, accurate diagnosis and detection of clarithromycin- and levofloxacin-resistant strains is essential for proper management of infection. The objective of this study was to develop and optimize multiplex quantitative PCR assay for detection of mutations associated with clarithromycin and levofloxacin resistance in directly from the gastric biopsies.

MATERIALS AND METHODS

Specific primers and probes were designed to amplify and mutations in 23S rRNA and 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 by conventional methods and 23.8% (136/571) were positive by the -qPCR with 96.4% sensitivity and 88.5% specificity, while the +LR and -LR were 8.72 and 0.04, respectively. The -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 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 , 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 infection at a much greater pace.