4

Potential Technologies for the
BioWatch Program

Having been provided with an overview of the BioWatch program and input from public health officials that run local BioWatch programs and use its information for critical, potentially high-regret1 decision making, the workshop participants discussed the current state of the art, exploring the potential uses of four families of technology in the BioWatch program, and considering how these technologies might be strategically combined or deployed to optimize their contributions to an effective environmental detection capability. For each of these four sessions, the writer of a commissioned paper presented an overview of the technology being discussed (see Appendixes GJ). Each presentation was followed by three or four talks on potential next-generation technologies and then an open discussion among the workshop participants.

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. Panel members were also asked to identify the major integration issues in making these systems field-deployable and capable of detecting organisms of interest with the required sensitivity and specificity to meet the needs expressed earlier by public health officials. This chapter summarizes those presentations and the ensuing open discussions among the workshop participants. Key points during the four sessions include

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1In this context, “high-regret” refers to a situation in which a decision maker would encounter large negative consequences for being wrong.



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4 Potential Technologies for the BioWatch Program Having been provided with an overview of the BioWatch program and input from public health officials that run local BioWatch pro- grams and use its information for critical, potentially high-regret1 deci- sion making, the workshop participants discussed the current state of the art, exploring the potential uses of four families of technology in the BioWatch program, and considering how these technologies might be strategically combined or deployed to optimize their contributions to an effective environmental detection capability. For each of these four sessions, the writer of a commissioned paper presented an over- view of the technology being discussed (see Appendixes G–J). Each presentation was followed by three or four talks on potential next- generation technologies and then an open discussion among the work- shop participants. Each of the panels in the four sessions focused on autonomous, ful- ly 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. Panel members were also asked to identify the major in- tegration issues in making these systems field-deployable and capable of detecting organisms of interest with the required sensitivity and specificity to meet the needs expressed earlier by public health offi- cials. This chapter summarizes those presentations and the ensuing open discussions among the workshop participants. Key points during the four sessions include 1 In this context, “high-regret” refers to a situation in which a decision maker would en- counter large negative consequences for being wrong. 41

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42 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH  Potential challenges to consider: o Integration of multiple components or technologies (“detec- tion is done”). o Effectively maximizing sample concentration and minimizing sample contamination (sample to environmental background). o High-quality databases, bioinformatics tools, and assay vali- dation are needed for all technologies.  A multidimensional approach is encouraged: o Technologies using immunoassays and mass spectrometry are fast; nucleic-acid approaches and genome sequencing can provide specificity and identify novel threats. o Orthogonal testing increases the reliability of a BioWatch Actionable Result (BAR) and confidence in the system.  Extensive and repeated field testing will be necessary for a de- tection system.  It is important to involve the end user (public health) in system development and testing.  The Department of Homeland Security (DHS) was encouraged to look beyond the familiar technologies and development pathways. AUTONOMOUS DETECTION SYSTEMS USING NUCLEIC-ACID SIGNATURES In the first of the four sessions, Raymond Mariella, Jr., a senior sci- entist at Lawrence Livermore National Laboratory (LLNL), provided an overview of his commissioned paper reviewing the state-of-the-art tech- nologies available for detecting organisms using nucleic acid signatures (see Appendix G). The following topics were then discussed by the pan- elists: lessons that could be learned from other industries in autonomous detection and how those lessons might apply to BioWatch; the evolution of assays to include and exclude various threats; the current state-of-the- art system used by the U.S. Postal Service (USPS) and next-generation autonomous detection system based on multiplex polymerase chain reac- tion (PCR) assays; and systems concepts and integration issues. Nucleic-Acid Signatures at Three Levels of Readiness In his interpretation of the three tiers of readiness, Mariella said that any system that would be deployable at TRL 6-plus by 2016 would have

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POTENTIAL TECHNOLOGIES FOR THE BIOWATCH PROGRAM 43 to consist of off-the-shelf components and be running today. For Tier 2— those ready by 2020—it should be possible to improve sample prepara- tion and add another level of identification to the automated assays, per- haps through some sort of targeted amplification of virulence regions. For Tier 3 systems, those available after 2020, technology developers still have years of vigorous research ahead to create and validate assays, develop the necessary informatics software and data interfaces, and per- form extensive integration work. Mariella then provided his notional overview of the system compo- nents and their states of readiness (see Figure 4-1). For Tier 1 readiness, the technologies that are available today and are at TRL 8 or TRL 9 (see Appendix E for TRL descriptions) include those for high-volume aerosol collection into an aqueous medium, sample collection, and primary de- tection. For collection, virtual impact collectors can reduce sample con- tamination and determine the particle size distribution of the particles that it collects. It should also be a simple matter to add a video camera to the system to monitor external events that might explain a positive sig- nal. Wetted-wall and other high-volume particle collectors can increase sample yield and deposit particles directly into aqueous media that can be divided into aliquots for multiplexed analyses. Collecting samples directly into a liquid would also increase the odds that any microorganisms would remain alive for subsequent viability testing. Automated systems that in- clude PCR for nucleic acid analysis, which require the lysis of spores to release nucleic acids, are currently the only technologies ready for 2016 deployment. For primary detection, Tier 1–ready technologies can divide samples into aliquots for single-target, real-time PCR or can conduct mul- tiplexed assays using bead-based capture probes, Mariella said. Tier 2 systems could incorporate a light-scattering unit that would log the ambient particle count, size distribution, background fluores- cence, and other information that provides context for any positive sig- nal. 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 communica- tions with monitoring. One potential technology that Mariella mentioned that could be available after 2020 would use a surface molecular recogni- tion reagent or a pool of reagents to capture organisms of interest to en- rich samples for subsequent analysis (Hou et al., 2013).

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44 FIGURE 4-1 Notional: General system overview. NOTE: TRL = technology readiness level. SOURCE: Mariella presentation, June 25, 2013.

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POTENTIAL TECHNOLOGIES FOR THE BIOWATCH PROGRAM 45 Today, Mariella said, two systems are ready for deployment: the Au- tonomous Pathogen Detection System (APDS) developed largely by LLNL (Regan et al., 2008) and the Microfluidic Bioagent Autonomous Networked Detector (M-BAND) developed by Microfluidic Systems Inc., a subsidiary of PositiveID (Sanchez et al., 2011). Mariella said that both systems have been tested successfully in the field. “If you want to put something in the field this afternoon, you have those two to choose from,” he said. He characterized a suite of aerosol-characterizing equip- ment as being mature and at TLR-8 or higher, including an inexpensive simple aerodynamic particle sizer and more expensive systems that use fluorescence measurements to characterize particles. 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. In his opinion, he said, it should be feasi- ble to use a panel of 20 or 30 reagents on magnetic beads to capture all of the threat organisms, separating them from the background atmospheric microbial content and thereby improving the performance of the down- stream analytical technologies. He also mentioned the AmpliSeq tech- nology (Murphy et al., 2005; Towler et al., 2008), which can be used to search for targeted amplification of virulence regions or other identifiers, including plasmids in bacteria, which again would provide better signal- to-noise performance. Emulsion or digital PCR could also improve both single and multiplexed amplification and provide intrinsic quantification of starting copy number, offer real-time detection in fewer cycles with less amplification bias, and be available by 2020 (Kiss et al., 2008; Shiroguchi et al., 2012; Vogelstein and Kinzler, 1999; Whale et al., 2013; Woyke et al., 2010). Cindy Bruckner-Lea from the Pacific Northwest National Laboratory commented that one driver of technology that had not been mentioned was the need to analyze indoor samples more efficiently. She suggested that particle detection could be used to trigger the more costly sample analysis. Mariella questioned this suggestion, saying that, in his view, it would not provide the best return on investment.

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46 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH Issues and Opportunities for BioWatch Autonomous Detection According to Allen Northrup, sample processing and fluidics are the two areas related to next-generation BioWatch systems that need the most attention. “In my opinion, detection is done,” he said. “The point is to get the sample into the detector, and there are plenty of state-of-the- art, FDA [Food and Drug Administration]-approved detection technolo- gies for doing that.” Fluidic control has its challenges—mainly related to situations in which the output of one module may not match the input needs of another module—but industries other than biodefense, particu- larly the food and beverage and pharmaceutical industries, have solved this problem and have created proven commercial systems using fluidics. The key challenges for any system, he said, are to define specificity within a defined limit of detection and to then define sensitivity within the context of that specificity (see Figure 4-2). False-positives and false- negatives, he said, also need to be defined within a predetermined limit of detection, with the caveat that, at the limits, false-positives and false- negatives are inevitable with all analytical systems. “The question be- comes what can we live with? What is realistic?” PCR is a well-proven, reliable technology that can provide quantita- tive results in a multiplexed system. The challenge for the next- generation BioWatch system, Northrup said, is to automate it and im- prove handling of the complex samples that are collected in the field. “However, the thing I learned today is that BioWatch is more than in- struments and threshold cycles,” he said. “It is a basis of human interac- tion and communication, informed decisions, and ancillary information. Whatever we build and deploy is going to have to have that human interac- tion.” In his opinion, a lab-in-a-box system that inputs a sample and outputs a result will not be well accepted by public health, which will want to see the direct result of any analysis and understand thoroughly the analytical param- eters. Northup recommended that system developers provide public health agencies and laboratories with the subcomponents of their systems for eval- uation and approval early in the development process. To conclude his remarks, Northrup said that the BioWatch communi- ty 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 concern- ing which processes they would like to see automated. The resulting au- tomated modules should then be provided to the laboratorians for

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POTENTIAL TECHNOLOGIES FOR THE BIOWATCH PROGRAM 47 FIGURE 4-2 Fundamentals of all instrument analyses. SOURCE: Northrup presentation, June 25, 2013; Northrup Consulting Group. 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. Life Sciences, Inc., focused his re- marks on the question: How will the technology evolve signatures as in- creasing knowledge of targets and neighbor strains reveals both false- positive and false-negative situations? As background, he described how such signatures are developed today, starting with identifying genes that are distinctive to a target organism and developing probes or primers to detect those genes. This deductive approach, which has been used success-

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48 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH fully for more than 25 years, relies on deep knowledge of the pathogen in question and works well for targeting key pathogenic determinants, such as toxin-coding genes. However, this approach ignores most of the genome as a potential signature candidate. The newer genomic approach, which has been developed in the past decade or so in parallel with the revolutionary advances in genome se- quencing technology, starts with a target organism’s full-genome se- quence data and compares that sequence with the full-genome sequence data from all other organisms. The result is a set of sequences that are unique to the target. This approach requires no knowledge of the patho- gen, and the analysis is done completely in silico, yielding multiple tar- gets. Knight noted that this approach relies on coherence between the in silico world of genome databases and the real world, where false-positives and false-negatives arise. As the number of microbial sequences continues to increase, he said, signature designs will continue to improve, lowering the probability of false-positive and false-negative results. During the past decade, this type of approach has also yielded a new understanding of bacterial and viral genetics. One thing that has become apparent, Knight said, is that there is not a clear-cut situation in which a sequence is found in the target and not in the background genomes. “What we have learned is that microbial genomes form a continuous ge- netic background,” he said. Instead of each species having a completely unique genome, species share what Knight called pan-genomes, core ge- nomes that are common to all members of a particular genus of bacteria, with various additions and subtractions of genes and gene families across the species and strains of species in that genus (Hu et al., 2011) (see Fig- ure 4-3). 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. “We need multiple targets to be able to assess differences on this continuous genome space,” Knight said. Moreover, target sets will have to be adaptive, and analyti- cal systems will have to accommodate changing signatures on what Knight suggested could be a quarterly basis. “We’re going to want to change the signatures so that we can have greater accuracy in our detec- tions,” he said. For the 2020 scenario, Knight said, it will be necessary to sequence not just signature regions but the entire genetic complement of a sample in order to provide a probability-based analysis of the genomic content of the sample.

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POTENTIAL TECHNOLOGIES FOR THE BIOWATCH PROGRAM 49 FIGURE 4-3 Pan-genomes of the genus Burkholderia. SOURCE: Knight presentation, June 25, 2013 (adapted from Hu et al., 2011). 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. The big challenges ahead, he said, will involve system integration and workflow. For example, how will reflex- ive testing, in which an initial screen for a single target provides a poten- tial positive result that triggers additional, deeper testing, be integrated into an autonomous system? Knight also noted that consumables han- dling has not yet reached the required level of maturity for it to be ready for widespread deployment in an autonomous system in a way that would enable laboratorians responding to a positive result to instantly trace the history of the consumables in an instrument all the way back to

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50 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH their manufacturers. Similarly, systems monitoring and preventive maintenance procedures will need to be developed. Finally, reiterating Northrup’s earlier recommendation, Knight said that all of these systems and procedures must from the beginning be worked out with the laboratorians involved. “If public health laboratorians are going to be involved in decision making, they are going to want to know and under- stand the quality system that was put in place for that automated system. They are going to want to see that the quality system is the same or simi- lar or analogous to the quality system that they are used to in their la- boratories 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, radiologi- cal, nuclear, and explosive] Defense at Northrop Grumman, focused on two systems: the Biohazard Detection System (BDS) that the USPS has been using for more than a decade and the Next Generation Automated Detection System (NG-ADS) that the company has developed and field tested under a Generation 3 Phase 1 contract with the Department of Homeland Security (DHS). NG-ADS, Tilles explained, evolved from LLNL’s APDS device, which Mariella had discussed earlier. BDS, which was developed in the wake of the anthrax letter incident in 2001, screens mail as it enters the USPS network and provides a warn- ing before contaminated mail is distributed through the mail network. In a sense, Tilles said, BDS can be considered “Lab-in-a-Box 1.0,” an au- tonomous system that collects samples adjacent to a USPS mail pro- cessing machine. Samples are taken over the course of an hour, during which time between 30,000 and 40,000 letters could have been processed. The USPS can track those letters to isolate them in the event of an alarm. At the end of an hour, the sample moves into a reservoir for PCR testing, while the instrument starts collecting the next sample. Sample prepara- tion and PCR are performed using a disposable cartridge operating in a Cepheid GeneXpert machine, and the results are then reported. So far, the USPS BDS system has conducted more than 11 million tests with no false-positives. Tilles said that the system has been validat- ed by a number of federal agencies outside the USPS, including the De- partment of Defense and the Centers for Disease Control and Prevention (CDC). The USPS also collaborated with the U.S. Army’s Edgewood

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POTENTIAL TECHNOLOGIES FOR THE BIOWATCH PROGRAM 51 Chemical Biological Center to create a test bed to assess system perfor- mance under simulated operational conditions. National deployment of the system was completed in 2005, and more than 1,000 detectors have been installed at hundreds of sites. These detectors are networked in a fully integrated support infrastructure that not only transmits data but also monitors the health and status of the instruments; manages consum- ables, spares, and repairs; and conducts trend analysis for continuous improvement. The National Field Monitoring Center, run by Northrup Grumman, oversees operations of the system. NG-ADS, Tilles explained, is similar to BDS in that it is an automat- ed system that collects, processes, and analyzes samples. “But because of the requirements of the BioWatch mission, there are some pretty signifi- cant differences as well,” he said, particularly in terms of assay flexibil- ity. NG-ADS uses a separate fluidic module to perform sample prep- aration and the Luminex bead array technology to perform multiplexed PCR assays that can detect up to 50 discrete targets from a single sample. Northrop Grumman has built more than 50 units and subjected them to multiple tests at third-party and government laboratories. Tilles charac- terized NG-ADS as being close to, if not at, TRL 8 (see Table 4-1). As it currently stands, NG-ADS is running 22 signatures, including control signatures, to ensure that there are no non-specific reactions occurring. He added that, guided by experience with the BDS and the Postal Ser- vice, NG-ADS has added diagnostic features. In closing, Tilles empha- sized the importance of iterative testing, noting that NG-ADS has undergone extensive testing that revealed some issues that the company has since addressed. 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 mul- tiplexed sample analysis to avoid the need to split a sample into aliquots.

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70 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH TABLE 4-3 Monitoring Abundances of Target Environmental Organisms in Oil- Exposed Sediment Using Next-Generation Sequencing Percent of Bacterial Sequences Found With and Without (W/O) Exposure to Oil Relation to Oil Rhode Island Alaska Organism Degradation W/O With W/O With Aerobic Pseudoalteromonas gammaproteobacteria; 0.10% 13% 1% 9% spp. several species consid- ered oil degraders Aerobic Alcanivorax spp. gammaprotebacteria; principal carbon 0.40% 2% 0.07% 0.20% source is linear-chain alkanes Facultative anaerobic Escherichia coli gammaproteobacteria; 4% 0.20% 0.60% 0.80% not associated with oil degradation SOURCE: Young presentation, June 26, 2013. tional field specificities or false-positive rates. Furthermore, the cost of sequencers and sequencing consumables are still too high for routine use. The DNA inputs required for sequences are an order of magnitude too high for practical field use without amplification, and adding amplification would essentially turn these instruments into high-end PCR detectors. Metagenomics could prove to be disadvantageous in high-background scenarios in which heavy pollen or mold spore loads would consume se- quencing bandwidth and reduce sensitivity to pathogens. Developing tech- niques for removing eukaryotic DNA background could solve this problem, Young noted. In terms of technology readiness, Young estimated that the integration of aerosol collection with next-generation sequencing, autonomous opera- tion of sample preparation, and autonomous operation of sequencing are “TRL low.” Data analytics, he estimated, are actually more developed and at TRL 7, and he showed results from tests using the CosmosID system as an example of how far data analytics has come over the past few years. In closing, Young said, “What I’d like to leave the audience with is that no matter what technology gets deployed in [autonomous detection], ge- nomics sequencing has to be in the equation somewhere. You’re going to

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POTENTIAL TECHNOLOGIES FOR THE BIOWATCH PROGRAM 71 want it in the lab to do the attribution analysis; you are going to want it in your system for the interrogation that ultimately will happen.” As a result, he said, any technology that gets deployed should at the very least preserve a sample or not destroy the sample so that it can be sent to the laboratory for sequencing. Discussion John Vitko asked if any of these technologies can meet the BioWatch goal of reporting within a few hours of sampling. Detter replied that Tier 1 technologies will have a turnaround time of about 10 hours after sample collection is complete. Tier 2 technologies should reduce that to about 6 hours, and Tier 3 should drop that further to below 4 hours. The big un- known, 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) that PCR-based detection does not, given the extra cost and time of sequencing the PCR products. Detter said that instead of mere- ly getting a band (indicative of presence/absence of a PCR product), se- quencing identifies the one or more components of that band. He acknowl- edged that it may not be necessary to do this in the field or as the primary application in Tier 1 devices but rather as a confirmatory test. In response to a question from Colwell about the quality of sequence data in public databases, Detter said that there is an effort under way to create what he calls a trusted database. This database would have quality scores associated with the data, and sequences would be checked against near neighbors to generate a set of curated pathogen sequences. The diag- nostics community is leading this effort. Thomas Companion from the Office of Standards in the Science and Technology Directorate at DHS asked how next-generation sequencing technologies will deal with the various structural variations of DNA that are found in organisms and with genome dynamics, such as in the case of organisms that edit their DNA as they use it. Detter replied that he does not see those as issues that are beyond solving with well-curated databases to a level that will be satisfactory for BioWatch purposes. Cebula agreed with that assessment. Schloss added the issues of variability and change are also being addressed by those who are studying complex human diseases. He said that as the research community builds its databases, it will be possible to identify the normal variation seen in environmental samples.

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72 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH Raymond Mariella, Jr., asked about the problem of contamination re- sulting from carryover from one run to the next. Detter replied that today’s sequencers use disposable slides or chips, which solves what was an issue with older capillary sequencers. Schloss said that this issue is being ad- dressed by the clinical community because it has the same concerns about carryover in sample preparation and sequencing. Mariella then asked why there seems to be so little work on viruses, which can also be important biothreat agents. Cebula said that part of the reason is that the taxonomy of viruses is still poorly understood, making it harder to correlate characterization work with a particular species or strain. Detter replied that another advantage of metagenomics is that it will allow for more efficient investigation of viruses using the DNA and RNA se- quencing technologies that he and Schloss described. He added that the Defense Threat Reduction Agency is now working to expand the reference database of viral sequences. Stephen Morse from Columbia University said that while virologists may argue about the fine points of virus classifi- cation, the known viral threat agents can be identified today. He said that he also believes that some of the new technologies offer great potential for better understanding viruses. Allen Northrup asked if BioWatch would be able to afford next- generation sequencing, given the prospects that full deployment would require several hundred million PCR reactions per year. Schloss replied that part of the motivation for using solid-state nanopores is that they would be manufacturable using industry-standard complementary metal– oxide–semiconductor (CMOS) technology that would drive down the cost significantly, although he acknowledged that the effort is still in its infan- cy. 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 com- ment that Detter seconded. Cindy Bruckner-Lea commented that using sequencing in the laboratory to confirm results from the field makes a great deal of sense. She then asked the panel to comment on the major challenges in sample preparation. Cebula replied that work still remains to be done to develop a reproducible and automated procedure, but that PCR had the same issues to deal with before it could be approved for routine clinical use. Young said that what is really needed is a program to field test various methods to see what hap- pens with environmental samples. “You don’t know what inhibitors you’re going to see, you don’t know when you’re looking at minor components in

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POTENTIAL TECHNOLOGIES FOR THE BIOWATCH PROGRAM 73 a complex background, you don’t know how hard or easy it’s going to be to detect that until you get out in field testing,” he said. Thomas Slezak from LLNL asked what the BAR of the future will be in the context of next-generation sequencing, given how difficult it was to agree on a definition with PCR. Young replied that the BAR problem for sequencing will be two-dimensional. One dimension will be the depth of evidence or the number of reads needed, and that is an issue shared with PCR. The second dimension is how far across the genome the analysis will have to proceed before the evidence is convincing enough to declare a BAR. Detter noted that the BAR will not be declared in a vacuum and that field results can be confirmed in the laboratory. AUTONOMOUS DETECTION SYSTEMS USING MASS SPECTROMETRY For the final technology session, A. Peter Snyder and Rabih Jabbour prepared the commissioned paper (see Appendix J). The panelists dis- cussed, quality issues that are critical for mass spectrometry, the use of single-particle aerosol mass spectrometry (SPAMS) for biothreat detec- tion, and the use of a miniature mass spectrometry system for analyzing microorganisms. Current State of Mass Spectrometry Peter Snyder began by explaining that the fundamental principle of all mass spectrometry methods is that they separate gas-phase ions ac- cording to their mass-to-charge ratio. There are many methods for ioniz- ing samples and a variety of analyzers for moving the ions from the region where they are created to a detector, where they produce an am- plified signal. Mass spectrometry is capable of detecting substances at the low-attomole level, Snyder said, and it generates huge datasets in a short period of time. On a Tier 1 time frame, there are two mass spectrometry systems that are mature enough to identify organisms based on their structural compo- sition and that meet the DHS criteria: the Hamilton Sundstrand chemical biological mass spectrometer (HSMS) and the SPAMS. The HSMS sys- tem, which measures the fatty acid content of a microbial sample, has been refined and tested extensively over the past 20 years, but tests have yet to generate quantitative figures of merit for false-positives and the

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74 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH probability of false-positives. Doing so, said Snyder, will merely require a larger body of experiments that will take time to complete. The SPAMS instrument is an improvement on the bioaerosol mass spectrometer developed at LLNL, Snyder said. It uses multivariate data analysis that generates a huge dataset of both positive and negative ions from sampled particles after a preliminary ultraviolet fluorescence analy- sis conducted as particles enter the system. Because of this prescreen, the system can handle 10,000 particles per second. SPAMS was tested at an international airport and produced no false-positives in a 7-week period, during which time it tracked approximately 1 million particles. The down- side to this technology is that the mass spectrometer is large and expen- sive; however, operating costs are low and a comprehensive set of per- formance data has generated figures of merit for prediction of false- positives and false-negatives. Tier 2 systems, Snyder said, cannot begin to address the DHS analyt- ical figures of merit. Though there are many mass spectrometers and sample processing technologies that have the potential to meet those fig- ures 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 engineer- ing 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. He noted that biochips have been manufactured to include microcolumn liquid chromatography and electrospray ioniza- tion and that work is being done on a microfluidic proteomic reactor that can digest a protein sample efficiently to produce a sample suitable for analysis by mass spectrometry. In a protein-based system, the peptides resulting from a bacterial pro- tein digest would be analyzed by mass spectrometry and the output would be matched to a complete theoretical list of peptides in a bacterial database generated from genomic sequencing data. The U.S. Army Edgewood Chemical Biological Center has been developing this type of technology for its ABOID (Agents of Biological Origin ID) system and has shown that it can identify specific bacteria in a mixture using mass spectrometry proteomics data generated in the laboratory. Snyder noted that this approach can identify bacteria at the genus level when a specific species is not in the database. The advantages of proteomics-based mass spectrometry as a biothreat detector are that no prior information about the sample is re- quired for analysis, Snyder said. In addition, no specific reagents are

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POTENTIAL TECHNOLOGIES FOR THE BIOWATCH PROGRAM 75 needed in the analytical process, and it can classify an organism when a primer or probe set is not available. Proteomics mass spectrometry, he added, can provide a presumptive identification of a true unknown or- ganism by mapping its phylogenetic relationship with other known path- ogens (Jabbour et al., 2010). He concluded his summary of the field by stating that there is no sys- tem yet envisioned for Tier 3. He voiced the opinion that PCR or an antigen-antibody technology could be interfaced with mass spectrometry to fill this gap in the market. Critical Quality Issues Rudolph Johnson, acting manager, Emergency Response Branch, at CDC’s National Center for Environmental Health, began by saying that from his perspective as someone who is interested in technology that can be put in the field, the two areas of focus for microbial mass spec- trometry are to develop fingerprint spectra for bacteria and to produce interpretable results on a representative number of samples as quickly as possible. Both of these areas are being tackled by the clinical micro- biology community, he said, and that community has the money to conduct the necessary research. According to Johnson, the BioWatch community needs to be more concerned about quality in the field set- ting than in the laboratory, using environmental samples rather than clinical samples. One of the advantages mass spectrometry has over some of the other technologies that have been discussed is that it is both mature and robust. Mass spectrometry itself is about a century old, and most instrumentation is conserved between applications, Johnson said. 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 de- veloping 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. He said that mass spectrometry is portable, although he noted that “portable” does not mean “stand-alone,” as mass spectrometry is usually done with human input. One unusual feature of mass spectrometry is that a smaller sample usually produces better sensitivity and specificity. Sample quality is important, then, to the veracity of the data from a mass spectrometer, particularly in terms of competition for ions during the ion- ization stage. Johnson offered a caveat regarding the selection of instru-

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76 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH ments, specifically concerning low- versus high-resolution systems. “The one thing I want to tell you about mass spectrometry is that better is the enemy of good enough,” he said. “If you are looking at mass spectrome- try, look for something simple.” Lower-resolution systems, he explained, are more flexible, less expensive to purchase and to operate, and more reliable. Data processing is an area in which advances have brought big changes in the field, and Johnson stressed that the goal for BioWatch should not be data interpretation but rather fingerprint identification, a much simpler task. He said that bacterial databases are now being used in hospitals for routine diagnosis, which suggests that these databases have reached a level of maturity and reliability that bodes well for the biodetection field. He also remarked that these databases are transferable between instruments, which is an important development. In closing, Johnson said that mass spectrometry has reached a stage where it is largely a black box for the average user. However, it is still important to understand the workings of these instruments and their key quality issues. Otherwise, he said, “you will end up with poor-quality data, and the system that you deploy in the field will not be effective.” The questions that every user needs to answer include  How do you verify instrument performance daily?  What are your positive and negative controls?  How do you characterize fingerprint spectra, and who controls database entries?  What is a minimum threshold criteria for a positive result?  What is your matrix? Instruments placed in different locations will have different matrices.  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. Instead, aerosol particles are pulled directly from the atmosphere and analyzed independently in a time that is of the order of milliseconds. This short time frame allows for multiple measurements that can improve detection sensitivity and detec- tion statistics (Steele et al., 2008).

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POTENTIAL TECHNOLOGIES FOR THE BIOWATCH PROGRAM 77 The collector for this device is an aerodynamic lens that focuses par- ticles and directs them past tracking lasers that provide information on size, speed, and location of particles as they move through the system. A fluorescent stage excites the particles and measures fluorescence emis- sions as an indicator of whether a particle is likely to be biological. 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 posi- tive 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. As Snyder men- tioned, the system was first deployed at an international airport for ap- proximately 7 weeks to test background monitoring in a realistic environ- ment. The second field deployment, which occurred at Fort Irwin, evalu- ated how it would perform in a dusty environment combined with heavy equipment emissions. Finally, the Applied Physics Laboratory at Johns Hopkins University conducted independent, laboratory-based simulation tests in the presence of high concentrations of highly interfering back- grounds. Over the course of running 24 hours per day in full autonomous mode for 1 week on two separate occasions, the instrument was able to detect relevant target concentrations of up to 100,000 particles per liter of air within the size range of interest. Detection time was on the order of 1 minute with a greater than 90 percent probability of detection. There was one false-positive during the 14 days of testing, and the instrument failed one time when the inlet clogged as a result of the extremely high particle concentration used in the test. From these data, Gard concluded that this technology is at TRL 5, perhaps approaching TRL 6. Describing the weaknesses of SPAMS, Gard said that the harshness of the ionization method reduces the information content of the finger- print, restricting it largely to metabolic signals. The system does not have predictive capability for signatures in this mass range; therefore, the sys- tem needs to be trained for each signature, given the lack of understand- ing of how genomic and proteomic information translates into metabolic products. Finally, the cost and size of this system are too great for wide- spread deployment. The strengths of the system are that it has a broad range of potential signatures, given the generality of the ionization technique, and that analysis times are in the microsecond range with detection times of less than a minute. Contextual information provided by the system in terms of

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78 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH particle size, concentration, temporal profiles, spatial distributions, and the potential to see other additives can indicate whether a release was intentional. In addition, SPAMS can detect other threats, such as chemi- cal and radiological releases. With its low false-alarm rate and the ability to generate data rapidly, SPAMS could be used to initiate modeling and low-level data-gathering activities in preparation for a potential BAR event, Gard said. In the near term, he argued in closing, the atmospheric context that this technology produces and the speed with which it produces that information can sup- port decision making and response activities that support data from high- confidence detectors. By preserving the aerosol characteristics of the sample, this technology allows one to “translate” between the biology and its implications in terms of transport and potential consequences. This information would fill what Gard said is an important gap in the BioWatch system. “I think this intermediate detection specificity has not really been looked at in terms of how it could be useful within the net- work and supporting the decision makers,” he said. Miniature Mass Spectrometry System for Microorganisms Mass spectrometers do not have to be large, said Zheng Ouyang, as- sociate 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. For example, an instrument that he and his colleagues developed is about the size of a large box of tissues and weighs about 4 kilograms (Gao et al., 2008). However, this device func- tions at atmospheric pressure, and so any biomarker larger than a fatty acid molecule would not be volatile enough to enter the mass spectrome- ter for analysis. He said that he does not believe it is possible to miniatur- ize a universal mass spectrometry platform, but it is possible to miniatur- ize a system for a specific application. As an example, he described a system that his group developed for monitoring therapeutic drug levels from a drop of blood in about 60 seconds. How might this approach be applied to the analysis of microorgan- isms in aerosol droplets? Zheng proposed starting with a disposable fur- nace filter rated for microorganisms fitted inside a small cartridge. Air would be drawn through the filter, which would collect particles. After a suitable collection time, the cartridge would be inserted into the machine for paper spray mass spectrometry analysis (Yang et al., 2012).

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POTENTIAL TECHNOLOGIES FOR THE BIOWATCH PROGRAM 79 Before discussing what might be observed using such an approach, Zheng said that there are more than 40 research systems available today that can do ambient ionization—that is, ionization without any sample preparation—up from 2 that were available in 2005. More than 10 compa- nies have produced miniature mass spectrometers, but most of these can only handle gas samples. The key questions, he said, are whether any of these systems can identify biomarkers that can be efficiently sampled and ionized by ambient ionization methods and that can be used for high- specificity characterization and identification of bacteria. He believes that lipid profiles may be the answer, and he presented data experiments demonstrating real-time identification of bacteria grow- ing in a biofilm using both positive and negative ion modes to create li- pid fingerprints (Song et al., 2009). These experiments demonstrated that it is feasible to differentiate subspecies, too. 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 ambi- ent ionization on small mass spectrometers could be Tier 2 or Tier 3. What is needed to start this development effort, he said, is the provision of standard samples of microorganisms to generate a large body of spec- tra in order to optimize statistical data analysis methods. From there, it would be necessary to optimize sampling ionization conditions, charac- terize matrix effects and detection limits, and determine if the method can identify multiple microorganisms in the same sample. Discussion Raymond Mariella, Jr., asked if either fatty acid esters or the low mass-to-charge ratios measurable with relatively simple instruments have the ability to generate fingerprints that can meet the BioWatch criteria of detecting a thousand organisms of interest against a background of 1 to 10 million other organisms with a false-positive rate of 10−7 or better. Gard replied that for the system he has been working with, the answer is no, and that is one reason that he sees that particular method as an or- thogonal technology that would provide atmospheric context and other information that would benefit BioWatch. Gard also said that he has not seen any mass spectrometry system that can meet those specifications. Mariella agreed that these systems would have value for orthogonal de-

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80 TECHNOLOGIES TO ENABLE AUTONOMOUS DETECTION FOR BIOWATCH tection, but he was curious if they could serve as primary detection sys- tems as well. Rabih Jabbour said that proteomic mass spectrometry is more likely to reach the needed level of specificity, and Johnson added that what he likes about whole-organism protein mass spectrometry is that this field is moving forward without requiring government investment. He predicted that these instruments, which now cost about $200,000 and a $2 cost per sample, will be miniaturized to a point-of-care level. “If you want to move into environmental detection 4 or 5 years from now, you would probably do that much more easily than trying to design a system for this purpose now,” he said. Cindy Bruckner-Lea pointed out that SPAMS could be valuable for monitoring indoor releases because it provides the possibility of real- time detection, which could be used to trigger follow-on analysis. She indicated that a cost-benefit analysis would be needed, however, and a major limitation is the very high cost of these systems. She questioned whether SPAMS would be able to have enough sensitivity for outdoor settings, because the limit-of-detection requirements are more challeng- ing for outdoor compared with indoor releases (unless there is a very high density of detectors outdoors). Thomas Companion raised the point that mass spectrometry is one of the primary technologies used to detect trace explosives, which are of very high molecular weight and with virtually no vapor pressure, so there are some similarities between that and detecting large biomolecules. What DHS found was that how sampling was done was more important than the device that was used to analyze the sample. He suggested that investments should be made in the area of sampling. Snyder said that, given the complexity of environmental samples compared with clinical samples, he supported that idea.