BioWatch PCR Assays Building Confidence, Ensuring Reliability: Abbreviated Version (2015) / Chapter Skim
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Pages 3-20
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, and intelligence-gathering activities. The BioWatch system aims to complement these efforts in order to provide public health, emergency management, and law enforcement with an alert to the presence of one of the tested biothreat agents prior to the onset of large numbers of clinical symptoms.
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Similarly, if the result of the BioWatch PCR assays is negative, individuals with responsibility for public health and safety need to understand the limits
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An important element of this confidence is that the PCR assays used in the program have performance characteristics that have been validated and that provide proper and sufficient information to program stakeholders. This issue is the central topic of this report and is the focus of the committee's statement of task (Box S-1)
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To be applicable to BioWatch, a standard to characterize PCR assay performance and conduct assay validation would need to • Establish that the assay has sufficient sensitivity to detect the release of a tested biothreat agent at a program-relevant concen tration above the baseline of environmental background (i.e., it is fit for purpose) ; • Establish that the assay has sufficient specificity to detect patho genic strains of concern but not to cross-react with other strains and organisms, within acceptable program false positive and false negative rates;1 and • Determine that the assay is sufficiently robust for routine opera tional use by jurisdictional laboratories and is otherwise accept able to program users.
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. A BIOWATCH ASSAY STANDARD CAN BE INFORMED BY EXISITING GUIDANCE The report reviews existing guidance on the process for conducting laboratory validation of the performance of PCR assays.
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In concert with third-party contractors, DHS and the BioWatch program have designed laboratory arrangements for undertaking assay and device testing, assembled and quality-controlled DNA from a number of applicable inclusivity and exclusivity panel organisms, conducted testing to obtain system operational data, and established a quality assurance program with the jurisdictional laboratories in the network. These approaches represent appropriate options for characterizing real-time PCR assays for detection of biothreat agents.
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BioWatch particularly relies on the expertise of public health laboratorians to conduct and interpret assay results within their jurisdictions, and these experts need to be aware of the available performance data and be engaged in discussions surrounding assay performance issues that arise. BETTER UNDERSTANDING ENVIRONMENTAL BACKGROUND WOULD IMPROVE THE UNDERSTANDING OF ASSAY PERFORMANCE The current BioWatch approach focuses on testing for the presence of a subset of known biothreat agents considered to be of particular priority.
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However, the committee notes that evaluating performance of PCR amplification starting with purified strain nucleic acid represents a simplified and only partial view of a complex problem. The committee encourages the BioWatch program to consider the critical roles of the steps surrounding the PCR amplification and concludes that it would strengthen a BioWatch PCR assay performance standard to explicitly consider guidance on steps such as sample extraction and preparation as a standard is developed: Recommendation 2.
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PCR involves polymerization of nucleotides in a reaction per formed and mediated by enzymes and thermal conditions, resulting in a theoretical doubling of the amount of targeted nucleic acid in each cycle. In the BioWatch program, PCR is the basis of the targeted amplification of specific nucleic acid sequences in order to identify whether nucleic acids from biothreat agents tested by the program are present on collected filters.
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BioWatch should strive to test its PCR assays against as many known strains of its target agents and relevant near neighbors as possible. Undertaking all such testing through laboratory analysis alone is likely to be cost, time, and feasibility prohibitive.
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Recommendation 8. Federal agencies, including the Department of Homeland Security, the Department of Defense, the Department of Energy, the Department of Health and Human Services, and other partners, should collaborate to produce a reference document or database identifying which repositories contain strains or strain nucleic acids relevant for testing pathogen detection assays.
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Nevertheless, some reasonable compromise must be achieved in order to undertake performance characterization and validation work on assays deployed or being considered for deployment in the program. Understanding and clearly communicating the strengths and limitations of the laboratory data obtained from the PCR assays is crucial to interpreting results and using them as a basis for taking action.
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It should also be noted that the BioWatch program currently employs an initial screening assay, provided through the DOD's Critical Reagents Program, to detect a single target nucleic acid sequence per tested pathogen. If this screening assay yields a positive detection, a secondary assay panel, provided through the CDC Laboratory Response Network, is used to test for three or more additional pathogen sequences.
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Multiplex PCR assays could be used by BioWatch and have the potential to reduce the labor incurred by jurisdictional laboratories as they conduct routine filter testing. Appropriate standards and validation guidance recently have emerged to address the additional issues that arise when characterizing and validating multiplex assays.
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In the relatively near term, targeted NGS approaches may become useful to the program in place of current secondary PCR assays because of the ability to analyze more regions for identification and characterization, thereby improving specificity of detection. Metagenomic sequencing, on the other hand, could assist in characterizing environmental background and might eventually enable the implementation of universal assay protocols that would minimize the effort and costs needed to continually update and revalidate PCR primers, probes, and assay conditions.
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TABLE S-1 Applications of Next-Generation Sequencing for BioWatch Key Challenges / Barriers to Implementation Related Terms Purpose Advantages Complexity of Turnaround Challenges of Cost Bioinformatics Time Interpretation Analysis Primary BioWatch assay Most inclusive X X X X Environmental and detection Enable detection of "natural" positive filter background X X X controls (fungal spores, etc.) measurement Shotgun sequencing, Metagenomic Better understand sources of false random sequencing, sequencing positive signals unbiased sequencing Improve design of primers and probe targets used for primary screening Identification of novel or divergent Novel pathogen discovery pathogens that would go undetected X X X by existing tests Optimize design of primers Will identify new targets for PCR and Whole-genome X Microbial whole- and probes improve primer / probe design resequencing, de novo genome sequencing genome assembly Will better capture target diversity and Expand reference databases X X inform primer / probe design Investigate false-positive Better understand sources of false Amplicon sequencing, X X X results positive signals biased sequencing, Targeted sequencing conserved region More informative than "yes-no" result sequencing Primary BioWatch assay from real-time PCR; more inclusive of X X diverse strains 18
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THE IMPORTANCE OF COMMUNICATION TO PROGRAM SUCCESS Finally, one of the primary messages of the report is that the network of BioWatch stakeholders should cooperate and be integrally involved in the process of establishing standards that will meet the community's needs and should have access to the relevant data to help them understand the performance and limitations of the assays in order to enable appropriate interpretation of the results. Building additional, specific mechanisms into the program to share assay and system performance results, discuss what these performance results reveal about the limits of the assay and the translation of assay results into actions, and share applicable detailed assay performance information across agencies such as DHS and CDC and with appropriate individuals within the network of jurisdictions appears to the committee to be a critical need.
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The BioWatch program, relevant federal agencies, and local and state jurisdictions should expand the communication and data-sharing that occurs among the network of federal and nonfederal partners involving both the screening and the secondary PCR assays. This effort would assist in establishing acceptable performance standards, enhance informed data interpretation and decision making, improve the ability to undertake root cause analysis of assay issues encountered by jurisdictions, and enable the collective identification and dissemination of actions as part of robust quality assurance.
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