Science Needs for Microbial Forensics Developing Initial International Research Priorities (2014) / Chapter Skim
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6 Validation and Reference Materials for Microbial Forensics
6 Validation and Reference Materials for Microbial Forensics
Pages 117-142
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From page 117... ...
Microbial forensics is a relatively new discipline and the science and technologies it uses are evolving rapidly. Because of the demands to produce and apply reliable and robust capabilities, validation measures, requirements, and protocols are essential.
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From page 118... ...
The latter is not readily achievable with currently validated genetic typing capabilities; and 2.) o integrate the microbial forensic findings with other forensic and T investigative evidence to address the ultimate question of guilt or lack of guilt.
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2. Internal validation refers to accumulation of test data within the laboratory that intends to use the method to demonstrate that established methods perform as expected.
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From page 120... ...
Microbial forensics relies not only on microbial evidence but also on additional forensic evidence, and the collection of microbial evidence can sometimes be incompatible with other types of forensic evidence. Given a choice between a fingerprint and a spore, the level of individualization offered by the fingerprint could make it the more desirable piece of evidence to preserve.
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From page 121... ...
Simple strategies, such as pre-labeling tubes before beginning sampling due to difficult manipulation of labels when wearing protective gear, is an example of a simple consideration. Collection validation must address • Recovery, • Stability, • Integrity, • Target -- organism or analyte (e.g., DNA, RNA, protein, toxin, agar)
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From page 122... ...
The basic criteria for validation during the analytical stage appear in Box 6-1A, and additional criteria may be warranted. The amount of target that can be analyzed by an assay should be defined.
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An important issue that Budowle believes receives too little attention is the process for handling conflicting results or considering alternative hypotheses. Conflicting results will occur.
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One would expect that at some point, one technology may resolve better than the other and that observation should not be considered discordant data in itself. If assessing an entirely new technology for assaying a target and generating results with no comparable existing data, one must define the criteria by which to test it, develop the test, and validate the performance.
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It is human nature to want to accommodate data to scenarios that make sense to us, regardless of alternative hypotheses or explanations. This human nature affects how we perceive false positives and false negatives.
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The sample analysis flowchart in Figure 6-3 provides an idea of the variety of microbial analyses one might consider. Investigators might perform classic taxonomic analysis, for example, and analyze for weaponization engineering.
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Higher throughput can result in greater depth of coverage, potentially allowing for detection of low-abundance or trace-level targets. Therefore, NGS will likely become one of the primary tools for microbial forensics.
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From page 128... ...
tularensis; however, only two were positive, and there was no clear interpretation for a partial profile and what actions to take. Budowle believes the concept of "missing data" is also an important factor to be considered when interpreting results and should be part of the validation equation.
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We do not know what is endemic, and this can be an issue in the overall interpretation process. If we identify a "match" or a "similarity" or the most common ancestor, the significance of these associations must be supported by something defensible.
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Forensic questions based on genetics-based analyses appear in Box 6-4. Investigators want to know, "Is the information provided with genetic markers probative?
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However, the rapidly evolving technology environment must be embraced or there will be a risk of reducing capabilities. Deep sequencing generates substantial data, and sequencing capabilities are enabling scientists to develop elaborate databases tailored specifically to the demands of a case.
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. He suggested that neither by itself may be sufficient, and validation is necessary to guide the user on how best to use quality metrics.
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databases on pathogens A and near neighbors and their background occurrence with epidemiological his tory, when available; and • ata management with the capability to access and process large amounts of D diverse genetic data and to communicate data rapidly with stringent informa tional security (i.e., fully functioning information interoperability)
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The magnitude of the problem of developing material standards is illustrated by the wide variety of targets, which include but are not limited to genes, proteins, morphology, physiology, and biochemistry. Reference materials are needed for all developers and analysts.
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Budowle also posed the question of who will prepare these standard reference materials and who will maintain them? Is it government's, industry's, or an individual laboratory's responsibility?
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Computationally it should be feasible to define barcodes, but depending on how they are used may limit the numbers of samples that can be indexed. New reference materials must be generated, but old ones must be maintained, or there may be no good way to compare back with existing or former methods going forward.
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From page 137... ...
• centralized, comprehensive physical archive of reference materials would A facilitate o Implementation of a standardized characterization system, o Uniform QA/QC, o Development of standard typing techniques, o Standardization of new techniques and analytical methods, o Reference samples for high-resolution genomic comparisons, and o Duplication. SOURCE: Budowle presentation, 2013.
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From page 138... ...
Microbial forensics investigators need increased access to reference resources, yet there is a real conflict: One position may demand that access be restricted for security reasons while another position is that tests and countermeasures cannot be developed without access to the materials. To make any progress, we must consider and understand both positions.
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From page 139... ...
The WDCM also provides access to the data, serving as an online gateway to international databases on microbial diversity, culture collection catalogs, services, and molecular data relating to microorganisms. The WDCM was developed under the auspices of the World Federation of Culture Collections and UNESCO's Microbial Resources Centres (WFCC-MIRCEN)
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to develop the WDCM Reference Strain Database for all microbial resources in conformance with ISO standards, and all of this information is available to the public. Ma believes that the WDCM can make contributions to microbial forensics and perhaps serve as another model for information on culture 4 More information is available at http://ibol.org/; accessed November 23, 2013.
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. free information-service platform to help culture collections to manage, dis A seminate, and share information related to their holdings.
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• Papers • Patents SOURCE: Ma presentation, 2013. ible, and pointed out that it is much easier to begin cooperative efforts among stakeholders when working in the information field.
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