Why There Hasn’t Been An Anthrax Outbreak

SINCE SEPTEMBER 11, 2001, LABORATORIES ALL OVER THE United States have been growing, feeding, and studying extremely dangerous anthrax germs. Scientists at the Institute for Genomic Research in Rockville, Maryland, have grown batches of anthrax to sequence the entire genome of the germ that killed the photo editor Robert Stevens, the first person to die in the bioterrorism attacks. The Justice Department has subpoenaed a dozen laboratories to submit their Ames strains of anthrax to the U.S. Army Medical Research Institute of Infectious Diseases at Fort Detrick, Maryland, for genetic fingerprinting studies that might yield clues to the identity of the anthrax mailer. On a different tack, the Sandia National Laboratories in New Mexico are developing disinfecting foams to evict unwelcome anthrax spores from buildings.

With billions of anthrax germs growing in laboratory incubators or stoppered away in vials, how can the scientists and technicians who handle them daily be confident they will not accidentally inhale the potentially fatal microbe? For an explanation, one must first go back a century to Dr. Robert Koch, a founder of the science of bacteriology and a 1905 Nobelist. The German scientist discovered the germs that cause tuberculosis and cholera and was the first to observe the life cycle of the anthrax bacillus, showing how it could transform into a dormant spore capable of rejuvenating years later as an infectious bomb. Despite his hazardous researches, Koch never caught any of the diseases he investigated.

At some point during his experiments, Koch realized that germs could float on air, and he likely was the first scientist to put forth the notion of “biocontainment.” With a penchant for homemade laboratory equipment, he constructed a prototype cabinet in which to conduct experiments involving highly pathogenic bacteria. His nineteenth-century invention was nothing more than a glazed tabletop box with two openings fitted with oilcloth sleeves for the user’s arms.

As it turned out, Koch escaped infection more because of luck than on the merits of his cabinet. Both the sleeves and the seams leaked. Even worse, a hinged lift-door at the top acted like a bellows, forcing sometimes contaminated air out of the box each time it was opened or closed. Koch’s idea of biocontainment was seminal, but his execution was poor. Still, versions of his safety cabinet were installed in many bacteriological laboratories in the mistaken belief that they protected the worker.

Dr. Howard Taylor Ricketts wasn’t as lucky as Koch. Ricketts was a modest and unassuming pathologist from the University of Chicago who built a laboratory in Mexico City to study a kind of typhus called tabardillo. Typhus was a worldwide scourge. In armies it caused epidemics that were usually far more devastating than enemy ordnance. Napoleon Bonaparte lost 30,000 French soldiers to typhus as his army retreated from Moscow in the winter of 1812, and 62,000 of the pursuing Russian soldiers also fell to the disease. Typhus germs caused massive deadly epidemics among civilians as well, usually infecting the poorest of the population. In fact, a century later, Austria postponed its invasion of Serbia following Arch- duke Franz Ferdinand’s assassination because of a raging typhus epidemic there that eventually claimed 150,000 lives. During the four years of World War I, Russia counted about 30 million cases of typhus, with nearly three million deaths.

Ricketts’s monumental discovery was that body lice transmitted typhus, but the elusive germ escaped his cultivation methods. It also escaped his primitive safety cabinet, and Ricketts died of typhus on May 3,1910. Stanislaus von Prowazek, a Polish bacteriologist, suffered the same fate in 1915 while investigating typhus in Turkey. Obituaries rightly called them medical martyrs, and the typhus-causing germ, finally isolated and identified in 1916, was named Rickettsia prowazekii in tribute.

Around the time of Ricketts’s death, the quality of biological containment took a small step forward. In 1909 the W. K. Mulford Pharmaceutical Company, of Glenolden, Pennsylvania, hit upon the idea of maintaining the interior of the cabinet under constant negative pressure and designed the first commercial biological safety cabinet, which had a ventilated hood and a pump exhausting the air through a bottle of disinfectant. But this provided only an illusion of safety. Like Koch’s model, it had leaky seams, and the bubbling disinfectant puffed out aerosols of both “disinfected” and viable germs each time a bubble broke the surface in the fizzing bottle. To make matters worse, without professional or governmental guidelines defining laboratory safety, scientists continued to install their own homemade cabinets in their laboratories.

That’s probably why laboratory-acquired infections in the United States climbed rapidly in the decades after Ricketts’s death. By 1940, as many as 2,456 laboratory workers had become infected with the germs they were handling, and 164 of them had died. Medical historians are convinced that many more infections went unreported. It was becoming a worrisome general rule that the isolation and identification of a microorganism infectious to humans would be followed in less than 15 years by laboratory-acquired infections with that organism. Laboratory workers were coming down with the bloody cough of tuberculosis, the black skin ulcers of tularemia, the hot, pounding headache of Q fever, and even bubonic plague.

The situation became so grim that in November 1953 it prompted Dr. Arnold Wedum, the chief scientist at the U.S. Army’s Camp Detrick Biological Laboratories, to say, “We take a well justified pride in our martyrs to public health research- those who have sacrificed their lives to save others from disease. But the time has come for us to take an equal pride in our efforts to prevent such martyrdom. … It is out of date to expect or permit the principal investigator and his technical or non-technical assistants to contract the disease under investigation.” More than 40 years had had to pass from the time of Ricketts’s death for the notion of medical martyrdom to be considered passé.

But before cases like Ricketts’s could become a thing of the past, the industry would have to fix two deadly design flaws in the safety cabinets: the inability to contain aerosols and the inefficient decontamination of exhausted air. In the 1940s British and American inventors tried to solve the latter problem by incinerating the exiting gases. They fitted a metal duct containing a gas-fired furnace or an electric heater to the top of each cabinet. Updrafts of contaminated air passing through the duct on their way out of the cabinet were forced into a hot zone where temperatures reached a withering 300 to 600 degrees centigrade—hot enough to incapacitate any pathogen, given a long enough exposure time. Although the idea sounded good on paper, the heaters proved unreliable even without the problem of power failures. Germs could escape in cool eddies or survive the few short seconds of intense heat unscathed. That’s why operating-room autoclaves for sterilizing surgical instruments today subject contaminants to intense heat for minutes at a time.

Designers next tried to filter the air exhausted from a cabinet. They turned first to spun glass and calculated its efficiency for removing particles suspended in an aerosol to be about 95 percent. But the other 5 percent was a huge worry to laboratory scientists who worked with deadly bacteria and viruses.

Then World War II came to the rescue. As with countless other nonmilitary technologies, the U.S. wartime research effort found a solution to the decontamination problem—the high-efficiency particulate air, or HEPA, filter.

SCIENTISTS IN THE MANHATTAN PROJECT’S ATOMIC-BOMB laboratories were detecting aerosolized microscopic particles that had become contaminated with radioactive material. Particles less than a micron in diameter were especially dangerous because they could easily travel deep into bronchial air passages and lodge there. (A micron is a millionth of a meter, or about a hundredth of the width of a human hair.) With scientists desperate for a decontamination system, the U.S. Army Chemical Corps made it one of its highestpriority tasks to analyze captured German gas-mask canisters and learn how their glass fibers provided such excellent filtration. Their investigation resulted in a classified government research contract awarded to the Arthur D. Little Company, as part of the Manhattan Project, to develop a high-efficiency glass-fiber filter.

This research gave birth to the remarkably efficient HEPA filter, which could remove 99.97 percent of particles as small as 0.3 microns. Elegant in design, the filter is composed of randomly oriented glass microfibers woven into a paperlike sheet. Contaminated air must travel a circuitous path through the dense glass jungle, and during its passage large particles are sieved by smaller openings between the fibers. The erratic path through the jungle causes the tiniest particles to collide with and adhere to the glass fibers, drawn by inertia or electrical attraction between the molecules, so that air exiting the HEPA filter is virtually sterile. In fact, air from biological safety cabinets handling all but the most dangerous germs can be safely vented back into the room.

The second major hurdle on the path to the safe bio lab was the reliable containment of aerosols. An aerosol is a suspension of droplets or powder in a gas. The most famous example is the giant plume of microscopic dust particles that spewed out of the crater of Mount St. Helens. The most deadly biological aerosol to date was the cloud of anthrax accidentally liberated from a Soviet military installation in the Urals in April 1979. After a worker failed to turn on the safety filters in a laboratory known as Compound 19, reserve officers downwind at Compound 32 began dying. In the end, at least 68 people died. (A KGB disinformation operation attributed the outbreak of anthrax to “tainted meat,” but Izvestiya published a truthful account 13 years later.) Another aerosol —a spray of droplets of deadly germs out of a Philadelphia hotel’s air-conditioning vents—caused the first outbreak of Legionnaires’ disease, in 1976.

In the nineteenth century, aerosols intrigued such great theoretical scientists as Faraday, Lister, Kelvin, and Maxwell because aerosol particles represented the smallest bits of matter then known. There was so little practical interest in them, however, that the first international conference on the topic wasn’t held until 1936. So it’s understandable that not fully comprehending the aerosol problem, biocontainment scientists in the 1950s designed cabinets with open fronts. After all, the cabinet users frequently needed to insert or retrieve glassware, chemicals, or experimental animals, and cabinet doors were cumbersome and arm ports inconvenient. The open front seemed justifiable because cabinet designers relied on an exhaust fan to create a constant negative pressure inside the cabinet. They didn’t realize that aerosols could sneak out the open front. “These cabinets were nothing but glorified fume hoods,” says Scott Christensen, national sales manager of NuAire, Inc., a leading manufacturer of safety cabinets in suburban Minneapolis. (Anyone who has ever worked in a college chemistry laboratory remembers the pungent odor of ether or the acrid, rotting-cabbage smell of butyric acid that could leak out of the fume hood even with the exhaust fan running.) Laboratory-acquired infections continued to plague not only the scientific personnel in front of the cabinets but other downwind workers such as janitors and clerical staff.

To make matters worse, scientists didn’t understand that seemingly innocuous laboratory procedures could stir up deadly aerosols. In 1957 the British researcher Andrew Tomlinson discovered that merely opening a screw-capped bottle of tubercle bacilli could liberate an infectious tuberculous cloud. Similarly, handling live experimental animals could be treacherous even if they didn’t bite or scratch. For example, inoculating the nose of a guinea pig with bacteria could cause the animal to breathe puffs of infected droplets into the technician’s face. In fact, a South African laboratory outbreak of tsutsugamushi disease, a relative of typhus that can cause delirium and convulsions, started that way in 1945. Something as innocent as a gentle draft can lift rickettsial organisms from lice feces; some historians speculate this is how Ricketts himself became infected.

The solution came from the budding space program. By the late 1950s NASA’s contractors were having problems manufacturing key miniature components because even when they used HEPA filters, tiny particles buffeted by air turbulence contaminated the works. NASA asked Willis J. Whitfield, of the Sandia Corporation, to find a better airflow system. He discovered that when he generated a current of HEPA-filtered air that moved in a single direction and at a uniform speed from a wall or ceiling, airborne contamination caused by people or machinery would be blown away from the worktable. Since the air was moving in only one direction, the contaminants could not be swept back, and cross-contamination was all but eliminated because the particles had a tough time getting past the unidirectional airstream.

NASA put the new concept, called laminar airflow, into practice in 1961, and it quickly found a home in biological safety cabinets. Fans on either one side or the top of the cabinet blow parallel currents of filtered air to the opposite side or the bottom, where the air is filtered again and recirculated. Flow velocity is typically around 90 feet per minute. “When you make air flow in laminar fashion from top to bottom inside the cabinet, that body of air acts just like a piston,” Christensen says. “All the aerosols liberated inside the cabinet are plunged down into the grilles along the workspace and then get sucked through a HEPA filter for sterilization.” Scientists are even able to keep the fronts open, as air drawn through the opening serves as a protective curtain to prevent outflow. This kind of open-fronted workspace is called a Class II cabinet.

With the two major cabinetdesign problems on the way to solution, the incidence of laboratory infections declined between 1955 and 1964, and there were fewer still in the next ten years. The decrease came despite the discovery of new infectious organisms and increasing numbers of people employed in laboratory work.

But inhalation infections didn’t disappear completely. Why were some aerosols still leaking out of the open-fronted cabinet? Twentyfive years ago Dr. Barbara Rake, of the Baker Company, in Sanford, Maine, a leading producer of cabinets, investigated the problem and found the culprit to be the velocity of air currents outside the cabinet in the laboratory.

In an elegant set of experiments, Rake generated horizontal crossdrafts of varying velocities across the face of an open-fronted cabinet in which nonpathogenic bacteria were being aerosolized. She discovered that crossdraft velocities greater than 100 feet per minute sucked clouds of bacteria out of the cabinet’s open front and into the user’s face.

She also identified the common laboratory events that could produce dangerous crossdrafts. Opening or closing laboratory windows and doors, for example, can send air currents traveling in excess of 200 fpm. Heating and air-conditioning vents are worse offenders and can create exit velocities of 250 to 500 fpm. Even a laboratory worker walking past a cabinet at 264 fpm—3 miles per hour, a moderate pace—can push forward a column of air at almost that speed. “The solution is to install the cabinet in a remote area of the laboratory away from personnel traffic, doors, and vents,” Christensen says. “Another danger occurs when a cabinet user inserts or withdraws his arms through the front opening too rapidly. The turbulence causes aerosolized particles to escape.” Even the body heat of the user seated in front of the cabinet turned out to be a coconspirator. Plumes of warm air lifting off the warm surface of the body can carry downwind any aerosol liberated through the front of the cabinet.

What made those seemingly minor leaks so significant was the fact that some microbes are infectious even in very low doses. Francisella tularensis , the bacterium that causes tularemia, can trigger an infection after inhalation of as few as 10 organisms, a property that makes it attractive as an agent of bioterrorism. Coxiella burnetii , the rickettsial germ responsible for Q fever, is remarkably resistant to drying and other adverse environmental conditions. The infectious dose, calculated from animal experiments, turns out to be as small as a single organism.

The protection that an open-fronted Class II cabinet provides obviously depends on the virulence of the organism being handled inside it. Some organisms are so dangerous that the U.S. Department of Heath and Human Services recommends instead the cumbersome Class III cabinet, a closed, gas-tight system. Work inside the cabinet is performed through arm-length rubber gloves. Clean equipment enters through a double-door autoclave, a double-door air lock, or a dunk tank filled with a proven microbicidal disinfectant. Exotic organisms, such as Ebola virus or Congo-Crimean hemorrhagic fever virus, for which there are no cures, no vaccines, and an extraordinary risk of infection by aerosols, are restricted to Class III cabinets.

WHILE BIOCONTAINMENT WAS FAST BECOMING A SOPHIS ticated science of air currents, airflows, velocities, and gravitational effects, something strange was happening in the pharmacological industry. Compounds created to fight disease were posing contamination problems startlingly similar to those in the bio labs. Beginning in the 1960s, nurses and pharmacists mixed vials of new and powerful antibiotics into bottles or bags of saline solutions for intravenous administration to patients. Personnel known to be allergic to some of these antibiotics were developing allergic reactions when they prepared the mixtures. Lacking containment cabinets, they were obviously breathing in aerosols, or the aerosols were settling on their skin and being absorbed as easily as if these people had been using drug patches. An equally dramatic problem occurred among pharmaceutical workers who manufactured oral contraceptives. Male workers developed breast enlargement from exposure to the female hormones in the pills they were making.

An even more worrisome situation surfaced in the 1980s with the wider use of sophisticated anticancer drugs that are toxic to cells and can cause mutations and even cancers themselves. Pharmacists who mixed anticancer drugs into bags of intravenous solutions were accumulating mutation-causing substances in their urine. Pregnant nurses who were exposed to the drugs during their first trimesters experienced alarmingly high rates of malformed infants and even stillbirths. After stringent tests showed undetectable levels of mutation-causing substances in the urine of personnel who mixed the drugs inside a Class II safety cabinet, the U.S. Occupational Safety and Heath Administration recommended that the cabinet be considered essential equipment for this kind of procedure.

More recently, safety cabinets have become a mainstay in yet another sector, the young and innovative biotechnology industry. Recombinant DNA technology allows the creation of novel bacteria by inserting foreign genes, turning organisms into miniature factories to produce scarce biological drugs. New genes are also often spliced into viruses for human gene therapy. With all these developments emerging rapidly over the last 30 years, there’s a healthy fear that engineered microbes with new genetic material will wreak a new kind of havoc in the lab. “These companies are some of my biggest customers for safety cabinets,” Christensen says. “Since they aren’t sure exactly what they’re working with in the early stages, they want to be protected from it.” They’re showing a caution that proves that after a century medical martyrdom has finally become truly passé.