Evaluation of the Xpert Carba-R assay for quantifying carbapenemase-producing bacterial load in stool samples.

ABSTRACT

BACKGROUND: The spread of Carbapenemase-producing Organisms (CPO) remains a major threat globally. Within clinical settings, the existing method of determining gene load involves traditional culture to determine bacterial load and polymerase-chain-reaction-based Xpert Carba-R Assay to determine carbapenemase gene type. However, there is a need for a fast and accurate method of quantifying CPO colonisation to study the risk of persistent CPO carriage. OBJECTIVE: This study evaluated the accuracy of Xpert Carba-R Ct value in estimating carbapenamase producing bacterial loads in stool samples. METHODS: Stool samples were obtained from an ongoing study investigating the household transmission of CPO in Singapore. Stool samples lacking carbapenemase producing organisms were spiked with organism carrying a single carbapenemase gene (blaKPC, blaNDM, blaVIM, blaOXA-48(-like) or blaIMP-1) and serially diluted before being subjected to Xpert Carba-R assay and traditional culture. Standard curves with regression lines showing correlation between Ct values and plate counts were generated. The standard curves were validated with stool samples collected from patients. RESULTS: The limit of detection of blaNDM, blaKPC, and blaOXA-48 was approximately 103 cfu/mL, while that of blaIMP-1 and blaVIM was approximately 104 cfu/mL. Validation of the blaNDM and blaOXA-48 curves revealed average delta values of 0.56 log(cfu/mL) (95% CI 0.24-0.88) and 0.80 log(cfu/mL) (95% CI 0.53-1.07), respectively. CONCLUSIONS: Our validation data for stool positive for blaNDM and blaOXA-48-type suggests that bacterial loads can be estimated within a reasonable range of error.