Cracking the anthrax case

Within two months of the 2001 anthrax attacks, researchers helping the FBI were able to exclude Iraq and Russia as likely suspects.

They also identified the one of 89 known anthrax strains that infected the first victim before he died.

Progress on anthrax came fast early on, but then hit major snags. Ten million dollars would eventually be spent on the scientific investigation, and it took a lucky insight from an investigator to narrow down suspects in this seven-year-old medical mystery.

More details of the scientific anthrax probe keep surfacing after the prime suspect, Bruce Ivins, committed suicide on July 29. The FBI held a briefing on Aug. 18. And in the few last weeks, scientists have been freer to discuss their role in aiding the FBI.

Anthrax germs of the same strain offer almost no genetic variation - which made it seem impossible at one point to use DNA to narrow the hunt for whomever mailed the deadly spores in the tense weeks following 9/11.

The spore-bearing letters killed five people and sickened 17.

Paul Keim, a leading expert on anthrax, was the first outside scientist to get involved. As soon as tabloid photo editor Bob Stevens was diagnosed in Florida with inhalation anthrax in early October 2001, health officials sampled his spinal fluid and grew a culture of anthrax bacteria in a test tube.

"They shipped that to us in a CDC corporate jet," said Keim, a geneticist at Northern Arizona University, near the Grand Canyon. It might seem an unlikely place to start investigating, but in the 1990s Keim had invented the first tests capable of DNA-fingerprinting anthrax spores to distinguish among strains.

"The sample arrived here on a Thursday night," he said. "We worked all night long and by the start of business Friday we told them it was Ames," a deadly strain isolated in 1981 from a Texas outbreak and erroneously named for the Iowa city. It's now commonly used by many U.S. researchers.

In the months after Stevens' death, Keim and his colleagues pulled more all-nighters as dozens of samples of spores were flown in. "Every time there was an outbreak, a jet would take off and bring samples," he said.

Federal authorities also asked him to analyze spores obtained from the Russian and Iraqi stockpiles.

The Iraqi spores came from shells that had been weaponized, said Keim. They were picked up in the 1990s by United Nations weapons inspectors in the midst of dismantling Saddam Hussein's bioweapons program. Keim found none that came from the Ames strain.

"It's quite possible that would have been used as justification for the Iraqi war," he said.

But virtually ruling out Saddam Hussein took scientists only so far. Hundreds of U.S. researchers had access to Ames anthrax.

So investigators turned to The Institute for Genomics Research (TIGR) in Maryland, where scientists were trying to read the entire genetic codes of human pathogens.

Claire Fraser-Liggett founded TIGR along with her then-husband Craig Venter, and is its director. Venter had become famous for spearheading the privately run competitor to the federal $3 billion Human Genome Project - an effort to read every code character in human DNA.

In 2002, TIGR scientists began reading the genetic code characters of anthrax spores. The FBI also began collecting a sample of every known flask of Ames anthrax in the world. That came to 1,070 samples.

Someone just needed to find genetic mutations - distinguishing marks in the DNA - that would rule out some and connect others to the attack.

Since the anthrax from Stevens' spinal fluid may have developed mutations during the infection, Keim isolated DNA from spores recovered from envelopes used in the subsequent attacks. In spore form, the germs don't divide and so could not collect any new mutations.

Once isolated, the DNA was flown to Fraser-Liggett's lab in Maryland. There she, Jacques Ravel and David Rasko read out the DNA sequence and compared it to a sequence made from what they called original Ames spores.

By early 2003, Fraser-Liggett and her colleagues were stuck: Out of more than five million code characters in the DNA, the envelope anthrax looked identical to the original Ames strain. It had no distinguishing mutations.

But then a researcher at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) at Fort Detrick in Frederick, Md., made a key discovery that would turn suspicion on his own colleagues.

The insight, Fraser-Liggett said, came from observation. Spores from the attack envelopes had been cultured on special plates, revealing several sub-populations of bacteria that looked different to the naked eye - some more yellowish, others growing in rougher or smoother blobs.

Fraser-Liggett said she and other geneticists knew instantly that the different-looking colonies probably carried mutations they could use.

By early 2004, she and her colleagues turned up a handful of key mutations - four of which were turned into tests to screen the 1,070 samples.

Seven samples tested positive for all four mutations. Since the samples' history was recorded in lab notebooks that the FBI had collected, investigators could see that all seven had been derived from the same source - a set of two flasks labeled RMR-1029 in Ivins' lab at USAMRIID.

But the investigators got a negative result from the sample Ivins sent in, according to affidavits released Aug. 6. Since all other samples derived from RMR-1029 tested positive, Ivins' sample should have as well.

The affidavits say FBI agents raided Ivins' lab in 2004 and seized his flasks of RMR-1029. Those tested positive for all four mutations, as did an earlier sample Ivins had made from RMR-1029 that was stored in Keim's lab.

Did Ivins alter the official sample he submitted? Keim said the test was sensitive enough that it should have picked up the signature of the mutant spores had Ivins followed the FBI's directions for making the samples.

"Ivins may simply have failed to collect a representative sample," he said, adding that "the FBI is implying he did it on purpose."

Keim notes that a conviction of Ivins would have required more than just this genetic connection and would be dependent upon other investigative evidence.

Still, he said, much good will come from the money that poured into the investigation.

"Now we can reconstruct how outbreaks occur with a level of precision far beyond what we've had," he said.

"If you used this in the recent e-coli outbreak," he added, "you'd be able to track it back to the exact pig that pooped on the spinach."