|
Transcript of the Aug. 18, 2008, roundtable
discussion of scientific aspects of the Amerithrax investigation
Annotations by Ed Lake www.anthraxinvestigation.com - Important sections
are highlighted in red.
Annotations are in blue boxes like this box. Links to key discussions: NATURALLY OCCURRING, AFIP ERRORS, USAMRIID ERRORS, SILICON UNDER THE EXOSPORIUM, THE 2002 SCIENCE PAPER, "10 TO THE 12TH", EDX PEAKS, CARBON DATING, REVERSE ENGINEERING, THE SIZE OF PORES IN ENVELOPES, BUOYANT PARTICLES, BACILLUS SUBTILIS, SLANTS, FEW EXPERTS IN POWDERS, DANGERS WITH DRIED SPORES, BEHAVIOR OF UNWEAPONIZED SPORES, TIME NEEDED TO PREPARE THE POWDERS, THE GOLD STANDARD, MILLING, AFFIDAVITS, RMR-1029, THE ERLENMEYER FLASK, MUTANT BACTERIA, THE M&M ANALOGY NOTE: Some obvious transcription errors have been corrected. Examples: APHID changed to AFIP, "exosporian" changed to "exosporium." The original transcript can be viewed by clicking HERE. |
UNITED STATES DEPARTMENT OF JUSTICE
Monday, August 18, 2008
Federal Bureau of Investigations
J. Edgar Hoover Building
William H. Webster Conference Room
935 Pennsylvania Avenue, NW
Washington, D.C. 20535-0001
BACKGROUND OFFICIAL: Thanks, everybody, for coming today. It's great to see so many Justice regulars flanked by their best science friend. As advertised, today's briefing will be designed to shed more light on the scientific aspects of the investigation into the Anthrax mailings. And what we said before, and I'll say today, it's difficult to separate the investigation from the intelligence from the science, we are, in fact, going to attempt to do that today because we're limited.
At the same time that the DOJ and FBI are moving toward making more documents available, it's still an open investigation and we remain limited today what we can discuss, which will be emphasized, I'm sure, time and time again; and that for a number of reasons. And when that status changes, obviously, there'll be more information available to you.
As stated on the advisory, today's briefing is on the record with respect to the individuals who are identified here. At the same time, we have a number of investigators, prosecutors, and other scientists who are going to be available to clarify points, or, in some cases, make sure we stay within the boundaries we need to stay today.
Leading today's discussion is Dr. Vahid Majidi and Dr. Chris Hassell of the FBI. Dr. Majidi is our Assistant Director for the Weapons of Mass Destruction Directorate. He comes to us from the Los Alamos National Laboratory. Dr. Hassell is even newer to us. He heads the FBI laboratory. He came from Oklahoma State University as well as Los Alamos. And our format today will be Dr. Majidi will open the program, give a brief overview; Dr. Hassell will introduce the other panel members.
DR. MAJIDI: Thank you. Good afternoon, ladies and gentlemen. I am Vahid Majidi, the assistant director responsible for the FBI's Weapons of Mass Destruction Directorate. I would like to start today's session with a brief opening statement and define the scope of our roundtable discussion.
After nearly seven years of investigation, we have developed a body of powerful evidence that allows us to conclude that we have identified the origin and the perpetrator of the 2001 Bacillus Anthracis mailing. The attribution process and identification of the specific perpetrator relies on the confluence of intelligence, investigations, and forensic information.
It is the forensic information that determined the source of the 2001 Bacillus Anthracis mailing to be derived from a unique pool of spore proporation known as RMR-1029 that was maintained at U.S. Army Medical Research Institute for Infectious Disease, Fort Detrick, Maryland. From now on I will refer to that USAMRIID.
While there were countless investigative hours spent narrowing the field of suspects, we are here today to focus on the scientific aspects of this case.
First of all, let me dispel some frequently repeated erroneous information. For example, there were no additional additives combined with the Bacillus Anthracis to make them any more dispersible. The purity of samples obtained from the four letters was sufficiently different, which allowed us to conclude that at least two different Bacillus Anthracis batches were prepared from the original RMR-1029. This indicates that alloquots of RMR-1029 were removed and cultured in at least two separate batches to produce the materials used in the mailings.
| I'm told that an “alloquot” is a "subsample" which evidently means part of a sample. |
The FBI began this complex investigation by coordinating analysis of the spore powders contained in the 2001 Bacillus Anthracis mailing. We enlisted the help of more biodefense experts to assist our examination, including those who have previously developed and tested strains of Bacillus Anthracis to identify the spores in the letters of the Ames strain.
Other strategies were employed to target the chemical and elemental profiles of the spore powders. Specific techniques included scanning and transmission electron microscopy, energy-dispersive x-ray analysis, carbon dating by accelerator mass spectrometry, and plasma optical illusion and mass spectrometry.
Additional scientists under the Department of Defense and the Center for Disease Control examined the spore materials and it was determined that there were many phenotypic variants within the samples. The general support that both the National Institutes of Health, the National Science Convention, and other government agencies, FBI scientists worked with the Institute for Genetic Research to determine if genetic mutations were responsible for the altered appearance of the variant found in the Bacillus Anthracis letter. Several genetic mutants were discovered in these studies.
FBI microbiologists contracted the assistance of several laboratories to develop highly specific assays to detect four specific genetic mutations found in a Bacillus Anthracis letter. The mutation detection assay were validated and used by the FBI laboratory to examine the repository of Bacillus Anthracis Ames that was collected through the course of this investigation. And we'll give you more information on that repository. This unprecedented scientific approach allowed the FBI to identify potential sources of the Bacillus Anthracis used to produce the 2001 spore powder.
For a comprehensive analytical approach the investigators were provided with validated scientific data which linked the materials in the 2001 attacks to materials from USAMRIID identified as RMR-1029. It is important to emphasize that the science used in this case is highly validated and well accepted throughout the scientific community. The novelty is in the application of these techniques for forensic microbiology.
Today I'm very confident that the significant lessons learned from the 2001 Bacillus Anthracis case have been rigorously evaluated by the FBI and appropriate actions have been taken to safeguard the American public. The FBI laboratory has revolutionized the approach to non-traditional forensic samples and developed robust capabilities to collect and examine evidence containing biological, chemical, radiological, or nuclear materials. We have developed a strong partnership with the U.S. Government Laboratory Complex, Public Health System, private industry and academia to significantly enhance our capabilities dealing with future investigations.
The creation of the Weapons of the Mass Destruction Directorate is another example of FBI's progressive approach focusing on prevention, as well as investigations of all issues involving chemical, biological, and radiological as well as nuclear materials.
Please note that there were many dedicated individuals including prosecutors, scientists, investigators, analysts, and support personnel that have worked on this case. For the purpose of this meeting, the science and technology community within the Bureau has a lead, and if individuals from the sidelines are asked to provide additional background to any of your questions, please make sure to attribute all answers to the FBI Laboratory Director, Dr. Hassell. You can also use “FBI scientist” as a notation as well.
|
They
evidently don’t want to
identify every scientist or person at the session, since that could
result in
those people being hounded by the media or by private citizens with a
bone to
pick. |
Finally,
I'm asking you to understand that this is the first step toward
broader dissemination of the scientific information surrounding this
case. Additional information will be available through peer
review publications, and I ask you to please respect the integrity of
that process.
In fact, several research projects related to the FBI's investigation have already resulted in peer review publications and we will provide you with that list. Additional publications will be available for peer review as more information from the investigation is released.
Before we open the floor for question and answer, we would like to introduce you to our distinguished panel. Today we have with us a small group of individuals representing a large cadre of non-bureau scientists that helped us chart and navigate our scientific path through this unprecedented case.
In the near future, after we work through each non-disclosure agreement and privacy issues, we will release the names of those key individuals who tirelessly worked with us on the 2001 Bacillus Anthracis mailings.
To my left is the current FBI laboratory director, Dr. Chris Hassell. Dr. Hassell will introduce our panel members.
His research focuses on molecular genetics for a wide variety of organisms, including bacteria, plants, and animals. His work in support of the FBI included identification of the spore powders as the Ames strain of the Bacillus Anthracis.
Next -- to his left -- is Dr. James Burans, who's currently the associate laboratory director of the National Bioforensic Analysis Center. He has been in the forefront of the development of diagnostic assay techniques to identify and characterize biological threat agents. He led several of the scientific working groups that were assembled from the National Academy of Sciences, National Laboratories, and other federal R&D facilities in support of this investigation.
To his left is Dr. Rita Colwell. She is currently a distinguished professor both at the University of Maryland, College Park as well as at the Johns Hopkins University Bloomberg School of Public Health. She's also senior advisor to Canon U.S. Life Science Incorporated.
From 1998 to 2004 she served as director of the National Science Foundation, which provided funding for much of the genetic sequencing efforts in support of the FBI investigation. She has served as president of American Association for the Advancement of Science, the American Society for Microbiology, and she is a member of the National Academy of Sciences. In July of 2007, she received the National Medal of Science.
To my left -- immediate left -- is Professor Claire Fraser-Liggett. She's a professor of medicine and director of the newly created Institute for Genome Sciences at the School of Medicine, University of Maryland in Baltimore, Maryland. She was previously the president director of the Institute for Genomic Research, sometimes referred to as TIGR, where she led teams that sequenced the genomes of several microbial organisms including important human and animal pathogens. TIGR performed genetic sequence analysis in support of this investigation.
To her left is Professor Jacques Ravel, who is also at the same institute. He's an associate professor of microbiology and a member of the Institute for Genome Sciences at the University of Maryland School of Medicine. He also was formerly with the Institute for Genomic Research. His research focuses on the application of microbial genomics to several key areas including microbial genomes, sequence, comparative analysis, to the special emphasis on human microbial pathogens including Bacillus Anthracis. His work included genetic sequence analysis and characterization of genetic mutants in support of this investigation.
And finally, to my far left, is Dr. Joseph Michael. He is the distinguished member of the technical staff Sandia National Laboratories in Albuquerque, New Mexico. He currently works in the materials characterization department of the Materials Science Center there, where he develops and applies electron and ion microscopy to the characterization of materials. He is co-author of the leading textbook on scanning electron microscopy. He assisted mineral analysis, electron microscopy of the samples and with development of the strategies for analysis for analysis of chemical and physical properties of the spore powders.
The next step we'd like to actually go through the panel and have each member of the panel talk in a little bit more detail about their specific participation in this investigation. So if I could start with Dr. Keim.
DR. KEIM: So as Chris mentioned I'm a professor of microbiology at Northern Arizona University and director of the Pathogen Genomics division of TGEN. I'm also an affiliate at Los Alamos National Laboratory in the biosciences. My laboratory began studying Bacillus Anthracis prior to 9-11 and the Anthrax letter attacks by developing methods for precisely identifying -- what we considered precisely identifying at the time -- different strains of Bacillus Anthracis and it was those relatively crude pre-genomics type technologies that we employed in October 2001 to identify the Ames strain from the letters.
After that, we continued to analyze populations of Bacillus Anthracis around the world to try to understand exactly what the Ames strain is and what it isn't. And in the post-genomic era and at a period of time after we had whole genome sequences generated by TIGR, we were able to develop more sophisticated, more sensitive methods for identifying what the Ames strain is and what it isn't. And so my laboratory worked on that aspect.
We
worked on the repository that the FBI established for Ames strain,
which was collected from around the country, and indeed around the
world, providing biosafety support for the Bureau early on in the
investigation as they began to stand up their own facilities in that
area.
DR. BURANS: My name is Jim Burans. I'm a pathogenic microbiologist. I'm a retired naval officer and come from the U.S. Naval biodefense community. In the early ‘90s I provided bioforensic support to several biocrime investigations for the FBI as well as supporting bioforensic analysis for the U.N. Special Commission to Iraq.
In the November time frame of 2001 I became a scientific consultant to the FBI in the early stages of the Anthrax investigation, and in 2003, I helped to establish the National Bioforensic Analysis Center to deal with the analytical investigative challenges in bioforensic analysis to support homeland security and the FBI. And since that time we have supported the FBI in analyzing evidentiary samples from biocrime investigations.
DR. COLWELL: -- microbial systemitist, microbial ecologist, and I've worked my entire career on cholera in many countries, as well as the few cases that occur in the U.S. and its ecology. I've been long interested in the molecular systematics and evolution of microorganisms. I was director of the National Science Foundation when the Anthrax event occurred. The National Science Foundation received a proposal which we were able to fund under the program of small grants for innovative or exploratory research. This permitted us to act very swiftly and to have the sequencing done rapidly. The work was done by TIGR, about which you will hear in a moment. We, at that time, a consortium of agencies worked together. The NIH, the NSF, Department of Energy, Homeland Security, the intelligence community, Department of Justice, the FBI, the USDA, DOD, including USAMRIID. We met on a regular basis and we served as a source of advice and advisory for the FBI, but also to establish the capacity to expand the few sequences that were available at the time. And so from the work, as you will learn this morning, has emerged a legitimate new discipline, namely microbial forensics.
DR. FRASER-LIGGETT: I'm Claire Fraser-Liggett. I'm currently a professor of medicine and director of the Institute for Genome Sciences at the University of Maryland School of Medicine. Prior to last year I was the president and director of the Institute for Genome Sciences that was involved in essentially all of the genome sequencing work related to the Amerithrax investigation. I've been involved in the field of comparative microbial genomics since 1995, with the sequencing of the first microbial genome and out of that work have come an interest on my part in using these techniques to better understand microbial evolution and diversity.
At the time of the Anthrax letter mailings in October 2001, we were finishing up the first project to sequence Bacillus Anthracis Ames. It was a strain that was unrelated to the Anthrax letter mailings, but as soon as this news broke, as Dr. Colwell mentioned, we quickly put together a proposal to the National Science Foundation to ask the question as to whether or not genomics-based technologies could be used to identify polymorphisms, that is DNA sequence variance, in different samples of the same isolate of Bacillus Anthracis. The isolate that we were working on for genome analysis also happened to be Bacillus Anthracis Ames as was reported in a paper published in Science in 2002. Indeed, that initial comparative study identified a small number of high-quality snips and we were very encouraged by that initial finding that, indeed, this might be a very useful approach as one part of a large toolbox to be brought to bear on the Amerithrax investigation.
As you've also heard in the opening statements, we were then subsequently asked to work with the Bureau to process a large number of samples. This gave us an opportunity to further develop a number of both experimental and computational approaches that had utility not only in the investigation, but I think will also have broader utility of microbial forensics. Many of those approaches were developed by my colleague sitting to my left, Dr. Jacques Ravel.
<>DR. RAVEL: My name is Jacques Ravel. I'm an associate professor at the University of Maryland School of Maryland at the Institute for Genome Sciences, which Claire is the director of. My laboratory is focusing on several things, but to this matter today the part of my laboratory is the one that studied human pathogens, so we're applying comparative genomics to study human pathogen and what leads a non-pathogen to become a pathogen. So the part that the team that I led at TIGR back in 2002 took in the investigation were sequencing the genomes, some of the variant we'll be talking about later, and using comparative genomic approaches to identify specific polymorphism, DNA sequence differences in those variants, to develop a specific assay to query those for the presence of those variants in other samples.
DR. MICHAEL: Good afternoon. My name is Joe Michael. I work at Sandia National Laboratories. My background is material science, and specifically I use electron and ion microscopies to characterize materials over range of length scales expanding from millimeters down to the nanometer scale.
In this case, I got involved back in January of 2002, when we first started to receive samples from the investigation, and our interest was to determine if there were or were not any additives added to the samples as far as weaponization.
|
Later in this
transcript, we’ll see that the
first testing done on the attack anthrax at AFIP and at USAMRIID
resulted in a
lot of false assumptions. Going to
Sandia might have avoided requiring those people at AFIP and USAMRIID
to rethink mistakes
they made. That's always a problem. |
DR.
HASSELL: As was mentioned earlier, many people are seated
behind us that all work for Bureau -- many for the laboratory itself
-- have done a tremendous amount of work on this whole
investigation. To start out, since a lot of the questions we
kind of anticipate, it might be helpful to just give an introduction
on some of the work that went on to led us up this point. So
I've asked one of our chief scientists, actually, to sort of open
that introduction.
BACKGROUND OFFICIAL: I'm going to give you in a nutshell --
<>QUESTION: Can we get a name, please?
|
An analysis
of comments by
this “Background Official” indicates that he or she is most likely a
prosecutor from
the Department of Justice who was preparing to prosecute the Amerithrax
killer. |
BACKGROUND OFFICIAL: -- my statements are not for attribution. They're for background only. Thank you. To give you a little bit of a background as to the process that was followed for the scientific part of the investigation. The investigators early on worked very hard to try and identify the source of the Anthrax, but the first job was to identify which strain was used, and Dr. Keim, his work was really a central work in identifying it as the Ames strain. So the investigators had to then identify what laboratories had the Ames strain. That was a process that took a lot of work, and over the course of several years through the process of subpoenas and the execution of warrants for search, a collection that we call the FBI repository, was collected. This amounted to well over a thousand samples of the Bacillus Anthracis Ames that was collected both domestically and internationally. That repository is the evidence that we used, that we screened, for forensic signatures to assist the investigation. It was noted early on in the examination of the Anthrax powders that there were some unusual characteristics of the Anthrax in those powders. There were many different variants of the Ames strain that were noted in the Anthrax powders that had a different appearance.
Those colonies with different appearance had their DNA purified by Dr. Keim's lab and that DNA was sent to the Institute for Genomic Research, for sequencing of the entire genome. And through those efforts it was noted that many of those variants had mutations that were most likely associated with the change in the characteristics. Some of those mutations were further exploited in the development of very specific assays, genetic assays, to identify those mutations in an overwhelming background of wild-type Ames. And it was those scientific assays that were used to examine that repository of over a thousand Ames strains that were collected throughout the course of the investigation. The results of that scientific work and the screening of the repository resulted in the identification of eight samples that had the combination of four genetic markers that were all characteristic of the Anthrax in the letters. And that's the background for what you've been hearing of the genetic screening of the Anthrax.
DR. MAJIDI: All right. We'll open the floor for questions. I will moderate the question and answers. The majority of the questions will be answered by myself or Dr. Hassell. If you have specific question for any of our panel members, please voice them and we'll get that answered appropriately. There are areas that are clearly not within our expertise. So if you ask a question that falls within one of our panelists' expertise we will defer those questions to them.
QUESTION: It would be helpful to know, especially for the background, some timeline or some understanding of when certain things were done. You know, if you would, the scientist who just gave us the background, could give us a little bit of an idea when certain identifications were made. That would be helpful.
BACKGROUND OFFICIAL: In 2002, or early on in the investigation, it was noted that there were variants of Ames that were detected from the Anthrax powders. And it took some time because each one of those had to be confirmed to make sure that we didn't have a mixed culture, that it was, in fact, still Bacillus Anthracis, and it was still, in fact, Ames. And that's what Dr. Keim was very instrumental in doing for us. Once they were identified as Ames then it fell to TIGR to help us identify what those mutations were. Most of that was completed between 2002 and late 2003. Then subsequent to that we contracted the assistance of other scientists, experts in the biodefense arena who have experience with Anthrax, and contracted them to assist us in developing those assays. We felt there were a number of assays that needed to be developed. If the FBI laboratory took on that responsibility itself it would have taken a long time so we contracted the help of the biodefense community to help us build those assays. We validated those assays and then over the many years of this investigation, the repository was also being built. So from roughly 2003 to 2006 is when we completed the repository and the screening of the material in that repository.
QUESTION: Could I ask a little more about the timeline? I saw in the paper that colleagues published in 2002 in Science describing a large number of markers. Did you not have sufficient information there to select the four markers and thereby match the source to the strain; and if not, what was missing and when did you acquire those missing pieces?
BACKGROUND OFFICIAL: What was the reference you quoted?
|
I’ve
encountered people who
believe the
2002 paper in Science Magazine described all that was needed to
locate the
source of the anthrax. The point being
made here is that is very definitely NOT true. |
DR. FRASER-LIGGETT: That was the comparison of the Porton strain that we had sequenced initially with the Florida strain; and that was just -- I think to answer your question, Nick, that was a comparison of one sample that had been collected as part of the investigation and I think what's been made clear is that in the interest of being thorough what was essential to do was to look at as many different samples as possible that had been collected from the multiple letters in order to get a broader sense of whether the markers that had been identified initially were seen in all samples from the investigation.
QUESTION: In the 2002 paper, there are a large number of markers described. It does include the four markers that were --
DR. FRASER-LIGGETT: Some of them turned out to be found in other samples, but not all of them.
QUESTION: So you --
DR. FRASER-LIGGETT: Correct.
QUESTION: When were the final four markers --
BACKGROUND OFFICIAL: In the 2002 paper we had not yet fully characterized the mutants that were isolated from the letters, so those four genetic markers were not referenced in that paper.
QUESTION: -- what the markers were --
DR. FRASER-LIGGETT: No, ultimately, no.
DR. KEIM: Do you understand that those markers are not relevant to the four? Those markers in the 2002 paper were only relevant to the Porton Down strain of Ames versus the Florida strain; and those did not exist within the core lineage of the Ames strain so they're not relevant to the current investigation. What they were important for was demonstrating that we could discover these small number of markers. There weren't a large number, it was a small number of markers, and so we knew -- and TIGR knew -- that this is a strategy that was going to be used.
QUESTION: So when you characterize the attack strain in the 2002 paper, so again, what needed to happen then to make the match to the source? Could you describe the sequence itself -- events, the logic tree you went through to get to the June 2002 paper to the match of the attack strain?
BACKGROUND OFFICIAL: One thing that's important to note is that when you look at wild-type Ames, when you have a sample that dates back to 1981, which is the probably the closest sample that we have to the isolations from the cow. And this was provided to a scientist in 1981; and that is what we referred to as the wild-type Ames. That sequence was published in --
DR. RAVEL: It's not published. It's available in GenBank.
BACKGROUND OFFICIAL: -- right, but it is identical to the published sequence, I believe.
DR. RAVEL: That's exactly the point to make here is that this paper referred to the comparison to the Florida isolate to Porton. Porton is a highly modified Ames isolate. It was cured of its plasmid so it doesn't have any plasmid. It underwent mitogenesis to get rid of the plasmid, so the mutation you're actually looking at are actually more looking at what happened to Porton instead of Florida. And when you only have one strain like this, if you start using those polymorphisms and discover you're looking polymorphisms that Porton has and try to tie them under the Anthrax from the attack would not make any sense. We needed more isolates from the attack to make the comparison to and comparing to the Ames ancestor, not the Ames Porton, to discover the real polymorphism.
DR. FRASER-LIGGETT: And I think another important point is that the Florida strain which was related to the Anthrax attacks was a sample that came from the first individual to die of inhalation Anthrax. This was not a sample that came from the spore powders. This was a sample that was obtained from either the blood or the CSFs, I don't remember, the spinal fluid of the first Anthrax victim. So in that sense, this was a sample, if you will, that was yet one step removed from the powder in the envelope.
The importance of the paper that you're referring to is the point that Dr. Keim made just a few moments ago; that this was really a proof of principle demonstration that in fact using whole genome sequencing at a sufficient level of coverage, it was possible to identify high-quality polymorphisms. Had we not found any differences whatsoever, that would have put the potential to use these approaches in a whole different light.
DR. MAJIDI: Paul, did you want to make a comment?
DR. KEIM: No, I was just going to say exactly what Claire said was, this was coming out of a victim, and so this is a very purifying process, and many of these variants will not come through a victim or through a host.
BACKGROUND OFFICIAL: Okay, so getting back to the timeline, the Anthrax powders, themselves, were investigated. And I'm going to give you a little background with Ames. If you look at the 1981 Ames isolate and what was isolated from the victims from their samples, there's virtually no difference. So that is -- the genome sequencing of those samples gave us an idea of what Bacillus Anthracis Ames is supposed to look like, what it looks like.
Naturally, as you mentioned, the Porton Down strain looked quite different from that and that was explained by the fact that they did manipulate it a lot and were trying to mutate it in their laboratory. But when we looked at the examination of the Anthrax powders, what we noticed was something that we don't normally see with Ames, and that is a large amount of colony variation within those samples. It's very unusual for Anthrax, not just Ames. So we exploited the findings of those phenotypic variants, things that looked different, first identified that they were Ames and then TIGR, using their genome sequencing capabilities were able to compare those strains to the wild-type sequence just as they did the Porton strain, and determined that there were significant mutations in many of these colony variants that were from the letter powder.
The FBI then decided that we would exploit those genetic differences for forensic purposes and develop assays to detect those mutations and screen the entire repository of samples that we collected to try to identify possible sources for the production of the Anthrax letters.
QUESTION: How many of the samples in your database match three of the markers?
BACKGROUND OFFICIAL: None.
QUESTION: None of them matched three?
BACKGROUND OFFICIAL: None.
QUESTION: Okay. And what level were the mutants present at? Was it 1%?
BACKGROUND OFFICIAL: Yes, very low percentages.
QUESTION: Do you have a number?
BACKGROUND OFFICIAL: You mean percentage --
QUESTION: Yeah, I mean, I assume most of them are one type of Ames and there are some small mixture of these mutants in there. What level is --
BACKGROUND OFFICIAL: Oh, yes, in many of the samples, the mutant was well below 1%.
QUESTION: Was there one mutant or four mutants, one with each --
BACKGROUND OFFICIAL: We used four in the --
DR. MAJIDI: There are more than four mutants. Four was used specifically --
QUESTION: -- but each of those are for a separate mutant. It's not four the identify
one --
DR. MAJIDI: Exactly.
QUESTION: Okay.
QUESTION: What's the level of confidence? If something hits, matches all four mutations, what’s the level of confidence that's a real match and not a random chance?
DR. HASSELL: Well, it's very high because since there were eight of the thousand that matched, that gives us a high degree of confidence, and then also the way that the investigation side of this kicked in, we found those eight out of the thousand. We were able to trace those back. The investigation in this whole exercise shows that they were traced back to a single flask.
DR. MAJIDI: So from 1,000-plus material that we have in our repository, roughly eight of them had the four markers, and then when you add the investigative approach, you find out where those samples came from. They were all daughters, or directly driven, from the RMR-1029.
QUESTION: Where were those samples taken from? I understand that you are saying they all came from one flask.
DR. MAJIDI: Right.
QUESTION: What victims or other areas did you get them from?
DR. MAJIDI: To help me understand the question again, I'm not sure exactly what you're asking.
QUESTION: So you pooled the samples from labs and then compared them; right? And then were able to match them back to USAMRIID; correct?
DR. MAJIDI: Right.
QUESTION: Okay, so those eight that matched; where did they come from?
DR. MAJIDI: Those locations -- it is not eight laboratories. I got to be clear about that. They came from different locations. A good number of them came from USAMRIID itself. And we're not disclosing the location.
QUESTION: How many were outside of the United States, and how many were non-governmental labs?
DR. MAJIDI: None outside the United States.
QUESTION: Were they all government labs?
DR. MAJIDI: There's a fine distinction there and I don't know really what we call government and what we call quasi-governmental, so we're going just going to leave that as is.
|
The Battelle
Memorial Institute in Columbus, Ohio, would likely be
referred to as “quasi-governmental.” It’s
privately owned, but they do a lot of work for the
government. |
QUESTION: When you said that eight have them had four markers --
DR. MAJIDI: Roughly eight of them had four markers.
QUESTION: -- so what were the four markers? Were they snips? Were they tandem repeats? What are the four markers and what is the statistical confidence in the uniqueness of these four markers as some type of Ames Anthrax?
DR. HASSELL: There are actually more than four that were found, but because, for example, there were issues and less confidence in the snips being used in this, so those were not used.
QUESTION: You didn't use any snips?
DR. HASSELL: No.
DR. MAJIDI: They're all --
QUESTION: So they're insertions?
DR. HASSELL: Insertions or deletions.
QUESTION: I'm sorry, I couldn't hear you.
DR. HASSELL: They're all insertions or deletions.
QUESTION: And do you have a statistical confidence number? I mean, you said you were able to generate something for the uniqueness for these four markers.
DR. MAJIDI: Well, the question of statistics is tough because the processes I'm going with this, roughly about 1,000 sample, eight of them had the markers but all eight were driven from the same source, so if you're going to do the statistical validation, it's really skewed because the source is a similar source. Now, the fact that from the universe of the 1,000-plus samples that we analyzed, we found these four markers in eight and we were able to drive the eight samples back to a single source through validation -- investigative validations, it's really difficult to assess the statistical value, but that's what it is.
QUESTION: How did you tie it back to that source? I mean, just by --
DR. COLWELL: If I may, I think it's important to note that the samples were all blind samples. In other words, those being tested didn't know where they came from.
QUESTION: So where I see we’re getting to is a whole idea of an error rate. How many eyes were doing this and you've got people like Frances Kohn saying why don't you just make all this public so that scientists outside of your cadre can say, look --
DR. HASSELL: So let me actually pull on that string a little bit. I'm going to answer a couple of questions. Number one, we have had a large body of scientists, very well-known scientists, working with us and helping us throughout this process. You see a handful here today, but the universe of folks that were really expert in this area and helped us, it exceeds 60-plus and we'll provide those as soon as we get their okay to release their names. So the fact that we have had peer review Red Team validation, rigorous validation of our data throughout the process by external scientists, that has happened.
Number two, we've had a number of peer review publications, and that list is available and you can see it. So the fact that those have already been published in a peer review journal outside the context of Anthrax, that is once those papers were out we didn't advertise that these were directly related to our case, but there were many methodologies that was actually used to go into the analysis of our case. So we have done many of those things, and the answer is -- today is another example of we're trying to provide as much information as possible to help you identify some of these methodologies.
One other aspect of this is that we're trying to preserve the peer reviewed scientific publishing process, so we've identified a number of papers that will come out of this also, so again, these are multiple layers of validation. We talked about the various ways that -- we had the working groups that advised on the approach, how we develop the process; we had many people work on the actual samples themselves and on the repository. There were so many people involved in this that participated we want allow them another layer of validation, which is the peer review process. So this will be made public. We have more than 10 papers that we have tentatively identified to be published on this. We're just preserving the ability to do that. If we disclose everything here then we will not be able to publish those papers.
QUESTION: I guess the big question then is, once you have the one set of eyes, the one lab that said okay, it's these eight and we trace it back, how many other sets of eyes -- how many other independent science labs went and retraced everything and said you're right, and did any lab retrace those and say wait a minute, they have a problem?
DR. MAJIDI: To the best of my knowledge we've had nobody telling us that they had a problem with it. We've had many discussions that actually helped us fine-tune our approach to many of these methodologies. And, again, I've got to say, it's not a new science. It's not a new technique. It's the application of well agreed-upon set of standards that's used in human genome factored into microbial forensics.
QUESTION: So how many groups verified your technique and your results?
BACKGROUND OFFICIAL: Let me interject here a little bit. As I said, when we developed these assays, we did not do this -- you know, without the participation of several laboratories. So there were, in fact, there were four laboratories that independently developed assays and actually looked at several of the genetic mutations that we had and took a crack at developing assays. Of course at the end of all that, we chose the four best that were the most sensitive and the most specific for identifying those mutations in our evidence. Those were all blindly validated before they were applied to the evidence and over the course of the analysis we called in expert scientists from throughout the community, and we conducted reviews. We had scientific working groups. The FBI laboratory always had scientific working groups that participate in all of its forensic disciplines.
We had to create a new scientific working group in this area of forensic microbiology. In addition, when all the data came in, we invited a cadre of scientists to conduct a Red Team review of the science that we performed; and we took their suggestions, we made additional experiments and the data available to those Red Team at their suggestion. And so all of the science that went behind this was well-reviewed.
QUESTION: I'd like to ask a question about the chemical and physical analysis, elemental analysis. Can I actually ask a question unburdened by an advanced degree here? Can you explain something to me about -- as I understand it, every time there's a generation, every time you take a spore and culture it and make a new spore, you create the possibility for a mutation. So if you think of the flask, it's one generation of the powder and then I guess another generation that comes out of the victim in Florida, Dr. Stevens. That's what, two generations removed from the flask? Is that as far away as you got with the Anthrax attack material that you looked at; and was the specific four things you were looking for present in every generation?
DR. MAJIDI: Go ahead, Paul.
DR. KEIM: We've done a lot of work on mutations and how they occur in Bacillus Anthracis. In fact, there are many more generations than you've just described. Even in a very small colony on a plate, there's probably a billion -- almost a billion generations, and so because of that --
QUESTION: From the flask, you mean?
DR. KEIM: -- no, the flask --
QUESTION: Or this goes back before that?
DR. KEIM: -- the flask, itself, contains a very large number of spores, perhaps 10 to the 12th.
QUESTION: Generations?
DR. KEIM: Spores.
QUESTION: Oh, I'm sorry.
|
"10
to the 12th"
means 1,000,000,000,000 or one trillion spores. Dr. Keim has
advised me that the number he used was just a spur-of-the-moment
estimate, and he had no specific knowledge of the number of spores in
the flask. The real number is undoubtly many times larger,
since there
were almost a trillion spores in each of the two senate letters.
Several people are trying to track down the actual number of spores
produced for RMR-1029. Dr. Keim's intent when making the estimate was to show that there was a very large number of spores in RMR-1029, and, since mutants are very rare when growing Bacillus anthracis bacteria, it took a very large number of bacteria to produce the mutants that were found in the flask and in the attack anthrax. |
DR. KEIM: Okay? And if you have 10 to the 12th spores, it turns out that you have almost 10 to the 12th generations. It's actually 10 to the 12th minus one. So that's a very large number of generations, and so mutations, while they're rare, and that's an important part of biology, a part of life, is that you're progeny don't totally mutate like crazy otherwise your kids won't look like you do; so mutations tend to be rare but when you're working with very large numbers, like a trillion, something that only happens once in a billion, happens. So that means when you end up with that many spores you have the possibility of having these mutations occur and then an astute microbiologist perhaps can see the evidence of those in the way they look. So when you're describing the generation of a spore batch out of the original stock, that's not a single generation. That's a single amplification and there's many generations going on there.
QUESTION: All right, but the point is that every time that you find this Anthrax that came from the flask, whether it's from Dr. Stevens, the victim in Florida, or from the letters, the buoyant letters or the granular letters in the early mailings, you find these markers in all of these samples?
DR. MAJIDI: Well, let me correct that. The sample that's obtained from victims, when you sequence that you find the wild-type genome information because the predominant concentration of the material is the wild-type so the mutants don't have a opportunity to outgrow the wild number. It's only when we look at the letters and only when we look at the RMR-1029 is that when we see that ensemble variation of mutation within those samples.
DR. KEIM: And I think in response to David's question a moment ago, he mentioned that these variants are at a low percentage, like 1% or below. Those are very rare. And so if you look at the mass it will look like the progenitor and you won't see those variants. You have to go in and pick out the morphological variants and concentrate on those to find these mutations.
QUESTION: Okay. On the elemental and chemical analysis, what extra stuff was there in the spores? For example, we've heard about silicon. Was there material from the growth media? And also, were you able to tell something about the water that was used to develop the growth media? Did it come through into the spores?
DR. MICHAEL: First of all, we did find we did find that the spores contained silicon and oxygen. Our quick SEM analysis, that's Scanning Electron Microscopy, we detected silicon and oxygen within the spores. Later when we had thin sections for high resolution microanalysis in the scanning transmission electron microscope we then could localize that silicon and oxygen to the spore coat, which is a layer on the spore that's within the spore itself.
QUESTION: I'm having trouble hearing what you're saying.
DR. MICHAEL: The spore coat is a layer, as I understand it, that's within the spore and it's not the outermost layer of the spore. So the spore had sequestered silicon and oxygen in the same location on the spore coat. We found no additives; no exogenous material on the outside of the spores. We did have the opportunity to look at weaponized material to compare it to the letter material and they were very different. And the weaponized material the additives appear on the outside of the spore. Again, in the letter materials the silicon and oxygen were co-located on the spore coat, within the spore. In fact, we found some vegetative cells that were going through the sporulation process and the spore within the mother cell had this same signature.
|
The above statements very clearly explain the evidence that the attack anthrax was NOT weaponized with silica or silicon. There was no silica, and the silicon was under the outer coating of the spore (the exosporium) and was inside the spore coat. That means it would have NO effect on keeping spores from clumping or enabling them to aerosolize. The silicon had NO “weaponization” purpose. |
QUESTION: Did you develop a theory as to how each of those two additives came into contact --
QUESTION: I'm sorry, can I just --
QUESTION: -- sure.
QUESTION: Did you develop any theories on where the silicon and oxygen came from, and do you think it played any role in making the spores super buoyant?
DR. MAJIDI: If I can actually pass that question to Dr. Burans, because he's our expert on processing.
DR. BURANS: In essence, as Dr. Michael described, the silicon associated with oxygen that was found within the spore, not on the surface of the spore, being present within the spore coat, which is covered by something called an exosporia, the silicon would not have contributed to the fluid-like qualities of the Anthrax powders.
QUESTION: And as to where it came from?
DR. BURANS: It's known that Bacilli are capable of mineralizing different types of elements including silicon, so as early as 1982 Bacilli species Bacilli species have been shown to localize silica within their spore coat.
|
In reality,
it’s been known
since at least 1964. In that year, a
scientist named M. A. Rouf had a paper published that was titled “Spectrochemical
Analysis of Inorganic Elements in Bacteria” which described how
silicon was
often found in spores as a result of absorption from the environment. |
QUESTION: Can I ask a follow-up?
DR. MAJIDI: It could have been within the growth media. It could have been within --
DR. BURANS: It was a natural occurrence.
DR. MAJIDI: -- natural occurrence, yes.
QUESTION: Dr. Peter Jarling and Dr. Tom Geiserd of USAMRIID said that they both saw silica on the exosporium, and Dr. Frank Johnson and Dr. Florabel Mullick of the Armed Forces Institute of Pathology both said that they found silica, not -- you know, in their elemental analysis at AFIP. I went back to them several times and they both -- all these scientists insisted it was silica on the surface of these spores. So I was wondering what --
Can you please account for the discrepancy between your findings and those of two U.S. Army laboratories?
BACKGROUND OFFICIAL: I can answer that for you. They did not have the technology to make those statements. They would not have been able to give an elemental analysis using the technology --
QUESTION: You're telling me energy-dispersive x-ray fluorescence spectrometry is not capable --
BACKGROUND OFFICIAL: -- I'm not aware that they --
QUESTION: -- of doing elemental analysis?
BACKGROUND OFFICIAL: -- performed that.
DR. HASSELL: It's not capable of locating where it is. It could -- if there is bulk silica in there, but x-ray fluorescence is not capable of doing location.
BACKGROUND OFFICIAL: The Armed Forces Institute of Pathology [AFIP] used scanning electron microscopy to do a gross examination of the spore preps to see if there was exogenous material mixed with the spores. So it's Dr. Michael who did the x-ray analysis on the spores and showed that --
QUESTION: Wait, wait, wait. AFIP published a newsletter saying that they did energy-dispersive x-ray fluorescence spectrometry on the spores.
BACKGROUND OFFICIAL: Right, they did a bulk analysis of it. They could not tell where the presence of the elemental signature was coming from. They couldn't tell whether it was coming from the outside of the spores or the inside of the spores. The type of analysis they did was a bulk elemental analysis.
|
For me, this
could be the
most important information to come out of the roundtable discussion. It explains how prior information was wrong. Prior
information was often just assumptions
based upon
incomplete information. Tom Geisbert and
and Peter Jahrling at USAMRIID and the people at AFIP didn’t possess
the technological
capability to come to the conclusions they made. Instead,
they made false assumptions based
upon what they considered to be the most likely
explanations, plus they
misunderstood the data they did have. This confirms what
I wrote in Chapter 15 of my book. The
title of that chapter is “To Err Is Human.” |
QUESTION: Can you tell us what the dry weight percentage was on the silicon and the oxygen?
BACKGROUND OFFICIAL: There was no exogenous silicon in the spores.
QUESTION: I appreciate that, but can you please tell me what the dry weight percentage was of the silicon?
BACKGROUND OFFICIAL: It was high.
QUESTION: It was high?
BACKGROUND OFFICIAL: Yes.
QUESTION: Which was, according to Dr. Frank Johnson of AFIP, consistent with the silica signature, because they did a reference sample of silica before they examined the spores.
BACKGROUND OFFICIAL: Nobody is saying that there was no silicon, elemental silicon.
QUESTION: But he said they were silica.
BACKGROUND OFFICIAL: Okay, we're going to get to that. We're going to let Dr. Michael answer that. But I'm telling you what AFIP did was a bulk analysis. They would be able to tell that there was silicon and oxygen present in the prep, which would be hypothesized as silica. But their gross examinations did not show any exogenous silica --
| "Hypothesized as silica" is a euphemism for
"mistakenly assumed to be silica." "Exogenous" means
"externally originating," or in this case: "deliberately added." |
QUESTION: I appreciate that but obviously what Dr. Jahrling and Dr. Geisbert said they actually saw the silica on the surface of the spores.
DR. MICHAEL: But that's just not possible. It's not possible.
QUESTION: You're saying they're mistaken?
BACKGROUND OFFICIAL: Yes, they are mistaken.
BACKGROUND OFFICIAL: -- and there have been presentations at the American Society for Microbiology where some of those photographs were presented and --
QUESTION: Can you tell me what they saw then? How did they make such a mistake?
BACKGROUND OFFICIAL: -- I don't know that. I don't know where their statements came from.
| The statements came from Richard Preston's
book "The Demon In The Freezer." Preston interviewed
USAMRIID personnel just after they made their false assumptions, and
the false assumptions were printed. Either no one bothered to
contact Preston to tell him of the errors, or a clampdown on talking
with the media prevented further contact with Preston. |
DR. MAJIDI: What we can do is we can stay within the scope of what we know. Telling you what other people say on our samples, based on our knowledge of what they have done is -- this is the list of methodologies that we have; it's unlikely with the methods that they used they were able to actually derive that conclusion that silica was added. And again, it becomes very critical to look at those electron microscopies with energy-dispersive x-ray to be able to do the spatial location identification of the silicon signal you see.
QUESTION: May I say something? Manufacturers of the EDX machine, ThermoNORAN, disagree with you. They say that they can detect -- they can differentiate a silica signal.
BACKGROUND OFFICIAL: Let me ask Dr. Michael to address that. He did the analysis.
DR. MICHAEL: The first thing you have to understand is the scanning electron microscope is not a surface-sensitive technique. When we use an energetic electron beam on the order of 20 kv, which I believe is what they used in that study, we are sampling over a micron deep into the sample. Now, the average spore size that I have seen is on the order of a micron to a micron and a half --
DR. MICHAEL: They have no indication of exactly where that silica or that silicon oxygen signal -- I hesitate to call it silica, because we don't know how it's bound together, and EDX does not tell us how it's bound together. So when they sample they're sampling through a thickness of a micron or so. And again, the average spore size is a micron and a half or so. So they're sampling a large fraction of that spore. Now, what we did was after we looked at it in the SEM and said, "Yes. We see silicant oxygen signal here," using our ThermoNORAN system, by the way, since you brought it up, we then went and made thin sections of these materials. And then we took them to a scanning transmission electron microscope. Now, the stem -- or the TEM that you may be familiar with --
| In
other words, when AFIP examined a sample of the Daschle spores with
their EDX (Energy Dispersive X-Ray Spectrometer), they couldn't
tell if the information they got represented something on the surface
or something inside the spores. They were looking at
everything down to a depth of a micron in an object that is only about
a micron in diameter. |
QUESTION: Yes.
DR. MICHAEL: allows us to focus our probes down to the nanometer scale. And if we look at a thin section, we now are looking at spatial resolutions on the order of a few nanometers. In those samples we can localize the silicant and the oxygen signal to the spore code.
QUESTION: And can you tell us what the dry rate percentage was of your analysis?
DR. MICHAEL: My analysis does not --
QUESTION: Of the silicant -- that gentleman over there, who has not identified himself, said that it was a very significant spike, which is what Maj. Gen. John S. Parker, the commander, former commander, of USAMRMC said upon seeing the AFIP analysis.
DR. MICHAEL: It was a significant peak in the x-ray spectra. Yes.
QUEST