Transfusion-associated septic reactions (TAS), that occur due to transfusion of blood and blood components contaminated with bacteria often result in fatal consequences. Red blood cells (RBC) which are stored at 40C, are most frequently contaminated with gram negative bacteria such as Yersinia enterocolitica, Serratia liquefaciens, Pseudomonas fluorescens etc. Bacteria associated with platelet concentrates, which are stored at room temperature, most often are the skin associated gram positive coccis, such as Staphylococcus epidermidis, Bacillus cereus and Staphylococcus aureus. Methods for rapid and sensitive detection of these bacteria are lacking, as is knowledge of the true incidence rate of bacterial contamination of blood products. The existing methods depend on a bacterial concentration of about 104 - 107 bacteria /ml, before the bacteria can be detected. Nucleic acid based testing for detection, such as the polymerase chain reaction (PCR) is extremely sensitive and has the potential of detecting one bacterium within hours. Moreover it can identify the species of contaminating bacteria. I have directed my present efforts to develop a highly sensitive PCR assay for detecting Y. enterocolitica in blood samples stored up to 42 days at 40 C. Y. enterocolitica accounts for 50% of the clinical sepsis caused by RBC contamination. For this I have chosen the fluorogenic 5'-nuclease assay, the TaqMan PCR assay, which has the following advantages. (1) It is a real time PCR assay, and uses a fluorogenic oligonucleotide probe in the reaction. Thus, it has the potential of detecting targets in less than 2 hours. (2) It is a highly quantitative PCR that can indicate how many copies of the gene are present in the unknown sample. I have designed primers and probe targeted to the 16S rRNA gene region. There are several copies of this gene per cell and during active cell growth the abundance of the rRNA can be as high as 104 copies per cell, meaning that fewer target bacteria are needed to detect a contaminating unit. Several kits for purifying chromosomal DNA from blood that has been spiked with bacteria have been evaluated so that hemoglobin, which is a potent inhibitor of Taq polymerase, is efficiently removed, and so that the extraction reagents do not interfere with the fluorescence. Preliminary data indicate the sensitivity with the TaqMan PCR assay to be 10 bacteria/ 200 ul of blood sample. Efforts are in progress to increase the sensitivity of the assay in terms of the number of bacteria detected and to shorten the assay time. In the second phase of the project the Y.enterocolitica assay model would be used to detect a broader spectrum of contaminating bacteria. The rRNA gene has conserved DNA regions interspersed with variable sequences, in related bacteria. By using the same forward and reverse primer set directed towards the conserved regions shared by different bacteria and using specific probes directed towards the variable region of a given species, a wide range of bacteria contaminating RBCs could be detected in a single TaqMan PCR assay. The forward and reverse primers as well as the TaqMan probes are being developed now. Probes from different species will be labeled with a different colored fluorogenic dye, allowing definitive identification of the species. The probes will be tested in a survey of febrile reactions occurring during transfusion to quantify the frequency of and the bacterial species responsible for contamination. This information would help FDA to identify risk factors such as processing, storage, and donor selection. To this end, future projects would include seeding of platelets with the gram- positive bacteria mentioned above, and designing primers and probes directed toward the 16S rRNA to identify these bacteria using the sensitive TaqMan assay.
