BioWatch PCR Assays Building Confidence, Ensuring Reliability: Abbreviated Version (2015) / Chapter Skim
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1 Introduction
1 Introduction
Pages 21-36
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From page 21... ...
The National Biosurveillance Science and Technology Roadmap (NSTC 2013) , for example, describes over 20 programs, networks, and activities from across the government that contribute to the National Strategy's goals.
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From page 22... ...
BIOWATCH DETECTS AIRBORNE RELEASES OF SPECIFIC BIOLOGICAL AGENTS The BioWatch program is part of a layered approach to rapidly detect and respond to exposure to airborne biological agents of concern. A brief overview of the program is presented below and additional information is provided throughout the report.
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From page 23... ...
By 2013, reportedly more than 7 million polymerase chain reaction (PCR) assays had been conducted during routine daily operations to monitor for genetic material from the set of microbial agents for which BioWatch tests (IOM and NRC 2014)
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The laboratories that analyze the filters collected from BioWatch air samplers are generally co-located with state and local public health laboratories, which maintain a dedicated BioWatch capacity. Most of these public health laboratories are members of the Centers for Disease Control and Prevention's (CDC's)
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From page 25... ...
If this initial screen yields a positive result, a secondary panel of assays directed against three or more additional nucleic acid signatures is used to verify the presence of the agent.4 The BioWatch screening and secondary PCR assays currently are used in an integrated fashion by laboratories in the program (e.g., a BioWatch Actionable Result [BAR] generally is not called only on the basis of the results of the screening assay)
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From page 26... ...
For BioWatch, there are multiple steps from a pathogen in the air to a sample of extracted DNA used in a PCR, and so the relative amount of DNA in the PCR sample can be informative but does not directly provide the original pathogen concentration. During a PCR assay, a false positive detection could result from a number of factors, such as cross-reactivity of assay primers and probes (both types of short oligonucleotides that hybridize to target DNA)
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From page 27... ...
Responding to a detection that turns out to be a false positive places considerable drain on jurisdictional resources and could undermine confidence in the system when a true positive occurs. Over the history of the
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From page 28... ...
Addressing these concerns poses a greater challenge and is discussed as part of PCR assay performance validation in Chapter 2. A point is sometimes raised that the PCR assays used by BioWatch do not provide information on agent viability, and thus positive detection results do not necessarily indicate the presence of an active risk to public safety and health.
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From page 29... ...
Because real-time PCR is the core detection method currently used by BioWatch, assay performance characteristics, including false positive and false negative rates, provide valuable information to the program's managers and users. BioWatch Actionable Result The responsibility for declaring a BAR following the positive results of the PCR assays rests with the director of the local laboratory that conducted the sample analysis, or an appropriate designee, and technical consultation with partners in DHS and CDC can occur before making the determination.
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From page 30... ...
• Sample preparation • Movement and dispensing of prophylaxis • Federal and jurisdiction • Preliminary screen notifications • Protective actions such as limitations on movement • Agent-specific test • Jurisdictional conference call • Mass notification • Results review • Initial situational assessment • BioWatch National conference call BAR = BioWatch Actionable Result NRF=National Response Framework FIGURE 1-2 The BioWatch program maintains routine operations in support of preparedness goals and operates in the early notification and assessment stages of a potential event involving release of an aerosolized pathogen. Another important component of response and recovery is cleanup.
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From page 31... ...
The BioWatch program can leverage existing data and guidance to inform the development, testing, and validation of the PCR assays it uses. Relevant recommendations on the development and validation of PCR assays for biothreat agent detection have been developed by the Stakeholder Panel on Agent Detection Assays (SPADA)
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From page 32... ...
Despite federal multiagency efforts on assay performance and assay performance equivalency, there currently does not appear to be a clear and well-described set of PCR standards, associated validation protocols, and reference materials that are agreed upon and accepted by DHS, the BioWatch program, and the relevant stakeholder partners including the CDC, public health laboratories in the LRN, and those entities involved in developing BioWatch technology. DHS REQUESTED THAT THE NATIONAL RESEARCH COUNCIL AND THE INSTITUTE OF MEDICINE EXAMINE OPTIONS FOR A BIOWATCH PCR STANDARD Given the fundamental nature of the BioWatch program's reliance on PCR performance characteristics, the significant investments that the United States makes in BioWatch as a defense against large-scale releases of biological pathogens, and the need for the system's users to interpret and have confidence in its results, DHS asked the National Research Council and the Institute of Medicine to examine the types of performance data, performance criteria, and data analyses that the community requires.
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From page 33... ...
Most of the performance characterization and validation strategies BOX 1-2 Statement of Task An ad hoc committee will be convened to conduct a study and prepare a report that will evaluate and provide guidance on appropriate standards for the validation and verification of polymerase chain reaction (PCR) tests and assays in order to ensure that adequate performance data are available to public health and other key decision makers with a sufficient confidence level to facilitate the public health response to a BioWatch Actionable Response (BAR)
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From page 34... ...
However, the report does not make recommendations on • he utility or effectiveness of the method of sample collection, such as air T filters or air collectors; • articular sample processing methodology, including specific methods to P extract and purify the nucleic acids from the sample, other than to suggest rigorous validation of all processes; • ow to address the effects of materials that can affect a PCR, such as road or H brake dust, pollen, dirt, heavy metals, or other substances, or how to ddress a the effects of background biological organisms and nucleic acids that may or may not be amplified in the PCR process, other than to recommend a rigorous examination of environmental background; • articular thermal cycling conditions or instrumentation; P • pecific sources of PCR reagents, or specific methods of storing or dispens S ing reagents; • ethod of detection of the PCR products, or the size or other particularities M of the PCR products; and • he algorithm for interpreting the results from the PCR.
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From page 35... ...
Rather, the report discusses performance characteristics of PCR and explores the implications for the BioWatch program and its stakeholders of different types of assay standards approaches. The report seeks to provide guidance to the program to inform decisions made on standards for the PCR assays it uses.
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