role of law enforcement response and microbial forensics in investigation of bioterrorism

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Role of Law Enforcement Response and Microbial Forensics in

Investigation of Bioterrorism

Te risk and threat o bioterrorism and biocrime have become alarge concern and challenge or governments and society to enhancebiosecurity. Law enorcement plays an important role in assessingand investigating activities involved in an event o bioterrorismor biocrime. Key to a successul biosecurity program is increasedawareness and early detection o threats acilitated by an integrat-ed network o responsibilities and capabilities rom government,academic, private, and public assets. o support an investigation,microbial orensic sciences are employed to analyze and character-

ize orensic evidence with the goal o attribution or crime scene re-construction. wo dierent molecular biology-based assays realtime polymerase chain reaction (PCR) and repetitive element PCR are described and demonstrate how molecular biology tools maybe utilized to aid in the investigative process. echnologies reliedon by microbial orensic scientists need to be properly validated sothat the methods used are understood and so that interpretation oresults is carried out within the limitations o the assays. Te threetypes o validation are preliminary, developmental, and internal.Te rst is necessary or rapid response when a threat is imminent oran attack has recently occurred. Te latter two apply to implementa-tion o routinely used procedures.

1Scientic Analysis Section, Federal

Bureau of Investigation,

Quantico, Va, USA2The Microbial Genetics and Genomics

Center, Department of Biology,

Northern Arizona University,

Flagstaff, Ariz, USA

Bruce Budowle1, Jodi A. Beaudry2, Neel G. Barnaby1, Alan M. Giusti1, Jason D.

Bannan1, Paul Keim2

437www.cmj.hr

Bruce Budowle

FBI Laboratory

2501 Investigation Parkway

Quantico, VA 22135, USA

[email protected]

> Received: April 12, 2007

> Accepted: June 27, 2007.

> Croat Med J. 2007;48:437-49

> Correspondence to:

Forensic ScienceForensic Science
http://MAILTO:%[email protected]/http://MAILTO:%[email protected]/


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Te use o bioweapons throughout history hasbeen well-documented (1,2 and reerences with-in); however, it was the delivery o Bacillus an-

thracis through the US postal system in 2001that raised to a new level public awareness o theneed or biosecurity (3,4). Te risk and threato bioterrorism and biocrime are greater con-cerns today because o the relative ease o acquir-ing and preparing a pathogenic organism and therecognition that methods o dissemination neednot be sophisticated or complex. Tese concernsor biosecurity have been exacerbated due to therecent natural outbreaks o Severe Acute Respi-ratory Syndrome (SARS), avian inuenza, and

monkeypox. Yet, the letter attacks o 2001 andsubsequent exaggerated reactions by the pub-lic revealed the need or law enorcement orga-nizations to be able to respond to such an attackmore eectively to identiy the perpetrator o thecrime, to exclude those not associated with thecrime, to interdict, to deter, and to prevent u-ture crime, and to maintain public saety and se-curity.

Tis paper describes the role o law enorce-

ment in investigative and assessment activities in-volved in a bioterrorism or biocrime event. Tenan overview is presented o how the eld o mi-crobial orensics can assist investigations by ana-lyzing and characterizing orensic evidence, withthe goal o attribution or crime scene reconstruc-tion, and includes some o the challenges to meetthat goal. For the analytical part, two very dier-ent molecular biology-based assays are described:real time PCR (R-PCR) (5-11) and repetitiveelement PCR (rep-PCR) (12-19); these proce-

dures are used to exempliy how molecular bi-ology tools may aid in an investigative process.Te ormer approach oers high throughput,sensitivity, specicity, and resolution. Te lattermethod is less likely to be used routinely but ismore generic in application, oering a tool use-ul in characterizing evidence when genomic sig-natures are not known, and is available as a com-mercial kit. Finally, the need or validation is

stressed, to ensure that the methods used are un-derstood and that interpretation o results is car-ried out within the limitations o the assays and

existing supporting data.

Role of law enforcement

Te use or potential use o biological agents ortheir by-products as weapons to inict harm orto terrorize creates biological security risks (Fig-ure 1). Law enorcement must prepare to ad-

dress these risks by taking established approachesand adapting them. Tis is based on an appreci-ation o novel issues that arise with the growthand preparation o microorganisms, the prepara-tion o toxins, the various approaches or weap-onization and dispersal o biothreat agents, andthe use o synthetic biology. Because o the dualuse o biotechnology, ease o access to technologyand inormation, and low cost o development,it may never be possible to detect all threats andto prevent anti-societal actions. However, to

best meet the needs o biosecurity, governmentsand the public should work together and takeresponsibility to prevent and minimize threatsarising rom the malicious use o microorgan-isms. Increased awareness and early detection othese threats are keys to acilitating investigationand attribution as well as deterring some poten-tial criminals. Foremost, an integrated networko responsibilities and capabilities should be de-

Figure 1. Flowchart for law enforcement for identifying and reducing risk from

bioterrorism and biocrime.


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rensic evidence, a robust microbial orensics eldhas been developed which is dedicated to thecharacterization, analysis, and interpretation o

evidence or attribution purposes rom a bioter-rorism act, biocrime, hoax, or inadvertent agentrelease (3). Attribution in this context is the in-ormation obtained regarding the identicationor source o a material to the degree that it can beascertained (3,25). Te ultimate goal o attribu-tion is the identication o those involved in the

perpetration o the event, which is necessary orcriminal prosecution, to prevent additional crim-inal acts, or or useul background or national

policy decisions and actions.

Te eld o microbial orensics is multidisci- plinary, relying heavily (but not exclusively) onthe oundations o traditional orensic sciences,epidemiology, microbiology, and molecular biol-ogy. Forensics laboratories have well-established

procedures or all aspects o investigating crimescenes through the scientic analysis o evidence.Similar methods are being developed or micro-bial orensics cases. Te goal is to develop an in-rastructure so that microbial orensic evidence

will be collected, stored, analyzed, and interpret-ed in a manner that is scientically robust andthus legally deensible.

Intentional bioattacks can be classied as ei-ther overt or covert (1,26). Te dierence be-tween them is that an overt attack is oen recog-nized immediately; while a covert attack may notbecome known or some time, i at all. Covert bi-ological attacks are by their nature more difcultto discover than are overt attacks. Complicatingactors include the background o commonly oc-

curring ood-borne illnesses and endemic cas-es o emerging and re-emerging inectious dis-eases. Separating naturally occurring outbreaksrom those that are the result o an intention-al attack requires an in-depth understanding ocommon domestic pathogens and their epide-miologies. Tereore a signicant challenge existsin developing awareness, surveillance measures,and methods o detection (1,27-29). Te type

o orensic inormation sought in such cases mayevolve over the course o the investigation, butevidentiary collection is initiated at the rst sign

o an attack or potential attack. Indeed, the an-thrax letters attack began as a covert attack andbecame an overt attack with the discovery o theanthrax tainted letters. Regardless, whether anattack is overt or covert, public (which includesagriculture) health ofcials will likely be the rstones involved. Law enorcement and microbialorensic scientist involvement would likely ol-low the discovery o a criminal act.

Once collected and preserved, the biologi-cal evidence is sent to a laboratory or analysis.

Within the laboratory, a core group o analyticaltools is available to aid in characterization o theevidentiary material. Because microbial orensicsocuses on tracking and linking microorganismsto individuals and locations, dierent strategiesmay be implemented, depending on the natureo the attack and the type(s) o evidence collect-ed. In an overt attack, or example, the package,the weapon, and associated materials, in additionto traditional orensic evidence (eg, hairs, bers,

ngerprints), may be analyzed. In a covert attack,the evidence may be more limited to medical his-tories, diagnoses, and isolates taken rom victims.In the case o the 2001 attacks, a combination othese methods has been and is being utilized inthe investigation. As such, an investigative ana-lytical plan may involve many and diverse strate-gies (Figure 2).

While many methodologies are available,molecular biological tools gure prominently inthe repertoire o the microbial orensic scientist.

Comparative genetic sequence analyses o SelectAgent pathogens and their near neighbors are animportant part o the attribution process. Meth-ods that enable recognition o nucleic acid sig-natures will be relied on or screening potentialcandidate sources to acilitate an investigation.Tere are a variety o genetic markers (able 2)and methods that allow highly specic and accu-rate characterization o microbial diversity (30).


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For orensic purposes, assaying rapidly evolvingmarkers enables better afliation to recent com-mon sources, while more stable markers providebetter lineage-based evolutionary interpretations,such as strain and sub-strain denition. Sincebacteria, viruses, and some ungi reproduce asex-ually, their genomes are considered to be clonaland portions o their genomes may be very sta-ble and uninormative or distinguishing sam-

ples. Tereore, it may not be possible to individ-ualize the source o a sample by genetic analysisalone (as oen is accomplished in human DNAidentity testing). Since many microbial genomes

have relatively short generation times, in an over-night culture, a single microbe could have repro-duced its genome over a million times, increasingthe chance o mutation that may be seen withinthe culture. Tus, some variation, and hence aorensic signature, may occur during asexual re-

production. However, there also may exist sexu-al reproduction, horizontal gene transer, conju-gation, transduction, lysogeny, gene conversion,

mobile elements, recombination, reassortment,gene duplication, rearrangements, and mutation-al hotspots that can be exploited or the purposes

o seeking orensic attribution (30,31).Te orensic comparison o a genetic pro-le rom a reerence sample with that o an evi-dentiary sample can have three possible generaloutcomes: match or inclusion, exclusion,or inconclusive. With microbial genetic inor-mation, it is less likely to have a prescribed in-terpretation policy or what constitutes a matchand what does not. Some questions may be di-cult to answer unequivocally based on extantdata (able 3). Uncertainty is greater than what

is experienced or human DNA identity testingbecause o unknown diversity, limited databas-es, unknown manipulations, and limited genetictesting. However, the power o microbial oren-sic tools is increasing rapidly with ever advanc-ing technology. Still, the questions, such as thoselisted in able 3, must be addressed to utilize theull extent o microbial orensic analyses.

Even with the current degree o uncertainty,capabilities exist that enable analysis and inter-

Figure 2. A hypothetical sample microbial forensic analysis flowchart for

characterizing evidence from an overt attack. The flowchart will vary depend-

ing on the type, quantity and quality of the microbial forensic evidence. An

alternate flowchart may be considered for traditio nal forensic evidence.

Table 2. Genetic markers that can be selected for analysis for

attribution (29)Genetic marker:

Single nucleotide polymorphisms

Repetitive sequences

Insertions and deletions

Mobile elements, including bacteriophage, insertion elements,transposons, integrons, and plasmidsPathogenicity islands

Virulence and resistance genes

House keeping genes

Structural genes

Whole genomes

Table 3. Selected questions that genetic analyses might ad-

dress (25,27,29,30)

What might be deduced about the nature and source of the evidentiarysample?

Is the pathogen detected endemic or introduced?

Do the genetic markers provide a signicant amount of probativeinformation?

Does the choice of markers allow the effective comparison of samplesfrom known and questioned sources?

If such a comparison can be made, how denitively and condently can aconclusion be reached?

Is it possible that the two samples have a recent common ancestor, orhow long ago was there a common ancestor?

Can any sample be excluded as contaminants or recent sources of theisolate?

Are there alternative explanations for the results obtained?

Where does the divergence occur within an individual gene and what are

the mechanisms of variation?What mechanisms are responsible for the genetic changes thatare detected (to include horizontal gene transfer, gene conversion,recombination, gene duplication, random mutation, mutation hot spots,mobile elements, etc.)?

Have host selective pressures contributed to the genetic composition ofa sample?

What are the effects of laboratory stresses and manipulation on geneticvariation?

Is there evidence of genetic engineering?

Can natural mutational events be discerned from creative or subtlegenetic engineering?

*The degree to which these questions can be addressed depends on the context ofthe case and the available knowledge of the genetics, phylogeny, and ecology of thetarget microorganism.


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pretation o results that can be meaningul orinvestigation purposes. At times, interpretationso genetic data can be made qualitatively (such

as strain identication, re. 1). ypically, qualita-tive statements are made based on characteristicsthat are shared or not shared between evidentia-ry and reerence samples. Genetic analysis meth-ods used by epidemiologists to track inectiousdisease outbreaks employ interpretation guide-lines to evaluate the results and to determine caserelationships in epidemics (32). Forensic analy-ses can oen use the same or similar methods butmay require additional criteria, such as identica-tion o individualizing characteristics or higher

resolution source attribution.

Technology

Methodologies are available to enable attributionto the level possible based on existing geneticdata. Samples may be viable microorganisms and,i culturable, there will be available copious mate-rial or orensic analyses. For non-viable materi-als, the challenge is greater. Te evidence can be

limited in quantity and quality, sometimes being partially or totally degraded, contaminated withinhibitors and/or other background microorgan-isms rom the environment. PCR analysis willgure heavily in the molecular analyses o suchgenetic evidence because o the exquisite sensitiv-ity and specicity that those particular assays a-ord. Producing PCR amplicons o lengths o 60-80 base pairs make it more possible to type highlydegraded samples. Te markers most suited oranalysis with small length amplicons are Single

Nucleotide Polymorphisms (SNPs). A variety omethods is available or SNP detection (30); theone described briey below is R-PCR (5-11).

Real-time PCR

Te PCR approach used or many assays is de-signed or end-point analysis. Tus, the PCR

products are examined at the yield plateau o

the reaction. Endpoint assays, although eective,tend to have limited dynamic range, are at bestonly semiquantitative, and tend to be laborious

in that they require several manipulations. TeR-PCR assay involves continuous monitoringo the generation o PCR product, particularlyduring the linear reaction phase. Tis providesboth qualitative identication and quantitativeestimation o target amounts in a single tube bycalibrating reaction kinetics to known standards(6,7,11). Te aqMan PCR assay (8,10), a 5nuclease based approach, makes R-PCR acileand reliable. Tis assay relies on the uorescentdetection o unquenched reporter dye moieties

rom displaced digested probes (by aq poly-merase) that were hybridized to a target sequenceo interest. Te uorescence signal should be pro-

portional to the hydrolyzed probe within the re-gion dened by the primers in any one cycle othe PCR, and this is directly correlated with theamount o PCR product.

For SNP detection, probes can be designedto identiy specic SNP states such that anynon-complementarity between the probe and

the template will disrupt the duplex conorma-tion and probe hybridization, so that digestion isless avored (5). Te use o minor groove binder(MGB) probes has greatly enhanced the peror-mance o R-PCR SNP assays because the m(melting temperature) o a probe is increased, ascompared with equivalent non-modied probes(9). Te stability allows or MGB probes to beshorter and yet still achieve an acceptable probem. Te benets o shorter probes during R-PCR are: 1) specicity is enhanced because a

single mismatch will more likely destabilize the probe/template duplex and 2) uorescent sig-nal-to-noise ratios are improved because the ef-ciency by which the uorescence o unhybridized

probe is quenched is directly related to the dis-tance between the quencher moiety and report-er dye. Shorter probes tend to allow or a closer

proximity o the reporter dye to the quenchermoiety, thus reducing background uorescence


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without aecting the signal yield that results a-ter cleavage. Other modications, such as use oPeptide Nucleic Acid molecular beacons, have

been developed to enhance R-PCR parametersor genotyping (33).

The primary benets of the RT-PCR assay are:

Extremely low limits o detection (ap-proaching single copy detection);

Inherent specicity that enables design ohighly discriminating assays that are capable odistinguishing closely related molecular species;

A broad dynamic range o quantication(up to 7 orders o magnitude);

A single step reaction contained within aclosed tube, which reduces manipulations andlaboratory contamination; and

Rapid automated detection in a 96-well or384-well ormat.

For most diagnostic applications, a SNP is astable marker likely to have occurred only oncein the phylogenetic history o the species (partic-ularly at the population level). Tis stability canbe invaluable or dening strain groups, such asmajor phylogenetic divisions, as well as speci-cally dening a particular terminal branch strain(eg, B. anthracis Ames strain). Moreover, whenavailable, multiple SNPs along a branch providethe same phylogenetic inormation but are diag-nostically redundant. When multiple SNPs thatdene a branch are available, then an assay canbe optimized by choosing the SNP that contrib-utes to the most robust (based on sensitivity andreproducibility) assay. Alternatively, multipleSNPs may be needed to increase condence in

interpretation when knowledge on populationdata and diversity are somewhat limited.

In addition to their use as phylogenetic re-search tools, R-PCR SNP assays also have beendeveloped or microbial orensic application(4,34-37). Te most notable have been SNP as-says designed or identication o B. anthracisstrains/isolates (34,38-40). Tousands o SNPshave been discovered in B. anthracis through

whole genome sequencing (40). Indeed, Keim etal (34,41) have identied a number o SNPs thatcan dene major genetic groups in B. anthracis.

Tese diagnostic canonical SNPs (canSNPs)identiy particular phylogenetic points in the evo-lutionary history oB. anthracis (1). Both Ampli-ed Fragment Length Polymorphisms (AFLP)(42) and Variable Number o andem Repeats(VNR) (43) markers provided a phylogenet-ic ramework across several major clades (38,43)or assessing the inormative value o potentialcanSNPs (against a diverse set o 26 strains roma collection o more than 1300 B. anthracis iso-lates). One representative SNP marker rom each

o the major evolutionary branches was then se-lected as the dening SNP or that clade (Figure3). Finally, each canSNP candidate was testedagainst the strain collection to support or reutethe validity o its canonical designation (34).

Six SNPs specic or theB. anthracis Amesgenetic cluster (ie, the strain identied in the an-thrax letters attacks o 2001) were identied.Four chromosomal SNPs (designated Br1-7,Br1-26, Br1-28, Br1-31) and one plasmid SNP(designated PS-52 on pXO2) are diagnostic or

the Ames strain (34) (ables 4 and 5). Another

Table 4.B. anthracis Ames specic Single Nucleotide Polymor-

phisms and genome position

Ames Specic SNP SNP Genome position GENBANK accession No.

PS-1* (SNPO1) A-G 7452 NC_003980

PS-52 (SNPo2) A-C 72924 NC_003981

Br1-7 C-A 433277 NC_003997

Br1-26 T-C 4624132 NC_003997

Br1-28 T-G 4929186 NC_003997

Br1-31 G-A 2749543 NC_003997

*PS-1 does cross react with isolates closely related to Ames.

Figure 3. Phylogeny based upon SNPs with branch designations.

Branch designations are indicated in bold text and strains are compa-

rable to previous phylogenetic hypotheses (33). Highlighted numbers

refer to strains in reference 33.


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SNP on the pXO1 plasmid (PS-1) shows slightlyless specicity as it shares SNP identity with ourother closely related strains (able 5); but thisSNP shows greater sensitivity o detection.

A dual probe allelic discrimination assay us-ing R-PCR (and MGB probes) has been devel-oped or typing these canonical SNPs. Te assaysrely upon dierential cleavage o allele-specic

probes to score the SNP state o unknownB. an-thracis DNA templates (Figure 4). Te real-timeand endpoint analysis is perormed on an AB7900H instrument (Applied Biosystems, Fos-ter City, CA, USA). uantication o DNA by

PicoGreen (Invitrogen, Carlsbad, CA, USA)analysis has demonstrated that as little as 10 g(approximately 1.6 genome equivalents) o start-

ing DNA is sufcient to obtain robust and reli-able real-time amplication results (based on thePoisson distribution). Te Ames canSNP assaysappear robust and have been successully per-ormed under a range o validation criteria (ourunpublished results).

Repetitive element PCR for bacteria

identification

Repetitive sequences have been ound in many

bacterial genomes (12-15). Some stable and con-served repetitive elements occur in lengths o 33and 40 bp and comprise about 1% o the bacte-rial genome. Tis translates into 500 to 1000copies per genome (16,17). Another repetitiveelement, known as enterobacterial repetitive in-tergenic consensus or ERIC, is 124 to 127 bpin length and occurs at 30 to 150 copies per ge-nome (18). Tese repetitive elements are stable

Table 5. Ames specic Single Nucleotide Polymorphisms com-

pared to close genetic relatives (33)

SNP proles for selected isolates ofBacillus anthracis

SNP

A0462Ames

A1115Texas

A1117Texas

A0394Texas Goat

A0728China

A0584China

A0488Vollum

PS-52 A C C C C C C

Br1-7 C A A A A A A

Br1-26 T C C C C C C

Br1-28 T G G G G G G

Br-31 A G G G G G G

PS-1* G G G G G A A

*PS-1 does cross react with isolates closely related to Ames.

Figure 4. (A) The real-time PCR analysis of A mes strain template ranging from 1.0 ng to 10 fg. (B) The endpoint analysis of Ames strain (y-axis) and

non-Ames strain DNA tem plates (x-axis) ranging from 10 pg-10 fg.


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but dier in their copy number and chromosom-al locations within and among bacterial species.Tis variation can be used or species, subspecies,

and at times strain dierentiation by a procedureknown as rep-PCR (12-14).Te rep-PCR procedure exploits the variable

distribution o these repetitive elements through-out the genome o the microorganism by usingoligonucleotide primers complementary to therepetitive element(s) in a PCR amplication othe microbial genomic DNA target. Because othe variation in number and location o repeti-tive elements within a bacterial genome, manydierent length amplicons will be generated. Te

various length rep-PCR amplicons are raction-ated by electrophoresis. DNA patterns rom di-erent isolates or strains can then be comparedto enable some degree o resolution within andamong bacterial species. Rep-PCR is not as ran-dom as is AFLP, since its primers target knownconserved regions (19).

Commercially-available kits (Bacterial Bar-codes, Athens, GA, USA) are available to acili-tate analyses and oer increased complexity othe repetitive element proles by using primersequences or several distinct repetitive elements.Tereore, more ragments may be generated, re-sulting in better dierentiation o strains.

In the United States, there were 33 589 doc-umented cases o Salmonella inection in 2003(44). Tus, Salmonella is an epidemiologicallyimportant microorganism. Tis organism alsohas been used previously as a bioweapon to inten-tionally contaminate public ood sources (2,45).Consequently, Salmonella is well-characterized

(46-48) and is a good model or testing the e-cacy o genetic tools or attribution. Te rep-PCR analysis is not expected to be used routine-ly to examine Salmonella, but Salmonella typingcan exempliy the degree o resolution that maybe attained (49).

Figure 5 displays proles rom several strainso Salmonella that can be dierentiated along

with a tree derived rom the data using Diversi-

lab version 3.1 (Diversilab, Athens, GA) analyti-cal soware. Tus, by using this rapid assay somestrain resolution is demonstrated. For example,

the discrimination potential between S. newportand S. inantis is very good. However, our dis-tinct strains oS. typhimurium have very similarrep-PCR proles, making it difcult to dier-entiate these strains, and one strain o S. inan-tis is more similar to the S. typhimurium strainsthan to another example oS. inantis. Tereore,

while the rep-PCR technique can generally dis-tinguish dierences between strains, the presenceo the same prole may not provide strong evi-dence o common origin.

Tere may be times when an urgent needor a quick characterization o a pathogenic or-ganism is needed; in such situations the rep-PCR may be one viable option. Te advantageso the rep-PCR are that it does have some dis-criminatory power, can be implemented rap-idly so results can be obtained quickly, does notnecessarily requirea priori knowledge o geneticsignatures, and the cost o implementation anduse is relatively low. Te primary disadvantagesare the lower discriminatory power o the analy-sis compared with more specic assays, and theact that the rep-PCR process is more reracto-ry to template quality and quantity. In contrast,the R-PCR ocuses on specic sites o high res-olution or lineage or or orensic discriminationand is more likely to provide results rom highlydegraded and limited quantity samples. Howev-er, R-PCR requires substantially more develop-ment time to identiy the markers o interest and

validate the methodology.

Validation

Te reliability and proper interpretation o re-sults rely substantially on understanding the per-ormance and limitations o an assay. Validityand rigor are essential or the tools o microbialorensics, or determination o quality and or es-tablishing condence in results attained in anal-


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yses. uality Assurance guidelines have been developed (3,25) and are similar to those adoptedor human DNA orensic analyses (50). Te sec-tion (Section 8 o the guidelines) related to vali-dation is listed below:

8.1 Te laboratory should use validated meth-ods and procedures or analyses.

8.1.1 Developmental validation should beappropriately documented and should address

specicity, sensitivity, reproducibility, bias, pre-cision, alse-positives, alse-negatives, and deter-mine appropriate controls. Any reerence data-base used should be documented.

8.1.2 Preliminary validation is the acquisitiono limited test data to enable an evaluation o amethod used to provide investigative support toinvestigate a biocrime or bioterrorism event. Ithe results are to be used or other than investiga-tive support, then a panel o peer experts, exter-nal to the laboratory, should be convened to as-

sess the utility o the method and to dene thelimits o interpretation and conclusions drawn.

8.1.3 Internal validation should be perormedand documented by the laboratory.

8.1.3.1 Te procedure should be tested usingknown samples. Te laboratory should monitorand document the reproducibility and precisionand dene reportable ranges o the procedure us-ing control(s).

8.1.3.2 Beore the introduction o a new pro-cedure into sample analysis, the analyst or exami-nation team should successully complete a qual-iying test or that procedure.

8.1.3.3 Material modications made to an-alytical procedures should be documented andsubjected to validation testing commensurate

with the modication and have documented ap-proval.

Validation is an essential process by which a procedure is evaluated to determine its efcacyand reliability or analysis. Te undamental cat-egories o validation are developmental valida-tion, internal validation and preliminary valida-tion. Developmental validation is the acquisitiono test data and the determination o conditionsand limitations o a newly developed methodolo-gy or use on samples. Internal validation is an ac-cumulation o test data within the laboratory todemonstrate that established methods and pro-

cedures perorm within determined limits in thelaboratory. Tese two types o validation are cru-cial or addressing the reliability and robustnesso any method routinely implemented in the lab-oratory.

Preliminary validation, however, is not de-scribed in the human DNA orensic arena, butis essential when addressing biodeense and bios-ecurity. One cannot predict which microorgan-

Figure 5. Rep-PCR profiles, relatedness tree, and similarity index for ten Salmonella enteritidis serotypes. Note lack of diversity in 4 typhimurium

strains and greater similarity between an infantis strain and typhimurium strains than with another infantis strain. Also, note discriminatory power for

two Newport strains and two infantis strains. All isolates were collected from unrelated events. The DNAs were kindly provided by Eugene LeClerc

(FDA).


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ism, virus, or toxin will be used in the next attack(planned, attempted, or successul) or the nexthoax. Yet, authorities will need to respond expe-

ditiously to protect the public and the countrysassets. I there are no established validated stan-dard operating protocols to identiy or character-ize the bioweapon, it is incumbent upon the mi-crobial orensic scientists and others to identiyany tools that are available to assist in the investi-gation even i they have been used previously orresearch purposes only. It would be irresponsibleto wait or months or years (as is done or humanidentity testing) or validation o an analytical

procedure when an attack is under way and bi-

osecurity is threatened. However, quality o themethods used and understanding the limitationso a methodology should not be overlooked.Tereore, the concept o preliminary validation

was developed. Preliminary validation is the ac-quisition o limited test data to enable an evalua-tion o a method used to assess materials derived

rom a biocrime or bioterrorism event. Te eval-uation is based on peer review o extant data, typ-ically by a panel o experts that determines theextent o use and limitations o the technologyor methodology and makes recommendations oevaluations or studies that may be needed priorto processing evidentiary material or studies thatmay be carried out subsequent to analyses andresults are obtained. Te goal is to be able to re-spond expeditiously, eectively, and efciently

while maintaining scientically valid and rigor-

ous approaches.Validation o a procedure includes addressing

many parameters. Some o these are listed in a-ble 6. While not all parameters may be applicableor any one assay, they should be considered toascertain whether they apply. By using appropri-ate validation criteria, the reliable conditions canbe determined or the methodology or or the in-terpretation o the analytical results. In addition,conditions can be dened under which the re-sults or the standard interpretation are not valid.

Conclusion

Many nations are aced with the possibility o abiological attack, and discovering, attributing,and prosecuting these cases pose serious chal-lenges or law enorcement. Te best approachor preparedness to detect and/or respond to anevent is an eective integrated network with gov-ernment, academia, industry, and the public all

contributing to this common goal. Te eld omicrobial orensics has been implemented to en-able better condence in scientic analyses o o-rensic evidence and to gain greater condence inthe interpretations o the results obtained. Prog-ress in microbial orensics is currently ocused ona number o areas to address (52-54), including:

Sample collection and preservation strat-egies,

Table 6. Minimum validation c riteria (24,50,51)

Sensitivity is the minimum amount or concentration of analyte requiredto generate a reliable result

Specicity is the ability to measure the intended target analyte orsignature

Reproducibility is the closeness of agreement between the results ofsuccessive measurements of the same analyte under similar but notnecessarily identical conditions

Precision is the degree that measurements are similar

Accuracy is the degree that the measured material or analyte is similar

to its true value*Resolution is smallest difference between measurements that can be

meaningfully distinguishedReliability is the closeness of agreement between the results of

successive measurements of the same analyte under the sameconditions of measurement

Robustness ability of analytical performance under challengedconditions or when analyzing challenged samples

Specied samples are those necessary to test the performance ofthe assay (eg, reference panels and mock or non-probative materials)corresponding with the intended application of the assay

Purity is the quality required for extracted analyte to be analyzed

Input values is the range of quantity of analyte that can be analyzedreliably

Quantitation is the amount or concentration of material for input

Dynamic range is the range of values or limits where precision is held

Limit of detection is the lowest concentration of analyte that can beconsistently detected

Controls are dened test materials for the measured analyte (includesblind samples, negative and positive controls)

Window of performance for operational steps of assay parameterssuch that slight analytical condition variation will not substantially affectperformance or reliability

Critical equipment calibration are those requiring calibration prior totheir initial use and on a regular basis thereafter

Critical reagents are determined by empirical studies or routinepractice to require testing on known samples prior to use withevidentiary materials in order to prevent unnecessary consumption offorensic samples

Databases a collection of data to be used to support interpretation ofresults

*The term analyte is used here in a broad sense and can range from intact viablemicroorganism to an ion.


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448

Novel methods and analyses or typingnon-viable and/or trace materials,

Development o validated methodologies

that dene the limits o an analysis, Identication o genetic markers and match

criteria that can establish baselines or comparingreerence and evidence samples, and

Interpretation criteria,

Acknowledgment

Tis is publication number 07-04 o the Laboratory Di-vision o the Federal Bureau o Investigation. Names ocommercial manuacturers are provided or identica-tion only, and inclusion does not imply endorsement bythe Federal Bureau o Investigation.

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