Direct Detection of Bloodstream Pathogens from Whole Blood Using the Workflow from the DNAe BSI/AMR Test: The First NGS Sample-To-Result Solution
INTRODUCTION
Sepsis is a significant life-threatening condition with 48.9 million cases per year leading to 11 million deaths worldwide1. Patient outcomes are directly related to the timely diagnosis and identification of the pathogen and administration of the appropriate antibiotic treatment. However, traditional standard of care methods like blood culture are labor intensive, slow and take days for actionable results, and limited to detection of organisms that can grow in blood culture. See Figure 1. DNAe is developing the first rapid, sample-to-result, next-generation sequencing (NGS) solution, the DNAe bloodstream infections/antimicrobial resistance (BSI/AMR Test), for identification of bacterial and fungal targets directly from whole blood within a work shift compared to days for the current standard of care.
Figure 1: Typical timeline for pathogen identification in sepsis cases using current standard of care methods
The BSI/AMR Test can detect a comprehensive range of bacterial and fungal targets with select antibiotic resistance markers. The test workflow includes sample preparation, library preparation, cluster generation, sequencing, and bioinformatic analysis to generate a clinically actionable test result. The test is designed to work on DNAe’s proprietary sample-to-result NGS platform using the LiDia-SEQTM platform. The entire workflow is completed within a work shift. See Figure 2. In this study, DNAe have evaluated the initial performance of the DNAe BSI/AMR Test workflow on low-level spikes, blanks, and clinical samples.
Figure 2: For Research Use Only. Not for use in diagnostic procedures. For illustrative purposes only.
MATERIALS AND METHODS
Materials: The DNAe BSI/AMR Test uses proprietary sample preparation, library preparation, and sequencing reagents. A NEBNext® UltraTM II DNA Library Prep Kit from Illumina for library preparation and MiniSeqTM Mid Output Kit (300 cycles) for sequencing was used for the orthogonal baselining study. Organisms were purchased or obtained from multiple banks as shown in Table 1 below. Clinical samples were obtained through a research clinical study using UCSD Hillcrest and CALM Lab sites.
Table 1: Source of Spike Organisms
Methods: Spiked samples and no template controls were prepared by using 3 mL whole blood and spiked at 100 CFU/mL 10 CFU/mL and 3 CFU/mL with qualified bacterial and fungal strains. Samples for clinical studies used de-identified clinical samples which included the standard of care results and isolated organisms. DNAe BSI/AMR Test Method: All samples were processed through the BSI/AMR Test workflow on the bench or on LiDia-SEQTM Platform by lysing the sample, binding the probes to a magnetic bead, washing inhibitors, and concentrating the targets. The eluate was amplified, and the copies of the targets were controlled by DNAe’s proprietary copy control method. The targets were clustered on our semiconductor flowcell and were sequenced using DNAe’s patented ISFET technology which rapidly detects protons released during nucleotide addition. DNAe’s proprietary analysis pipeline, the DNAe Assay Caller, was then used to analyse the sequencing data to generate test results. Orthogonal Test Method: All DNAe results were compared to an orthogonal test method that used the DNAe sample preparation, library preparation and data analysis steps combined with an orthogonal sequencing method using a library prep kit to add daptors. The orthogonal sequencing data was analyzed using the NAe Assay Caller to generate the orthogonal test result.
RESULTS
The DNAe BSI/AMR Test workflow successfully detected all spiked samples for all 10 tested organisms down to ≤ 3CFU/mL (Table 2). The blank samples detected all the correct internal controls and had minimal or low-level contaminants that were used to threshold the analysis. All 10 clinical samples were successfully tested by the BSI/AMR Test workflow. The test successfully detected organisms at ≤3 CFU/mL from the clinical samples and had 90% positive percent agreement (PPA) with the standard of care/clinical isolate results. All the clinical samples were also tested with the orthogonal testing method and there was 100% PPA between the test result of the BSI/AMR Test and the orthogonal test method (Table 3). Lastly, spiked whole blood samples were tested by DNAe BSI/AMR Test on the LiDia-SEQ TM platform and the test successfully detected the spiked organisms and reported a genus/species level call (Table 4).
Table 2: Spiked Samples tested at three concentration (3, 10, 100 CFU/mL) with the DNAe
BSI/AMR Test workflow
Table 3: Clinical Samples tested by DNAe BSI/AMR Test Workflow and Orthogonal test
method
Also detected at a lower level in the background: Bradyrhizobium spp., Comamonas spp., and Pseudomonas spp.
**Also detected at a lower level in the background: Bradyrhizobium spp. and Pseudomonas fluorescens.
Table 4: Spiked Samples tested at 50CFU/mL by the DNAe BSI/AMR Test on the LiDia-SEQ
platform
CONCLUSION & DISCUSSION
The DNAe BSI/AMR Test workflow can comprehensively detect bacterial and fungal targets directly from whole blood at low, clinically relevant levels of detection. These represent the first performance data of the LiDia-SEQTM BSI/AMR Test and demonstrate the potential of the technology to deliver clinically actionable results within a work shift compared to days with standard-of-care methods.
REFERENCES
1. Global, regional, and national sepsis incidence and mortality, 1990–2017: analysis for the Global Burden of Disease Study. Rudd, Kristina E, et al. The Lancet 2020, Volume 395, Issue 10219, 200 – 211
Acknowledgement
This project has been supported in whole or in part with federal funds from the Department of Health and Human Services; Administration for Strategic Preparedness and Response; Biomedical Advanced Research and Development Authority (BARDA), under contract number HHSO100201600017C.
Disclaimer: The DNAe BSI/AMR test and LiDia-SEQ platform are under development and have not been approved or cleared by the FDA or any other regulatory agency.
