Strategies for Effective Improvements to the BioWatch System Proceedings of a Workshop (2018) / Chapter Skim
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3 Developing Effective Biological Detection Systems
3 Developing Effective Biological Detection Systems
Pages 32-64
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From page 32... ...
Rather, they focused their review on detection and characterization of biological agents, with an emphasis on nucleic acid amplification and sequencing technologies that would be ready for deployment in 5 to 10 years. For a BioWatch system improvement to be ready for deployment, he explained, it has to be hardened, scaled, standardized, deployed, and validated across a network.
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From page 33... ...
The first set of technologies Merlin discussed were those that detect nucleic acid signatures, and, in his opinion, there is a real opportunity to upgrade and expand the current primary polymerase chain reaction (PCR) process.
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From page 34... ...
The ThermoFisher IonAmpliSeq and Ion Chef systems, used in conjunction with IonTorrent sequencers, are highly sensitive and good for complex or difficult samples, said Merlin, but the minimum turnaround time is currently around 10 hours. Illumina's TruSeq system, which feeds into Illumina's sequencers, has similar advantages with a turnaround time of 6 to 27 hours.
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From page 35... ...
A second metagenomics approach uses shotgun sequencing, which involves extracting nucleic acids from a sample, sequencing everything present, and using bioinformatics to identify what is most likely present. Shotgun sequencing is useful for microbial ecology studies that aim to identify predominant organisms, but it is not good for finding organisms present in small amounts.
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From page 36... ...
FIGURE 3-1 Current BioWatch Generation 2 aerosol collector. SOURCES: Kadavy slide 2, http://www.sfgate.com/superbowl/article/That-mysterious-Homeland-Security-box-plugged -6790510.php#photo-9317069 (accessed March 16, 2018)
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From page 37... ...
"The endgame here is to make the process more effective and decrease the time to answer." She also wondered if BioWatch might be missing an opportunity to include targets beyond biologicals, even though the BioWatch mission is exclusive to biological agents. Kadavy also wondered if it would be useful to evaluate alternative materials for the filters in the portable sampling units.
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From page 38... ...
Improvements in PCR Before addressing approaches for improving the PCR step in the BioWatch process, Henry Erlich, session moderator, reviewed some of the relevant performance metrics used to characterize PCR performance. One obvious metric, he
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From page 39... ...
Inclusivity panels would include the set of pathogen strains the assay should detect, while exclusivity panels would include a set of closely related species and strains the assay should not detect. Reference collection standard backgrounds are needed to determine how environmental background properties affect PCR performance, and should include a panel of other organisms in the environment the assay should not detect, as well as background substances such as dust and pollen that might interfere with an assay.
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From page 40... ...
Public databases, said Brown, could be screened against private databases to determine which portions of the private databases are not public and can be used for primer design by those who are trying to design biological weapons. In addition, he said, public databases can be used to expand at relatively low cost the secondary "reflex" panel screening for distinguishing between pathogens and nonpathogenic microorganisms.
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From page 41... ...
His suggestion was to consider developing multiple primer pairs. One action Brown suggested that BioWatch might take is to use informatics tools to postprocess multiple false-positive events to distinguish endemic organisms from signals.
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From page 42... ...
The participant then asked if the current BioWatch assays could be multiplexed to include several agents in a single reaction given that there are now numerous fluorescent detectors available for use with TaqMan PCR. Erlich replied that he could not answer the question specifically because he was not sure what the current reactions are, but he has great confidence that it is possible to develop robust multiplex PCR assays for almost any targets.
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From page 43... ...
Budowle wondered if it would be possible to develop a PCR assay for antibiotic resistance genes in the known target organisms that would be used after a first signal or a second panel signal, rather than having to culture the identified organism. Everyone on the panel thought that was a good idea.
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From page 44... ...
An open discussion followed the three presentations. Mass Spectrometry A mass spectrum, explained Wayne Bryden, is the pattern of molecular fragments produced when a source of energy, such an electron beam, ion beam, or laser, strikes a molecule, ionizes it, and causes it to break into smaller fragments.
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From page 45... ...
The technical challenge with this approach is making sure the trigger can sense the presence of the targeted agent in the outdoor environment, though Bryden said it does work well in the indoor environment. The combined trigger-MALDI-TOF system can analyze a sample in a few minutes.
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From page 46... ...
Point-of-Need PCR Lyle Probst, who was part of the team that developed the first-generation BioWatch system, discussed his company's work developing a battery-powered microfluidic system with an integrated and automated sample preparation module that performs point-of-need PCR in 30 minutes. This system, which the company has named FireflyDx, can detect a variety of pathogens, including those of interest to BioWatch, without the need for highly trained personnel.
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From page 47... ...
In a separate test program, FireflyDx was able to detect a marker for genetically modified organisms, which is relevant to the corn and soybean export market, with the same performance as the industry-standard laboratory PCR system. FIGURE 3-5 Evolution of the FireflyDx technology produced reductions in size, cost, and time to result.
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From page 48... ...
It also makes possible assays that are less sensitive to genetic drift and shift and that might have a reasonable chance of detecting something someone designed using synthetic biology, issues that several participants raised in earlier discussions at the workshop. One advantage of a sequencing-based assay, said Reed, is that even if it detects something that was not anticipated, it still generates sequence data for further analysis.
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From page 49... ...
There are, however, some significant limitations to next-generation sequencing. Turnaround time from collecting a sample to generating sequence data typically requires days or even weeks, in large part because of the extensive sample preparation needed before sequencing can begin.
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From page 50... ...
DNA Electronics' goal is to refine amplicon-based sequencing so that it produces results from as little as one target per milliliter, directly from raw specimens, in 3 to 4 hours, including sample preparation and analysis. The resulting system also needs to be user friendly, robust enough that a general-purpose user can operate it, and provide broad coverage of organisms, strains, and antibiotic resistance.
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From page 51... ...
An unidentified participant asked Reed about the length of the reads his technology generates and the error rates for homopolymer regions. Reed replied that, while the technology can generate sequence reads of hundreds of bases, many applications will only require 50 to 75 base reads, which would optimize turnaround time.
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From page 52... ...
James Liljegren, research scientist at Argonne National Laboratory (ANL) , discussed modeling; Chuck Burris, deputy chief of the Department of Security at the Metropolitan Transportation Authority/New York City Transit, then spoke about using BioWatch in a transit environment; and Suzet McKinney, executive director of the Illinois Medical District Commissions, addressed the public health implications of an indoor release of a biological agent.
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From page 53... ...
Natural airflows, driven by winds and temperature differences and mechanical ventilation, serve as the principal drivers of particle transport in other indoor environments. FIGURE 3-7 Particle distribution by size in the ambient air in a Washington, DC, subway station and the air outside of the station.
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From page 54... ...
is the standard modeling tool for transit systems, and field studies in several transit systems have validated the model. The BGM predicts transport and fate of a chemical or biological agent in an underground subway system and predicts the amount of material released to the aboveground environment via vents, station exists, and portals.
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From page 55... ...
Turning to the subject of fomite transport, Liljegren explained that subway passengers become fomites for biological agent particles, and these particles can resuspend and deposit elsewhere in the subway system and beyond. In addition, passengers can track particles on their shoes to points distant from where they are exposed to the biological agent.
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From page 56... ...
56 FIGURE 3-10 Particle transit modeled 1 hour after a 10-gram release of Bacillus anthracis using a combination of the BGM subway model and the QUIC outdoor model showing dosage (milligram-minute per cubic meter, mg-min/m3) and Bacillus anthracis probability curve values for untreated lethal infections corresponding to colors on the map.
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From page 57... ...
To illustrate the complexity of his job fulfilling the "safe" part of that mission, he explained that the Metropolitan Transportation Authority (MTA) , which serves 12 counties in downstate New York and two counties in southern Connecticut, comprises six subsidiaries: the Long Island Railroad, Metro-North Railroad, MTA Bus, Capital Construction, Bridges and Tunnels, and New York City Transit, which by itself is the largest subway and bus system in the United States.
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From page 58... ...
He noted that the results of one exercise simulating a biological attack on New York City Transit was so sobering that he had an easy time convincing the MTA executive staff that it should pursue a monitoring system. Another analysis showed that the economic cost of investing in a detection system paled in comparison to the cost to get the transit system running again once it is contaminated.
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From page 59... ...
government has spent some $90 billion since 2001 to prepare for a biological attack, of which approximately $12 million went to New York City Transit to develop the first subway-vetted Detect-to-Protect prototype that was eventually validated by researchers at the Massachusetts Institute of Technology. His department has worked closely with Liljegren and the ANL team to conduct a range of pathogen dispersion studies in the city's subway system to help with modeling and portable sampling unit siting.
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From page 60... ...
Public Health Implications of an Indoor Release One indoor environment that the previous speakers had not mentioned, said Suzet McKinney, is the interconnection between buildings. In larger cities such as Chicago, tunnels and walkways connect a number of buildings.
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From page 61... ...
Decision makers also have to consider the safety of first responders who have to go into a contaminated indoor environment.
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From page 62... ...
Moving forward, McKinney stated BioWatch will continue to be a critical component of the U.S. biodefense effort, one that requires increased coordination among local, state, and federal officials to frame the local response in the context of an event of national significance.
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From page 63... ...
Another unidentified participant asked the panelists what they would do to mitigate the current lack of a real-time detection system. McKinney said more training exercises and public announcements to educate the public about biological weapons and possible responses to an attack would be a help as far as speeding the decision-making process.
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From page 64... ...
"When the field test was completed, I felt as though I had enough experience with the system and had had enough coordination and involvement with the team from Homeland Security that I could at least provide information and advice to elected officials in my jurisdiction about how well protected we were with that technology," she said. "My advice to those who have the responsibility for determining what the new technology might be would be to involve public health as soon as you can." Referring to her experience with the Generation 3 system test, McKinney said that there were only a few people from public health involved, which created problems when trying to go to other cities to discuss the proposed system.
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