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Last Updated

05 Dec 2002

Source: Newsday, December 27, 2001.

Scientists at Loss in Anthrax Probe

Powder from attacks is best clue, but labs lack tools for a positive ID

By Laurie Garrett and Earl Lane, STAFF WRITERS

The White House openly discussed last week the possibility that the anthrax that has sickened 18 people and killed five was made domestically, and FBI investigators concentrated on labs involved in military biodefense research.

But scientists knowledgeable about anthrax said the tools may not yet be in hand to solve the mystery of who is responsible for this fall's attacks - at least, not based on analysis of the single best clue: the anthrax powder itself.

Anthrax spores mailed to Capitol Hill offices reportedly have the same genetic fingerprint as the virulent anthrax variety, named the Ames strain, that has been used in biodefense studies at the U.S. Army Medical Research Institute on Infectious Diseases at Fort Detrick, Md., and distributed in recent years to a handful of other labs, including the Dugway Proving Ground in Utah and Battelle Memorial Institute in Columbus, Ohio.

Homeland Security Director Tom Ridge said last week that, based on the nature of the substance found in the letter sent to Sen. Tom Daschle (D-S.D.), "There are multiple agencies within government that have for many years, for many reasons had access to this strain of anthrax. That connection [allegedly to a U.S. military laboratory] could very well exist. The fact is we have multiple leads."

But some scientists question whether it will be possible to definitively trace the mailed anthrax spores back to a specific laboratory, military or otherwise. The genetic analysis may not be sensitive enough, they said. Analysis of genetic "markers" can distinguish between strains of anthrax but may not be able to reliably identify some of the subtle mutations that occur within subcultures of the same strain, they said.

Chemical analysis might hint at the process by which the spores were filtered and whether they were treated with chemicals to remove electrostatic charges and prevent clumping, experts said. That could provide clues to whether the spores originated in a government lab or were prepared by someone using government-taught methods.

Little is known publicly about the chemical analysis so far, and some experts said if the finely powdered spores were prepared in an ad hoc way by a knowledgeable loner using off-the-shelf materials, the spores might be difficult to trace chemically.

Even as investigators search for ways to narrow the probe, science is providing information that challenges some assumptions about how lethal anthrax spores behave. Recent studies suggest that even spores prepared without anti-clumping chemicals - if small enough - may be able to spread more efficiently in a closed space than had previously been thought possible. Further, scientists said, the fine-grained, readily inhaled character of the Daschle anthrax sample need not require production in a state-sponsored lab.

For now, without more information on the analysis being done on the available spore samples, experts said it is too soon to narrow the search for suspects only to government laboratories and their contractors.

The notion "that only the U.S. Army can build biological weapons is not based on fact," Dr. Tara O'Toole of the Johns Hopkins Center for Civilian Biodefense said last week in a speech in Manhattan to the United Hospital Fund.

"They are obscuring the facts," said Dr. Cecil Fox, a microbiologist who heads Molecular Histology, a private company in Gaithersburg, Md., that conducts for the National Institutes of Health the sorts of studies the FBI, presumably, has performed on the anthrax samples. "And I don't see how anybody can shed light on this. And there may be people out there in the public who would have valuable information if they knew what they were supposed to look for. So far the FBI has painted a picture of a Unabomber-type guy in a starched white lab coat skulking around government labs, which doesn't tell us anything."

Investigators have determined that the spores in the anthrax attacks in Florida, New York and Washington, D.C., were of the Ames strain, one of the most widely studied and used form of the bacillus in the United States. It was isolated from a cow in Iowa in 1979 and eventually given its name by researchers at the medical research institute at Fort Detrick in a 1986 scientific paper.

Paul Keim, an anthrax specialist at Northern Arizona University, said via e-mail that the Ames strain "is pretty unique," with all of the closest related isolates found in North America.

Until recently, it had been hard to tell the difference between strains of anthrax. The organism changed so slowly that virtually all of the samples isolated from dead animals looked to have exactly the same DNA. Techniques developed by Keim and others have allowed identification of distinct strains based on slight changes in patterns of repeated base pairs in the organism's DNA. Using eight such "markers," Keim's group reported in May 2000 that it was able to fingerprint 426 anthrax isolates from around the world and identify 89 distinct strains.

Keim's team, now reportedly involved in analysis of anthrax samples from the mailed attacks, can use 50 or more such markers. Whether that will be sensitive enough to connect a sample to a specific lab is not yet clear, several specialists said.

Other scientists at The Institute for Genomic Research in Rockville, Md., are doing the painstaking identification of every base-pair building block in a reference sample of the Ames strain DNA and of an Ames strain isolate from the attack on America Media, a publishing company in Boca Raton, Fla. Such sequencing may show whether the bioterrorist Ames strain has subtle mutations that distinguish it from other Ames cultures.

But such analysis also has its limits, experts said. The error rate, about 1 in every 100,000 bases examined, can overwhelm the finding of rare differences in base pairs that might point toward a unique sample.

All the bioterrorism anthrax samples examined so far are said to be identical to the reference Ames strain that has been in research use and distribution by Fort Detrick (and originally obtained from a U.S. Department of Agriculture lab in Iowa). In itself, however, that finding may not be surprising, nor does it significantly narrow the field of suspects, scientists said. It is difficult to say how many labs might have obtained copies of the reference Ames strain by informal exchange among scientists before the 1996 federal Centers of Disease Control and Prevention rules on transfer of select agents such as anthrax. Some scientists said it could be a dozen or more.

There are other ways to narrow the search. The most dangerous parts of the anthrax DNA are free-floating bits of DNA called plasmids. They reproduce separately from the rest of the microbe's genes and are far more prone to mutation. The plasmids carry the instructions enabling the anthrax organism to invade the human lymphatic system and release toxins into the bloodstream. It is common practice at anthrax laboratories, except those that are specifically studying the characteristics of virulence, to pop the plasmids from the organisms or mutate them into nonvirulent forms. Thus, it is unlikely that samples of virulent anthrax can even be readily found in most U.S. or European labs that study the bacillus.

The methods of anthrax storage also can provide clues. Most scientists store anthrax bacteria the way Louisiana State University anthrax expert Martin Hugh-Jones does. They generally are kept in vials of a nutrient called agar rather than as dry spores, he said. If they are freeze dried, he said, "the quality of all these is very rough, indeed."

The Army's medical research institute only works with agar-stored anthrax, according to a spokesman, and never has dried spores in the facility. At Dugway Proving Ground, the anthrax samples used by the institute are grown and then shipped to Maryland, "in paste form in hermetically sealed containers ... and then returned," having been inactivated using radiation at the institute, a Dugway statement released last week said.

All of which further lowers the likelihood that a legitimate American research laboratory had samples of virulent, dried anthrax spores lying about, ripe for theft by a would-be assailant.

After the Oklahoma City federal office building bombing in 1995, most anthrax labs significantly increased their security, but before that time many - particularly those in academic settings - were quite open and freely shared their samples with anybody requesting them as a scientific colleague. If the strain was shared widely, experts said, there may be no way to pin down all possible sources.

Growing a quantity of stolen Ames anthrax and then cleaning and drying the sample sufficiently to produce deadly disease would, some experts said, be within the reach of a terrorist with sufficient scientific background. William Patrick III, a leader of the old U.S. biological weapons program, said it could be "done by a very decent graduate student." He also told The Associated Press it could be done in a crude home laboratory "as long as you are dealing with small quantities of material."

The only real difficulty posed by the entire process is protecting the health and environment of the maker. The FBI indicates on its Web site, probably for this reason, that the assailant is likely to have been taking antibiotics for some time.

Fox and others also question whether electrostatic treatment needs be considered a technologically advanced feat. Fox said proper use of fabric softener will do the trick.

A recent Canadian study, as well as published research from the University Of Cincinnati's Aerosol and Air Quality Research Laboratory question whether anthrax spores even need to be so treated to disperse widely and prove insidiously lethal.

In February, after an anthrax hoax aimed at a leading Canadian political figure, a team at the Defence Research Establishment in Suffield, Alberta, ran tests aimed to determine how dangerous an envelope full of spores might be. Kent Harding and his group used a well-known, harmless substitute for dried anthrax spores, Bacillus globigii, which they prepared "in a routine manner," Harding said, using classic textbook methods. They did not treat the spores with any chemicals, or exotic filters, and their preparation dried into a clumpy, undistinguished mess. The only step they took to refine the powder was to run the mess through a sifter that separated the largest clumps from those that were roughly the size of anthrax spores.

Using that crude method, Harding said, they were surprised to see "there was a high proportion of singular spores" in their final powder and, after being placed in a sealed envelope, readily dispersed when a man opened the letter in a windowless room with standard ventilation.

"We found there was a rapid and immediate release of spores just simply with the opening of the envelope," Harding said. "Spores did move about the room very quickly. We only ran the experiment for 10 minutes and they moved to fill the entire 18 feet. The distribution was purely because of air currents."

Had the spores in use been anthrax, Harding said, their studies, which were repeated several times, showed the man who opened the envelope would have inhaled a lethal dose.

The University of Cincinnati team recently published results of studies of the movement in air currents of spores similar to anthrax. They found that regardless of whether the spores were electrostatically treated, the microbes dispersed widely in an indoor airspace. They discovered that the key factor affecting the amount of electrical charge on spores was the amount of shaking and agitation to which the microbes had been subjected. Any spore sample that has been handled gently and subjected to a minimum of abrasion in its production would be likely to have sufficiently low electrical charge to allow it to drift freely.

Even if a crude sample could drift in the air, would it do so in sufficient doses to cause disease?

"Certainly, there are many infectious diseases for which some people are exposed, but uninfected and others become infected and rapidly develop disease," said Dr. Steven Wolinsky, chief of infectious diseases for Northwestern University School of Medicine in Chicago. "For those who are susceptible to a particular pathogen, the chances of infection with that microbe increase with the size of the inoculum. Underlying immunosuppression and extremes of age contribute to the likelihood of microbial disease."

"Theoretically a single spore could infect if it could get past immune system macrophages and begin germinating and reproducing," bioterrorism expert O'Toole said. An older person or someone else with a less robust immune system would be more vulnerable to a small number of spores, she said. The state of the science of anthrax is in rapid flux. Truisms spouted, based on quite old data, in October have since proved less than accurate. In many ways, the scientists who are scrambling to track down the anthrax perpetrator and make guesses about the production of his deadly samples are in the same position researchers and doctors faced in New York City in the 1980s and early 1990s when tuberculosis suddenly raged through the region's hospitals and jails. Having long forgotten TB, with few scientists or doctors on hand who had ever seen the disease or studied the microbes, the CDC and local health officials were hard-pressed to reckon how the microbe spread through the air, who was at risk for infection and how best to stop TB airflow inside hospitals and subways. It took the CDC four years to come up with recommended standards for TB-proof airspaces.

"That we don't have all the definitive answers is frustrating for all of us," said D.A. Henderson of Johns Hopkins University and the top adviser on bioterrorism to Health and Human Services Secretary Tommy Thompson.