Performance Assessment Real-time PCR For Detecting Bacterial Pathogens In Cultured Blood Sample From Patient With Suspected Bacteremia

[Objective] The purpose of this study is to evaluate the performance of the real-time fluorescence multiplex polymerase chain reaction (FM-PCR) tests developed in our laboratory in previous research for detecting common pathogens cultured blood specimen from patient with suspected bacterial infections.[Method] (1) Three methods for extracting DNA from bacteria were compared. They were alkali/heat lysis, lysozyme digestion and commercial DNA extraction kit. The efficiency of each method was estimated with analysis the products of the species-specific convenient PCR by gel electrophoresis. (2) The analytic sensitivities of four FM-PCR assays were assessed with different concentrations of bacterium (including 1.5×101CFU/ml,1.5×103CFU /ml,1.5×105CFU/ml and 1.5×107CFU/ml) of staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Stenotrophomonas maltophilia, and Acinetobacter baumannii in simulating bacteremia blood samples. (3) Following assessment the clinical performance of application simultaneously of one broad-range primers real-time fluorescence PCR assay and four FM-PCR assays,191 cultured blood samples (comprising of 134 bottles with positive alarm and 57 negative bottles incubated for five days) were evaluated by comparing the FM-PCR results to that of traditional methods for bacterial identification. (4) We also estimated the early diagnosis bacteremia value of combined utilizing of those five PCR assays by detecting bacterial pathogens in 293 clinical blood samples incubated for 8 hours.[Results] 1. The results of PCR amplified products gel electrophoresis show that the methods of alkali/heat lysis was better than other two methods for drawing DNA from both Gram-negative and Gram-positive bacterium.2. The low limits of detecting Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii and Stenotrophomonas maltophilia of FM-PCR assays were 1.5×101CFU/ml. For Escherichia coli and Staphylococcus, it was 1.5×103CFU/ml. But for Enterococcus faecalis, Staphylococcus epidermidis and Enterococcus faecium, the low limits increased to 1.5×105CFU/ml and 1.5×107CFU/ml, respectively.3. Of 191 samples examined,134 were positive and 54 were negative by both methods, and 3 were positive by PCR assays but negative by blood culture. The sensitivity, specificity, positive and negative predictive values of PCR tests were 100%,94.7%,97.8%andl00%, respectively. (2) 58.3%(83/143) strains organisms identificated by traditional methods were detected by species-specific FM-PCR. Both methods identified the same organisms in these 83 cases. The coincidence rate of both methods was 87.3%. (3) The average time of around turn (TAT) of the PCR assays was significantly shorter than that of traditional blood culture.21.6±18.7h(5-117h) vs.96.2±24h(48-228h),p<0.001.4. Pathogens were detected from 7of 293 blood samples incubated for 8 hours by PCR tests. At the same time there were no positive alarms for blood culture. But blood specimen were incubated continually for 5 days, microorganisms were identified froml8 of 293 by traditional method.38.9%(7/18) bacterium were found with PCR assays in 12 hours after obtained the blood samples[Conclusion]1. It is alkali/heat lysis method that suit for extracting DNA from clinical pathogens.2. The analytic sensitivity of the four FM-PCR developed in our laboratory can meet to detect bacterium in cultured blood specimen from patient with suspicious sepsis.3. Combined application of one broad-range primers real-time fluorescence PCR assay and four FM-PCR assays have high sensitivity and specificity for examination of clinical common pathogens in cultured blood samples, and it is a rapid protocol for early diagnosis bacteremia and could be widely used.