A dangerous, often deadly, type of bacteria that lives in soil and water has been released from a high-security laboratory at the Tulane National Primate Research Center in Louisiana. Officials say there is no risk to the public. Yet despite weeks of investigation by multiple federal and state agencies, the cause of the release and the extent of the contamination remain unknown, according to interviews and records obtained by USA TODAY.
The incident has raised concerns that bacteria from the lab may have contaminated the facility's grounds and though initial, limited tests didn't detect it, some officials are pressing behind the scenes for more action, records show. The safety breach at Tulane's massive lab complex 35 miles north of New Orleans is the latest in a recent series of significant biosafety accidents at some of the most prestigious laboratories in the country where research is performed on bacteria and viruses that are classified as potential bioterror agents.
"The fact that they can't identify how this release occurred is very concerning," said Richard Ebright, a biosafety expert from Rutgers University in New Jersey, who testified before Congress last summer in the wake of lab incidents at federal agencies involving anthrax, smallpox and a deadly strain of avian influenza.
The Tulane incident involves the release, possibly in November or earlier, of a bacterium called Burkholderia pseudomallei, which is primarily found in Southeast Asia and northern Australia and is spread to humans and animals through direct contact with contaminated soil and water where it can live and grow. Tulane's research, which has been halted by federal officials, was part of an effort to develop a vaccine against the bacteria. It was conducted mostly with rodents inside a secure biosafety level 3 laboratory with multiple layers of safety equipment that were supposed to ensure the pathogen couldn't get out.
Yet at least four monkey-like rhesus macaques — that were never used in the experiments and were kept in large outdoor cages in another part of the 500-acre facility — have been exposed to the bacteria, initial tests have found. Two of the macaques became ill in November; both eventually had to be euthanized. Meanwhile, a federal investigator, who became ill 24 hours after visiting the facility in January as part of the ongoing release investigation, has also tested positive for exposure to the bacteria — though it remains unclear whether her exposure may have occurred during international travel and not at the lab.
"We're taking this extraordinarily seriously. It's very disturbing to us," said Andrew Lackner, director of the Tulane primate center. "Right from the beginning we've spent an enormous amount of time trying to figure out how this could have happened."
Lackner said the investigation so far indicates that the four macaques were exposed to the bacteria while being cared for in the complex's veterinary hospital and he emphasized that tests of 39 soil and 13 water samples from the center's grounds have not detected the presence of the bacteria.
"There has never been a public health threat," Lackner said.
Yet studies reviewed by USA TODAY indicate too few samples were taken to detect what can be an elusive bacterium. The Louisiana Governor's Office of Homeland Security and Emergency Preparedness, which is leading the joint federal-state response, expressed concerns about "whether the organism has escaped the compound and whether livestock and domestic animals are at risk," in a Feb. 20 letter from the state to the U.S. Environmental Protection Agency. The state provided a copy to USA TODAY on Friday.
The letter formally asked the EPA for help addressing potential soil contamination where sick macaques were housed, including with testing and assistance "to perform soil remediation in area(s) identified as high-risk as soon as possible to prevent further transmission/transference."
The primate center is located in Covington, La., near wetlands and a river, across the street from a school and close to a neighborhood.
Officials at state and federal agencies responded to some questions via e-mail but did not grant interviews. The Centers for Disease Control and Prevention, which is leading the investigation of the laboratory breach because the bacterium is classified as a potential bioterror agent, said its investigation is ongoing and that at this time "there is no known public health threat."
The U.S. Department of Agriculture, which is jointly investigating the lab breach with CDC, said in an e-mail: "USDA is still evaluating if a risk to Louisiana agriculture exists."
SICK MACAQUES SIGNAL A BREACH
The first indication of trouble came in early November, when Tulane staff discovered two sick macaques kept among 4,000 animals in a huge outdoor breeding colony on the compound's South Campus. The two sick animals lived separately in different chain-link enclosures that are among dozens of "field cages" on the campus. They were transported by van to the primate center's veterinary hospital in Building 21, about a five- minute drive away on the facility's North Campus where several buildings with labs and offices are located.
Initially, there was no reason to be concerned, Tulane officials said. "Animals get sick all the time, just like people do," said Lackner, the primate center director.
Yet despite extensive clinical workups, including exploratory surgeries, a diagnosis was initially elusive. One of the macaques became so ill it had to be euthanized on Nov. 26. The other animal initially appeared to recover, but relapsed and was euthanized Feb. 19.
After a series of increasingly specific tests on the two animals indicated that they may be infected with Burkholderia pseudomallei, Tulane sent specimens to CDC's labs, which confirmed the diagnosis. "Things really ratcheted up after that," Lackner said.
By mid-January, additional testing by CDC scientists determined that the strain of bacteria that sickened the two macaques was identical to the strain Tulane was using in its research in a highly secured lab elsewhere on the property. The strain — known as Strain 1026b — was originally recovered from a rice farmer sickened in Thailand in 1993, CDC told USA TODAY. Rice farming is a common way people are infected because the bacteria live in contaminated soil and water and can enter the body through cuts or sores on the skin.
With a lab release confirmed, CDC ordered Tulane's primate center to stop all research involving Burkholderia pseudomallei and all other pathogens that are classified as "select agents," the federal government's term for bacteria, viruses and toxins that pose significant threats to public health or agriculture and have the potential to be used as bioweapons.

The research will remain suspended until the lab breach investigation is completed and any problems are corrected, said the CDC, which jointly runs the Federal Select Agent Program with the USDA. About 10 research projects involving a variety of pathogens have been halted as a result, Lackner said.
Tulane's select agent labs were last inspected in December 2013, CDC said, and no significant problems were cited at that time. The primate center has never been suspended or subjected to regulatory enforcement actions before.
The diagnosis of Burkholderia pseudomallei infections outside Tulane's lab was of special concern because the two animals had been living in outdoor cages and the bacteria have the potential to establish colonies in soil and water — potentially contaminating their cages or spreading into the environment.
The state homeland security agency activated a command center to help coordinate a response. From Jan. 20-24 a team of federal investigators visited multiple locations on the primate center's campus seeking answers to what had happened.
Then, in a surprising development, a day after leaving the facility, one of the USDA select agent investigators became ill and was hospitalized. Results of a Feb. 6 blood test showed she had antibodies indicating a possible current or prior exposure to Burkholderia pseudomallei. She has since recovered from her illness.
Burkholderia pseudomallei can cause a potentially serious disease in people and animals called melioidosis, that has a wide range of non-specific symptoms, such as fever, headache, loss of appetite, muscle and joint pain, and infections are often mistaken for other diseases such as pneumonia or tuberculosis. The time between exposure to the bacteria and the development of symptoms can range from one day to many years, according to the CDC, though most human infections do not cause symptoms.
Several countries have studied using the bacteria as a bioweapon because strains can be obtained from the environment and engineered to be resistant to multiple antibiotics, according to the UPMC Center for Health Security, an independent biosecurity think tank.
In Thailand, where the bacteria is endemic, the fatality rate for patients with melioidosis is up to 50%; in Australia it can be up to 20%, according to published studies. Confirmed infections are relatively rare. There were 176 culture-confirmed cases of melioidosis in Australia's north Queensland during the 10-year period 2000-2009, according to that country's health department.
CDC officials noted that the USDA investigator had a history of international travel to an undisclosed region that could have been the source of her exposure. Blood tests so far show a stable immune response, indicating she likely was not exposed at Tulane, the CDC has said. But further testing is needed.

Richard Ebright, the Rutgers microbiologist and biosafety expert, is skeptical that the USDA investigator was exposed to the pathogen through travel rather than her job inspecting research laboratories. Select agent program inspectors, just like researchers working with these dangerous pathogens, should regularly have blood serum samples taken and stored so they can be checked if an exposure is suspected.
"They shouldn't even need to be speculating that this is probably a prior exposure," Ebright said. "If they don't have reference samples, it's a sign of gross negligence."
The USDA said it does not collect such samples from its select agent inspectors; the CDC says it stopped doing it a few years ago because it felt they weren't as useful as testing after an incident.
After the USDA investigator's potential exposure, federal officials returned to the primate center Feb. 9-12 to take an even closer look at the facility and its procedures, state records show.
By Feb. 25, ongoing tests had identified two additional animals from the outdoor breeding colony that had antibodies to Burkholderia pseudomallei, indicating they had been exposed even though they were not showing any signs of illness. Only two of the four exposed animals shared the same outdoor field cage, Lackner said.
But all four have one thing in common: All had been in the center's veterinary hospital around the same time, which has led CDC and Tulane to say the hospital is the leading suspect for where the animals were exposed to the bacteria.
Even though the two euthanized animals became ill while living outdoors, Lackner said they may have been sick with something else before going to the hospital and their weakened immune systems made them more susceptible to a secondary infection by the bacteria once there. The two other animals that weren't sickened but show signs of Burkholderia exposure were in the hospital because of injuries and not illness, he said.
While investigators suspect the hospital may be ground zero for the macaques' infections, there are no tests from surfaces or equipment in the hospital showing the bacteria was ever present there. It had been decontaminated before the location became a prime suspect.
SEARCHING FOR THE BREACH
The deadly bacteria should never have been in the hospital — or anyplace else where the outdoor macaques could have been exposed to it.
Burkholderia pseudomallei should only have been inside the specific Tulane lab that was doing vaccine development research.
That research was being conducted in a biosafety level 3 laboratory — the second highest containment level — with a wide range of high-tech safeguards, physical barriers and procedures that are supposed to ensure dangerous pathogens can't escape.
The lab, as Tulane describes it, is essentially a "box-within-a-box within a box." The research was being done in a completely contained lab under negative air pressure, inside Building 5. Air leaving the chamber passes through multiple HEPA filters before leaving the building.
Access to the BSL-3 lab is strictly controlled. To enter, staff must have an authorized access card and procedures call for employees to change into protective clothing and use personal protective equipment while in the laboratory. Contaminated gear can't leave a BSL-3 lab without being sterilized. Everything that goes in — cages, animal bedding, supplies — can't come out without being sterilized. Research animals that go into the primate center's BSL-3 labs do not come out alive and never go to the hospital, the center said.
The veterinary hospital, where the macaques may have been exposed, is in Building 21 about a five-minute walk from Building 5, according to Tulane. The breeding colony where they lived on the South Campus is about a five-minute drive, and access to the area is controlled with proximity cards and a double-fence and gate system. The individual field cages are locked.

So how did the bacteria get out of the lab?
"Human error is always the first explanation you should consider for a laboratory accident," Ebright said. "The easiest way to transfer something from one place where it's supposed to be, to another where it isn't supposed to be is by a worker moving from one place to the other."
Sloppy biosafety practices can result in pathogens being tracked out of labs on shoes, coats, gloves, hands or other materials that weren't properly sterilized before being removed, he said.
Federal officials did not provide any information on how they suspect the bacteria got out.
IS THE SOIL, WATER CONTAMINATED?
Despite the focus on the hospital, Louisiana officials are concerned about the potential for the bacteria to have contaminated soil or water on the primate center grounds, according to their Feb. 20 letter to the EPA.
Tulane officials emphasize that tests of soil, air and water have not detected the bacteria outside. And people who live nearby have been reassured by the test results to date.
However, it appears that far too few soil tests were taken to draw any conclusions. Studies have found that that Burkholderia pseudomallei is difficult to detect in contaminated soil without taking a large number of samples because the bacteria form colonies that are like invisible ant hills. They also aren't evenly distributed across an area. So unless a shovel is put in the right spot, the test will yield a false-negative result.
According to the EPA contractor's sampling plan and location report, a total of 39 soil samples were taken in a variety of locations on the primate center's grounds.
In field cage G12, described in the EPA report as the field cage of "an affected macaque," records show just four soil samples were taken. Tulane says its individual field cages are 100 feet by 200 feet — more than half the size of a football field.
Yet to detect this type of bacteria in a cage of this size, more than 100 samples would have been needed, according to a 2010 study of the bacterium's uneven distribution in soil in Thailand. The study recommends that a minimum of 100 sampling points should be taken for an area of land measuring about 98 feet by 98 feet.
EPA provided USA TODAY with a copy of the sampling plan and locations, but it gave little scientific justification for the number of samples done. In a series of e-mails last week, EPA deflected many of USA TODAY's questions and sought to distance the agency from the soil and water sampling plan — often referring the newspaper to state or CDC officials. None of the agencies would provide answers to questions about the scientific basis for the sampling that was done or whether it was adequate to detect the bacteria if it is present outdoors.
When USA TODAY on Wednesday initially asked the EPA Region 6 office in Dallas about its soil sampling at the primate center, the agency said: "Tulane did the water and soil sampling." EPA press secretary Liz Purchia in Washington, in an e-mail to USA TODAY on Friday night, again re-emphasized that Tulane collected the samples.
But Tulane primate center officials said they lack the expertise to do soil and water sampling. For reasons that are unclear, Tulane says the EPA insisted that primate center staff collect the actual samples — but that the locations and numbers of samples was overseen by EPA team members who were looking over their shoulders.
Louisiana emergency response officials also appear to be having difficulty getting help from EPA. "It [has] come to our attention, that EPA requires a letter to ask for their active participation in this investigative process," wrote Kevin Davis, director of the Governor's Office of Homeland Security and Emergency Preparedness, in his Feb. 20 letter. "To this end, Louisiana is formally requesting EPA's assistance with the following" and he goes on to ask the agency to provide strategic direction and technical guidance to help develop additional sampling and soil clean-up plans for the primate center.
The EPA has not yet replied to Davis' letter, according to an e-mail late Friday afternoon from EPA Region 6 spokesman David Gray. The e-mail did address the soil sampling generally, saying it involved sending "rapid-response environmental samples to CDC for analysis" and that the number of samples was based on CDC's lab capacity. "EPA completed that work and demobilized on February 13," Gray said.
The EPA has provided Tulane with options for decontaminating potentially contaminated soil on the property. The agency has not made any recommendations, Gray said.
In addition to the soil tests, the EPA report shows 13 water samples were taken in adjacent wetlands, ditches and in the sewage- and stormwater treatment system areas, plus EPA did air sampling, although Tulane and others note that the bacteria isn't generally transmitted through the air. Some wipe samples from vans that transported exposed macaques also were tested. No bacteria were detected in any of the samples.
For now, Tulane is not planning to remove soil or fumigate the area. With no bacteria detected outdoors, the center sees "growing evidence that makes it increasingly unlikely that Burkholderia pseudomallei has been in our outdoor breeding colony," said Tulane spokesman Michael Strecker. Testing of the animals in the outdoor breeding colony, however, will continue.

For full coverage of USA TODAY's ongoing investigation of laboratory safety and security, go to biolabs.usatoday.com
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