Validation of Hexaplex Recombinase Polymerase Amplification Assay for the Detection of Carbapenemase Genes in Klebsiella pneumoniae Isolates From Pediatric Inpatients

BACKGROUND: The carbapenemase-producing Klebsiella pneumoniae (CRKP) is a global health problem associated with nosocomial and opportunistic infection and gastrointestinal colonization. A rapid detection method for carbapenemase genes is necessary for effective treatment and to prevent dissemination of carbapenemase genes. METHODS: ) and waaQ as a K. pneumoniae-specific target. The carbapenemase genes from 129 CRKP strains isolated from pediatric inpatients and 50 carbapenemase-susceptible strains were identified by hexaplex RPA and validated with the Modified Carbapenem Inactivation Method (mCIM) and PCR sequencing results. RESULTS: The duplex to hexaplex primer concentrations were successfully optimized. Single-tube RPA reaction could be completed at 37°C in 25-30 min. The limit of detection of each target varied from 0.1 to 10 ng. No cross-reactivity of hexaplex primers was observed against five Gram-positive and four Gram-negative pathogenic bacteria in pediatric infections. The sensitivity and specificity were 100% with perfect concordance with both reference methods. CONCLUSION: Therefore, the hexaplex RPA has potential for rapid carbapenemase genes detection in limited resource settings.