Simultaneous Immunodetection of Anthrax, Plague, and Tularemia from Blood Cultures by Use of Multiplexed Suspension Arrays

Dose-response curves of multiplexed immunoassays for the detection of spiked soluble disease biomarkers from blood cultures, implementing the 4-plex diagnostic array. Blood cultures (Bactec plus aerobic/F culture vials) containing naive human blood were centrifuged and spiked with recombinant F1 (light blue) and Vag (blue) for plague detection (a) or purified PA (Bordeaux) and soluble anthrax capsular antigen (CAP) (pink) for anthrax detection (b). Points are the averages of at least three independent sets of measurements. Signal-to-noise (S/N) ratios were calculated as described in Materials and Methods, and the limit of detection for each test was determined for S/N ratios to be ≥2 (black dashed line).

Detection of native disease biomarkers from inoculated blood cultures utilizing the 4-plex soluble diagnostic array. Blood cultures (Bactec plus aerobic/F culture vials) containing naive human blood were inoculated with Y. pestis EV76 bacteria (a) or B. anthracis Vollum spores (b) and incubated at 37°C. Blood cultures were then centrifuged and plague biomarkers, F1 and Vag, or anthrax biomarkers, PA and capsular antigen, were measured with the soluble 4-plex array. S/N ratios were calculated as described in Materials and Methods, and the limit of detection for each test was determined for S/N ratios to be ≥2 (black dashed line).

Blood culture processing procedures for bacterial detection. Blood cultures were processed either with a stepwise centrifugation approach (a) or with a Vacutainer (b). The stepwise centrifugation methodology consisted of a mild centrifugation (140 × g for 10 min) of 1.5 ml of culture fluids. The recovered supernatant (indicated as 1 and highlighted in yellow) was then sedimented (14,000 × g for 5 min), and the pellet (indicated as 2 and highlighted in yellow) was washed with 1 ml of PBS and resuspended in 200 μl of PBS. In the second approach, a 5-ml blood culture sample was centrifuged (1,400 × g for 10 min) in a Vacutainer. The resulting bacterial layer, located on the separating gel, was resuspended in 500 μl of PBS and placed in an Eppendorf tube (indicated as 3 and highlighted in yellow). All bacterium-containing blood fractions that were implemented in the test are indicated in yellow.

Evaluation of blood culture fractions for bacterial detection. Blood cultures (Bactec plus aerobic/F culture vials) containing naive human blood were inoculated with Y. pestis EV76 (green) (a) or F. tularensis LVS (red) (b) and incubated at 37°C to a final concentration of 1 × 10⁸ or 1 × 10¹⁰ CFU/ml, respectively (determined by viable counts). The resulting blood cultures were processed with the stepwise centrifugation approach or with a Vacutainer. Fractions 1, 2, and 3 (indicated in yellow in Fig. 4) were tested with the 2-plex diagnostic array, undiluted or at 10- and 100-fold dilutions. S/N ratios were calculated as described in Materials and Methods, and the limit of detection for each test was determined for S/N ratios to be ≥2 (black dashed line).

Dose-response curves of multiplexed immunoassays for the detection of spiked bacteria from blood cultures implementing the 2-plex diagnostic array. Blood cultures (Bactec plus aerobic/F culture vials) containing naive human blood were mildly centrifuged (140 × g for 10 min) and spiked with inactivated Y. pestis EV76 (green) (a) or inactivated F. tularensis LVS (red) (b). Points are the averages of at least three independent sets of measurements. S/N ratios were calculated as described in Materials and Methods, and the limit of detection for each test was determined for S/N ratios to be ≥2 (black dashed line).