March 1, 2001
LTG James B. Peake
The Surgeon General of the Army
5109 Leeseburg Pike
Falls Church, VA 22041-3258
Dear General Peake;
Although I risk being presumptuous, I felt compelled to report to you my impressions of the AIBS 26 February meeting and some confidential ideas relating to my own perspective. Since this was the first time I have attended in its entirety a formal evaluation involving the ‘players’ (both civilian and military principals) involved with the mycoplasma validation study, I believe my perspective may contribute and my recommendations may facilitate an advantageous outcome.
I. Friction amongst the participants was based upon what I perceive to be philosophical differences in the approach to the design, methodologies, workscope, and reporting of results. The lack of sufficient interdisciplinary training amongst certain participants was necessary for the accurate assessment of experimental design and may have presented handicaps that were potentially obstructive to initial success. This problem is not uncommon when we consider that most of the investigators were trained during an era of intense specialization in science and medicine. These factors are presented against a background of historical acrimony amongst the investigators, which is not uncommon in a highly competitive, and often special interest motivated arena.
II. Basic flaws in the experimental design and execution of the study, which involves a continuing evolution and perfection of the methods employed in the study compounded by a lack of communication appropriate for professional colleagues may have contributed to observed discrepancies.
III. Complexities associated with a historical political pattern compounded by the necessity to eventually present our military establishment in a positive light to the public while considering historical security related issues and judgments implemented during the Cold War may have effected the study. It is imperative that all participants comport themselves in a manner that will not further dampen public confidence in our military and undermine the very essence and existence of a healthy and powerful Armed Forces. We all face pioneering obstacles and challenges on all fronts (defense, scientific, political, and global relations relating to the task at hand).
Solutions to (I-III)
In conversations with LTC Friedl and Engel, I am absolutely confident that the clashing personalities of some of the players can be overcome. I have spoken to Drs. Garth Nicolson and Joel Baseman to advise them that academic approaches to the scientific dilemma we are experiencing need to be placed in a defense related perspective. It is difficult for people who read about a war to truly comprehend being in combat and actively fighting a war. I am aware that a matrix organization such as the Pentagon has numerous advisors from the academic and corporate sectors, and historically while this kind of marriage worked well with the Manhattan Project during WWII, perhaps it would be timely and prudent to re-evaluate the communication channels and potential influences of special interests amongst these sectors.
In terms of philosophical problems relating to the study discussed, reviewed, and analyzed at the AIBS review, my impression is that LTC Engel has reacted strictly in terms of a military perspective possibly shadowed by a sense of urgency to report the initial results and discrepancies in the study. I hypothesize that internal and external pressures that are tantamount to a military setting could theoretically result in a proclivity to report observations in a manner that would not pass muster in an academic setting but would be more conducive to the quick reaction of the former. On the other hand, the academician accustomed to the University setting or private sector approach of Drs. Garth Nicolson and Joel Baseman, and to a lesser degree myself necessitates that the publication of data is precluded if there are serious methodological problems and discrepancies. Of course academicians are subject to time constraints by granting agencies and corporate entities are subject to completing milestones in a timely manner, but the normal standard operating procedure that characterizes the academic setting and corporate setting usually does not have to consider directly the defense of our nation on a daily basis.
The major discrepancies and cause for contentious interactions evident on the 26th were focused on the fact that other highly qualified laboratories that were not participants in the study, have reproduced and published observations corroborating the observations of the Nicolsons. The observations noted by the Nicolsons and 4 civilian laboratories outside the scope of the study indicated that a subset of Gulf War Veterans (~40%) was infected with mycoplasmas utilizing polymerase chain reaction (PCR) molecular biological and other methods. The results reported from these laboratories were within the appropriate degrees of freedom considered necessary for statistical agreement. In contrast, the laboratory of Dr. Shyh Ching Lo at the Armed Forces Institute of Pathology, a pioneer in the field of mycoplasma and anthrax research, has reported consistently negative (0%) results. These observations by Dr. Lo are somewhat puzzling as previously he had reported informally that he had noted ~14-21% positive tests for Gulf War veterans several years ago using an antibody-based methodology. In fact, an initial cause for contention at the training workshop in January 1997 was that antibody based testing was more sensitive than PCR as suggested by Dr. Lo. This statement was strongly objected to by Drs. Garth Nicolson and Baseman on the grounds that antibody based tests are not reliable for the conclusion that an active infection is present. This same sentiment was corroborated by COL Friedlander at Fort Detrick when the Nicolsons visited with Major General Parker and his research team.
Faced with discrepancies of the positive observations noted by Dr. Garth Nicolson and Dr. Baseman compounded by the fact that Dr. Lo consistently has reported no positives contributed to a highly charged atmosphere of disagreement and an adversarial tone of interaction amongst the participants at the 26th review. The following observations describe the polarization of disagreement that I observed at the 26th February meeting.
A. Dr. Garth Nicolson argued that other labs outside the scope of the study were able to reproduce and publish results in peer-reviewed journals that corroborated that a subset of Gulf War Illness patients had mycoplasmal infections. He then went on the show that there are a subset of civilian Chronic Fatigue Illness patients as well as patients suffering from a variety of autoimmune disorders who also appear to have mycoplasmal as well as other bacterial and viral infections, and that as we move further away from the Gulf War Era, the civilian and military patients begin to exhibit a similar pattern of multiple infections. On the other hand, the closer we were to the Gulf War era at the time of the testing for infections, the military patients appeared to be infected with primarily one type of mycoplasmal infection, Mycoplasma fermentans, which was not found in civilian chronic illnesses.
B. Drs. Garth Nicolson and Baseman then argued that probable reasons for discrepancies involving a lack of reproducibility of positive observations in split DoD samples in the Nicolson and Basemen laboratories may have resulted from poor sample quality due to handling and shipping. In fact, the Cowans described these kinds of difficulties involved in the sample handling, and all parties were particularly perturbed that the participating laboratories were not introduced to SRA prior to the execution of the study. The Cowans replaced Dr. James Lane in the management capacity of the samples.
C. LTC Engel then opined that it was unconscionable that none of the participants voiced their objections to the quality control of the samples until after the initial study was completed. Dr. Garth Nicolson and one of the review boards, Dr. Clarke asserted that the wording of the contracts awarded to the Nicolson and Baseman lab legally precluded them from rejecting the samples. Dr. Garth Nicolson stated that in spite of this he and Dr. Baseman as well as Dr. Lo had noted hemolysis in the samples and very low amounts of DNA and reported this during the course of the study. Dr. Garth Nicolson further stated that the samples in the study would never have been approved for assay in the Nicolson’s certified reference laboratory and that in instances when there was a question of quality or methodological problems we automatically re-run the assays at no charge to the customer.
D. Dr. Lo then interjected that Drs. Nicolson and Baseman were observing false positives, a statement that was hotly contested. Dr. Baseman presented a series of elegant immunno-confocal microscopy studies elegantly showing the presence of mycoplasma in Gulf War Veterans white blood cells. He further showed a series of analyses comparing chelex and Quiagen-extracted DNA that are used in studies involving PCR technology and showed the reduction of recovery of extracted materials at various phases of purification. He then concluded that the samples used in the study were at the threshold of sensitivity detection and were therefore unreliable for a comprehensive analysis. Dr. Garth Nicolson stated that you cannot analyze “garbage.” My own opinion is a technique is as only as good as the sensitivity of the measurement. In PCR one has to optimize the conditions of the reaction for uniformity and sensitivity considerations as well as provide a continual scrutiny for contamination in all reagents. In fact we routinely test both solid and liquid reagents that we purchase for we have noted a recurrence of contamination in commercial products when we first opened the reagent bottles.
E. Dr. Lo went on to state he could not find Mycoplasma fermantans in any of the samples and did allow that perhaps there were shipping problems. He presented two papers that he said showed that a study involving 30 laboratories using PCR indicated that there was disagreement in observations amongst some of the laboratories with respect to the positive test for Chlamydia. Dr. Baseman asked “are you sure that all the laboratories used the same optimization conditions?” Dr. Lo answered “Yes!” In fact, the laboratories involved in the study in the manuscript cited by Dr. Lo were not uniformly consistent in aspects of the techniques.
My impression of the assertions of all the participants of the study is that they all have valid points and are all partially right as well as wrong (including myself). My opinion based upon experience since I was in graduate school (1979-1982) is that there are problems in any field, including molecular biology. At the time I was a graduate student those students who could not ‘pass muster’ went on to be technicians at companies that manufactured molecular biological reagents. At the time I told my professors that one day this might come back to haunt us as we may eventually use substandard reagents. Since my graduate days I have always been leery of commercial reagents, even when they are presented as being of the highest level of purity. For this reason I demand constant testing for contaminants in our laboratory. We also spend thousands of dollars on laboratory duct cleaning, and enforce absolute stringency in sterile techniques. We also do different steps of the purification, mixing, amplification, separation, etc. in different isolation rooms. It is my opinion that there is a lack of standardization in the molecular biological community regarding particular assays such as PCR. All PCR assays must be optimized according to the primers and materials as well as source of materials to be assayed. Our lab uses many built in internal standards as we feel that there is no such thing as too many controls. We should have communicated this better to the participants. In our defense we are constantly refining our techniques. Since the onset of the study, our techniques have been continually updated and are part of our proprietary information that is the basis for our company being evaluated by a conservative Wall Street Firm at $40 million after nine months in operation with the projection of $160 million by summer. I must reiterate that there are no sufficient guidelines to enforce proper standards and controls, and companies that manufacture molecular biological reagents may have flaws in the execution of their product development which can impact upon the quality of the reagents with the possibility of introducing lot variations. This possibility would definitely make it a requirement that all laboratories participating in a blinded study should use the same “batch” solutions for uniformity as well as making certain all solutions are absolutely fresh. This could reduce at least some variables that may be introduced into the assays. In defense of those manufactures of molecular biological reagents, the field is still in a pioneering phase in spite of the advances of the last 20 years. Unfortunately, the participants in the study did not communicate these concerns to one another in an effective manner appropriate to a colleague interaction.
The analysis of blood is complicated because it is composed of complex macromolecules that fall into a statistical mechanical ensemble system (a multi-bodied system that is comprised of complex macromolecules and often polymers that adapt a distribution of conformations but tend to favor a distribution equivalent to an intermediate energy state). Stringent guidelines need to be enforced for the investigator to successfully replicate the appropriate statistical mechanical average. Since the conformation of macromolecules such as DNA, RNA, deoxyribonucleoproteins, etc. can be influenced by the purity of the solution, the type of test tube used, the ionic concentration, the volume as compared to solute/solvent concentrations, etc. needs to be considered. Thus it is mandatory that a PCR assay be designed to maximize successful reproducibility. If strict sterile procedural requirements are not followed along with scrutinizing the kinetics of the reactions (stopwatch logistics), the likelihood of a successful outcome will be compromised. This means that if the Armed Forces Institute of Pathology is performing the assay at 9 AM EST, the Nicolson laboratory should be performing the assay at 6 AM PST. Communication needs to be open at all times amongst the labs. This did not occur in the study.
The “shoulda-coulda-woulda” undertones and overtones pervasive throughout the February 26th proceedings, in my opinion, painted all the participants in a somewhat ridiculous vein. However, pioneers are always subject to hindsight revelations. I cannot emphasize how important sample integrity is to the successful outcome of the study. Quality assurance, open communication, lack of consistency in shipping, handling, and perhaps a uniform stringent adherence to sterile procedures inhibited the logistics of this study. There was also a lack of continuity with the change in personnel at SRA (Dr. Jim Lane departed, the Cowans replaced him, and the fact that no one interacted in a consistent manner with SRA) presented a huge obstacle. Another onerous consideration, which is difficult to voice, is that a person/s unknown to all participants may have interfered with the sample coding. I do not want to create or project unnecessary paranoia, but many of us have had experiences relating to this type of work that do not fall into the category of normalcy. Based upon advice from professional intelligence operatives, there is a strong suspicion and allegation that subterfuge may have been enacted by an unknown element. It has been suggested that there are individuals unknown to Dr. Lo, Dr. Baseman, Drs. Nicolson, LTC Engel, Dr. Cowan and Ms. Cowan as well as trusted technical staff who may be interfering. The only information we have been given is that the potential suspect’s use a moniker and various alias names. We do not know who they are at this time nor do we believe that these are ‘real’ names.
We recommend that security measures need to be implemented. We also recommend that past attitudes be completely put to rest. Everyone needs to ‘bury the hatchet.’ We are in a new millennium and the Cold War must finally end. Any remnant of Cold War agendas needs to be put to rest if we are to implement directives that may have resulted from the psychological climate of a very polarized era. In our laboratory we have scientists who grew up and were trained in the former Soviet Union and Red China as well as scientists from every ethnic persuasion and religious background including Palestinians and Chasidic Jews. We even have the exclusive world-wide rights to distribute and manufacture an herbal formulation that the Red Chinese Army utilized against agent orange through our partnership with the Chaka Zulu whose former health minister grew up within a “communist” ideology. We all should be able to work together and realize we are facing bioterrorist mentalities that do not grasp that if your enemy is sick, eventually your friend will be also.
We recommend that there be a clarification of the need for cross-disciplinary trained professionals to be assessed in terms of an oversight role along side those professionals who are more specialized. Two of the participants (LTC Engel and Dr. Cowan) verbally reported that their training in their respective disciplines made them feel somewhat ill equipped to deal with complex molecular biological systems. Since it is likely that Gulf War illnesses are part of a category of chronic disease based upon multfactorial considerations we need to have a better understanding of how to put the psychiatric ramifications in terms of the epidemiology within the context of complex molecular biology and biochemistry. This is quite a challenge, but I believe it is achievable. The other professionals evidenced a frustration in communicating the details of the molecular biological systems to professionals of the aforementioned background. It is my feeling that Dr. Shyh Ching Lo has the training to move with ease between the disciplines. Dr. Garth L. Nicolson is also trained in multiple disciplines and excels at an engineering perspective as applied to experimental systems involving complex biochemical, molecular biological, and cell biological processes. Dr Baseman is a superb microbiologist and cell biologist who has a very open-minded perspective and is very conservative in his experimental implementation of the basic dictate of Roger Bacon’s carefully detailed treatise of experimentation and detailed observation. Therefore, both Drs. Garth Nicolson and Joel Baseman are extremely prudent in their conclusions as evidenced by the fact that their published peer-reviewed manuscripts always include multiple controls. Dr. Lloyd Old once stated “the art of science is the art of the proper control.” It is hard to be objective about oneself but in terms of cross-disciplinary training, I excel at discerning the fine details related to the chemical physics of molecular biological systems such as complex chromatin chemistry.
I know we can succeed to reconcile all these problems related to the study if we could develop a higher level of trust. I feel that problems in trust have arisen amongst the players because of negative experiences that are attributable to “the fog of war” as communicated to me in a letter from General Schwartzkoph. I know we can get together. LTC Engel and I emphasized this at the February 26th meeting.
Although I swore that I would never go into a lab again (I enjoy interfacing with the financial community), I would be willing to go into the lab side by side with all concerned especially Dr. Lo. I feel that together we can troubleshoot discrepancies in the technical execution of the assays by directing our technicians and possible ‘rooting-out’ of unconscious mistakes at the technical level. Many times, it has been my experience and observation that experimental discrepancies often boil down to what may be viewed as a trivial manipulation of the samples that can dramatically impact the results.
I have enclosed a sample of an e-mail we got from a truly bitter veteran, a former MAJ in the 3rd AD as an example of the negative image our military faces and needs to correct. We receive this type of communication too often. I often hear people at random in stores, on planes, at social gatherings, etc. who have completely lost faith, confidence, and trust in our government with the Pentagon often being the most singled out for derision. The reason I take this so personally is that my biological family who “placed me” to be raised away from them, are the most powerful secret and silent financiers and armaments dealers in the world and have been for a very long time. They did not want me to be stigmatized, and they did not want me to be accused of using their money for my education, business, etc. or being part of their milieu. I have never benefited from their enormous wealth. I love them, but I hate that they undoubtedly financed the lion share of unconventional weapons of mass destruction development through a complex financial web of banking and governmental associations that I suspect few comprehend or have knowledge. Although, they have voiced their regrets to me, one has to understand that in their type of world there are no loyalties to countries, ideologies, governments, or noble paradigms. Only profit rules, and since they always back adversaries at every level, they never lose financial power. Therefore, I feel personally responsible for every soldier that has suffered or lost his/her life in war. Because I am an American, my loyalty is to the United States. I believe that I can act as a creative bridge in this most serious arena to aid the Department of Defense concerning the issues and ramifications discussed in this letter.
Respectfully and with regards,
Nancy L. Nicolson, Ph.D.
Chief Executive Officer
Chairman of the Board
International Molecular Diagnostics, Inc.
Molecular Hyperbaric Medicine, Inc.