Strategies for Effective Improvements to the BioWatch System Proceedings of a Workshop (2018) / Chapter Skim
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Appendix H: Invited Paper: Technology-Focused Outlook for Future Biological Detection Systems
Appendix H: Invited Paper: Technology-Focused Outlook for Future Biological Detection Systems
Pages 155-190
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From page 155... ...
Furthermore, this paper was prepared for the September 18-19, 2017, workshop on Strategies for Effective Biological Detection Systems hosted by the National Academies of Sciences, Engineering, and Medicine. It does not necessarily represent the views of the National Academies, the Department of Homeland Security, or the U.S.
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From page 156... ...
Despite some methodological updates, the core technology of the BioWatch program has remained fundamentally unchanged since its inception. The system bears a heavy reliance on the manual retrieval of air filters and batched PCR testing, resulting in turnaround times from a potential release to a laboratory detection of 12 to 36 hours.
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From page 157... ...
While efforts were made to thoroughly review emerging and established biological detection technologies for the purposes of this report, the summary and examples herein are not meant to be a complete and comprehensive description of all possible diagnostic strategies for environmental biosurveillance. Rather, the authors took the specific operational requirements of the BioWatch program into account and selectively deprioritized technologies that were unlikely to be practical or cost effective within the next 5 to 10 years.
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From page 158... ...
In addition, the BioWatch program has other key factors that will impact the evaluation, applicability, and selection of next-generation technologies for biological detection, including the following: a. Sensitivity and specificity.
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From page 159... ...
Any new or proposed biological detection strategy must include careful consideration to ensure that the entire assay can be performed routinely and at scale, while remaining within existing or anticipated personnel, resource, and budgetary constraints.
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From page 160... ...
Specific details of the BioWatch assays, including primer and probe sequences, must also re main carefully guarded to prevent deliberate evasion. An optimal next generation biological detection system would either be completely pathogen agnostic, capable of detecting all microbial hazards in an air sample, or sufficiently broad and flexible to reliably detect both Tier 1 biothreat agents and other emerging or dangerous airborne pathogens in a sample.
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From page 161... ...
3. OVERVIEW OF BIOLOGICAL DETECTION TECHNOLOGIES In June 2013, the Department of Homeland Security commissioned the Institute of Medicine and the National Research Council to convene an expert workshop on potential future technologies for the BioWatch Program.
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From page 162... ...
approval for xMAP diagnostic panels for gas trointestinal and respiratory pathogens and emergency use authoriza tion for Zika virus. Of particular relevance, Luminex MAGPIX was a candidate plat form for recent research and development efforts that were under taken to update BioWatch detection technologies.
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From page 163... ...
First, both the TaqMan Array and OpenArray platforms require specific, high-end rtPCR instru mentation, so implementing these approaches across the BioWatch network would represent a significant new investment in terms of capital equipment costs, training/proficiency, and maintenance and support. Second, because these instruments partition the sample across thousands of nanoliter-volume wells, the limit of detection for low-abundance pathogen sequences in a sample is likely to be markedly higher than in a single-tube reaction.
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From page 164... ...
Second, like the ThermoFisher arrays, and depending on the sample extraction and preamplification workflow, low-abundance targets may not be de tected with sufficient sensitivity due to the partitioning of the sample across thousands of individual reaction volumes. While Wafergen stresses the extensive dynamic range of the SmartChip, and a bevy of preamplification options, it nonetheless recommends 3 to 10 ng/µL DNA input per 100-nL well, which would prove challenging in practice with environmental samples.
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From page 165... ...
3.1.3 Commercial rtPCR-Based Diagnostic Panels While an open and flexible detection strategy is likely going to be the most preferable option for BioWatch in the long term, com mercial PCR-based diagnostic panels are worth considering as a supplementary or transitional technology option, particularly if there is engagement and support from instrument manufacturers in the private sector. Most commercial assays for biothreat pathogens are designated for research use only and some, such as the Cephe id™ BA test, which provides identification and limited characteri zation of Bacillus anthracis alone, are very limited in scope.
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From page 166... ...
and other isothermal methods have been widely used in both commercial diagnostics and research applications and are favored due to their ability to amplify genetic targets with high specificity and with minimally sophisticated laboratory equipment. A number of studies have ex plored the use of isothermal amplification for field-portable diag nostics, including multiplexed identification of viruses, bacteria, and parasitic diseases.
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From page 167... ...
TABLE H-1 Performance Characteristics of Selected Nucleic Acid Amplification Technologies Instrument Targets/ Samples/ Platform Assay Com- Instrument Consumables Setup Time Runtime Sample Run Flexibility plexity Sensitivity Specificity Cost (Cost/run) Current/Baseline 1h 6-8 h -- <96 ++++ +++ ++++ ++++ $50,000 $60 Luminex/xMAP 1h 1h 50 <96 ++++ +++ +++ +++ $50,000 $100+ TaqMan Array 30 min 2.5 h 48 8 ++ ++ +++ +++ $85,000 $500 OpenArray 30 min 2.5 h 96+ <96 ++ ++ +++ +++ $185,000 $760 Wafergen SmartChip 30 min 2.5 h 120 42 ++++ ++ +++ +++ $100,000 $300 BioFire BioSurveillance 2 min <1 h 26 1 + + ++++ ++++ $40,000 $185 167
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From page 168... ...
. As these technologies improve, they may provide the basis for inexpensive onboard biodetection for air sampling devices in a future generation of the BioWatch program.
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From page 169... ...
This is discussed in more detail in Section 3.3.2. 3.2 Immunoassays and Protein Signature Detection The 2013 workshop and review of potential technologies for the BioWatch program identified several options for autonomous or semiautonomous biological detection using immunoassays and protein signatures, including multiplexed immunoassays, xMAP enzyme-linked immunosorbent assay (ELISA)
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From page 170... ...
NGS has already proven itself to be a powerful and flexible tool for the study of infectious diseases and is increasingly being used for outbreak investigations and large-scale laboratory-based surveillance programs alike. An important advantage of NGS is that most sequencing workflows can accommodate DNA and/or RNA from a variety of different sources, including host, vector, pathogen, and the environment.
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From page 171... ...
The relatively high output of these sequencers also allows for convenient multiplexing: in practice, most conventional microbial genomic sequencing appli cations can be safely multiplexed to run dozens of samples per flow cell lane. Multiplexing is also possible for metagenomic sequencing, depending on the nature of the sequencing (amplicon versus shot gun)
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From page 172... ...
USD/Mb) Error Rate Illumina MiniSeq Benchtop SBS $50k 2 × 150 bp 24hr/17hr 25M/8M 7.5 Gb/2.4 Gb $0.23/$0.20 0.1% a MiSeq(Dx)
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From page 173... ...
With these limitations, and the relative complexity of an environmental air filter sample, Illumina metagenomic sequencing would likely be limited to amplicon-based approaches. Even then, an optimistic turnaround time from sample to result would be on the order of 10 to 12 hours, and the sequencing workflow would need careful development, optimization, and validation of both tar get signatures and controls.
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From page 174... ...
In recent years, ThermoFisher has largely shifted product develop ment efforts away from general-purpose sequencing and toward highly multiplexed amplicon sequencing for oncology, infectious diseases, and other clinical markets. Their most recent lineup of in struments includes the Ion S5 and Ion S5XL, which effectively re place and deprecate the Personal Genome Machine and Ion Proton, although both are still available for purchase.
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From page 175... ...
, it is pos sible to computationally identify certain base modifications, in cluding 6-methyl adenine, 4-methyl cytosine, and Tet-converted 5-methyl cytosine, directly from the sequence data. The bacterial methylome is believed to play an important role in the restriction and modification of bacterial genomes and the gain or loss of genes related to virulence and antimicrobial resistance.
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From page 176... ...
. Nanopore sequencing is another more recent long-read sequencing technology, which relies on an array of membrane-embedded, protein nanopores to linearize and sequence molecules of nucleic acid.
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From page 177... ...
This streaming model enables rapid base calling and real-time analysis, allowing users to "read until" sufficient da ta have been gathered and to continuously assess the progress and quality of data generation, rather than analyzing the entire data set after the completion of a run. Basic sample prepara tion is also extremely fast compared to other platforms, with a streamlined 10-minute protocol, and an inexpensive sample preparation module (VolTRAX)
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From page 178... ...
As nanopore-based sequencing technologies continue to ma ture, and other systems enter the market, these flexible, cost effective sequencing instruments will almost certainly play an in creasing role in microbiological testing, particularly in small labor atories, clinics, and the field. 3.3.2 Metagenomics: Highly Multiplexed Amplicon Sequencing (HMAS)
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From page 179... ...
For routine biosurveillance, however, 10 hours is an important constraint, particularly since it is unlikely that the Ion Chef workflow can be shortened or optimized without sacrific ing quality or performance of the assay. Even so, because of the nature of semiconductor sequencing, this ~10-hour turnaround time is significantly faster than comparable Illumina-based HMAS.
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From page 180... ...
Perhaps more importantly, Flu idigm systems are not common in most microbiology laboratories and would require significant new capital investment, as well as ongoing commitments in terms of reagents, maintenance and sup port, and workforce training to accommodate any new task specific instrumentation. 3.3.3 Metagenomics: Shotgun Sequencing Shotgun metagenomic sequencing, or whole-sample sequencing, has increasingly been used for pathogen discovery and diagnostics, with several health care institutions in the United States now offer ing routine clinical metagenomic testing under CLIA.
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From page 181... ...
The bioinformatic analysis of shotgun metagenomic sequence data is also significantly more complicated than targeted sequencing, due to the much larger data volumes that are generated, and the absence of target specificity. Consequently, in order to compare and match sets
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From page 182... ...
Relative to targeted amplicon sequencing, the overall cost and complexity of shotgun metagenomics are both significantly higher. Barring significant technological advances, reductions in cost, im provements to laboratory infrastructure and informatics, and con certed efforts to build the necessary databases and bioinformatics capacity that are needed for routine large-scale sequencing and analysis, shotgun metagenomics are unlikely to be feasible or cost effective for large-scale surveillance applications within the next 5 to 10 years.
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From page 183... ...
These limitations are likely untenable for the BioWatch program, unless this sequencing strategy was some how paired with triggered sample collection, reserved for priority samples, or applied instead to targeted metagenomic sequencing, which could be more easily multiplexed.
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From page 184... ...
, and deployability. It also presents some critical advantages in terms of sequencing perfor mance, namely, sequencing speed, read length, simple sample prep and library construction, data output up to 20 Gb, and "read until" continuous data streaming that begins nearly instantaneously upon the start of sequencing.
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From page 185... ...
is another mass spectrometry (MS) technology that has been widely used for identification and genotypic characterization of biothreat agents, health care–associated pathogens, and other microbial diagnostic tasks.
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From page 186... ...
Single particle aerosol mass spectrometry (SPAMS) has also been discussed in the context of BioWatch surveillance, and has been successfully demonstrated for indoor bio-aerosol surveillance during a 7-week demonstration in an airport, with no issues or false positives (Hook-Barnard et al., 2013)
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From page 187... ...
doi: 10.3791/2536. Committee on PCR Standards for the BioWatch Program et al.
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From page 188... ...
2013. Potential Technologies for the BioWatch Program.
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From page 189... ...
Briefings in Bioin formatics 4:133–149. SPAMS Overview: Single Particle Aerosol Mass Spectrometry.
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From page 190... ...
190 Strategies for Effective Improvements to the BioWatch System Wuyts, V., Roosens N
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