Highly sensitive detection of Staphylococcus aureus directly from patient blood

Abstract

Background: Rapid detection of bloodstream infections (BSIs) can be lifesaving. We investigated the sample processing and assay parameters necessary for highly-sensitive detection of bloodstream bacteria, using Staphylococcus aureus as a model pathogen and an automated fluidic sample processing-polymerase chain reaction (PCR) platform as a model diagnostic system.

Methodology/principal findings: We compared a short 128 bp amplicon hemi-nested PCR and a relatively shorter 79 bp amplicon nested PCR targeting the S. aureus nuc and sodA genes, respectively. The sodA nested assay showed an enhanced limit of detection (LOD) of 5 genomic copies per reaction or 10 colony forming units (CFU) per ml blood over 50 copies per reaction or 50 CFU/ml for the nuc assay. To establish optimal extraction protocols, we investigated the relative abundance of the bacteria in different components of the blood (white blood cells (WBCs), plasma or whole blood), using the above assays. The blood samples were obtained from the patients who were culture positive for S. aureus. Whole blood resulted in maximum PCR positives with sodA assay (90% positive) as opposed to cell-associated bacteria (in WBCs) (71% samples positive) or free bacterial DNA in plasma (62.5% samples positive). Both the assays were further tested for direct detection of S. aureus in patient whole blood samples that were contemporaneous culture positive. S. aureus was detected in 40/45 of culture-positive patients (sensitivity 89%, 95% CI 0.75-0.96) and 0/59 negative controls with the sodA assay (specificity 100%, 95% CI 0.92-1).

Conclusions: We have demonstrated a highly sensitive two-hour assay for detection of sepsis causing bacteria like S. aureus directly in 1 ml of whole blood, without the need for blood culture.

Figure 1. Flow diagram of the fluidic steps involved in sample processing and PCR amplification for target genes.

Figure 2. Blood components study processing schematic.

CBC blood from patients with blood culture positive for S. aureus was divided into 1 ml each for detection of S. aureus load in plasma, white blood cells (WBCs) and whole blood. Plasma was processed using a column based resin (CBR) cartridge. WBCs and whole blood was processed in a filter based (FB) cartridge.

Figure 3. Analytical sensitivity of the nuc and sodA assays for the detection of Staphylococcus aureus genomic DNA (A) and S. aureus cells spiked in blood (B).

Figure 4. Detection of bacterial load in different blood components by nuc (A) and sodA (B) assays.

Each Ct data point (open circles) indicates individual patient samples tested in each blood component (one color for each patient sample). The horizontal line indicates median Ct values. Solid color bars in green (nuc assay) and orange (sodA assay) represent the corresponding % positive on the secondary axis. An “n”, indicates the number of samples analyzed. WBC, indicates white blood cells.