Background: Rapid detection of bloodstream infections (BSIs) can be lifesaving. We investigated the sample processing and\nassay parameters necessary for highly-sensitive detection of bloodstream bacteria, using Staphylococcus aureus as a model\npathogen and an automated fluidic sample processing ââ?¬â?? polymerase chain reaction (PCR) platform as a model diagnostic\nsystem.\nMethodology/Principal Findings: We compared a short 128 bp amplicon hemi-nested PCR and a relatively shorter 79 bp\namplicon nested PCR targeting the S. aureus nuc and sodA genes, respectively. The sodA nested assay showed an enhanced\nlimit of detection (LOD) of 5 genomic copies per reaction or 10 colony forming units (CFU) per ml blood over 50 copies per\nreaction or 50 CFU/ml for the nuc assay. To establish optimal extraction protocols, we investigated the relative abundance\nof the bacteria in different components of the blood (white blood cells (WBCs), plasma or whole blood), using the above\nassays. The blood samples were obtained from the patients who were culture positive for S. aureus. Whole blood resulted in\nmaximum PCR positives with sodA assay (90% positive) as opposed to cell-associated bacteria (in WBCs) (71% samples\npositive) or free bacterial DNA in plasma (62.5% samples positive). Both the assays were further tested for direct detection of\nS. aureus in patient whole blood samples that were contemporaneous culture positive. S. aureus was detected in 40/45 of\nculture-positive patients (sensitivity 89%, 95% CI 0.75ââ?¬â??0.96) and 0/59 negative controls with the sodA assay (specificity\n100%, 95% CI 0.92ââ?¬â??1).\nConclusions: We have demonstrated a highly sensitive two-hour assay for detection of sepsis causing bacteria like S. aureus\ndirectly in 1 ml of whole blood, without the need for blood culture.
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