Technologies to Enable Autonomous Detection for BioWatch Ensuring Timely and Accurate Information for Public Health Officials: Workshop Summary (2014) / Chapter Skim
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4 Potential Technologies for the BioWatch Program
4 Potential Technologies for the BioWatch Program
Pages 41-80
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From page 41... ...
Each of the panels in the four sessions focused on autonomous, fully automated, end-to-end systems that would be technology readiness level (TRL) 6-plus ready over three time frames: by 2016, by 2020, and beyond 2020.
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AUTONOMOUS DETECTION SYSTEMS USING NUCLEIC-ACID SIGNATURES In the first of the four sessions, Raymond Mariella, Jr., a senior scientist at Lawrence Livermore National Laboratory (LLNL) , provided an overview of his commissioned paper reviewing the state-of-the-art technologies available for detecting organisms using nucleic acid signatures (see Appendix G)
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From page 43... ...
Enrichment chemistries and amplification methods for nucleic acid target sequences could improve sample preparation in ways that allow for virulence assays that would increase the signal-to-noise ratio of the system. It may also be possible by 2020 to add 400-base sequencing of targeted sequences to an autonomous system and to incorporate resident expert systems for preliminary data analysis and two-way communications with monitoring.
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44 FIGURE 4-1 Notional: General system overview. NOTE: TRL = technology readiness level.
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The most advanced system that is ready for deployment uses an aerosol flow cytometer to capture particles and feed them into a mass spectrometer for further characterization. Mariella quickly described the use of selective reagents for capturing pathogenic bacteria and viruses using antibodies, aptamers, synthetic peptides, or nanolipoproteins.
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From page 46... ...
To conclude his remarks, Northrup said that the BioWatch community needs to test new systems repeatedly and be tolerant of early failures. He proposed that BioWatch should be developed as a modular system with significant, ongoing input from public health laboratories concerning which processes they would like to see automated.
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From page 47... ...
validation alongside currently accepted methods in an iterative process that yields a robust, proven module for incorporation into an automated system for which public health will have already developed familiarity and confidence. Assay Development and Evolution In the next presentation, Ivor Knight, senior vice president and chief technology officer of Canon U.S.
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From page 48... ...
. As a result of this new understanding of microbial genomes, it is now necessary to consider pan-genomes and to use multiplexed capabilities to interrogate more potential genomic signatures.
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. In his opinion, Knight said, PCR-based multiplex technologies using bead arrays and real-time PCR with or without hybridization probes are mature and will be ready to be deployed by 2016, while digital PCR will be ready for deployment by 2020.
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They are going to want to see that the quality system is the same or similar or analogous to the quality system that they are used to in their laboratories that has been developed and is tried and true over the years." State-of-the-Art and Next-Generation Autonomous Detection Systems Next, in a talk on Northrop Grumman's work in the biodefense field, David Tilles, vice president for CBRNE [chemical, biological, radiological, nuclear, and explosive] Defense at Northrop Grumman, focused on two systems: the Biohazard Detection System (BDS)
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Issues in Systems Concepts and Integration Stevan Jovanovich, cofounder and chief technology officer for IntegenX, Inc., discussed a series of potential problems associated with automating workflow in a way that is reliable and meets the sensitivity needs of the BioWatch program. The hardest part of integrating workflow, he said, is matching the input of one module to the output of another in a way that retains the highest possible sensitivity; to do this, an integrated system must process as much of the initial sample as possible and then use multiplexed sample analysis to avoid the need to split a sample into aliquots.
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Using technology developed to process human clinical samples combined with other technology that the company developed, IntegenX has built several integrated sample-to-sequence next-generation systems that produce results in a few hours. The RapidHIT 200 system, for example, uses paramagnetic beads to purify DNA and position it for PCR analysis, processing large volumes of sample into nanoliters of beads.
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Vitko noted that several of the talks stressed taking a modular approach and also stressed that it is critical to involve public health laboratorians early in the development and testing process for each module. Vitko said that Knight's discussion of pan-genomics and what it means to do genetic testing introduced him to concepts he had not thought about before and raised important questions for the future of detection based on genomics.
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Toby Merlin, director of the Division of Preparedness and Emerging Infection at CDC's National Center for Emerging and Zoonotic Infectious Diseases, asked if any of these technologies could be married to orthogonal technology2 to confirm positive results. While Mariella said that this was a big challenge and would probably occur on the 2020 time frame, Tilles said that multiple PCR signatures are, in effect, a lowtechnology approach to orthogonal testing and that designing in flexibility in the fluidics systems will provide the means of integrating orthogonal technologies in next-generation instruments.
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"We want the technology to work and we want to know that we're going to make good decisions based on that technology," said Pan. AUTONOMOUS DETECTION SYSTEMS USING IMMUNOASSAYS AND PROTEIN SIGNATURES To start the second technology session, Paul Schaudies, chief executive officer of GenArraytion, Inc., and author of the commissioned paper on immunoassays and protein signatures (see Appendix H)
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Immunoassays are inexpensive to carry out, largely because they are based on a well-developed technology platform and come without the patent licensing burden associated with PCR. Sample preparation is significantly less complicated than for PCR analysis, and immunoassays are less sensitive to dirty samples.
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What remains is for the device to undergo field testing to determine its real-world performance in an environmental test setting. She said that the company believes that the technology could be TRL 6-plus ready by 2016 if it can demonstrate the same immunoassay performance in an autonomous system that it does on the benchtop and if it is confirmed that stable reagents can be deployed and provide stable performance over the entire service period.
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Those tests have shown that the system is capable of identifying with 100 percent accuracy the biological agents in aerosol samples containing between 75 and 450 agent-containing aerosol particles per liter of air, which he said meets or exceeds the targets set by the BioWatch program. Detecting Biothreats with Raman Spectroscopy Andrew Bartko, principal scientist at Battelle Memorial Institute, said that the Resource Effective Bio-Identification System (REBS)
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Field tests have
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Tests on clinical samples for a wide range of substrates demonstrate that this technology routinely achieves sensitivities in the femtogramper-milliliter range, compared with the pictogram-per-milliliter limit of detection for other technologies. In one test, for example, SIMOA was as sensitive as PCR for detecting the human immunodeficiency virus prior to seroconversion.
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07/2013 NOTE: ABT = ambient breeze tunnel; ACPLA = agent containing particles per liter of air; ASEC = aerosol simulant exposure chamber; BWA = biological warfare agent; CBDS = Chemical and Biological Defense; CRADA = cooperative research and development agreement; CWA = chemical warfare agent; DHS S&T = Department of Homeland Security Science and Technology Directorate; DPG = Dugway Proving Ground; ECBC = Edgewood Chemical Biological Center; ID = identification; JBTDS = Joint Biological Tactical Detection Systems; JHU-APL = The Johns Hopkins University Applied Physics Laboratory; JPM-BD = Joint Program Manger Biological Defense; JPM-CA = Joint Program Manager Contamination Avoidance; JPM-G = Joint Program Manager Guardian; JPM-NBC = Joint Program Manager Nuclear Biological and Chemical; TRE = technology readiness evaluation. SOURCE: Bartko presentation, June 26, 2013.
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In a final comment, Jeffery Runge noted that attribution is important and that any autonomous detection system must have archiving capabilities. The panelists agreed and responded that their systems all have the ability to store samples for subsequent analysis.
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Colwell also said that she believes that the BioWatch program should shift its perspective, partner with the National Institutes of Health (NIH) , and bring public health laboratorians into the discussion.
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The first was rapid sequencing of the amplicons from PCR reactions to identify and characterize pathogens; this amplicon sequencing can readily test hundreds of samples. Shotgun sequencing of multiplexed environmental or clinical samples can identify and characterize both known and emerging pathogens.
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By 2020, improved next-generation sequencing technologies, in combination with bioinformatics, should enable metagenomic sequencing of entire environmental samples at TRL 6. These systems would be capable of detecting emerging and engineered pathogens.
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For any system to be adopted, "it must be responsive to public health officials' needs and provide acceptable and actionable information for their decision-making process." Challenges and Opportunities in Genome Sequencing Technologies Jeffrey Schloss, director of the Division of Genome Sciences and program director of Technology Development and Coordination at the National Human Genome Research Institute (NHGRI) , began by noting that most of the next-generation sequencing technologies were developed as a result of the NHGRI's realization toward the end of the Human Genome Project that sequencing the human genome was only the first step toward realizing the full potential of genomics, not just for the study of human diseases but also in many fields, such as agriculture and microbial ecology.
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By 2009, one machine using sequencing by synthesis on array technology could sequence a human genome in less than 1 month. By 2011, one machine could sequence three genomes simultaneously in 1 week.
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"The question is, When is the future? " Though the cost of sequencing has fallen significantly in the past decade, a major part of the expense arises from the need to provide multiple coverage -- on the order of 30-fold -- in order to be able to assemble a genome from the short reads generated by next-generation sequencing technologies.
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From page 69... ...
He echoed Cebula's comments that software and bioinformatics packages will be key to developing these types of applications for rapid identification purposes. On the subject of how metagenomic sequencing would fit into an autonomous detection system, Young said that it would be similar to the approach used with PCR.
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In closing, Young said, "What I'd like to leave the audience with is that no matter what technology gets deployed in [autonomous detection] , genomics sequencing has to be in the equation somewhere.
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The big unknown, he said, is sample preparation time. Cindy Bruckner-Lea, from the Pacific Northwest National Laboratory, asked what sequencing provides in the proposed Tier 1 device (sequencing of PCR products)
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Young agreed that costs must drop and was of the opinion that further development of new reagentless technologies would significantly affect cost. He added, though, that he found it hard to imagine a robust BioWatch program that does not use sequencing somewhere in the process, a comment that Detter seconded.
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AUTONOMOUS DETECTION SYSTEMS USING MASS SPECTROMETRY For the final technology session, A Peter Snyder and Rabih Jabbour prepared the commissioned paper (see Appendix J)
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Though there are many mass spectrometers and sample processing technologies that have the potential to meet those figures of merit, none of the current systems are at the point that they are ready for field testing, and all of them will require appropriate engineering to create a robust, autonomous system. Various microfluidic systems, for example, can concentrate pathogens into a relatively small volume of liquid at a potential operating cost that would be suitable for a large-scale deployment in the field.
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From page 75... ...
Mass spectrometry is a large field, and most of the research effort is aimed at improving the sample preparation, ionization methods, and database analysis, or at developing new applications for the existing detectors, rather than new mass spectrometers. "If you are looking for a new testing platform, you need to consider mass spectrometry," Johnson said.
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How do you compensate for charge competition? Single-Particle Aerosol Mass Spectrometry Eric Gard, scientist in the Defense Biology Division at LLNL, began by saying that one of the advantages of SPAMS is that it eliminates the need to process bulk samples of aerosols, a process that adds sample preparation delays into the analysis pipeline.
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From page 77... ...
Based on measurements of a particle's speed, the instrument knows when a prescreened particle reaches the center of the ion source, and at that point it delivers a high-powered laser pulse that produces both positive and negative ions that are then analyzed by the mass spectrometer. Gard explained that he and his colleagues had what he characterized as a fantastic opportunity over 3 years as different sponsors allowed them to put their instrument through a variety of field tests.
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"I think this intermediate detection specificity has not really been looked at in terms of how it could be useful within the network and supporting the decision makers," he said. Miniature Mass Spectrometry System for Microorganisms Mass spectrometers do not have to be large, said Zheng Ouyang, associate professor at Purdue University, but mass spectrometers cannot access samples directly and therefore need to be interfaced with sample preparation purification and injection modules that add to the size and complexity of the total system.
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More recent work has shown that the same lipid fingerprint is obtained from organisms collected on filter paper and analyzed using paper spray mass spectrometry. According to Zheng, none of these methods is ready for Tier 1, but he believes that with further work, lipid-based identification using ambient ionization on small mass spectrometers could be Tier 2 or Tier 3.
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Snyder said that, given the complexity of environmental samples compared with clinical samples, he supported that idea.
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