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
Introduction:
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.
Methods:
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.
Results:
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.
Conclusion:
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.