* Dr. Paul Keim answers questions about the anthrax investigation, some from CASE CLOSED blog participants
Posted by DXer on July 8, 2009
why the FBI failed to solve the 2001 anthrax case
* VIDEO – introducing CASE CLOSED
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Dr. Paul Keim answers questions
about the anthrax investigation,
some from CASE CLOSED blog participants
Paul Keim, Ph.D., is a Regents Professor of Biology at Northern Arizona University and the Division Director of Pathogen Genomics at the Translational Genomics Research Institute (TGen). Keim serves on the Scientific Working Group for Microbial Forensics (FBI) and the National Science Advisory Board for Biodefense to advise the U.S. government on issues of science and policy.
On July 6, 2009, Paul Keim answered selected viewer questions about the anthrax letter attacks and microbial forensics in general. Excerpts from these Q & A follow; the entire Q & A is found in the comment below.
Q: What are the limitations of using microbial forensics for attribution purposes? Are policymakers fully aware of these limitations? Jonathan B. Tucker, Washington, D.C.
A: … “The laboratory Ames strain can be precisely identified and differentiated from all other types of Bacillus anthracis, including those very close relatives isolated from the same geographic region of Texas.” This is a long ways from concluding that letter spores came from USAMRIID, but it is a start and eliminates a lot of possibilities. “Exclusion” due to a lack of a match is one of the most powerful conclusions that can be drawn in DNA fingerprinting analysis. In the Amerithrax case, the scientific evidence (morph typing) tying the letter spores to the RMR-1029 appears strong. The FBI repository of Ames cultures was extensive, and only cultures derived from RMR1029 or that culture itself have all four morphs. While this scientific conclusion excludes a very large number of possible perpetrators, it still doesn’t directly attribute the crime to an individual. I understand that there was more than one person with access to RMR-1029 spore preparation. I don’t know how many individuals had access, and I can only speculate that it could have been quite a few (10? 50? 100?). This limitation is well understood by the scientists involved but may or may not be understood by the public or policymakers.
Q: Did the FBI or U.S. Department of Justice consult with any genetics expert when they were asked in October 2001 whether it was okay for the [B. anthracis strains held at] Iowa State University and USDA Ames (at the strip mall) to be destroyed? Ross Getman, Syracuse, New York
A: I was not consulted nor am I familiar with the actual steps that lead to the destruction of these materials. During that time period (late 2001), I was doubtlessly the most engaged microbial geneticist working with the FBI. If I wasn’t consulted, then probably none others were either. Additional Commentary: It is hard to understand why this destruction was done and why it was allowed to occur. Clearly someone in Iowa panicked at being in the media bright lights and wanted to get rid of the material. If this was really authorized by the FBI, who that authority was has not been released, to my knowledge. If the investigation had eventually lead back to Iowa, this would have been viewed as destruction of evidence and obstruction of justice. Now that would have been a public relations nightmare!
The following are several of the many questions proposed at my CASE CLOSED blog, where a vibrant discussion of issues related to the anthrax investigation is active every day. Lew Weinstein, Collioure, France:
Q1: Did the 1,070 samples examined by the morphotype analysis accurately reflect the actual number of anthrax samples held in the U.S. in the 2001 timeframe?
A: This would not be representative of all the Bacillus anthracis samples, but rather Ames strain stocks. I don’t know how many B. anthracis cultures existed in the U.S. in 2001, but many more than this. The Ames strain is only one of many different identifiable strains.
Q2: Did the process of self-submission of anthrax samples by labs and individuals (per the FBI methodology) result in a reliable representation of the Ames samples that may or may not have existed prior to 2001?
A: Presumably, the 1,070 collection represents all the Ames cultures in the U.S. plus some from around the world. First, the Department of Justice subpoenaed all the U.S. anthrax labs for our inventory records with special attention to the Ames strain cultures. Next, the DOJ subpoenaed all U.S. laboratories that had the Ames strain to provide a sampling (a portion) of their Ames cultures. For example, my lab had only seven different Ames stocks, which we sampled and sent in duplicate to the FBI.
Q3: Did the FBI use any type of proficiency testing for their genetic analysis?
A: Definitely yes. Extensive proficiency testing was performed both prior to and during the genetic analysis at Northern Arizona University. The initial determination from the first Florida sample was done using normal research controls and standards, but soon afterwards very rigorous forensic-level protocols were implemented. There was no time for proficiency testing for the first analysis on the Robert Stevens isolate, as we had only a few hours notification. Immediately afterwards, the FBI forensic scientists began to review our procedures and help us to institute forensic-level operating protocols, including proficiency testing. Once these were in place, original analyses were repeated with the same result.
Note that there were no forensic standards for microbes and that we had to adapt the ones developed for human DNA testing to our new anthrax tests. This is a topic of great debate as to what the standards should be for a new technology.
Q4: Was the Bacillus subtilis contaminant found in the first batch of letters genetically identical to any forensic evidence collected from any lab? Also, was that contaminant tested against strains from Dugway Proving Grounds?
A: Disclaimer: My lab did not work on the B. subtilis contaminant, so what I am relating is secondhand knowledge. However, it is based upon my conversations with government experts and my participation in the press conferences and the public ASM symposium. Details of the contaminant were discussed in these forums. Background: The question refers to the non-hemolytic (B. anthracis is hemolytic) bacterial contaminant found in some of the letters. It proved to be a spore forming Bacillus subtilis bacterium. As the name implies, this is somewhat related to Bacillus anthracis and shares many biological properties. But it is not a disease causing organism, and there are many, many different types found in the environment. It would not be surprise to me to find a novel B. subtilis on my computer keyboard. The government investigators thought that perhaps this contaminant could be used to trace back the spore preps to a particular laboratory. If the contaminant was a common laboratory strain of B. subtilis, this might have been possible. In the end, this apparently proved impossible because this contaminant was different from any known lab strains. Likewise, Dugway Proving Grounds labs were intensely investigated, and I would assume that any Bacillus strains at that facility would have been investigated. Again, no lab strains matched the contaminant using DNA analysis.
Three questions from Marcia Ann Chambers, Topeka, Indiana
Q1: The mailed spores were compared to ancestral Ames (genotype62) and determined to be identical to it. In what lab and under what circumstances was the ancestral Ames stored?
A: The ancestral Ames culture was stored at USAMRIID until it was discovered during the Amerithrax investigation. It is the oldest known archival Ames culture discovered in the investigation and dates from early 1981 (see: Ravel et al. 2009 J. Bacteriology 191:445-6.).
Q2: When testing other sources to compare to the mailed spores, did you look for colony morphology differences?
A: The morphs themselves were useful for identifying minor genetic differences in the letter spores, but it would have been very tedious and unreliable to depend upon visual analysis of colony morphology across the entire repository or across all the evidence. In addition, the colony morphological differences can be subtle and subjective. (A lot of credit should be extended to the skilled microbiologists who originally spotted the morphs.) So, the final repository analysis was of the genetic differences controlling the morphological differences. These were genetic tests (PCR) developed after the full genomes of each morph were determined by TIGR [The Institute for Genomic Research]. These tests are rapid,
sensitive, and highly reproducible. The entire process was done in a blinded fashion where the labs used anonymous samples.
Q3: If Iowa State University destroyed its entire collection of anthrax, how can it be eliminated as a source for flask RMR-1029?
A: It is regrettable that ISU destroyed their archival B. anthracis isolates (see my commentary above), as this could have exonerated them. Our DNA analyses would have quickly determined whether they even had the Ames strain in their collection. The reason they were under such scrutiny was due to the misnaming of the Ames strain (see my discussion on this topic above).
Commentary: This is an opinion, but given the mailing location in New Jersey, it is unlikely that an ISU employee could have driven that far to mail the letters without being detected. It is also unlikely that ISU even had the Ames strain, let alone RMR1029. They did not have a vaccine development program, and there were no notable anthrax research teams there at that time. After all the unwarranted media attention, I’m guessing that the government investigated activities at ISU with great diligence and ruled out potential suspects.
NOTE: The entire post from Dr. Keim is contained in the comment below, submitted by DXer and reproduced below.
CASE CLOSED ... what really happened in the 2001 anthrax attacks? 
DXer said
“With the input from the U.S. Attorney’s Office, these isolates were not tested for the presence of the four genetic markers.”
Comment: The attorneys in the U.S. Attorney’s Office advised not to test the AMI for the four genetic markers. What was their reasoning?
DXer said
The NAS reports (and lead FBI genetics expert Paul Keim has long said):
Finding 6.3: Some of the mutations identified in the spores of the attack letters and
detected in RMR-1029 might have arisen by parallel evolution rather than by derivation
from RMR-1029. This possible explanation of genetic similarity between spores in the
letters and in RMR-1029 was not rigorously explored during the course of the
investigation, further complicating the interpretation of the apparent association between
the B. anthracis genotypes discovered in the attack letters and those found in RMR-1029.
Another challenge with determining the cause of the apparent association between some
of the B. anthracis genotypes in the attack letters and those found in RMR-1029 stems from the
possibility that the same mutations might have arisen repeatedly in other Ames strain
populations. Colony variants with similar or even identical mutations might arise repeatedly in
other populations for two reasons. First, we do not know the number and rate of possible
mutations that could produce similar phenotypes. Research by Worsham and colleagues (see, for
example, Worsham and Sowers, 1999) identified numerous oligosporogenous variants with
phenotypes similar to those described in the letters. In response to questions raised by the
committee, the FBI indicated that among the 296 Ames submissions to the FBIR by Worsham,
only the Morph D genotype was detected, and it was in three samples. The A1, A3 and E
mutations were not detected in any of the Worsham samples submitted to the FBIR (FBI, 2010a).
Second, under the conditions used to grow B. anthracis for the large-scale production of spores,
there may well have been inadvertent selection that favored oligosporogenic mutants, which
would cause the frequency of these mutants to be higher than expected for mutations that
conferred no advantage to the cells, thereby increasing the likelihood of parallel evolution in
replicate spore productions. The recent published work by Sastella and colleagues (2010)
highlights the possible role of repeated passage of B. anthracis in the enrichment of sporulationdeficient
mutants.
DXer said
The NAS report will be citing and relying on
Bacterial Population Genetics in Infectious Disease
D. Ashley Robinson (Editor), Edward J Feil (Editor), Daniel Falush (Editor)
ISBN: 978-0-470-42474-2
Hardcover
428 pages
April 2010,
I commend the alternative of quoting authoritative treatise. If anyone would like a copy for their personal use, let me know.
This book is a unique synthesis of the major concepts and methods in bacterial population genetics in infectious disease, a field that is now about 35 yrs old. Emphasis is given to explaining population-level processes that shape genetic variation in bacterial populations and statistical methods of analysis of bacterial genetic data.
• A “how to” of bacterial population genetics, which covers an extremely large range of organisms
• Expanding area of science due to high-throughput genome sequencing of bacterial pathogens
• Covers both fundamental approaches to analyzing bacterial population structures with conceptual background in bacterial population biology
• Detailed treatment of statistical methods
7 Population Genomics of Bacteria.
7.1 Introduction.
7.2 Classical Bacterial Population Genetics.
7.3 The Genomics Era.
7.4 Bacterial Population Genomics.
7.5 Next-Gen Bacterial Population Genomics.
7.6 Next-Gen Genomics Technology.
7.7 Next-Gen Genomic Data Analysis.
7.8 Conclusions/Future Prospects.
8 The Use of MLVA and SNP Analysis to Study the Population Genetics of Pathogenic Bacteria.
8.1 Introduction.
8.2 MLVA and Other DNA Fragment-Based Methods.
8.3 SNP and DNA Sequence-Based Methods.
8.4 Conclusion.
Part II Population Genetics of Select Bacterial Pathogens.
9 Population Genetics of Bacillus: Phylogeography of Anthrax in North America.
9.1 Introduction.
9.2 History of Anthrax in North America.
9.3 The Anthrax Districts after 1944.
9.4 Molecular Genotyping of B. anthracis.
9.5 Genotypes within the Anthrax Districts in North America.
9.6 Phylogenetic Resolution within the WNA Lineage.
9.7 Phylogeographic Resolution within the Ames Lineage.
9.8 Additional B. anthracis Genotypes in North America.
9.9 Conclusions.
DXer said
In a chapter in the Fall 2010 MICROBIAL FORENSICS, titled “MIcrobial Forensics: Educating the Workforce and the Community,” Dr. Steven E. Schutzer, Bruce Budowie and Paul S. Keim write:
“As the field of microbial forensics evolves, substantial developments in technology
and analytical capabilities have occurred. An equally important aspect
of preparedness involving microbial forensics that needs a similar commitment
is education and training. The scientific bases, applications, interpretations,
and lessons learned by those who have been intimately involved in the
early years of microbial forensics need to be documented and transferred to
the next generation of scientists and decision makers so that we can protect
society from potential harm resulting from bioterrorism and biocrime. Thus,
the burgeoning field of microbial forensics should be accompanied by a parallel
development of educational infrastructure and resources targeted at the
next generation of practitioners, as well as diverse elements for the policy,
research, and law enforcement communities. A microbial forensics education
program can be broad, providing information encompassing all aspects of
the field from science to policy, or more focused depending on its purpose
and target audience. On one end of the target audience spectrum is the student
at an academic center who desires to enter into the discipline of microbial
forensics and would like to have options for a career choice. This student
may become a forensic scientist analyzing crime scene evidence for a law
enforcement or intelligence agency. Alternatively, the student may become
an investigator who employs traditional law enforcement approaches merged
with those of epidemiology for attribution purposes or crime investigation.
An individual may become a law enforcement official whose responsibility
is to understand the scope of an investigation and what tools are available to
generate investigative leads. Policy makers must have a general understanding
of microbial forensics results and better appreciation of their implications in
order to effect sound and defensible policy decisions. Finally, an important
group that informs the public and government is the news media. They are
frequently the primary interface between the scientist and the public, making
their observations, insights, or inaccuracies of great importance and impact.
Educational efforts will better prepare such individuals to be informed and
responsible and must be varied in depth and scope to match the target audience
of various entities involved in microbial forensics.
***
Conclusion
Disciplines related to microbial forensics are evolving rapidly. This evolution
includes technology, analytical capabilities, and, equally as important, education
and training. This book is one form of education that should accompany
advances in the field of microbial forensics. Other forms of education should
include didactic lectures, practical demonstrations, and discussions at specialty
societies. The target audience may include college students, bench scientists,
law enforcement agents, medical care and first responder personnel,
lawyers, and judges. Those who fulfill teaching roles, whether by profession or
indirectly as reporters and even entertainment writers, can become informed
so that their writings are founded in facts.”
Anthrax and Al Qaeda: The Infiltration of US Biodefense
http://www.blurb.com/bookstore/detail/1443811
Comment: Alternatively, if decision-makers wait until the next attack to become informed so that
their writings are founded in facts — which is the present course set — the analysis can be done
in a 911 Commission-style report issued after a mass aerosol attack on D.C. and NYC or
another targeted assassination attempt on Congressional leaders. The brilliant, hard-working
and hands-on FBI Director Mueller has assured the public that the FBI is committed to doing everything
possible to keep our Congressional leaders safe and so should reopen the investigation given that the evidence
does not support its closing.
DXer said
Paul S. Keim, Bruce Budowle, and Jacques Ravel explain the whole genome sequencing of the “Florida” Ames strain in a chapter in the treatise published late last Fall MICROBIAL FORENSICS (p. 19) that
“In 2001, whole genome sequencing was a slow and expensive process, but the importance of the strain identification was high. Thus, a project was initiated quickly at the Institute for Genomic Research (TIGR) in Rockville, Maryland. Ironically, the genome sequence of the isolate from the index case, also known as the Florida Ames strain, which was completed in early 2002, provided little investigative value as there was no genome database for comparison. At the time, the only other B. anthracis genome available was that of an attenuated strain, the Porton Down Ames isolate, which was partially completed and was found to be highly similar to that of the Florida Ames strain. This genomic
comparison identified a few single-nucleotide polymorphisms (SNPs) and indels, but they were of little value because the Porton Down strain had been cured of its plasmids by mutagenic treatments and the differences could have been the results of this treatment. Subsequently, the whole genome sequence of B. anthracis Ames Ancestor (12) demonstrated that these differences provided no investigative value when compared with the Porton Down sample; they were proven to be unique to the Porton Down strain and, thus, provided no insights into the origin of the Florida Ames strain, and the Florida Ames strain showed no differences from the ancestral Ames isolate.”
The lead FBI genetics expert helping Dr. Keim in his lab, Kimothy Smith, had provided the BL-3 to the former Zawahiri associate at LSU. LSU received its Ames from Porton Down from Peter Turnbull.
DXer said
The experts, all highly expert and leaders in their field, continue to explain the “development of the Ames-specific assays:
“While early genome sequences provided no investigative leads, they were used to develop highly robust assays for SNP-based markers for the Ames strain (13–15). As mentioned before, the MLVA8 subtyping system was not 100% specific and even the addition of more VNTR markers (MLVA15) improved resolution only slightly. In addition, the MLVA was a multiplex polymerase chain reaction (PCR) system that involved several manipulations and acrylamide gel or capillary electrophoresis that was tedious to perform. The resulting fragment sizes required careful scoring by an experienced team to assign alleles. Subsequently , the Ames strain genome was compared
to sequences of other strains (e.g., Sterne) to identify 32 SNP marker alleles that might be unique to the Ames strain. These potential private alleles (i.e., autapomorphic characters) separating the genomes of these two closely
related strains were converted into real-time PCR assays and then validated against a larger strain panel to verify their ability to identify the Ames strain. Many of the 32 SNPs proved to be less than 100% specific, but four in particular
had alleles unique to all Ames strains within the data set tested. A globally representative panel of 88 strains was genotyped to validate the “Ames specificity.” Even when a panel of isolates from Texas, the origin of the
Ames strain, was examined, these four SNP markers were able to differentiate the laboratory Ames strain from any other strain. Real-time PCR SNP assays for the Ames strain were less expensive, faster, and
had greater specificity than the MLVA15 system. This result was expected, as SNPs are far more stable markers than VNTRs. Along with their allele specificity, these assays were also subjected to multiple other challenges to test
their robustness, sensitivity, and fidelity. Several of these assays proved capable of correctly genotyping from single DNA molecules . When inhibitors such as melanin and humic acid were added to the reactions, inhibition
did occur and the sensitivity was affected, but they did not affect specificity. Likewise, an increased or decreased Mg2 concentration did not affect genotyping fidelity. Thousands of single-molecule reactions were performed
to test for stochastic misgenotyping events, but none was observed. Altering the allele-specific probes was the only factor that would generate a false genotyping result, necessitating standard controls for Ames and
non-Ames alleles. Extensive validation and ease of use were great assets to the investigation due to the large number of samples collected.”
The lead FBI genetics expert helping Dr. Keim in his lab had provided the BL-3 to the former Zawahiri associate at LSU. LSU received its Ames from Porton Down from Peter Turnbull. It will require the experts on the NAS panel to figure out whether this affects the validation of the FBI’s science. It is unlikely that the NAS has been given information necessary to address such an issue and so that will require the GAO.
DXer said
The authors Keimk, Budowie and Ravel, on the issue of the FBI repository, explain “that it cannot be verified that this repository was 100% complete.” (pp. 20-21)
DXer said
In the MICROBIAL FORENSICS treatise published last Fall, Dr. Keim, Budowie and Ravel, all world renown experts, explain the “morphological variants” at p.21:
Morphological Variants
Morphological variation was observed among colonies grown from spores
found in the anthrax letters early in 2002. Handling and culturing of these
spores were carried out at USAMRIID by experienced microbiologists. It was
fortuitous that these staff members were very experienced at spotting and
characterizing morphological variants of B. anthracis colonies. As the spores
were being cultured, they observed morphological variants similar to what
had been characterized previously at USAMRIID (18). The color of the colonies
differed from the gray/white appearance of the wild-type Ames strain
toward yellow and yellow/gray. Some variant colonies also had a more spreading
and flat morphology than the wild-type Ames, with concentric rings of
growth. In another variant, the colonies were more opaque and shiny, with a
very compact shape. Perhaps most importantly was that these variants exhibited
altered sporulation phenotypes. All of the anthrax-letter variants studied
were poorly sporogenic when compared to the wild-type Ames ancestor. This
characteristic is referred to as an oligosporogenic phenotype, which has secondary
effects upon other phenotypes, including colony morphology (18). In
all, four morphological variants were purified, studied extensively, and used
investigatively to eliminate potential sources of the letter spores (Figure 2.3).
The morphological variants were purified by USAMRIID
staff and transferred to NAU for DNA extraction and then the
genome was sequenced at TIGR. The whole genome sequences
were compared to a very high-quality genomic sequence of the
Ames ancestor strain (12). The Ames ancestor was the earliest
known archived culture (May 1981) of the Ames strain and is
believed to represent the original stock from which all other
laboratory stocks were derived. Importantly, this sequence was
identical to the Florida Ames isolate. Indeed, when sequences
from the letter isolates were generated, they were all identical to
the Ames ancestor. In contrast, the genome sequences from the
morphological variants each contained one minor difference,
likely to be the basis of the phenotypic variations. This variation
included SNPs, indels, and large duplications. In three of the
four cases, this variation was in or near genes involved in sporulation:
phosphorylation of spoOF/spoOA, dephosphorylation of
spoOF, and near the spoOF gene itself. Sequence differences were not limited
to the chromosome as in one case the mutation was found on the plasmid
pXO1. Critically, these genomic variations were amenable to the development
of PCR-based assays.
The FBI contracted with both commercial and nonprofit laboratories to
develop highly sensitive, specific, and quantitative PCR assays to detect the
variants. These laboratories included TIGR, Midwest Research Institute, and
Commonwealth Biotechnologies Inc. After extensive validation to establish
protocols applicable to forensic analyses, development of reference standards,
and demonstrated staff proficiency, assays were performed on DNA from the
1077 FBI repository samples. All analyses were done blindly and independently
by the different contractors. Data interpretation and conclusions were
then made by FBI scientists, independent of the performance laboratories. All
DNAs extracted from spores recovered from the attack letters contained signatures
for all four variants. Critically, 8 of the repository samples contained
signatures for all four of the morphological variants. These 8 samples were all
derived from the spore stock at USAMRIID known as RMR1029 and included
RMR1029 itself.
RMR1029 was an unusual type of spore stock for many laboratories but not
for USAMRIID where vaccine challenge trials required large quantities of stabilized
spores (5). RMR1029 was a pooled and concentrated liquid spore suspension
derived from twelve 10-liter fermentation batches and an additional
twenty smaller batch cultures, a total of 164 liters of culture concentrated
by centrifugation and resuspended to a 1-liter volume. The use of multiple
cultures, which was intended to produce a large quantity of spores, apparently
gave ample opportunity for variants to arise and become a stable portion of
the total spore population. The spores were stored with phenol as a stabilizing
agent, which would kill any vegetative cells or nonspore-forming bacteria.
This stock was used as an inoculum for additional amplification culturing.
Physical characterization of the letter spores supported the hypothesis that
the actual spores found in the letters resulted from a subculture of RMR1029
and not from this stock directly.
DXer said
Ask the Expert
Paul Keim
Paul Keim, Ph.D., is a Regents Professor of Biology at Northern Arizona University and the Division Director of Pathogen Genomics at the Translational Genomics Research Institute (TGen). He works on the integration of population genetics and genomics for many diverse organisms, including Bacillus anthracis, the causative agent of anthrax. Keim initially became interested in how genetics could be used in forensic investigations when he was called upon to provide testimony in a murder trial, and he subsequently has worked on cases involving wildlife and crop plant DNA testing. Keim serves on the Scientific Working Group for Microbial Forensics (FBI) and the National Science Advisory Board for Biodefense to advise the U.S. government on issues of science and policy.
On July 6, 2009, Paul Keim answered selected viewer questions about the anthrax letter attacks and microbial forensics in general. Please note we are no longer accepting questions, but see our Links & Books section and History of Biowarfare for additional information.
Introductory Note from Paul Keim
Anthrax is a disease that is caused by a bacterial pathogen called Bacillus anthracis. It is important to note that the word “anthrax” is commonly misused in the popular media, where it is substituted for the pathogen name. If you listened closely, I misused the word “anthrax” in a similar way during the show.
Several of my answers below are based upon second-hand information. While I believe that my sources are credible, I have indicated this aspect where appropriate.
“Amerithrax 184” is the case name assigned by the FBI to the investigation of the anthrax letter attacks.
The National Academy of Science will be reviewing the scientific evidence developed in the anthrax-letter investigation. At this time, they have tentatively assigned a committee to investigate the investigation and issue a report to the government. This NAS report should be of great interest, as it will examine the validity of a very unique criminal investigation. It is unlikely that this committee will examine other investigative efforts aimed at attribution to a particular individual. This will be disappointing to many, but it is really beyond the scope of a scientific forum. This URL will link you the NAS website: http://www8.nationalacademies.org/cp/projectview.aspx?key=49105
Q: What are the limitations of using microbial forensics for attribution purposes? Are policymakers fully aware of these limitations?
Jonathan B. Tucker, Washington, D.C.
A: Limitations to Attribution: The conclusions drawn from any forensic evidence are very dependent upon the actual events. We frequently hear about the incredible odds against a human DNA profile (“fingerprint”) matching another by random chance. This is probably the most powerful evidence that is currently admitted in criminal prosecutions. But even here, the conclusions are dependent upon the actual events. The level of certainty provided with a DNA match between a semen stain and sexual assault suspect is amazingly high and almost always leads to a conviction or exoneration. However, if there is an identical twin involved, the probabilities are not overwhelming, and you might just have to flip a coin (50:50) to decide who did it. Thus, making an accurate general statement about limitations to microbial attribution is hard to do because the specific details are critical.
However, there are some powerful specific conclusions in the Amerithrax case. These would include, for example, “The laboratory Ames strain can be precisely identified and differentiated from all other types of Bacillus anthracis, including those very close relatives isolated from the same geographic region of Texas.” This is a long ways from concluding that letter spores came from USAMRIID, but it is a start and eliminates a lot of possibilities. “Exclusion” due to a lack of a match is one of the most powerful conclusions that can be drawn in DNA fingerprinting analysis.
In the Amerithrax case, the scientific evidence (morph typing) tying the letter spores to the RMR-1029 appears strong. (Disclaimer: My lab did not do this analysis, and my knowledge is secondhand.) The FBI repository of Ames cultures was extensive, and only cultures derived from RMR1029 or that culture itself have all four morphs. While this scientific conclusion excludes a very large number of possible perpetrators (including myself), it still doesn’t directly attribute the crime to an individual. I understand that there was more than one person with access to RMR-1029 spore preparation. I don’t know how many individuals had access, and I can only speculate that it could have been quite a few (10? 50? 100?). This limitation is well understood by the scientists involved but may or may not be understood by the public or policymakers.
Policymakers’ Awareness: In general, I would say that the limitations of any technology and science are not well understood by either the public or policymakers. Scientific results almost always get simplified when communicated to a non-scientific audience, and many nuances to the conclusions get lost as well. In the end, even a jury has to decide which scientific expert is the most credible. I and other experts struggle continuously to communicate with the public and with policymakers in an effective manner. It is typical that laboratory results get communicated to a low-level policy person; who then communicates it to the next level; then to next; etc.
In the Amerithrax case, many of our scientific results were eventually communicated to President Bush. Long before he was briefed on the results, someone below him interpreted and massaged whatever report I filed with the FBI. Any U.S. president is going to be smart enough to understand the limitations, if they have the time learn them. I would guess that they never have enough time do this and that the subtleties are lost.
On the positive side, there are some very intelligent scientists and analysts in the government who do appreciate the limitations of scientific evidence. I believe that they sincerely try to communicate the shortcomings, as well as the power of a result. I get the sense that policymaking is a very adversarial endeavor and that someone will be willing to argue against any conclusion!
Q: Did any lab using electron microscopes find bentonite, or did they see silica and thought it was bentonite because they saw a brown ring?
Anonymous
A: This is a bit outside my expertise, but I’ll relate what I have heard from the other experts: At the ASM [American Society for Microbiology] Biodefense meetings, Dr. Joseph Michael from Sandia National Laboratories reported that there were high levels of the element silicon within the spore itself. His high-resolution microscopic elemental analysis did not detect any silica or bentonite on the outside of the spores. The internal silicon signal is thought to be due to biological incorporation during the formation of the spore by the mother cell. As per Dr. Michael’s talk, there was no evidence that silica or bentonite was added to the spore preps. How the silicon is incorporated inside the spore coat is not well understood but has been observed in other Bacillus spore-forming bacteria.
There is also a bonus video on the NOVA scienceNOW website involving Dr. Michael’s work. [See Was It Weaponized?] Here is a good press release from Sandia National Laboratories on this topic:http://www.sandia.gov/news/resources/releases/2008/anthrax.html
Note that there has been great confusion on this topic. A very early report by Dr. Peter Jahrling (a friend of mine) with a lower-resolution technology also detect silicon in the letter spores. This silicon signal was then reported “as probably silica,” which lead to speculation that bentonite was added to the letter spores to “weaponize” them. Again, this is outside my field of expertise, but so-called experts claim that bentonite can be added to spores to make them more readily dispersed. This resulted in extensive speculation that the spores were from a “state-sponsored” source and that they were of a very high “weapons grade” production. All this speculation now appears unfounded due to the over-interpretation of the early silicon signature data.
Additional Commentary: “Weaponization” is a tricky term. Most people use the term to indicate some special additive or a manufacturing process that increases the dispersion and lethality of the spores. I frequently get asked if the spores in the letters were “weaponsized.” The answer has to be yes! But recognize that a brick is a weapon as soon as someone throws it at another person. That act of throwing is, in essence, “weaponizing” the brick. From what I have heard, I don’t believe that the spores in the letters were “weaponized” through special milling or the additional of chemicals to increase their dispersal. I think that these are just plain purified spores that were dried and put in an envelope. Unfortunately, five people died from these “bricks.” But these letters were not weapons of mass destruction. What they did, instead, was to create a major terrorism event.
Q: Did the FBI or U.S. Department of Justice consult with any genetics expert when they were asked in October 2001 whether it was okay for the [B. anthracis strains held at] Iowa State University and USDA Ames (at the strip mall) to be destroyed?
Ross Getman, Syracuse, New York
A: I was not consulted nor am I familiar with the actual steps that lead to the destruction of these materials. During that time period (late 2001), I was doubtlessly the most engaged microbial geneticist working with the FBI. If I wasn’t consulted, then probably none others were either.
Background: This question refers to the destruction of archival B. anthracis strains held at Iowa State University during the early weeks following my lab’s identification of the Ames strain from the first victim. B. anthracis cultures were fairly common in veterinary teaching colleges (ISU is a good one), and their presence at ISU is not surprising. It is a real disease, and veterinarians need to know how to diagnose and treat it. Educators would need to have examples available for teaching and reference purposes.
As we now know, the Ames strain was not from Ames, Iowa but rather from a cow that died in Texas in 1981. Even I didn’t know this in October 2001 and mistakenly thought it had been a part of the U.S. bioweapons program from the 1950’s. Since 1981, the Ames strain has been used for vaccine testing and, as such, is commonly used in research laboratories. Its wide use for vaccine challenges lead to the misconception that it had been used previously as a part of the weapons program. Martin Hugh-Jones and I wrote a paper in 2000 (J. Bacteriology. 182:2928-2936) where we mistakenly stated this. Martin had worked at USAMRIID and thought that this was the case when we wrote the paper. We now know that this is false.
The misnaming of the “Ames” strain was due to confusion over its original source. Dr. Greg Knudson, who was at USAMRIID at the time, has stated that it was named “Ames” because of recycled packing material used by the Texas veterinarian to ship the 1981 isolate to USAMRIID. The packaging material had originally come from Ames, Iowa, where Iowa State University and the USDA Animal Disease lab reside. This is a credible report, and we have confirmed that other B. anthracis isolates from parts of Texas are very closely related to the laboratory Ames strain (Kenefic et al, Emerging Infectious Disease 14:1494-6; Simonson et al, BMC Microbiology. 9:71doi:10.1186/1471-2180-9-71). So, we now know that the Ames strain originally came from Texas, not Iowa.
Additional Commentary: It is hard to understand why this destruction was done and why it was allowed to occur. Clearly someone in Iowa panicked at being in the media bright lights and wanted to get rid of the material. If this was really authorized by the FBI, who that authority was has not been released, to my knowledge. If the investigation had eventually lead back to Iowa, this would have been viewed as destruction of evidence and obstruction of justice. Now that would have been a public relations nightmare!
I used to work at Iowa State University, and I lived in Ames, Iowa. I still have many friends there. So that they don’t get too mad at me, I would like to point out that this criticism is in hindsight, and I imagine the pressure was intense. I wasn’t there in October 2001. (I had my own stress at the time.) But I hope that I would have made a different decision.
Q: The following are several of the many questions proposed at my CASE CLOSED blog, where a vibrant discussion of issues related to the anthrax investigation is active every day:
1) Did the 1,070 samples examined by the morphotype analysis accurately reflect the actual number of anthrax samples held in the U.S. in the 2001 timeframe?
2) Did the process of self-submission of anthrax samples by labs and individuals (per the FBI methodology) result in a reliable representation of the Ames samples that may or may not have existed prior to 2001?
3) Did the FBI use any type of proficiency testing for their genetic analysis?
4) Was the Bacillus subtilis contaminant found in the first batch of letters genetically identical to any forensic evidence collected from any lab? Also, was that contaminant tested against strains from Dugway Proving Grounds?
Lew Weinstein, Collioure, France
A: [Editor’s Note: The questions above have been numbered to match the corresponding answers below.]
1) This would not be representative of all the Bacillus anthracis samples, but rather Ames strain stocks. I don’t know how many B. anthracis cultures existed in the U.S. in 2001, but many more than this. The Ames strain is only one of many different identifiable strains.
2) Presumably, the 1,070 collection represents all the Ames cultures in the U.S. plus some from around the world. First, the Department of Justice subpoenaed all the U.S. anthrax labs for our inventory records with special attention to the Ames strain cultures. Next, the DOJ subpoenaed all U.S. laboratories that had the Ames strain to provide a sampling (a portion) of their Ames cultures. For example, my lab had only seven different Ames stocks, which we sampled and sent in duplicate to the FBI.
3) Definitely yes. Extensive proficiency testing was performed both prior to and during the genetic analysis at Northern Arizona University. The initial determination from the first Florida sample was done using normal research controls and standards, but soon afterwards very rigorous forensic-level protocols were implemented. There was no time for proficiency testing for the first analysis on the Robert Stevens isolate, as we had only a few hours notification. Immediately afterwards, the FBI forensic scientists began to review our procedures and help us to institute forensic-level operating protocols, including proficiency testing. Once these were in place, original analyses were repeated with the same result.
Note that there were no forensic standards for microbes and that we had to adapt the ones developed for human DNA testing to our new anthrax tests. This is a topic of great debate as to what the standards should be for a new technology.
4) Disclaimer: My lab did not work on the B. subtilis contaminant, so what I am relating is secondhand knowledge. However, it is based upon my conversations with government experts and my participation in the press conferences and the public ASM symposium. Details of the contaminant were discussed in these forums.
Background: The question refers to the non-hemolytic (B. anthracis is hemolytic) bacterial contaminant found in some of the letters. It proved to be a spore forming Bacillus subtilis bacterium. As the name implies, this is somewhat related to Bacillus anthracis and shares many biological properties. But it is not a disease causing organism, and there are many, many different types found in the environment. It would not be surprise to me to find a novel B. subtilis on my computer keyboard.
Answer: The government investigators thought that perhaps this contaminant could be used to trace back the spore preps to a particular laboratory. If the contaminant was a common laboratory strain of B. subtilis, this might have been possible. In the end, this apparently proved impossible because this contaminant was different from any known lab strains. Likewise, Dugway Proving Grounds labs were intensely investigated, and I would assume that any Bacillus strains at that facility would have been investigated. Again, no lab strains matched the contaminant using DNA analysis.
Q: Did the Bacillus subtilis contaminant found in the New York Post and NBC anthrax letters raise speculation that it could have been residue from aerosol powder equipment used by military labs to manufacture bioweapon (BW) simulants? For example, Edgewood Army labs routinely produce aerosolizable Bacillus subtilis var. niger strain bioweapons simulants. See link here: http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA483822&Location=U2&doc=GetTRDoc.pdf. Did the Bacillus subtilis found match the niger strain or any other strain associated with BW simulant production? Was this considered?
Anonymous
A: DNA analysis of the B. subtilis contaminant did not match any known strain of bacteria (see my previous answer).
Background: The question refers to a fairly common lab strain of B. subtilis that has been used as a non-pathogenic biological weapons simulant of B. anthracis to test detector systems in environmental releases of spores. This strain forms a very identifiable colony upon plating, which is probably why it was chosen as the BW simulant.
Everyone in the investigation is well aware of the use of this B. subtilis niger as a simulant, and many of us have this strain in our labs. Upon DNA analysis, the letter-contaminant strain was considerably different from the simulant.
Not to belabor this point, but this BW simulant bacterium has been renamed several times as scientists have learned more about. It was originaly named Bacillus subtilis var. niger, but was then deemed distinct and different from B. subtilis and was then given its own species name, Bacillus globii. Later it was renamed again to Bacillus atrophaeus. I mention this just to re-enforce the idea that this BW simulant bacterium is very distinguishable from the B. subtilis found amongst the B. anthracis spores.
Q: [Editor’s note: The following questions have been numbered to correspond with the answers below them.]
1) The mailed spores were compared to ancestral Ames (genotype62) and determined to be identical to it. In what lab and under what circumstances was the ancestral Ames stored? 2) When testing other sources to compare to the mailed spores, did you look for colony morphology differences? 3) If Iowa State University destroyed its entire collection of anthrax, how can it be eliminated as a source for flask RMR-1029?
Marcia Ann Chambers, Topeka, Indiana
A: 1) The ancestral Ames culture was stored at USAMRIID until it was discovered during the Amerithrax investigation. It is the oldest known archival Ames culture discovered in the investigation and dates from early 1981 (see: Ravel et al. 2009 J. Bacteriology 191:445-6.). 2) The morphs themselves were useful for identifying minor genetic differences in the letter spores, but it would have been very tedious and unreliable to depend upon visual analysis of colony morphology across the entire repository or across all the evidence. In addition, the colony morphological differences can be subtle and subjective. (A lot of credit should be extended to the skilled microbiologists who originally spotted the morphs.) So, the final repository analysis was of the genetic differences controlling the morphological differences. These were genetic tests (PCR) developed after the full genomes of each morph were determined by TIGR [The Institute for Genomic Research]. These tests are rapid,
sensitive, and highly reproducible. The entire process was done in a blinded fashion where the labs used anonymous samples.
3) It is regrettable that ISU destroyed their archival B. anthracis isolates (see my commentary above), as this could have exonerated them. Our DNA analyses would have quickly determined whether they even had the Ames strain in their collection. The reason they were under such scrutiny was due to the misnaming of the Ames strain (see my discussion on this topic above).
Commentary: This is an opinion, but given the mailing location in New Jersey, it is unlikely that an ISU employee could have driven that far to mail the letters without being detected. It is also unlikely that ISU even had the Ames strain, let alone RMR1029. They did not have a vaccine development program, and there were no notable anthrax research teams there at that time. After all the unwarranted media attention, I’m guessing that the government investigated activities at ISU with great diligence and ruled out potential suspects.
Q: Dr. Keim,
In the segment on microbial forensics June 30th, the morphs were eventually found, which distinguished eight of the original 99 lab samples as being identical to the Ames strain RMR-10-20-9. When the initial comparison of the >5 M nt were done, comparing the anthrax in the dead journalist and the original cow anthrax, why was the original verdict that they were exact matches? Why were the morphs not detected in early 2002? Had the one scientist not seen a slight color variation, would the morphs never have been found? Were all the morphs found in the same petri dish together? Using today’s technology, would we be able to find the morphs via the sequencing matches?
Also, can you tell me what minimum undergraduate and/or graduate courses would have to be completed in order to be considered as a graduate student candidate in microbial forensics? I believe yours is the closest university that offers such a program in the Southwest U.S. Thanks.
Diana, Albuquerque, New Mexico
A: [Addressing the first paragraph:] The morphs were observed very early in the investigative process. I wasn’t there when it happened but probably in 2001 and 2002, the morphs had been observed. The observation probably occurred when a microbiologist was plating for single spore colonies on a blood agar petri dish. Hundreds of colonies could be grown in this fashion, side by side. Subtle difference in a few colony morphology were spotted at this time. We are told that they were in low frequency (1%?), so a fairly large number of spore must have been platted (grown) in order to see the few that were different.
With today’s genomic technology, it might be able to identify minor component genetic differences without first spotting the morphological mutants. The morphs themselves are of little value other than pointing to genomics differences that could be used for characterizing the samples. Today we could simply sequence a large number of colonies to find differences.
[Addressing the second paragraph:] Microbial forensics is a new scientific field but closely related to the field of epidemiology. In particular, forensic analysis similar to molecular epidemiology analysis was important in the Amerithrax investigation. A graduate program that covers infectious diseases, microbiology, molecular biology, genetics, genomics, epidemiology, evolution, statistics, and public health would be excellent training for a budding microbial forensic scientist. There is a great need for molecular epidemiologists to understand and solve public health problems. I am hoping that there will not be a great need for microbial forensics in the future. If so, some of the talent pool will come from epidemiology.
Q: Something did not make sense in the documentary. You narrowed the source to a particular bottle, but also stated that about 10 labs had tapped it. Why then was Bruce Ivins, the keeper of the master bottle, the only suspect? Why not one of those other labs or Ivins’ superior or coworker?
Also, the NOVA piece did not speculate about motives for the attacks. Surely that would also be a way to narrow down culprits. As Seneca put it: Cui prodest scelus, is fecit (The one who derives advantage from the crime is the one most likely to have committed it).
Roedy Green, Victoria, British Columbia, Canada
A: The scientific evidence from the morphs only narrows the search to RMR1029 and cultures derived from it. In the 1,070 FBI repository samples, there were eight samples that contained all four morph signatures, most of which were at USAMRIID. It is my understanding that there was only one other lab, besides USAMRIID. This is secondhand knowledge, on my part.
I have no special information concerning the attribution to a particular individual and can’t answer the second two questions.
I have no special information concerning the motives of any individual and can’t answer the final question.