Winning Abstracts from the 2009 Medical Student Abstract Competition: MRSA Detection of Colonized Needless Catheter Ports Using Real-Time PCR
Author: Jason R. Sebesto, third year medical student at Des Moines University, College of Osteopathic Medicine and Health Sciences
Each year, over 19,000 patients die from invasive methicillin-resistant STAPHYLOCOCCUS AUREUS (MRSA) infections related to hospital stays. A common route of entry for MRSA into the blood stream is via needless catheter ports (NCs), which while reducing needle-stick injuries to healthcare workers, have been associated with an increased incidence of MRSA blood stream infections in patients. The current "gold standard" for detection of NC colonization by MRSA is a standard culture, which takes 48-72 hours for results. This extended time period often results in immediate removal of the indwelling catheter and initiation of empiric antimicrobial therapy if clinical suspicion is high for colonization of the NC (and thus, the catheter). We are developing a rapid MRSA detection method using real-time PCR (qPCR) that could potentially detect colonization of NCs by small quantities of MRSA.
Three NC types were selected for this study: Clave, ICU Medical; Q-Syte, BD Products; and MaxPlus, Medegen. Each NC type had their lumens colonized for 72 hours with 90.0 cfu/µL and 9.0 cfu/µL of blood-based inoculum using a clinical MRSA isolate (G001). A constant-flow apparatus was utilized to prevent clotting due to hemostasis of the blood-based inoculum. After the incubation period, an achromopeptidase bacterial lysis protocol was used to collect DNA from the biofilms formed within the NC lumens. The collected DNA underwent qPCR to detect the genes FEMA, which is unique to S. AUREUS, and MECA, which incurs methicillin resistance to bacteria. Control samples without DNA were subjected to qPCR, as well. Cycle thresholds (CTs) were used to determine adequacy of detection. Results were analyzed for sensitivity and specificity.
During test development, criteria were established that a true-positive result for MRSA was indicated by a CT of 30 or less for both FEMA and MECA genes. Using a constant flow application with human blood introduced, the assay was found to detect MRSA routinely from inoculated ports. The limit of detection for the assay with inoculated ports was 9.0 cfu/µL. For all port types combined, the sensitivity of the assay was 81% and the specificity 100%. However, the sensitivity among port types differed. The time from the point of removing the NC from a treatment line to final assay results was 3 hours.
The qPCR assay and harvesting procedure developed here indicates this is a sensitive, specific and very rapid approach to identify intravascular catheter ports which are or are not contaminated with MRSA. This approach will aid clinicians in determining whether to leave a catheter in or remove it due to serious MRSA contamination.