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Feasibility of a Novel Approach for Rapid Detection of Simulated Bloodstream Infections via Enzymatic Template Generation and Amplification (ETGA)–Mediated.

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Presentation on theme: "Feasibility of a Novel Approach for Rapid Detection of Simulated Bloodstream Infections via Enzymatic Template Generation and Amplification (ETGA)–Mediated."— Presentation transcript:

1 Feasibility of a Novel Approach for Rapid Detection of Simulated Bloodstream Infections via Enzymatic Template Generation and Amplification (ETGA)–Mediated Measurement of Microbial DNA Polymerase Activity  Daniel R. Zweitzig, Bruce I. Sodowich, Nichol M. Riccardello, S. Mark O'Hara  The Journal of Molecular Diagnostics  Volume 15, Issue 3, Pages (May 2013) DOI: /j.jmoldx Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2 Figure 1 A novel approach for detecting microbes in blood culture samples via differential cell lysis followed by ETGA assay analysis. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3 Figure 2 ETGA assay and qPCR analysis. A: Erythrocyte-depleted HCs taken from two independent blood donors were analyzed by the core ETGA assay. The resultant assay curves indicate the level of ETGA signal detected from these cells. Substitution of ddCTP for dCTP within parallel DPE reaction mixes blocks the generation of ETGA assay signal, thus confirming that the HC-dependent background signal is indeed derived from DPE activity. B: qPCR was performed for the human genomic DNA marker BRAF, to verify that equivalent amounts of HCs were present in the ddCTP-containing DPE reactions shown in panel A. C: Representative ETGA curves demonstrate that the proposed differential cell lysis procedure dramatically reduces the ETGA assay background signal derived from erythrocyte-depleted HCs, while still enabling detection of S. aureus. D: The average CT values obtained from the triplicate non-spiked ETGA blood culture assay preparations shown in panel C were used to determine the fold reduction of ETGA signal that was achieved by the differential cell lysis procedure. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4 Figure 3 ETGA blood culture assay. A–D: Duplicate blood culture aliquots derived from 15 healthy donors were separated into groups A to D and were analyzed using the ETGA blood culture assay. ETGA assay curves indicate the level of background (Bkgd) signal obtained from the various donors. E: The data obtained from ETGA blood culture assay analysis of the 15 healthy donors were reassembled using Microsoft Excel. An ETGA blood culture assay model system positivity threshold value was subsequently generated, calculated as the average background CT minus three times its SD. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5 Figure 4 ETGA blood culture assay analysis of bacterial spikes. A and C: ETGA curves for seven Gram-positive (A) and six Gram-negative (B) bacterial species spiked into blood culture aliquots and processed in parallel with an NSC, using the ETGA blood culture assay protocol. B and D: To relate CFU input to ETGA CT values, the ETGA assay data shown in panels A and C were used to graph approximate CFU input for each of the Gram-positive (B) and Gram-negative (D) bacterial species. CFU values are listed above the bar for each species. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6 Figure 5 Time-course ETGA data analysis (S. aureus). A: CFU input, resultant ETGA curves, and BACTEC TTD for time-course aliquots taken from a blood culture bottle spiked with S. aureus and analyzed by the ETGA blood culture assay. B: ETGA curves and BACTEC results for the parallel time-course analysis from a donor-matched NSC bottle. C and D: Parallel plating was performed, using 0.1 mL from each of the time-course blood culture aliquots, for both spiked (C) and donor-matched NSC (D) blood culture bottles. E and F: qPCR curves. S. aureus–specific qPCR analysis was performed on the same lysates used to generate the time-course ETGA data presented in panels A and B. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

7 Figure 6 Time-course ETGA data analysis (E. coli). A: CFU input, resultant ETGA curves, and BACTEC TTD for time-course aliquots taken from a blood culture bottle spiked with E. coli and analyzed by the ETGA blood culture assay. B: ETGA curves and BACTEC results for the parallel time-course analysis from a donor-matched NSC bottle. C and D: Parallel plating was performed using 0.1 mL from each of the time-course blood culture aliquots, for both spiked (C) and donor-matched NSC (D) blood culture bottles. E and F: qPCR curves. E. coli–specific qPCR analysis was performed on the same lysates used to generate the time-course ETGA data presented in panels A and B. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

8 Figure 7 Time-course ETGA data analysis (C. albicans). A: CFU input, resultant ETGA curves, and BACTEC TTD for time-course aliquots taken from a blood culture bottle spiked with C. albicans and analyzed by the ETGA blood culture assay. B: ETGA curves and BACTEC results for the parallel time-course analysis from a donor-matched NSC bottle. C and D: Parallel plating was performed using 0.1 mL from each of the time-course blood culture aliquots, for both spiked (C) and donor-matched NSC (D) blood culture bottles. E and F: qPCR curves. C. albicans–specific qPCR analysis was performed on the same lysates used to generate the time-course ETGA data in panels A and B. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

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