Quantification of methicillin-resistant Staphylococcus aureus in blood by a real-time polymerase chain reaction assay and its application in monitoring the treatment of methicillin-resistant Staphylococcus aureus bacteremia
Date Issued
2007
Date
2007
Author(s)
Ho, Ya-Chi
DOI
en-US
Abstract
During the last decade, methicillin-resistant Staphylococcus aureus (MRSA) has become an increasingly important pathogen in both nosocomial and community - acquired infections. The recent report that MRSA bacteremia, a poor prognostic factor for MRSA infections, was responsible for persistent infections, recurrent episodes, prolonged hospital stay and high mortality rate, suggested the importance and the need of developing new methods and good parameters to monitor MRSA bacteremia. With the advancement in molecular technologies, several real-time polymerase chain reaction (PCR) assays were reported to detect MRSA in various clinical samples. However, the possibility of using a quantitative real-time PCR to quantify and monitor MRSA bacteremia has not been explored.
For the first specific aim of this study, we have established a quantitative real-time PCR assay for mecA gene by using known copy numbers of a plasmid containing mecA DNA as standard and the previously described mecA specific primers and probes (Francois et al., J. Clin. Microbiol. 2003;41:254). A real-time PCR assay for femASE was also established to exclude the possibility of contamination by Staphylococcus epidermidis. A linear relationship was found between mecA DNA copy numbers detected and the colony forming units (CFU) of a MRSA reference strain (correlation coefficient = 0.9967) and in MRSA-spiked whole blood samples (correlation coefficient = 0.9995). The sensitivity of the assay was estimated to be 100 copies of mecA DNA per mL whole blood. The levels of mecA DNA detected for DNA extracted from MRSA in whole blood stored at 4°C for up to 4 days remain stable, suggesting the feasibility of quantifying mecA DNA derived from MRSA in stored whole blood samples.
For the second aim of this study, we employed the quantitative real-time PCR assay for mecA gene to investigate the mecA DNA load in patients with culture-confirmed MRSA bacteremia and its relationships to various clinical parameters and disease outcome. This prospective observational study enrolled patients with culture-proven MRSA bacteremia in the intensive care units of National Taiwan University Hospital between July 1, 2006 and January 31, 2007. A total of 250 blood samples from 20 patients were collected, and 87 blood samples of them had concomitant blood culture performed. Among the 20 patients, 7 patients died of MRSA infection (non-survivor group) and 13 patients survived the episode (survivor group). The underlying illness was not significantly different between the two groups except for a higher percentage of mechanical valve implantation in patients of the non-survivor group than those of the survivor group (43% vs. 0%, P = 0.03). The foci of MRSA infection, the choice and delay of initial anti-MRSA therapy, delay in catheter removal were not statistically different between the two groups.
The level of mecA DNA levels were higher in samples with concomitant positive blood culture than those with negative blood cultures (1.65x105 vs. 3.49 x104 copies/mL, P = 0.00002), in samples taken during febrile period than those taken during afebrile period (9.38x104 vs. 2.81x104 copies/mL, P = 0.00005), and samples from patients receiving less than 3 days of anti-MRSA therapy than those from patients receiving >= 3 days of anti-MRSA therapy (9.26x104 vs. 3.07x104 copies/mL, P = 0.002). This trend was variable in other clinical parameters, such as white blood cell counts and C-reactive protein. Of note, the concomitant mecA DNA levels in the blood culture-negative samples were not zero (interquartile range (IQR), 5.45 x103-7.56 x104 copies/mL).
On the day of first positive blood culture when the treatment was not initiated, non-survivors and survivors had similar mecA DNA levels (2.34 x105 vs. 3.98 x104 copies/mL, P = 0.07). mecA DNA levels on the third day of treatment (7.17x105 vs. 2.00 x104 copies/mL, P = 0.02) and on the seventh day of treatment (1.62x105 vs 2.31x104 copies/mL, P = 0.04) were higher in the non-survivors than those in the survivors. There was a trend of persistence or increase of mecA DNA levels in the non-survivors and decreasing mecA DNA levels in the survivors at day 3 (0.49 vs -0.37 log, P = 0.06) and day 7 of treatment (0.00 vs -0.42 log, P = 0.07), though this has not reach statistical significance.
The overall objective of this study is to develop a quantitative real-time PCR assay for mecA gene and employ this assay to investigate the mecA DNA load in patients with culture-confirmed MRSA bacteremia and its relationships to various clinical parameters and disease outcome. Our study suggested that this mecA real-time PCR assay can be used to monitor MRSA bacteremia at day 3 and day 7 of treatment and has potentials to provide novel and useful parameters to evaluate treatment of MRSA bacteremia. Future investigation with a larger number of patients of MRSA bacteremia with different clinical outcomes would validate and extend the findings from this study.
Subjects
methicillin抗藥性金黃色葡萄球菌
mecA
即時性聚合酶
連鎖反應
菌血症
MRSA
real-time PCR
bacteremia
Type
text
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