1177 QUANTITATIVE QPCR-BASED BIOBURDEN TEST FOR THE DETECTION OF CARBAPENEM-RESISTANT ENTEROBACTERIACEAE (CRE): NOVEL TEST FOR SURVEILLANCE AND RISK-STRATIFICATION

Abstract

Current strategies to combat duodenoscope-associated carbapenem-resistant Enterobacteriaceae (CRE) transmission, a low-prevalence but high-risk clinical problem, are either capital intensive (ethylene oxide sterilization) or time- and resource-intensive (reference laboratory PCR or culture and sequester strategy). PCR tests do not discriminate live vs. dead organisms and quantify them. Our aim was to develop and perform initial analytical verification and clinical sample testing of a bioburden qPCR test that quantifies the number of live CRE present on duodenoscopes or biofluids within 3 hours of sampling. We designed TaqMan assays against each intended target gene sequences (16S rRNA, Klebsiella pneumonia carbapenemase (KPC) and New Delhi metallo β-lactamase (NDM)) that can detect and quantify genomic DNA (gDNA) across 5-9 log concentrations with >95% qPCR amplification efficiency. We developed an optimum gDNA extraction method to extract gDNA with maximum efficiency. Propidium monoazide (PMA)/ enhancer concentrations and incubation/light exposure time was optimized to yield maximum cycle threshold (Ct) difference in qPCR quantification between live and dead CRE (Figure. 1). We successfully tested and quantified live CRE from serial dilutions of intentionally spiked (with different CRE concentrations) patient-derived bio-fluids (bile and gastrointestinal secretions) acquired during ERCPs (n=15). Duodenoscope samples were collected by flushing 10 ml of sterile saline through the accessory channel and dipping and agitating the end of the duodenoscope and performing ten up and down movements of the elevator in a container of sterile saline Propidium monoazide (PMA)/enhancer can detect, differentiate and quantitate 10% of spiked-in live bacteria among the dead. Our DNA extraction method outperforms commercially available DNA extraction kits that we tested, in time, cost and efficiency. Assay reproducibly detects >100 intentionally spiked NDM1+ and KPC1+ CFUs either within patient derived biofluids or endoscope wash fluids (Saline) (figure 2). There is no detectable PCR inhibition from patient derived abdominal fluids, and biliary specimens. We report on the successful development and initial testing of a rapid-turnaround quantitative PCR bioburden test that can detect and quantify small numbers of live CRE on duodenoscopes and biofluids. Experiments are underway to test the accuracy, precision, and the analytical sensitivity of the assay. Future studies will include test validation in a multi-center duodenoscope and other medical device-derived biofluid testing and creation of a digital PCR interface.Figure 2Amplification plot demonstrating detection of live CRE among intentionally spiked biofluids.View Large Image Figure ViewerDownload Hi-res image Download (PPT)