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Association of Occupational and Environmental Clinics

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1 Association of Occupational and Environmental Clinics
Worker Preparedness and Response to Bioterrorism CORE SLIDE Terrorism: The FBI’s definition of terrorism is action(s) of violence, often employing weapons of mass destruction (WMD), to coerce a population in order to accomplish their goals. One of the hallmarks of terrorism is that there are no clear front lines in such a conflict. Unfortunately all aspects and segments of society become involved in the conflict and therefore are at risk for injury or attacks from a potential terrorist. On 9/11 we have experienced both a terrorist attack in the form of the Trade Towers and the Pentagon being essentially bombed to also being exposed to biological weapons in the form of Anthrax in the mail. The most important aspect of Anthrax, as a biological weapon being used in this form, is that it targets indiscriminately. We have learned that a number of segments of our population are at higher risk for exposure to this biological weapon in the mail, namely postal workers certainly, but also other potential handlers of mail, both in the private sector as well as in the government sector. There has also been a case of laboratory worker-associated Anthrax. Furthermore, there are a number of cases of people being targeted in the media industry, which are certainly not to be considered combatants but have been designated as targets. Therefore the intention of this teaching module is to familiarize the worker with the concepts of biological weapons and terrorism as a whole, as well as familiarizing the worker with specific biological agents. Also we will discuss the means to detect a biological attack, what a biological attack may appear like, in terms of the clinical presentation of a person who has become infected with a biological weapon/agent as well as what might appear in the community or the workplace as a result of a biological attack. Finally we will address the potential responses to biological weapons attacks in terms of personal protective equipment, the use of antibiotics, the use of immunizations and some aspects of public health. In doing so we will review the clinical presentation of some of these illnesses including the recent attacks that took place in the United States in the fall of 2001. Pictures in slide: (right) plague bacilli (center) anthrax spores Edward W. Cetaruk, M.D. Toxicology Associates University of Colorado Health Sciences Center Denver, Colorado, USA

2 Section 1 An Overview of Biological Weapons
Objectives: To be able to list biological agents that may be weaponized To describe the process of weaponization To develop an understanding of the bioterrorist threat To be able to recognize a biological attack CORE SLIDE

3 Probability vs. Potential Impact
BIOLOGICAL AGENT NUCLEAR WEAPON IMPROVISED NUCLEAR DEVICE CHEMICAL AGENT OR TOXIC INDUSTRIAL CHEMICAL CORE SLIDE This slide illustrates the range of weapons of mass destruction (WMD) versus their potential impact on a population. On the vertical (left) axis we have “Potential Impact.” Potential impact can be measured in terms of casualties, property destruction, or overall impact of the population. This impact includes both immediate victims of a terrorist attack, as well as the effect it may have beyond those directly involved in the attack. Keep in mind that terrorists endeavor to get the most “bang-for-the-buck” from their attacks. Their attacks are likely to be against targets that have intrinsic value (such as an important government structure), symbolic value (such as a national landmark), and human value (I.e. destruction of the target will result in significant casualties). These target characteristics are not mutually exclusive and serve to project the impact of the attack beyond the local target to the general population. Nuclear weapons are considered the “holy grail” of WMDs. The possess all the features of an effective terrorist weapon of mass destruction: they can inflict huge numbers of casualties, they can cause catastrophic infrastructure and property damage, they have sufficient name recognition to terrorize a population far greater than they can affect directly. However, nuclear weapons are difficult to develop, test, and the technology to do so is highly regulated and/or monitored. The possibility of complete nuclear warheads being stolen from existing military arsenals does exist, but most agree that the likelihood of a nuclear weapon being used by terrorists is remote. In this slide we see the probability of a terrorist attack using various weapons with their respective potential impact. By definition weapons of mass destruction are weapons of large-scale that will injure a great many people indiscriminately. Nuclear weapons have long been considered the Holy Grail of weapons of mass destruction in that they are quite powerful for their size, they can extract huge numbers of casualties and also causing prolonged and devastating environmental damage. In the slide on the left vertical axis we see the potential impact of a given weapon and on the horizontal scale we see the increasing likelihood of a weapon being used. The nuclear weapon, although it is quite powerful, is also quite difficult to come by. The development of a nuclear device requires a great deal of skill and technology, all of which are quite regulated and monitored. So as much as it is a quite powerful weapon the likelihood of it being used by a terrorist agency is thankfully somewhat remote. Next on the graph we see the improvised nuclear device also known as a “dirty bomb”. This type of device certainly has the potential to cause a significant number of injuries because of its radioactive component. But it does not have the explosive power of a true nuclear detonation. Simply, a dirty bomb is an explosive device that is exploded in conjunction with nuclear material. Nuclear material may be waste fissionable material from nuclear reactors such as plutonium, it could be stolen nuclear fuel such as uranium or it could simply be radioactive waste. In this device we see an explosive being used that may cause immediate and localized damage as a result of blast effect. But it is compounded by the addition of radioactive contamination, which can increase the amount of property damage as well as increase the number and severity of injuries. At the lowest point on our curve we see simply nuclear material. This is nuclear material without the addition of an explosive component. The potential impact here is quite limited because if the nuclear material is even distributed the number of potential victims is somewhat limited just by its limited reach. However it is somewhat more likely than a dirty bomb just simply because it is somewhat less complex to manufacture. But again, more importantly, it is a lesser weapon of mass destruction simply because it is less able to create a large number of casualties. As we move further to the right on our scale we see chemical weapons. We often consider chemical weapons to include things such as nerve agents and mustard gas and other warfare chemical weapons. However, in reality a great many of the industrial chemicals that we use in our country on a regular basis and in large quantities are available to be usurped as chemical weapons as well. Case in point would be that phosgene, which was one of the earlier chemical weapons used quite successfully in WWI is manufactured at the rate of billions of pounds in this country as a precursor for the manufacturing of plastic. A likely scenario may be a tank car full of phosgene being exploded in a crowded metropolitan area. Likewise, there are huge quantities of cyanide anhydrous ammonia, chlorine, being transported on our highways and our rails at any given point in time in this country. They should all be considered potentially chemical weapons of mass destruction. Finally, at the highest point on the right side of the scale is our biological weapons. The potential impact of a biological weapon can be massive. Simply looking in the past history, when an outbreak of disease such as smallpox or plague took place a small outbreak can turn into epidemics and even pandemics, depending on the infectious nature of the biological weapon. Biological weapons however do have a technological challenge in producing them and the proper form that would make them highly infectious. But again the scenarios are only limited by the terrorist’s imagination and his choice of dissemination. However, we do find the likelihood is higher and that they are more attainable than a nuclear weapon. They also carry with them the potential of a great deal of impact over a large number of victims. POTENTIAL IMPACT RADIOACTIVE MATERIAL PROBABILITY/LIKELIHOOD

4 History of Biological Warfare
Oldest of the NBC triad of agents Used for > 2,000 years Sieges of middle ages Smallpox blankets given to Native Americans Germany in World War I Japan in World War II Modern Bioterrorism CORE SLIDE A brief review of the history of biological warfare is merited at this point. Biological warfare is considered the oldest of the nuclear, biological and chemical triad of weapons of mass destruction. There are references to the use of biological weapons over 2000 years ago. The Assyrian’s poisoned their enemy’s wells with ergot fungus, which contains hallucinogenic compounds. In the 14th century, the Tartars laid siege to the fortress city of Kaffa (also spelled Caffa) (in the Crimea) when they invaded from Asia. They brought plague with them, which was endemic to continental Asia. During the protracted siege, it became apparent that they were losing more soldiers to plague than they were to the battle. Therefore, a strategy was used to catapult plague victims into the city, thereby introducing plague into the city of Kaffa. Eventually the city of Kaffa fell, and plague was introduced to Europe leading to one of the great plague pandemics of Europe. Even here in the continental U.S. the colonial British used biological warfare. Smallpox victims will shed a great deal of the smallpox virus from their rash lesions, which will become lodged in their bedding, clothing, and such. During the French and Indian War, Lord Jeffrey Amherst, was known to have given smallpox infected blankets from their smallpox infirmaries to the Native American tribes in the Northeast, introducing smallpox to their communities and essentially wiping them out. More recently, Germany was known to use glanders to infect horses in WWI. One must remember that WWI was a pivotal war in which the movement of men and materials primarily was done with horses and only later was it more likely to be done with machines. The idea was to infect the horses with glanders in the U.S. prior to their shipment to Europe, such that when they arrived they were too ill to be of any military value. During WWII Japan’s unit 731 headed up by General Shiro Ishii experimented a great deal with biological weapons. During their occupation of Manchuria China, a great number of the Chinese were captured and imprisoned at Pin Fan. Pin Fan was a large biological weapons research center where human experimentation on prisoners, the local populace, as well as some allied prisoners. This unit experimented extensively with plague and actually developed a way to disseminate plague by releasing plague-infected fleas over towns and Mainland China causing localized outbreaks of plague. They worked on additional agents such as anthrax, viruses, and toxins. During WWII, the allies also experimented with the development of an anthrax weapon. This was a collaborative effort between the British and the Americans. These efforts did result in a weaponized form of anthrax, however it was never mass-produced, nor was it ever deployed beyond the developmental stages to the battlefield. The actual testing of this anthrax weapon took place on a small island called Gruinard Island off the west coast of Scotland. In more recent years terrorists have begun using weapons of mass destruction as well. To date the simple explosive device remains the #1 weapon of terrorists, however, increasingly they have acquired biological and chemical weapon capabilities, as well. The Aum Shinrikyo cult acquired technology to develop nerve agent in Japan and were responsible for nerve agent attacks in both 1994 and 1995 that resulted in a nineteen deaths and hundreds of injuries. It also is well documented that they had released botulinum toxin and anthrax on multiple occasions and but were unsuccessful in causing any injuries. The strain of anthrax they were using, derived from an attenuated Sterne strain used for vaccines, was not virulent enough to cause illness To date however there is no documented cases of injury or toxicity as a result of these releases. This underscores the necessity for a certain degree of technology to develop effective biological weapons. Finally, it is known that they sent “humanitarian aid” to Zaire during the Ebola outbreak in This was purportedly an aid mission to help contain the epidemic but in reality it was an attempt to acquire samples of the Ebola virus to bring back to the laboratories in Japan and weaponize. Notably, this same strategy was used by the Soviets on multiple occasions to acquire pathogenic agents from various outbreaks around the world and to bring them back to the Soviet Union to incorporate into their own biological weapons programs.

5 Aum Shinrikyo Cult Sarin Nerve Agent attacks 1994 and 1995
CORE SLIDE The Aum Shinrikyo cult maintained a relatively ambitious chemical and biological weapons, and is known to have released aerosolized anthrax spores, as well as botulinum toxin, in Japan on multiple occasions. However, these bioweapon attacks did not cause any casualties, as they weaponized a non-virulent strain of B. anthracis (Sterne 34F2) used for animal immunizations. The cult did successfully execute nerve agent attacks in 1994 and The recent use of anthrax as a terrorist weapon in the United States has underscored the need for the clinician to be able to recognize and to treat victims of bioweapons. Reference: Keim P, Smith KL, Keys C, et al: Molecular investigation of the Aum Shinrikyo anthrax release in Kameido, Japan. J Clin Microbiol 39(12): , 2001. Sarin Nerve Agent attacks 1994 and 1995 Attempted Botulinum Toxin release multiple times Anthrax released multiple times Attempted to obtain Ebola virus in Zaire

6 Anthrax Letters United States
CORE SLIDE In more recent experience we have the use of letters as delivery vehicles for anthrax as a biological weapon in September and October of There were a total of 5 letters mailed, 4 of which have been recovered and three of which we see in this photograph. There were two known mailing dates, one on September 18 and the second was October 9, The letter(s) to the AMI building in Florida was not recovered. The September 18 letters went to the offices of NBC Studios in New York City, the N.Y. Post in New York. The October 9 letters were mailed to Senators Daschle and Leahy of the U.S. Senate. The amount of anthrax in these letters was estimated to be 1-2 grams. The letter to Senator Leahy (which was unopened at the time it was discovered) contained approximately 2 grams of highly weaponized anthrax spores. There may have been 4 anthrax-laced letters sent to New York City in the mailing of September 18th - letters to Tom Brokaw at NBC, Dan Rather at CBS, Peter Jennings at ABC and The New York Post.  (There may have been others, but these are the only letters that are actually known to have caused cases of anthrax.)

7 Weaponized Biowarfare Agents
Anthrax Botulinum Toxin A Brucellosis Glanders Marburg Virus Plague Q Fever Salmonella Smallpox Staph Enterotoxin B Monkey Pox Ricin Tularemia VEE VHFs CORE SLIDE On this slide we see what are considered the primary agents that have been weaponized. At the top of the list we see anthrax. It is a prototypical biological weapon as it is a highly virulent agent, it’s very stable as a spore in the environment, and is deadly. A number of other agents that we see, including botulinum toxin, Marburg virus (one of the viral hemorrhagic fevers (VHFs)), plague, smallpox, all have a high mortality rate and are considered “lethal agents”. In the battlefield setting, these agents are intended to cause large numbers of fatal casualties. Similarly, a bioterrorist could cause a large number of fatalities by using these agents. We also see a number of other agents, including Q-fever, tularemia, Venezuelan Equine encephalitis (VEE), which are considered “incapacitating agents” as they have high infection rates, but relatively low mortalities. One might ask why would you make a biological agent that is not lethal. However, in the battlefield setting it is typically more advantageous to gravely injure your enemy as opposed to killing soldiers outright, requiring your enemy to use a significant amount of resources to take care of that sick soldier. Whether a lethal agent or incapacitating agent is employed in the battlefield setting, a biological weapon is an effective means to reduce the war fighting ability of an enemy force. It is also important to note that the toxin weapons on this list (botulinum toxin, Staphylococcal enterotoxin B, ricin) are NOT infectious agents. They are classified as “bioweapons” as they are naturally produced toxins (poisons) from living organisms. Salmonella is a common cause of food poisoning. It has been used successfully as a biological weapon in the United States. In 1985??? It was used by the Bagwan Rashneesh cult in The Dalles, Oregon to poison salad bars in a small community where they wanted to affect the outcome of a local election. The attack was intended to make a sufficient number of local resident too ill to make to the polls on election day, thereby improving the cult’s chances of winning. They succeeded in making 751 people ill but fortunately did cause any deaths. The cult’s role in the attack was only realized well after the fact when several members left the cult and confessed their role in the attack. Reference:

8 Biological Agents of Highest Concern Category A
Variola major (Smallpox) Bacillus anthracis (Anthrax) Yersinia pestis (Plague) Francisella tularensis (Tularemia) Botulinum toxin (Botulism) Filoviruses and Arenaviruses (Viral hemorrhagic fevers) ALL suspected or confirmed cases should be reported to health authorities immediately CORE SLIDE Category A “Special Action” CDC Slide These six diseases were chosen because: the potential public health impact if an outbreak occurred is huge; 2) they can be weaponized; 3) public health needs to be ready if such an outbreak occurs; and 4) the high level of perception the public has regarding these diseases Highly contagious and/or easily disseminated; High mortality; Name recognition The biological agents of highest concern (Category A agent) are typically the agents that have the greatest public health impact if an outbreak occurred. This typically has to do with their infectious nature (communicability), and these agents are agents known to be able to have been weaponized. Furthermore, they are relatively well known biological agents, such that they could instill a significant degree of fear and “terror” in a population should they be used. These agents are considered Category A agents (Special Action Agents) that are highly contagious and are easily disseminated. They have a high mortality rate and they also have name recognition as mentioned above. Agents on this list that are also highly contagious, meaning that if one person were infected that person could pass on that infection to another person include smallpox and plague. It is important to also note that botulinum toxin, although derived from a clostridium botulinum, which is a bacteria, it is indeed a toxin and not an infection. Because it is a toxin, a person exposed to this agent will develop the clinical syndrome of botulism but cannot infect any other people. The viral hemorrhagic fevers listed here include well-known agents such as Ebola virus and Marburg virus. Should any of these agents be discovered in a community or a medical setting they are, by definition, a public health emergency and all cases would need to be identified and reported to the public health system as soon as possible. It is important to note that any agent in this class could potentially affect a large population and therefore a great deal of resources will be needed to respond to any of these cases.

9 Incubation Periods of Selected Biological Agents
Anthrax Days++ Plague Days Q Fever Days Tularemia Days Smallpox Days Viral encephalitides V(2-6d); E&W (7-14 d) VHFs Days Botulinum toxin Days Staph. enterotoxin B 1-6 Hours CORE SLIDE Viral encephalitides: V – Venezuelan, E – Eastern Equine, W – Western Equine In this slide we see the incubation periods for selected biological agents. As mentioned above, you need to be exposed to a biological agent and receive a sufficient dose, or inoculum, of that agent to acquire an infection. However, just as when someone is exposed to someone with strep throat and does not develop symptoms until 4-5 days later, a person may be exposed to a biological agent and not develop an illness until days later. This intervening time period is called an incubation period. During this period the infecting organism gains a foothold in the host and multiplies. As the organism multiplies it creates an infection, and as time goes by causes a clinical illness. An incubation period will vary depending upon the choice of agent, virulence of the agent, size and route of the inoculum, and the underlying health of the victim.

10 Infective Aerosol Doses of Selected Biological Agents
Anthrax 8,000 (or fewer) spores Plague organisms Q Fever organisms Tularemia organisms Smallpox organisms Viral encephalitides organisms VHFs organisms Botulinum toxin ug/kg SUPPLEMENTAL SLIDE With the exception of the toxin weapons (botulinum toxin, ricin, and Staphylococcal enterotoxin B), biological agents are infectious agents. Therefore, we talk about infectious doses, or inoculum, when considering how potent an agent is. For example, we see that the estimated infectious dose (ID50) of anthrax is considered 8,000-10,000 spores. This number is based upon animal experiments. No human scientific human data is available to determine the ID50 for humans. But for the purposes of comparison of agents, the ID50 means that 50% of a population (animals) that inhale 8,000-10,000 anthrax spores will develop inhalational anthrax. This may sound like a large number of spores but this number of spores can fit on the head of a pin and are not visible to the naked eye. As the number of spores increases, the percentage of those exposed that will develop infection also increases. Likewise, as the number of spores falls, the percentage of exposed animals that will develop infection also falls, (e.g. ID10 = 120 anthrax spores in animals (William C. Patrick, III – personal communication), the ID10 is the infectious dose to cause infectious in 10% of an exposed population). We also note that some of the other organisms on this list have very small infectious doses, such as Q-fever, requiring only a few organisms to successfully infect a victim. This underscores that an effective biological agent is a highly infectious agent. An agent that requires thousands upon thousands of organisms, either bacteria or viruses, to successfully infect a victim, is a less effective agent than one that only requires a small dose. Botulinum toxin is an exotoxin produced and secreted from the bacterium Clostridium botulinum, and is considered the most poisonous toxin known to man. It is a toxin that causes poisoning, not an infection. Therefore, the amount needed to cause illness, the effective dose (ED50), is measured in micrograms per kilogram of body weight, NOT in “number of organisms” like we see for the other biological agents. The toxic dose is ug/kg. What this dose means is that a patient would need to ingest ug of botulinum toxin per kg of body weight to develop botulism. It is important to note that ingested botulinum toxin is 1400 times more toxic than botulinum toxin disseminated and absorbed in the aerosol form. This demonstrates that the route of exposure affects the dose required to cause illness. Several important notes: 1) Most of these infectious doses are based upon animal studies and/or very limited human data. The actual amount needed to cause an infection in the setting of a bioterrorist attack is not known. However, given that a number of the victims from the anthrax attacks of 2001 had no known exposure to any of the anthrax letters supports the idea that a very small number of anthrax spores may be sufficient to infect a given person. 2) The infectious doses for these organisms is dependent on a number of factors. These include the actual organism (there are many strains of anthrax that vary greatly in their ability to cause infection), the weaponization process used (i.e. were the spores (in the case of anthrax) prepared in a form and particle size that makes them easily inhaled and disseminated), the underlying health of the victim (i.e. immunosuppressed or elderly people are often more susceptible to illness than healthy individuals). In summary, the ID50 ‘s shown here are best used for comparison of various agents and cannot be extrapolated to actual patients. 3) The toxin weapons (botulinum toxin, ricin, staph enterotoxin B, or any other toxin) are not infectious agents. You cannot be infected with botulism from a person sick with botulism. These should be thought of as natural poisons. They are included with the other biological agents because the are produced and disseminated in much the same way.

11 Aerosol Size and Infectivity
Particle Size (Micron, Mass Median Diameter) Infection Severity The ideal aerosol contains a homogeneous population of 2 or 3 micron particulates that contain one or more viable organisms Less Severe More Severe 18-20 15-18 7-12 4-6 (bronchioles) 1-5 (alveoli) CORE SLIDE Slide: After William C. Patrick, III An aerosol is a fine suspension of particles or liquid that essentially acts as a gas or vapor, remaining airborne for long periods of time. Bioaerosols are aerosols of biological materials. Examples could include pollen or molds. A biological weapon is most effective if prepared as an aerosol because the terrorist could release it into the air and potentially expose and infect a large number of victims. When speaking of bioaerosols of biological weapons it is important to understand the relationship between an aerosol’s particle size and the infectivity of that agent. An ideal agent is a dry preparation such that it might be easily stored and effectively aerosolized. More importantly, this aerosol is best comprised of uniform particles within the 1-5 micron particle size. Particles greater than 5 microns in diameter are more likely to impact in the nasopharynx or in the upper airways. (Refer to the right side of the slide re: “particle size” and “infection severity.”) This would limit their ability to become settled in the lower respiratory tract and cause disease. Although they may still cause an infection, and possibly illness, in the upper airways, the efficiency for them to do so is much less than if they were to become lodged in the alveoli (air sacs) of the lung. Particles within the 1 to 5 micron size range are effectively much more infectious, and therefore much more virulent biological weapon, than larger particles sizes. There are multiple ways to achieve particle sizes within these ranges. A dry powdered form the most difficult to develop. One may also develop liquid bioweapons that may be disseminated by specifically engineered spraying devices, such that the sprayed particles are within the 1 to 5 micron range when inhaled. However, dry weapons are much more stable than liquids, therefore are much more easily disseminated in the environment. Maximum human respiratory infection is a particle that falls within the 1 to 5 micron size

12 Epidemiologic Clues Large epidemic with high illness and death rate
Immunocompromised individuals may have first susceptibility Respiratory symptoms predominate Infection non-endemic for region Multiple, simultaneous outbreaks Multi-drug-resistant pathogens Sick or dead animals Delivery vehicle or intelligence information CORE SLIDE To change perspective now for a moment, we must now address the more important question of how to recognize the fact that a biological weapons attack has occurred. As we learned earlier, there is an incubation period with biological agents between the time that a susceptible person is exposed, and the time at which that person develops an illness. Therefore, one may not see a large number of people get sick in a very short period of time (hours) as exposure to an agent may not cause immediate illness. The exception to this would be the use of toxin weapons. These are effectively poisonings as no incubation period is required for them to cause illness. What one is more likely to see is a pattern of people developing illness a few days following the release of a biological agent. Therefore, we must look at individual cases with an epidemiologic eye to see if we can recognize patterns of cases that suggest a deliberate release of a bio weapon. First: one clue may be a large epidemic with a very high illness and death rate. An attack rate is the percentage of a population that develops illness after a given exposure to a given biological agent (AR = number of “sick people” divided by number of “exposed people”). If every single person that was exposed gets sick, the attack rate would be 100%. A high case fatality ratio (CFR = number of fatal cases divided by number of total cases) should suggest that the infectious agent is a more highly virulent form than the natural form of that agent. If every person that gets sick, dies, then the CFR = 100%. It has been hypothesized that immunocompromised individuals in a population may have a higher susceptibility to a given infection and therefore may serve as an epidemiological “canary in the coal mine” warning. Unfortunately, these individuals may be more likely to develop the illness and after a shorter incubation period for a given exposure. As most biological weapons are likely to be released in an aerosol form, we may expect that respiratory symptoms may be prominent in the clinical presentation of ill victims. Although early symptoms of most biological weapons exposures are relatively non-specific and have a significant degree of overlap, respiratory symptoms are certainly an important area to focus However, in the case of anthrax, keep in mind that although aerosol cases may occur, one may also see other forms of anthrax, such as cutaneous anthrax. These “more recognizable” forms may accompany cases of inhalational anthrax. Therefore, one may diagnose the true etiology of a nonspecific respiratory infection as being anthrax by recognizing cutaneous cases that may also be seen in coworkers or family members. The pattern of cases also may provide clues to identify the source of a released bioweapon. Bioweapons often are comprised of agents that may not be native to the area of the release. Case in point would be a relatively rare infection, such as Venezuelan equine encephalitis, showing up in such a place as the American Midwest or the Pacific Northwest. In the absence of a known source of exposure, cases of non-endemic illnesses should generate public health investigations to rule an intentional bioterrorist attack. Most infectious outbreaks will emanate from a particular points source of exposure. As patients move away from that source of exposure the epidemic spreads. However, should multiple and simultaneous outbreaks of the same illness occur in geographically distant areas, one should suspect whether or not has been an intentional release of those agents in multiple places at multiple times. Furthermore, once the organism has been identified and undergoes laboratory study, the discovery that the organism has uncommon and/or multiple drug resistances, should make one suspect that it is an engineered pathogen, and therefore more likely to be a biowarfare agent. Many of these biological weapons are actually zoonotic diseases (diseases that they can infect both humans as well as animals). If a weaponized zoonotic organism is released as an aerosolized bioweapon it will indiscriminately infect both humans as well as animals. This was seen in the Sverdlovsk accident of After a release of weaponized anthrax from a Biopreparat facility, in addition to the large number of human cases in close vicinity downwind from the point of release, there were cases of anthrax in livestock many miles downwind. Should an epidemic outbreak of illness include both animals as well as humans, it should be considered an important epidemiological clue of an intentional release. Finally, it is important to remember that victims of a biological weapon are likely to initially present with relatively non-specific symptoms including fever, cough, nausea, headache, and many other constitutional symptoms. Therefore, it is more likely that a biological weapon attack will be recognized on a basis of a number of victims presenting and showing a pattern of illness, as opposed to seeing and identifying an attack as a result of a single victim.

13 Epidemiologic Information
Travel history Local Distant Infectious contacts Employment history Activities over the preceding 1 to 2 weeks CORE SLIDE Therefore, there are simple questions that one should ask when considering whether or not a biological weapon has been used. Has a potential victim has had any recent travel? If a patient presents with a relatively rare tropical disease, such as Ebola, but has also recently visited the continent of Africa (where Ebola is endemic) such that he may have been exposed, his infection may have followed a natural route. However, if he have remained local, have not traveled to any areas where the infection naturally occurs, one should consider that the infection may have been intentionally disseminated locally. Additionally, one should ask where a potential victim has been recently to identify other potential infectious contacts. If a patient is able to tell that he was at a particular sporting event, or works for a particular company, the investigation should include locating and questioning all contacts from these places to determine if they too are infected. Likewise, determining the patient’s employment history, where he has was on certain date and hours, where he may have recently visited on vacation, may help identify the location and time of a biological weapons release. Overall, one should attempt to account for all the patient’s activities and places visited over the preceding 1-2 weeks as a minimum. Please recall that incubation periods may vary widely between agents as well as between exposed individuals.

14 Section 2 Bioterrorism and the Workplace
Objectives: To be able to develop practices and procedures to defend workers and the workplace from a bioterrorist attack To respond the unique risks faced by first responders To be able to choose and use the correct PPE needed for biological weapons

15 Bioterrorism Educational Needs of the Worker
Awareness Fundamental understanding of biowarfare agents Recognition and handling of suspicious mail or dissemination devices PPE and workplace safety Recognition of bioterrorist attack Post exposure management CORE SLIDE Awareness Situational awareness means knowing the likelihood of a bioterrorist attack occurring at your workplace or in your community. The level of awareness, and the particular aspects of awareness, will vary depending on the worker’s job. For example, a mail worker, either in a private or government delivery service, needs to be vigilant for a threat from mail or packages passing through his/her work environment. Conversely, a first responder must also consider the possibility of a exposure to victims of a bioweapons attack and the risk of becoming infected by a communicable agent. By its very nature, terrorism targets any and all segments of society. The same biological weapons attack that occurs within the occupational setting for one person, may occur within the community for others (e.g. security or EMS personnel being exposed to an anthrax release at a concert). In a more general sense, all workers in all fields must adopt an ever present level of awareness to be able to recognize and respond to a bioterrorist event wherever it may occur. Fundamental understanding of biowarfare agents All workers should develop a working understanding of bioweapons, including how they may be disseminated, as well as the basic epidemiology of a bioweapon attack. The depth of understanding needed will vary with occupation of the worker (e.g. First Responder vs. Mail Handler vs. School Teacher). Most importantly, workers should know which agents are communicable (smallpox, pneumonic plague, viral hemorrhagic fevers). Recognition and handling of suspicious mail or dissemination devices The recognition and safe containment a possible bioweapon dissemination device will likely be the most important initial response in limiting the impact of that attack. PPE and workplace safety Inevitably, workers may be exposed either to bioweapon agents, or to victims of a bioweapon attack. Both circumstances involve an unavoidable risk of infection. Furthermore, the exposure may occur unrecognized. Therefore, workers in environments that have a credible risk for exposure to a biological agent need to adopt workplace practices and utilize appropriate PPE to minimize the risk of exposure. Recognition of a bioterrorist attack A bioterrorist attack will most likely first be recognized as an unusual epidemiological pattern. For example, a cluster of illnesses among workers in a certain industry, at a particular workplace, or at multiple places simultaneously. For example, recognizing the cutaneous form of anthrax on workers may help uncover cases of inhalational anthrax as well.

16 Bioterrorism: Who are First Responders?
Primary Care Personnel Hospital ER Staff Public Health Professionals Emergency Response Personnel Laboratory Personnel Law Enforcement Public Military CORE SLIDE It was mentioned earlier in this presentation one of the hallmarks of a terrorist war is the fact that front lines are poorly defined if at all. Therefore, first responders to a bioterrorist attack will cross many traditional lines of who are considered first responders. First responders are essentially those who would come in contact either with the agent itself, or victims of that agent. Certainly primary care personnel would be at the head of that list if patients develop an illness and present to their own doctors. Emergency department staffs at hospitals or clinics are certainly likely to see patients early as well. Being the traditional first responders, EMS personnel are at extremely high risk to become exposed to either biological agents or patients with exposure to agents. Keep in mind to emphasize again that an all hazards approach should be established. There is a great deal of focus on anthrax but there are a number of other agents that are secondarily infectious such as plague and/or small pox. These agents are much more worrisome for the first responder. Therefore, emergency response personnel should include in their practices the use of an appropriate mask (e.g. N95) to prevent inhalational exposure to a potential agent. Laboratory personnel at hospitals and diagnostic laboratories can also be considered first responders. It is important to note that a laboratory worker contracted cutaneous anthrax as a result of testing samples of patients who were the victims of the 2001 anthrax attacks. Therefore, great care should be taken at any time that any patient and/or material that is associated with a bioterrorist attack is worked with. Law enforcement and military personnel are certainly likely to be involved in a response to a bioterrorist attack and therefore are at risk for exposure as first responders. Finally, the public at large is probably at highest risk to be first responders as they are typically the most general targets for any bioterrorist attack. This means they may indeed be the person to recognize the fact that an attack has taken place, recognize the fact that people are developing illness that are consistent with a bioterrorist attack and obviously are at risk to develop illness themselves.

17 First Responders Often dealing with unknown agent(s)
May be exposed to infectious agent May be exposed to infectious patients May be targeted with secondary devices May be first to notice the epidemiological pattern of a bioweapons attack CORE SLIDE First responders, especially EMS personnel, are at high risk for exposure to a biological weapon. This is for several reasons. As I mentioned earlier, anthrax has been focused upon but there are other agents that are infectious. Therefore, it is likely that first responders (e.g. when responding to a call to a home for an ill patient) are often dealing with an unknown agents or agents. Therefore, they may be exposed to the infectious agent itself such as anthrax spores in the workplace, or they may be exposed to infectious patients, i.e. patients with infections that are contagious such as small pox, plague, or viral hemorrhagic fever. Sadly, EMS first responders have been targeted with secondary devices. There are multiple examples of explosive devices being planted in the vicinity of an initial device. These secondary devices are typically timed to explode at the time that EMS personnel are responding to the first explosion. Therefore when an EMS person enters the scene of a terrorist attack, whether it be biological, chemical, or conventional explosives, they should be very cautious as they are likely to be targeted again in the future. Finally, with their medical training, and exposure to the population at large, they may likely be the first to notice an epidemiological pattern consistent with a bioweapons attack. One of the most important rules about diagnosing any medical illness is to first consider the diagnosis to make the diagnosis. That being said, they may notice an unusual pattern of respiratory illness in otherwise young and healthy people on a particular weekend. On further questioning they may discover that a number of these patients work at the same place, or visited the same sporting arena a few days earlier. Therefore, first responders, with this unique capacity, are valuable tools to detect a bioterrorist attack early, and alert the appropriate medical response.

18 Additional Reference Material:
Emergency Plan All Hazards Approach Identify areas with risk of exposure Develop controls to minimize risk Engineering Controls Administrative Controls Housekeeping Controls PPE for workers Develop response and recovery plan Training and Exercises CORE SLIDE A bioterrorism preparedness emergency plan for workers should encompass an “all hazards approach”. This approach allows one to build upon emergency plans that already exist in many occupational settings. There has been a great deal of focus placed on anthrax in the past year since it was used as a biological weapon in the U.S. However, there are numerous other potential biological agents and, for that matter, chemical weapon agents that may be used by terrorists. Therefore, an emergency plan at a particular facility or institution should have a basis so that it can respond to any hazard, be it a chemical, biological, or radiological weapon of mass destruction. The emergency plan should identify areas with risk of exposure to a biological weapon. An example of this certainly would be the mailrooms of potential target organizations including the U.S. Postal Service or government offices. The emergency plan should identify ways to minimize the risk of exposure in these areas. To this end the plan should develop and include controls to minimize this risk, including engineering controls, administrative controls, and housekeeping controls. Examples of these engineering controls would be to protect the HVAC system of a particular building from tampering. Administrative controls would include the appropriate hiring practices and security access practices for a given facility. Finally, housekeeping controls would include the ways that certain areas are cleaned and maintained to minimize increased risk of exposure. A good example of this would be wet cleaning of surfaces and Hepa-filter vacuuming of surfaces that may potentially be contaminated and need to be cleaned. The Brentwood facility anthrax cases were complicated by the fact that the mail-sorting machine that was contaminated by the anthrax letters was cleaned with high pressure air hoses at 70 psi. Needless to say, the use of high pressure to clean up this machine certainly aerosolized a great deal more anthrax spores than would have been aerosolized had the machine be cleaned with vacuum systems instead. Even though numerous controls can minimize the risk of exposure ultimately personal protective equipment for all workers at risk for exposure is indicated and will be discussed later in this presentation. The emergency plan should not only include a robust preparedness portion of the plan, but also an appropriate and adaptable response portion of the plan. Finally, the plan should also include methods and plans to recover from a potential attack. These aspects of the plan will require input from many departments and key employees at a business or other facility. A good rule of thumb is: All persons, departments, and agencies that are written into the emergency plan should participate in the writing of the plan. Finally, once the plan has been developed and is on paper it MUST be tested both with training exercises. A plan on paper is of little use to an institution, or employees of that institution, if it has not been appropriately tested. This would include doing tabletop exercises, such that the plan could be discussed in a roundtable discussion using a realistic scenario with all members that would be affected by a bioterrorist attack. Typically, tabletop exercises provide valuable opportunities to revise and improve the plan. Finally, full-scale exercises with appropriate scenarios are necessary to test the functional ability of the plan to respond to a bioterrorist, or other types of, terrorist attacks. These exercises should be repeated on a regular basis. The frequency of these exercises is dependent upon the likely risk of an attack occurring at a particular facility, among other factors. These exercises should include realistic scenarios and should include all members of a particular business, institution or facility that would be involved in a response to a terrorist attack. Terrorist agents or strategies should be varied between exercises such that people will have the opportunity to test the ability of the plan to respond to all hazards. Additional Reference Material: CDC Interim* Recommendations for Protecting Workers from Exposure to Bacillus anthracis in Work Sites Where Mail Is Handled or Processed (*Updated from CDC Health Advisory 45 issued 10/24/01) These interim recommendations are intended to assist personnel responsible for occupational health and safety in developing a comprehensive program to reduce potential cutaneous or inhalational exposures to Bacillus anthracis spores among workers, including maintenance and custodial workers, in work sites where mail is handled or processed. Such work sites include post offices, mail distribution/handling centers, bulk mail centers, air mail facilities, priority mail processing centers, public and private mailrooms, and other settings in which workers are responsible for the handling and processing of mail. These interim recommendations are based on the limited information available on ways to avoid infection and the effectiveness of various prevention strategies and will be updated as new information becomes available. These recommendations do not address instances where a known or suspected exposure has occurred. Workers should be trained in how to recognize and handle a suspicious piece of mail (<>). In addition, each work site should develop an emergency plan describing appropriate actions to be taken when a known or suspected exposure to B. anthracis occurs. These recommendations are divided into the following hierarchical categories describing measures that should be implemented in mail-handling/processing sites to prevent potential exposures to B. anthracis spores: 1. Engineering controls 2. Administrative controls 3. Housekeeping controls 4. Personal protective equipment for workers These measures should be selected on the basis of an initial evaluation of the work site. This evaluation should focus on determining which processes, operations, jobs, or tasks would be most likely to result in an exposure should a contaminated envelope or package enter the work site. Many of these measures (e.g., administrative controls, use of HEPA filter-equipped vacuums, wet-cleaning, use of protective gloves) can be implemented immediately; implementation of others will require additional time and efforts. 1. Engineering Controls in Mail-handling/processing Sites B. anthracis spores can be aerosolized during the operation and maintenance of high-speed, mail sorting machines, potentially exposing workers and possibly entering heating, ventilation, or air-conditioning (HVAC) systems. Engineering controls can provide the best means of preventing worker exposure to potential aerosolized particles, thereby reducing the risk for inhalational anthrax, the most severe form of the disease. In settings where such machinery is in use, the following engineering controls should be considered: • An industrial vacuum cleaner equipped with a high-efficiency particulate air (HEPA) filter for cleaning high-speed, mail-sorting machinery • Local exhaust ventilation at pinch roller areas • HEPA-filtered exhaust hoods installed in areas where dust is generated (e.g., areas with high-speed, mail-sorting machinery) • Air curtains (using laminar air flow) installed in areas where large amounts of mail are processed • HEPA filters installed in the building’s HVAC systems (if feasible) to capture aerosolized spores Note: Machinery should NOT be cleaned using compressed air (i.e., “blowdown/blowoff”). 2. Administrative Controls in Mail-handling/processing Sites Strategies should be developed to limit the number of persons working at or near sites where aerosolized particles may be generated (e.g., mail-sorting machinery, places where mailbags are unloaded or emptied). In addition, restrictions should be in place to limit the number of persons (including support staff and non-employees, e.g., contractors, business visitors) entering areas where aerosolized particles may be generated. This includes contractors, business visitors, and support staff. 3. Housekeeping Controls in Mail-handling/processing Sites Dry sweeping and dusting should be avoided. Instead, areas should be wet-cleaned and vacuumed with HEPA-equipped vacuum cleaners. 4. Personal Protective Equipment for Workers in Mail-handling/processing Sites Personal protective equipment for workers in mail-handling/processing work sites must be selected on the basis of the potential for cutaneous or inhalational exposure to B. anthracis spores. Handling packages or envelopes may result in cutaneous exposure. In addition, because certain machinery (e.g., electronic mail sorters) can generate aerosolized particles, persons who operate, maintain, or work near such machinery may be exposed through inhalation. Persons who hand sort mail or work at other sites where airborne particles may be generated (e.g., where mailbags are unloaded or emptied) may also be exposed through inhalation. Recommendations for Workers Who Handle Mail • Protective, impermeable gloves should be worn by all workers who handle mail. In some cases, workers may need to wear cotton gloves under their protective gloves for comfort and to prevent dermatitis. Skin rashes and other dermatological conditions are a potential hazard of wearing gloves. Latex gloves should be avoided because of the risk of developing skin sensitivity or allergy. • Gloves should be provided in a range of sizes to ensure proper fit. • The choice of glove material (e.g., nitrile, vinyl) should be based on safety, fit, durability, and comfort. Sterile gloves (e.g., surgical gloves) are not necessary. • Different gloves or layers of gloves may be needed depending on the task, the dexterity required, and the type of protection needed. Protective gloves can be worn under heavier gloves (e.g., leather, heavy cotton) for operations where gloves can easily be torn or if more protection against hand injury is needed. • For workers involved in situations where a gloved hand presents a hazard (e.g., close to moving machine parts), the risk for potential injury resulting from glove use should be measured against the risk for potential exposure to B. anthracis. • Workers should avoid touching their skin, eyes, or other mucous membranes since contaminated gloves may transfer B. anthracis spores to other body sites. • Workers should consider wearing long-sleeved clothing and long pants to protect exposed skin. • Gloves and other personal protective clothing and equipment can be discarded in regular trash once they are removed or if they are visibly torn, unless a suspicious piece of mail is recognized and handled. If a suspicious piece of mail is recognized and handled, the worker’s protective gear should be handled as potentially contaminated material (See “Guideline For Hand washing And Hospital Environmental Control,” 1985, available at <> Hands should be thoroughly washed with soap and water when gloves are removed, before eating, and when replacing torn or worn gloves. Soap and water will wash away most spores that may have contacted the skin; disinfectant solutions are not needed. Additional Recommendations for Workers Who May Be Exposed through Inhalation • Persons working with or near machinery capable of generating aerosolized particles (e.g., electronic mail sorters) or at other work sites where such particles may be generated should be fitted with NIOSH-approved respirators that are at least as protective as an N95 respirator. • Persons working in areas where oil mist from machinery is present should be fitted with respirators equipped with P-type filters. • Because facial hair interferes with the fit of protective respirators, workers with facial hair (beards and or large moustaches) may require alternative respirators (such as powered air-purifying respirators [PAPRS] with loose-fitting hoods). • Workers who cannot be fitted properly with a half-mask respirator based on a fit test may require the use of alternative respirators, such as full facepiece, negative pressure respirators, PAPRs equipped with HEPA filters, or supplied-air respirators. If a worker is medically unable to wear a respirator, the employer should consider reassigning that worker to a job that does not require respiratory protection. • In addition, the use of disposable aprons or goggles by persons working with or near machinery capable of generating aerosolized particles may provide an extra margin of protection. In work sites where respirators are worn, a respiratory-protection program that complies with the provisions of OSHA [29 CFR ] should be in place. Such a program includes provisions for obtaining medical clearance for wearing a respirator and conducting a respirator fit-test to ensure that the respirator fits properly. Without fit testing, persons unknowingly may have poor face seals, allowing aerosols to leak around the mask and be inhaled. (See December 11, 1998, MMWR, available at < htm>

19 Emergency Plan Exposure to Biological Agent
Policies and Procedures for handling suspicious mail or packages Plan for facility response Plan for involving appropriate authorities Medical Surveillance Training and Exercises CORE SLIDE An emergency plan for mitigating a biological agent attack should include policies and procedures for handling suspicious mail and/or packages. It should also include a plan for the facility response as mentioned above. This plan should also include methods and ways to integrate the appropriate authorities ASAP. These would include, but not be limited to law enforcement, emergency medicine services, and HAZMAT response. Depending on the facility (e.g. hospital, school, nursing home), one may want to establish a liaison with public health agencies. Medical surveillance should be integrated into the response. Medical surveillance is a way of assessing patients who are at risk for developing illness to detect illness at the earliest possible time. An example of this would be having a system and a healthcare provider pre-arranged such that employees may be seen in a timely fashion to be assessed for exposure (e.g. nasal swabs for anthrax spores) and started on the appropriate antibiotic prophylaxis and/or treatment. Public health authorities may be able to address these concerns as well, but employees may likely be off work, out-of-town, etc., when the bioterrorist attack is detected. Finally, the emergency plan must undergo exercises to test the plan; the participants in the emergency response plan must undergo appropriate and continuing training to assure they are proficient in their duties; participants in the plan should be given duties as closely related to their normal jobs as possible; participants should cross train in other duties as well. Write the plan, test the plan, train all participants, exercise the plan – Repeat often!!

20 Handling of Suspicious Mail
Do not shake, empty contents Do not carry, show others, or allow others to examine it Do not sniff, touch, look closely at it, or any contents that may have spilled Leave on stable surface, alert others, leave area, close doors, shut off ventilation Wash hands with soap and water Notify law enforcement Create list of persons with potential contact CORE SLIDE There have been government recommendations on the handling of suspicious mail. Keep in mind that much of these recommendations involve simply common sense. If one should encounter a package that is suspicious - do not shake it, do not attempt to examine the contents or empty the contents of the envelope or package. Do not carry it around and show it to your co-workers or allow others to examine it. There is a good example of this from the anthrax letters that were sent to the news media offices in N.Y. where the person who initially discovered the anthrax letter did bring it to several co-workers for them to exam. On follow up examination of the office anthrax spores were detected at each and every place and desk to where the letter was brought. Needless to say, this practice greatly increases the numbers of people with potential exposure to the anthrax in the letter, as well as increasing the contamination of the office. In terms of the package itself - do not attempt to smell it. If you haven’t touched it - don’t. Don’t even get close to it in an effort to closely examine it. Anthrax spores, as well as other well-weaponized biological agents, have a remarkable ability to become aerosolized without significant disturbance. What people should do is simply leave it on a stable surface such as a tabletop. Walk away from it and alert others who may potentially have been exposed. Close the door to the office and also shut off any ventilation to the office if possible. If you are able to do so, gently put it in a zip-lock bag or some other sealable container. In no way should you attempt to move it or manipulate it in a way that would potentially release its contents or encourage aerosolization of its contents. Once you have removed yourself from the suspected package wash your hands with soap and water. If bactericidal or antiseptic-type solution available - even better. Notify the appropriate law enforcement agencies. This may simply be a 911 call. This may also involve notifying your in-house security system. One of the most important things is to identify persons, and create a list of these persons, who may have had potential contact with this envelope. This would include tracing the envelopes route back to the point at which it entered your facility. An example of this would include the mailroom staff, the delivery staff, and other potential co-workers in your office, secretaries, administrative assistants, and visitors.

21 Personal Protective Equipment
Level A SCBA, Encapsulation Level of protection for entering contaminated, unsecured scene Level B Level C Level D CORE SLIDE Personal protective equipment is important in any kind of response to weapons of mass destruction. We have traditional levels of personal protective equipment including Level A, B, C, and D. Level A is an encapsulated ensemble that includes a self-contained breathing apparatus. This would be the standard level of protection for entering a contaminated, unsecured scene. This means that a person would wear Level A to enter a chemically contaminated scene or a biological agent contaminated scene. Once a Level A equipped person has determined the scene to be secured and there is no significant risk of vapor contact or vapor risk, then lower levels of PPE can be employed. It is important to re-emphasize that the scene also needs to be declared “safe” from secondary devices such as explosives. Level B would include a self-contained breathing apparatus, as well as a chemical resistant suit, such as a Saranex suit. What it does lack compared to Level A is the fact that it does not protect the patient from a vapor exposure. Level C would include respiratory protection in the form of a respirator, either negative or positive pressure. Level D is essentially street clothing with a minimum of respiratory protection. Pictures: Left: Level C Right: Level A (level C in yellow suits) Interim CDC recommendations for personal protective equipment for responding to biological weapons The interim CDC recommendations for personal protective equipment, including respiratory protection and protective clothing, are based upon the anticipated level of exposure risk associated with different response situations, as follows: 1. Responders should use a NIOSH-approved, pressure-demand SCBA in conjunction with a Level A protective suit in responding to a suspected biological Incident where any of the following information is unknown or the event is uncontrolled: - the type(s) of airborne agent(s); - the dissemination method; - if dissemination via an aerosol-generating device is still occurring or it has stopped but there is no information on the duration of dissemination, or what the exposure concentration might be. 2. Responders may use a Level B protective suit with an exposed or enclosed NIOSH- approved pressure-demand SCBA if the situation can be defined in which: - the suspected biological aerosol is no longer being generated; - other conditions may present a splash hazard. 3. Responders may use a full facepiece respirator with a P100 filter or powered air-purifying respirator (PAPR) with high efficiency particulate air (HEPA) filters when it can be determined that: - an aerosol-generating device was not used to create high airborne concentration, - dissemination was by a letter or package that can be easily bagged. These type of respirators reduce the user’s exposure by a factor of 50 if the user has been properly fit tested.

22 Personal Protective Equipment Respirators
Powered Air-Purifying Respirator (PAPR) HEPA filter face masks (N95, N100) Respirators must be in compliance with OSHA respiratory standard (29 CFR ) Respirators must be fit tested CORE SLIDE However, the military kind of personal protective equipment is not likely to be practical in a civilian sector. Furthermore, OSHA has established personal protective equipment standards that are well recognized in the industry. Respirators include a self-contained breathing apparatus that provides a safe air source to the user, either by a tank supply or a hose. A powered air-purifying respirator, otherwise known as a PAPR, will provide the worker with a continuous supply of filtered air. Finally, we have simple face masks of varying efficiencies. These HEPA-filter face masks are most commonplace in the hospital setting, and are the recommended choice for respiratory protection for biological agents. Keep in mind that the use of these various respirators is regulated by OSHA standards and those using them should be in compliance with these standards including fit testing, training, and medical surveillance. Please refer to the detailed recommendations from the CDC and OSHA listed below: CDC, OSHA respirator recommendations for potential exposures to biological agents, such as Bacillus anthracis, in their facilities: Summary The goal of using a respirator is to reduce the exposure to the contaminant of concern to an acceptable level that will not adversely affect the wearer. According to the Centers for Disease Control and Prevention (CDC) and Occupational Safety and Health Administration (OSHA) all National Institute for Occupational Safety and Health (NIOSH) approved particulate respirators will help reduce exposures to biological aerosols such as B. anthracis, the bacteria that causes anthrax. Recently the CDC and OSHA have published respirator selection guidance, based on the expected risk of exposure, for individuals who may be potentially exposed to B. anthracis while engaged in mail handling, first responder, or investigative activities. However, no safe exposure levels (i.e. the amount you can inhale without adverse health effects) have been set for biological aerosols, including B. anthracis. Therefore, it must be recognized that respirators can reduce inhalation exposures but cannot eliminate the risk of contracting infection, illness, or disease. Each facility or individual must use the best available information in determining the appropriate respiratory protection for the level of exposure reduction that they feel is appropriate for potential occupational exposures to B. anthracis in their facility. Respirator Use Limitations A properly fitted respirator can only help reduce exposures when used immediately prior to and during the release of B. anthracis spores. Unfortunately, in the case of terrorist activity it is unlikely that you would have warning or knowledge of your exposure until symptoms started to appear in infected people. Once anthrax symptoms appear, a respirator will not be effective in helping to prevent the disease. Before selecting respiratory products for biological agents, such as B. anthracis, there are important considerations you must be aware of. The airborne concentration of these agents will be unknown; therefore it may not be possible to select the most appropriate respirator. In addition, NIOSH is the government agency responsible for testing and certifying respirators. NIOSH tests and certifies respirators for use against particles, gases, and vapors. NIOSH does not certify respirators for use against specific particles or biological agents, such as B. anthracis spores. Therefore, their efficacy against biological warfare agents is not known. Respirators may help protect your lungs, however, they will not prevent entry through other routes such as the skin (cutaneous), which would require additional personal protective equipment (PPE). Without proper decontamination, materials could create a hazard by bringing the spores into areas thought to be uncontaminated. Proper fit of the respirator to the face is extremely important. If it does not fit properly, you will increase your likelihood of exposure to the B. anthracis you are trying to filter. Individuals wearing tight fitting face pieces must be clean-shaven at all times when wearing respirators. Respirators are designed for occupational/professional use by adults who are properly trained in their use and limitations. Individuals with a compromised respiratory system should consult with a physician prior to use. In the event of a known or suspected biological warfare agent release; respirators should be used for escape only; leave the area immediately; do not remove respirator until going through decontamination and are in a clean environment; seek medical advice; and dispose of respirator immediately in accordance to your employers directions. Filtering Bacillus anthracis Biological agents such as B. anthracis are particles and can be removed by particulate filters with the same efficiency as non-biological particles having the same physical characteristics (size, shape, etc.), although their efficacy against biological agents is not known. According to the CDC the typical size of B. anthracis spores is between microns. Both OSHA and the CDC recommend a NIOSH certified class 95 or higher filter for use against B. anthracis spores. However, the type of respirator facepiece and filter class required does vary depending activities and risk of exposure. Consult the OSHA and CDC requirements before selecting a respirator for potential occupational exposures to B. anthracis. NIOSH class 95 filters are certified to be at least 95% efficient against a particle of 0.3 microns. Therefore, the filter will be 95% efficient or greater for particles in the 1 to 5 micron size range. A NIOSH certified class 100 or HEPA filter is 99.97% efficient against this most penetrating particle size of 0.3 microns. Importance of Proper Fit The fit of a respirator is equally as important as filter efficiency. While a respirator may be equipped with filter media to effectively capture a high percentage of airborne particles, excessive particles may enter the respirator through leaks around the facepiece of an improperly fitted facepiece. A tight sealing respirator, one where the sealing surface contacts the face, will not provide an adequate seal when placed over facial hair. A bearded worker will typically require a respirator where the wearer’s facial hair does not interfere with the face seal. In many instances this will consist of a powered air-purifying respirator (PAPR) with a hood or helmet. Assigned Protection Factors It is important to understand that since the safe level of exposure to B. anthracis spores has not been established, there is no assurance that any respirator will mitigate or prevent anthrax infection or disease. Respirators are traditionally selected after determining the airborne concentration of the contaminant, the exposure limit of the contaminant and the assigned protection factor of the respirator. Since the exposure limit and concentration are unknown for biological agents the traditional respirator selection method cannot be uniformly applied. All NIOSH certified respirators have an assigned protection factor (APF), which predicts how much the respirator may reduce a wearer’s exposure. The assigned protection factor is only applicable when the respirator is correctly selected, properly used by a trained and fit tested wearer and the respirator is maintained in good working order. A respirator with a higher protection factor will provide greater exposure reduction when the respirator is used properly and fitted to the individual. Here is an example of how to use assigned protection factors when choosing an appropriate respirator: Assume the contaminant concentration in the air is 10,000 particles. A person has passed a fit test and is wearing a half mask respirator with an assigned protection factor of 10. This means the person could expect to reduce their exposure by 10 times, resulting in a possible inhalation of 1000 particles. A full-face respirator would reduce the exposure by 50 times resulting in a possible inhalation of 200 particles. When a facility decides to make respiratory products a part of its emergency management or response plan, it is essential they follow all aspects of the OSHA respiratory protection standard, 29 CFR CDC and OSHA Respirator Recommendations The CDC has published three documents with respirator recommendations for mail handlers, first responders, and investigators who may be potentially exposed to B. anthracis. CDC recommends that a combination of controls be used to help reduce mail handlers potential exposure to B. anthracis. These include engineering controls (e.g., local exhaust ventilation), administrative controls (e.g., limiting number of people who could be exposed), and good work practices (e.g., wet cleaning). These recommendations for mail handlers do not address instances where known or a suspected exposure has occurred. For instances where a known or suspected exposure has occurred CDC recommends respirators with a higher assigned protection factor for the first responders and investigators who must enter into these environments to perform their duties. In addition, CDC states that in work sites where respirators are worn, a respiratory protection program that complies with the provisions of the OSHA Respiratory Protection Standard (29 CFR ) should be in place. CDC emphasizes the need for users to be fit tested to ensure the respirator fits properly. CDC states “Without fit testing, persons unknowingly may have poor face seals, allowing aerosols to leak around the mask and be inhaled”. The following table lists 3M respirators that satisfy the CDC recommendations. OSHA has published a workplace anthrax exposure guidance document titled the Anthrax Matrix. This document is intended to assist employers and employees in dealing with possible workplace exposure to B. anthracis in mail handling operations today. The matrix helps guide employers in assessing risk to their workers, providing appropriate protective equipment and specifying safe work practices for low, medium and high risk levels in the workplace. Following is a summary of the respiratory protection requirements outlined in this document. Note, the document also specifies engineering controls, work practices, and other personal protective equipment (PPE). Please consult the Anthrax Matrix for complete details of this guidance document. The following table lists 3M respirators that satisfy the OSHA recommendations. *Red zone (Workplaces Where Authorities Have Informed You That Contamination with Anthrax Spores Has Been Confirmed or Is Strongly Suspected). The employer is notified by law enforcement or public health authorities that a facility is strongly suspected of or confirmed as having been contaminated with anthrax spores. The employer is engaged in emergency response to and clean up of bio-terrorist releases of anthrax spores. *Yellow Zone (Workplaces Where Contamination with Anthrax Spores Is Possible). This zone is where workplace contamination is possible. Risk factors that should be considered in this zone include handling bulk mail, handling mail from facilities that are known to be contaminated, working near equipment such as high-speed processors/sorters that could aerosolize anthrax spores; workplaces in close proximity to other workplaces known to be contaminated; or workplaces that may be targets of bio-terrorists. *Green Zone (Workplaces Where Contamination with Anthrax Spores Is Unlikely). OSHA states this zone covers the vast majority of workplaces in the United States. Since October 2001, anthrax spores have only been discovered in a very limited number of workplaces. Additional Information Please use the links below to access the most recent governmental information regarding respiratory protection against Bacillus anthracis: OSHA Homepage CDC Homepage OSHA Guidance Document: The Anthrax Matrix (November 16, 2001) CDC Advisory: CDC Interim* Recommendations for Protecting Workers from Exposure to Bacillus anthracis in Work Sites Where Mail Is Handled or Processed (October 31, 2001) CDC Advisory: Interim Recommendations for the Selection and Use of Protective Clothing and Respirators Against Biological Agents (October 24, 2001) CDC Advisory: Protecting Investigators Performing Environmental Sampling for Bacillus anthracis: Personal Protective Equipment (November 6, 2001) Last updated: 11/21/01

23 Powered Air Purifying Respirator (PAPR)
CORE SLIDE The powered air-purifying respirator or PAPR is a good choice as a respiratory protection for first responders not entering a heavily contaminated or unsecured scene. The PAPR can be battery powered, belt-mounted respirator with an integrated overhood as well. If it is an overhood-equipped PAPR, and does not have an integrated respirator, it does not require fit testing. In general, this type of PPE is easier for the typical employee to use without significant amount of training. However, OSHA regulations do apply the worker will be using different levels of respiratory protection (e.g. face mask respirator). PAPR: Butyl rubber hood; Provides continuous flow of filtered air; Belt mounted battery; No fit testing required prior to wearing hood without face mask respirator; about 8 pounds. OSHA regulations require employers to train and fit test employees who use respiratory protection during the course of their workday. OSHA requires that each employee must be medically evaluated before the employee is fit tested. In summary, employers must: Maintain a written respiratory protection program with worksite specific procedures for fit testing and training. Provide instruction on the respiratory hazards to which the workers are potentially exposed during routine and emergency situations. Provide instruction on the uses and limitations of all respirators worn in the work area. Instruct and demonstrate to employees how to properly don and adjust any respirators worn according to the manufacturers' instructions.. Allow the employees an opportunity to practice these procedures. Provide user seal check instructions. Fit test each employee to be assigned a respirator. Instruct the employees in the procedures for the maintenance and storage of the respirators being used. Inform the employees how to recognize medical signs and symptoms that may limit or prevent the effective use of the respirators. Document the successful completion of training and fit testing for all employees wearing respirators.

PPE Respirators Respirators should be used in accordance with a respiratory-protection program that complies with the OSHA respiratory-protection standard (29 CFR ). CORE SLIDE N95 Minimum 95% filter efficiency N100 Minimum 99.97% filter efficiency CHANGING YOUR RESPIRATOR/FILTERS/CARTRIDGES If you are using either a:      Dust mask (also called a filtering facepiece or "N95" respirator) or a      Rubber or plastic facepiece respirator with replaceable particulate filters (e.g. N95, P100) Then: 1)   Replace the dust mask or change the particulate filters when you notice:      Increased breathing resistance, or      Physical damage to any part of the facepiece or filters, or      The inside of the dust mask becomes unsanitary      Time use limitations on the package require replacement. 2)   If you are using a half facepiece with replaceable chemical cartridges (e.g. organic vapors, ammonia), the cartridges must be replaced:      In accordance with a change schedule, or      Earlier, if smell, taste or irritation from the contaminant(s) is detected. N95 N100

25 Personal Protective Equipment Respirators
The respirator is properly positioned over your nose and mouth at all times The top strap or head harness assembly is positioned high on the back of the head The lower strap is worn at the back of the neck below the ears The straps are snug enough to keep the respirator from moving but not overly tight Nothing (beards, head coverings, etc.) passes between the skin of the face and the respirator’s sealing edge CORE SLIDE The requirements for the appropriate use of respirators is listed here. Some of these are common sense but everybody who is at risk for exposure, and is in need of a respirator, should be instructed in the appropriate use of those respirators. Quite simply, the respirator should be fitted to cover the nose and mouth at all times. The head straps should be appropriately positioned to keep the mask appropriately applied to the face. The mask itself should be fitted appropriately and the user should undergo appropriate fit testing to assure a proper size and type of respirator is used. Straps should be adjusted such as to keep the mask snugly applied to the person’s face. Furthermore, nothing should be allowed to be between the skin and the respirator’s sealing edge. This would include things such as beards as well as other protective equipment such as head coverings. Again, this means that the person using the respirator should be trained in its use and appropriately fitted. Additional Reference Material: Summary of the CDC Health Advisory on the Interim Recommendations for Protecting Workers from Exposure to Bacillus anthracis in Work Sites Where Mail Is Handled or Processed. (Distributed via the Health Alert Network, October 31, 2001) On October 31, 2001, the CDC issued a "revised" advisory titled Official CDC Health Advisory: CDC Interim Recommendations for Protecting Workers from Exposure to Bacillus anthracis in Work Sites Where Mail is handled or Processed.* This document is intended to assist personnel responsible for occupational health and safety in developing a comprehensive program to reduce potential cutaneous or inhalational exposures to Bacillus anthracis spores among workers, including maintenance and custodial workers, in work sites where mail is handled or processed. These recommendations do not address instances where known or a suspected exposure has occurred. The document stresses the need for engineering controls (e.g., local exhaust ventilation), administrative controls (e.g., limiting number of people who could be exposed), and good work practices (e.g., wet cleaning) to minimize worker exposure to Bacillus anthracis. Personal protective equipment recommendations including gloves, eye protection, and other personal protective clothing are also discussed. According to the CDC, in the event an employer chooses to provide respirators for employees handling or processing mail the advisory provides the following guidance for selecting respiratory protection products to help reduce potential exposures to Bacillus anthracis: Persons working with or near machinery capable of generating aerosolized particles (e.g., electronic mail sorters) or at other work sites where such particles may be generated should be fitted with NIOSH-approved respirators that are at least as protective as an N95 respirator. Persons working in areas where oil mist from machinery is present should be fitted with respirators equipped with P-type filters. Because facial hair interferes with the fit of protective respirators, workers with facial hair (beards and or large mustaches) may require alternative respirators (such as powered air-purifying respirators [PAPRs] with loose-fitting hoods). Workers who cannot be fitted properly with a half-mask respirator based on a fit test may require the use of alternative respirators, such as full facepiece, negative pressure respirators, PAPRs equipped with HEPA filters, or supplied-air respirators. If a worker is medically unable to wear a respirator, the employer should consider reassigning that worker to a job that does not require respiratory protection. In addition, CDC states that in work sites where respirators are worn, a respiratory-protection program that complies with the provisions of the OSHA Respiratory Protection Standard (29 CFR ) should be in place. CDC emphasizes the need for users to be fit tested to ensure the respirator fits properly. CDC states "Without fit testing, persons unknowingly may have poor face seals, allowing aerosols to leak around the mask and be inhaled".

26 PPE Dermal Protection Disposable Reusable
Overgarments, Booties, Hoods, Gloves All PPE should be decontaminated prior to leaving potentially contaminated area PPE should be removed and discarded prior to removing face mask CORE SLIDE Personal protective equipment should include dermal protection. Typically these include some sort of rubber glove, overboots, splash-resistant suit (e.g. Saranex). Depending on the contaminant, the choice of material should be tailored towards a chemical exposure or a biological exposure. For biological agent exposure, nitrile gloves are recommended over latex gloves as nitrile are more durable and without the risk of eliciting a latex allergy. Double gloving is also recommended. Furthermore, nitrile is disposable, allows good manual dexterity, and can be easily discarded at the completion of its use. If gloves are reusable (example butyl gloves) they must be decontaminated and disinfected according to an approved procedure. All personal protective equipment should be decontaminated if it is reusable prior to leaving a potentially contaminated area. Again, part of your emergency response plan or your bioterrorism response plan should include policies and procedures on how to appropriately doff PPE such that the worker does not risk respiratory exposure. All PPE should be removed and discarded prior to removing the face mask, which should protect the worker from any untoward respiratory exposure. Likewise policies and procedures for the decontamination and disinfection of reusable PPE must be developed and included in response plans. Whether the dermal protection is disposable or reusable, all PPE should be removed and appropriately disposed of, or decontaminated, prior to leaving a contaminated area and before removing respiratory protection.

27 Section 3 Anthrax as a Biological Weapon
Objectives: To understand the microbiology and epidemiology of anthrax To understand the pathophysiology of the different anthrax clinical syndromes To be able to recognize cutaneous anthrax To be able to recognize an intentional anthrax release To be able to treat patients with anthrax

28 Anthrax Microbiology & Epidemiology
Bacterium Spores may survive > 100 yrs Worldwide soil distribution Common disease of herbivores Herbivores in USA vaccinated Man infected via animal products Woolsorter’s Disease CORE SLIDE To discuss several of the primary biological weapons we will start with anthrax: Anthrax is a bacterium that forms a spore. It can be found in two forms. The “vegetative bacillus” form is the form of the bacteria that can multiply, produce toxins, and cause infection. When the bacterium is in a high oxygen, nutrient-poor environment, such that it won’t be able to survive as the fragile bacillus, it forms a spore. The spore essentially functions as a seed, containing all the important cell components to germinate another anthrax bacterium when conditions are right. The natural reservoir of anthrax is as a spore is in the soil and they are found in nearly all corners of the world. Anthrax is much less common in the United States due to widespread vaccination of animals and aggressive containment of outbreaks when they do occur. Spores are very resistant to environmental stressors and may remain viable in the soil for decades. In fact, tannery sites have been excavated, producing spores that have been buried for 100 years that will sporulate and grow when placed in culture medium. Historically, the livestock and wool industries have had high rates of exposure to anthrax. Man typically becomes infected after contact with contaminated wool, animal hides or other animal products. It has been noted that during relatively dry or drought periods that the incidence of animal anthrax in grazing animals such as cattle, goats, sheep, horses, and other livestock increases as they need to graze closer to the ground for forage, and are more likely to ingest soil that is contaminated with spores. Although it is a common disease of grazing animals, especially livestock, anthrax is relatively rare in the United States due to widespread vaccination of livestock as well as humans. As mentioned above, man typically becomes infected after coming in contact with these spores in the work environment. Woolsorter’s Disease is inhalational anthrax found in mill workers who sort and work with raw wool, or other animal hair, etc.. They are exposed to aerosolized particles of anthrax that are in the animal products. This is an epidemiological natural, and/or occupational, form of inhalational anthrax. There are reported epidemics of human inhalational and cutaneous anthrax in textile mills (e.g. Portsmith, NH, 1957). Additional Reference: Plotkin SA, et al: An epidemic of inhalational anthrax, the first in the twentieth century. American Journal of Medicine 29: , 1960.

29 Anthrax Worldwide Occurrence
CORE SLIDE When discussing anthrax we must realize that it is a naturally occurring disease. As mentioned above, its natural reservoir is in the soil and subsequently a large number of grazing animals become infected. Anthrax has a worldwide occurrence. There are areas of the world, primarily in Africa, Spain, and some areas of Asia, where it is considered epidemic (found naturally), or hyper-endemic (found naturally with frequent outbreaks). What this means is that it is in very high occurrence in these places, and occasionally, or episodically, there are large outbreaks of animal anthrax that can associated with human cases as well. A well described outbreak of Anthrax in Rhodesia, which is now Zimbabwe, in Africa, around 1980 involved infection of thousands of grazing animals that also lead to infections of hundreds and hundreds of humans. Current estimates vary widely but it is estimated that approximately 10,000-20,000 cases of human anthrax, primarily cutaneous, occur on a worldwide basis annually. The interested reader is referred to the World Health Organization’s Anthrax Data Site on the worldwide web for further updates on outbreaks of Anthrax and the current epidemiology of this disease on a worldwide basis. WHO World Anthrax Data Site: (Access confirmed 9/11/2002. Source: WHO World Anthrax Data Site

30 Anthrax Pathophysiology
Spore enters skin, GI tract, or lung Germinates in macrophage Transported to regional lymph nodes Local production of toxins Swelling and Tissue Death Toxemia CORE SLIDE Let’s review for a moment the disease course, or pathophysiology, of anthrax. When anthrax is disseminated as a biological weapon, it is disseminated as the spore form. Example: A weaponized powder of anthrax spores being dumped into the ventilation system of a building, or released from a crop duster. When that spore enters either an open wound in the skin, the gastrointestinal tract, or the lung of a victim, it undergoes germination into the bacillus form as mentioned above. Example: A construction worker removing anthrax-spore-contaminated carpet from a government building that received a anthrax laden letter may get skin anthrax through an abrasion while handling the carpet. He/she may develop inhalational anthrax he/she drops or throws the carpet aerosolizing spores in the fabric of the carpet. This inhalational risk can be reduced by wearing an appropriate respirator. In humans, especially in the inhalational form, this germination takes place in a white blood cell called a macrophage. The macrophage is a white blood cell that fights infection. Its primary job is to move around the body eating foreign organisms like bacteria, viruses, and destroying them in the lymph nodes (glands). Anthrax is able to resist this destruction. Once within this macrophage it is transported to a regional lymph nodes. This is much akin to the same type of process that one would have when they develop a sore throat and have tender glands in their neck. Once the macrophage arrives at the lymph node the anthrax multiplies much like we saw in the previous two pictures. Once it multiplies it produces two toxins, one called edema toxin and one call lethal toxin. These toxins cause massive amounts of tissue edema (swelling in the tissues and lymph nodes) as well as tissue necrosis (tissue death), respectively. The growth of this bacteria, and the production of these toxins, produces the clinical picture of anthrax, and the eventual death of the patient with inhalational anthrax. Toxemia is when the amount of anthrax toxin in the victim is enough to cause severe illness and/or death.

31 Anthrax Clinical Syndromes
Cutaneous Gastrointestinal Inhalational CORE SLIDE Anthrax can be contracted either as a cutaneous disease, a gastrointestinal disease, or an inhalational disease. Typically the inhalation and gastrointestinal forms carry a very high mortality rate. The cutaneous form of anthrax, which is the much more common and natural form of anthrax, typically has a very low mortality with antibiotic treatment. However, if left untreated, cutaneous anthrax can progress to creating a generalized infection that can also be fatal (approximately 20%). When considering anthrax as a biological weapon, the inhalational form of anthrax is the most worrisome threat for the reasons discussed earlier. These reasons include that biological weapons are much more effective when disseminated by an aerosol route and a much larger number of victims may be infected by an aerosol route. However, as exemplified by the anthrax attacks that took place in the United States in the fall of 2001, a significant number of cutaneous anthrax cases may be seen in conjunction with inhalational cases. Therefore, it is important to remember that even though the anthrax may be disseminated via an aerosol or inhalational route, one may still find cases of cutaneous anthrax. These cutaneous cases are typically less severe and less life threatening, but are much more recognizable if one is educated as to what to look for. Case in point would be to recognize a co-worker with a cutaneous anthrax lesion on their arm, which may not be causing significant illness in that patient. However, it may alert that person, and for that matter, that workplace or facility, to the presence of anthrax. This may shed a diagnostic light on other patients who may have symptoms consistent with inhalational anthrax, but are not yet recognized as victims of a bioterrorist attack. It also may alert that workplace to institute preventative measures and to notify law enforcement agencies. In summary, anthrax may appear in multiple clinical forms as the result of a single biological weapon attack. Multiple forms can be seen as the result of a BW attack

32 Anthrax Gastrointestinal
Abdominal pain, usually accompanied by bloody vomiting or diarrhea, followed by fever and signs of sever infection GI anthrax is sometimes seen as mouth and throat ulcerations with tender neck glands and fever Develops after ingestion of contaminated, poorly cooked meat. Incubation period: 1–7 days Case-fatality: 25–90% (role of early antibiotic treatment is undefined) CORE SLIDE

33 Anthrax: Cutaneous Begins as a papule, progresses through a vesicular stage to a depressed black necrotic ulcer (eschar) Edema, redness, and/or necrosis without ulceration may occur Form most commonly encountered in naturally occurring cases Incubation period: 1–12 days Case-fatality: Without antibiotic treatment: 20% With antibiotic treatment: 1% CORE SLIDE By far the most common form of anthrax is the cutaneous form. This is true for both the naturally occurring form of the disease, whether acquired in an occupational setting or in the environment. Likewise, with the anthrax attacks that took place in the U.S. in the fall of 2001 there were actually more cases of cutaneous anthrax than there were cases of inhalational anthrax. This largely had to do with the degree and route of exposure to the anthrax letters. The cutaneous form of anthrax is certainly less severe, and much less likely to be fatal, compared to the inhalational form. Of the 23 cases of anthrax that resulted from the anthrax in the letters of September and October 2001 there were 12 cases of cutaneous anthrax and 11 cases of inhalational anthrax. In fact the first 4 cases of anthrax resulting from the letters were all cutaneous. There were no deaths associated with the cutaneous cases and there were 5 deaths associated with the 11 cases of inhalational anthrax. The importance of cutaneous anthrax in the setting of anthrax being used as a bio-terrorist weapon is the fact that the cutaneous cases of anthrax are likely to be recognized before someone recognizes cases of inhalational anthrax. This is because the initial symptoms of inhalational anthrax are non-specific. Eschar – the black scab that develops after the initial lesion ulcerates. The name Bacillus anthracis is derived from the Greek for black or coal, anthrakos.

34 Cutaneous Anthrax Hospital Day 5 Hospital Day 12 2 months after
CORE SLIDE Photo (Left): Armed Forces Institute of Pathology Photos: (Right): Abigail Freedman, et al: Journal of the American Medical Association (JAMA) February 20, 2002, 287: In this slide we see a series of pictures of the arm of the patient from the previous slide. We see that in the top picture (after the patient had been hospitalized for several days) we see a lesion that is obviously very reddened with a significant amount of swelling and the lesion is weeping from its center. In the middle photograph on the right, hospital day 12, we see the development of the characteristic black eschar of cutaneous anthrax. This is important to recognize because it may be the means by which the anthrax exposure in a particular workplace is recognized. Finally, the bottom photograph taken two months after the child was discharged shows the lesion is well healed with a permanent scar. Additionally, on the left portion of this slide we see anthrax on the neck of a worker with a cutaneous anthrax lesion after beginning antibiotic treatment. The anthrax eschar again we see as being a shiny black scab. Also please note the edges of the eschar are erythematous and raised. Another important distinction to make regarding cutaneous anthrax is that these initial lesions may itch but are typically not painful. 2 months after discharge JAMA. 2002;287:

35 Inhalational Anthrax Clinical Presentation
Incubation Period: 1-6 days A brief prodrome resembling a “viral-like” illness, characterized by muscle aches, fatigue, fever, with or without respiratory symptoms, nausea, vomiting, abdominal pain Early Symptoms: malaise, fever, fatigue, non-productive cough, chest discomfort Confusion, neck stiffness, and headache suggest meningitis (seen in 50% of patients) CORE SLIDE Inhalational anthrax is an infection and therefore has an incubation period. It is important to remember that, because they are infections, nearly all biological weapons will have an incubation period. Toxin weapons are an exception as they have preformed and active toxins. Inhalational anthrax’s initial prodrome is essentially a non-specific febrile illness. This is going to be a recurrent theme with most biological weapons, in that their initial presentation is not really specific enough to say they have a specific agent as the cause of their infection. This once again emphasizes the importance of recognizing cutaneous anthrax as it may lead to the diagnosis of other victims with inhalational anthrax. It is also important to note that in all workplaces where workers that contracted anthrax as a result of the anthrax letters, there were workers with cutaneous as well as inhalational anthrax. The prodrome essentially resembles a viral-like illness. Patients will complain of fever. As in the cases that occurred in the fall of 2001 the fever may be as high as oF. Patients often initially complain of respiratory symptoms, a non-productive cough later, shortness of breath, dyspnea (difficulty breathing), and chest pain. They may also complain of other more non-specific symptoms such as myalgias, fatigue, nausea, vomiting, abdominal pain, and malaise. As symptoms progress, more significant respiratory complaints develop, and patients may become significantly short of breath with complaints of air hunger (hypoxia). This is often the point at which they seek medical care. Inhalational anthrax is associated with anthrax meningitis in as many as 50% of cases. In fact, the first case of inhalational anthrax reported in the U.S. in October 2001, Mr. Robert Stevens at the AMI Building in Florida, had meningitis on presentation. Therefore, patients may present with initial complaints more suggestive of meningitis, including confusion, headache, neck stiffness, and change in mental status. This is important as well because this patient may not be able to provide a complete history do to confusion or coma, and a lumbar puncture (spinal tap) with cerebrospinal fluid analysis will reveal that they have anthrax meningitis.

36 Inhalational Anthrax Clinical Presentation
After initial onset of illness, symptoms may remain mild or even improve slightly before worsening Terminal Phase: dyspnea, stridor, cyanosis, shock, chest wall edema, meningitis, widened mediastinum with effusion with overall toxic/septic clinical picture CORE SLIDE As mentioned above, the clinical presentation of anthrax typically begins with non-specific, febrile symptoms. However, after these initial prodromal symptoms, a patient’s symptoms often do not progress, and may even improve somewhat. Mediastinum – the middle of the chest; the space between the lungs that contains the heart, trachea and other large airways to the lungs, the large blood vessels entering and leaving the heart, and the esophagus. Most importantly, this is where anthrax grows and multiplies after the spores have germinated in the lymph nodes found here. See the next slide for additional anatomy information about the mediastinum. Effusion – an effusion is a collection of fluid in the chest cavity around the lungs; the fluid collects between the lung and the chest wall, and often needs to be drained out of the chest by a needle or tubes inserted through the chest wall between the ribs. All inhalational anthrax victims of the 2001 bioterrorist attacks had effusions. Unfortunately, this period is followed by what is considered a “terminal phase” which includes the precipitous worsening of their respiratory status with severe dyspnea (difficulty breathing). They may also present with significant stridor (upper respiratory tract sound from compression of the trachea – sound like the “barky cough” seen in a child with croup) because inhalational anthrax will cause enlargement of the mediastinal lymph nodes (the lymph glands around the trachea and large airways) obstruction their breathing. As these people get increasingly ill they essentially develop a picture of a severely toxic and/or septic patient. Unfortunately, once they enter this terminal phase the disease is essentially 100% fatal and death typically follows within 24 hours. The edema toxin we talked about earlier may even cause edema and swelling of the entire chest wall as the multiplying anthrax bacteria produce and secrete larges amounts of anthrax toxins.

37 Presenting Symptoms Symptoms n=10 Fever, chills 10
Sweats, often drenching 7 Fatigue, malaise, lethargy Cough 9 Nausea or vomiting Dyspnea 8 Chest discomfort or pleurisy Myalgias 6 Headache 5 Confusion 4 Abdominal pain 3 Sore throat 2 Rhinorrhea 1 CORE SLIDE The presenting symptoms are similar to the ones we discussed earlier. As we see here, all patients had fever and/or chills, or other non-specific symptoms such as fatigue, malaise, and lethargy. Also common were respiratory symptoms of cough, dyspnea, as well as chest discomfort. But it is important to note that approximately ½ the patients had symptoms of headache and confusion consistent with the meningitis that we also mentioned earlier. Emerg Infect Dis vol.7, no. 6, 2001

38 Anthrax Diagnosis Clinical picture of sudden onset of respiratory distress with mediastinal widening on x-ray A small number of patients may present with GI or cutaneous anthrax Gram stain of blood and blood cultures - but these may be late findings in the course of the illness ELISA, FA, PCR and immunohistology testing may confirm diagnosis but samples must go to reference laboratory CORE SLIDE The diagnosis of anthrax is typically going to be made on a clinical basis. This means the patient who presents with symptoms, including those mentioned in the last slide, but also with a history that includes a high risk occupation or activity that would have exposed them to a bioterrorist attack. A good example of this would have been a patient that worked in a mail handling facility presenting with symptoms of cough and fever in October This combination of physical signs and symptoms and history should have been suspicious enough to consider inhalational anthrax in the differential diagnosis for this patient. Unfortunately, a fatal case of inhalational anthrax with just such a presentation went undiagnosed in October 2001. IMPORTANT: The first rule for making the correct diagnosis is to consider that diagnosis in one’s differential. Keep in mind, even though there was an aerosol release of anthrax (with the anthrax powder being aerosolized from the envelops) some patients may present with cutaneous or gastrointestinal symptoms. Finally, once the patient does reach a healthcare facility, the diagnostic options include gram stain of blood (or other body fluids or tissues) as well as his blood cultures and advanced laboratory testing can confirm the diagnosis of anthrax: ELISA = Enzyme Linked-Immunosorbant Assay FA = Fluorescent Antibody PCR = Polymerase Chain Reaction For first responders, such as paramedics or EMTs, the absolute diagnosis of anthrax is not essential for the proper prehospital management of these patients. The care of the patient with inhalational anthrax is simply to maintain an airway, oxygenation and ventilation, and fluid resuscitation. The ABCs are STILL the ABCs. Supplemental oxygen, IV access are also indicated. The patient with inhalational anthrax is not contagious and therefore there is no risk of contracting anthrax from these patients. That being said, if the patient is picked up from a locale where there was thought to be a release of anthrax the first responder is still at risk for exposure to that same anthrax. Furthermore, the patient with inhalational anthrax certainly will not likely be definitively identified in the pre-hospital setting. Therefore, until it is known exactly what biological agent one is dealing with, the first responder should consider other agents that may have high contagious rates such as plague and maintain strict universal precautions.

39 Anthrax Treatment Post-exposure Acute Treatment Oral prophylaxis
Ciprofloxacin (500 mg PO q12 h) X 60 days and until 3 doses of vaccine Doxycycline (100 mg PO q12 h) X 60 days and until 3 doses of vaccine Vaccination Acute Treatment Usually futile in severe mediastinitis patients who inhaled or ingested spores Ciprofloxacin mg IV q 8 to 12 hr Doxycycline mg IV q 12 hr Vaccination CORE SLIDE We have talked about a number of cases that have presented with inhalational anthrax. Let’s discuss treatment for a moment. Unfortunately if patients present with significant symptoms of advanced inhalational anthrax, the mortality rate is quite high (approaching 100%). Nonetheless, the drug of choice is Ciprofloxacin. Doxycycline is effective as well. Please keep in mind that some bioweapons-grade anthrax may have antibiotic resistance. It is important to note that the Ames strain used in the bioterrorist attack of 2001 indeed did not have antibiotic resistance introduced. It was, in fact, sensitive to penicillin, which is the initial drug of choice for naturally occurring cutaneous anthrax. Additionally, there are recommendations that a second antibiotic be added. Typically, one such as clindamycin, which inhibits protein synthesis, is recommended. This is intended to minimize the production of the exotoxins lethal factor and edema factor. It is also indicated to start an anthrax vaccine regimen at the beginning of drug therapy as well. Needless to say, additional treatment would include intensive care support and mechanical ventilation as needed. On the other hand, if the patient is exposed to anthrax but has not developed any symptoms of illness, prophylaxis with antibiotics is also indicated. Again the drugs of choice are Ciprofloxacin or Doxycycline. These should be started immediately as soon as exposure has been suspected and not when symptoms develop. The regimen for Ciprofloxacin is 500 mg orally twice a day for 60 days. Doxycycline is 100 mg orally twice a day, for 60 days. The 60-day regimen for prophylaxis was determined in part by the late presenting cases from Sverdlovsk. Therefore, a period of 60 days of prophylaxis should cover a patient for any latent germination of anthrax spores that would otherwise cause illness.

40 Anthrax Vaccine FDA approved 1970
Cell Free filtrate (NO organisms, dead or alive) Adverse effects 1-3% Bioport Corporation CORE SLIDE The anthrax vaccine marketed in the U.S. is a cell-free filtrate vaccine. There are no organisms in the vaccine and it is derived from the protective antigen exotoxin produced by anthrax. It has been FDA approved for over 30 years and is currently produced by the BioPort Corporation. It is an immunization regimen that includes 6 injections. The initial one injection, then 2 weeks, 4 weeks, and then at 6 months, 12 months, then 18 months. For this vaccine to remain effective however the patient will require yearly boosters. To the presenter: This is an animated slide. A graphic of the anthrax immunization course will enter fro the left 1 second after the slide appears For more information regarding the FDA-licensed anthrax vaccine, please see:

41 Laboratory Workers Increased need for universal precautions
Increased number of highly pathogenic bacterial and viral samples Increased need for universal precautions Increased need for security, including maintaining chain of custody for forensic samples Increased need for decontamination procedures Laboratory Response Network (LRN) CORE SLIDE Laboratory workers in the setting of a biological terrorist attack are faced with a number of new risks associated with their workplace. First, of all they are likely to be asked to test an increased number of highly pathogenic bacterial and/or viral specimens. The risk of this is two pronged in the fact that they may simply have an increased work load of having to process a large number of both biological (i.e. patient specimens such as body fluid cultures, biopsies) or environmental samples (e.g. samples from workplaces) for anthrax or other biological agents. Also, they will be asked to process more highly pathogenic organisms than they typically handle.    It seems relatively obvious that there is an increased need for adhering to principles of universal precautions. Respiratory, and dermal protection should always be employed. Additional attention should be given to the possibility of aerosolizing samples in procedures such as centrifugation of specimens. Also, with all bioterrorist events, there will be an increased need for security both of the lab itself, as well as the specimens in the lab. Keep in mind that terrorists may target microbiological labs themselves in an effort to cripple our response to a bioterrorist attack. Additionally, they may also be targeted as a site where terrorist can obtain samples of pathogenic organisms for weaponization. Additionally, it is of paramount importance that biological samples from bioterrorist events be treated as forensic samples. This means that laboratory staff should be very familiar with the concepts of chain of custody, and policies and procedures should be initiated to maintain a chain of custody, both for patient specimens as well as environmental samples. They should also be very familiar with shipping requirements of bioterrorism-related specimens. Finally, there is an increased need for more fastidious decontamination procedures for the laboratories. Again, the pathogenicity of likely bioterrorist agents may much higher than the routine biological organisms that are processed on a daily basis. The laboratory response network (LRN) is an important resource for any biological laboratory in responding to bioterrorism. The LRN is comprised of four different levels of laboratories. Level A being the most common lab such as the clinical laboratories found in most healthcare facilities. This level of lab is likely to see initial samples from patients. Level B labs are considered the next level up and are Public Health labs. They will serve as reference labs to the Level A labs and typically have more sophisticated testing modalities available. Level C labs are also Public Health labs but are again more specialized in technology and able to do organism typing. Finally the Level D lab is primarily comprised of the CDC. The Level A labs use biosafety Level 2 protocols and procedures. Their role is to detect cases early, to rule out a bioterrorist agent or refer to the next higher level of labs, the Level B labs. Level B labs are recommended to use a biosafety Level 3 procedures and protocols. They perform susceptibility testing for different isolates and again they are able to either rule in the presence of a bioterrorist agent or refer it to the next higher level lab. Level C labs (Public Health Labs with rapid identification) always work at biosafety Level 3. Again, they are able to rule in the presence of a bioterrorist agent or refer to the final and highest level of laboratory, Level D. Level D labs work at biosafety Level 4 labs and perform high level characterization of particular agents and are able to access, as well as establish and maintain, archives of data to unequivocally identify certain agents, including strains and possible genetic origins. In summary: Level A labs will rule out organisms or refer the testing to biolevel B labs; Level B labs rule in or refer to Level C; Level C either rule in or refer to Level D; and finally Level D will confirm and validate testing and maintain archives of the results. As the level A labs are the most numerous and, as mentioned above, are likely to receive many specimens for analysis. They will be responding to either covert or overt bioterrorist attacks. Therefore, it is their responsibility to be aware and alert for the possibility of bioterrorist attacks. They should have a plan that they test frequently and keep updated on how to handle specimens that are suspected bioterrorist organisms. Furthermore, they need to provide training for their staff, and should have policies and procedures in place to do so. They need to have a plan for consultation and/or referral to the next higher lab in the LRN. Keeping with our philosophy of maintaining an all hazards approach biological laboratories should incorporate their bioterrorist plans into their standard operating procedures. Additionally, all laboratories at all levels should maintain a fundamental understanding of microbiology safety. They should be aware of the various levels of biosafety labs that are indicated for the testing of certain organisms. For instance, Bacillus anthracis is considered a BSL 2 organism. However, should activities with high potential for aerosolization of these specimens is undertaken, then a BSL3 facility should be considered. Conversely, small pox or viral hemorrhagic fever samples should always be worked with in a BSL 4 lab. There are 81 labs in the LRN. See: Additional Reference: MMWR. 2002;51:482, April 5, Suspected Cutaneous Anthrax in a Laboratory Worker --- Texas, 2002 On April 5, 2002, CDC reported a case of suspected cutaneous anthrax in a worker at laboratory A who had been processing environmental samples for Bacillus anthracis in support of CDC investigations of the 2001 bioterrorist attacks in the United States.1 Since the initial report, the worker had serial serology performed at the CDC laboratory. A greater than fourfold rise from baseline in the concentration of immunoglobulin G to protective antigen was demonstrated. The peak antibody level was observed 7-8 weeks after the onset of symptoms, and the time course and levels of detectable antibodies were consistent with those seen in other cases of cutaneous anthrax. On the basis of case definitions developed during the recent investigation, these additional findings confirm this as a case of cutaneous anthrax.2 This case brings the number of anthrax cases identified in the United States since October 3, 2001, to 23, including 11 inhalation and 12 cutaneous (eight confirmed and four suspected). This is the first laboratory-acquired case of anthrax associated with the recent investigation. The epidemiologic and environmental investigation of this case indicated that the probable source of exposure was the surface of vials containing B. anthracis isolates that the worker had placed in a freezer. The storage vials had been sprayed with 70% isopropyl alcohol, which is not sporicidal, instead of a bleach solution because bleach had caused labels to become dislodged. The worker did not wear gloves when handling the vials. A culture of the vial tops performed at laboratory A tested positive for B. anthracis. The vial top specimen was confirmed positive for B. anthracis at CDC. Multiple-locus variable-number tandem repeat analysis found this isolate to be indistinguishable from the culture of the worker's clinical specimen. This case underscores the importance of safe laboratory procedures and anthrax vaccination for workers routinely handling B. anthracis isolates.3 A number of events during the past several years have served to focus attention on the threat of terrorism and the use of biological or chemical weapons against civilian populations.1 A single or sentinel act of bioterrorism, whether announced or unannounced, would initially be recognized at the local or state level, as would the initial public health response. The Centers for Disease Control and Prevention (CDC) was designated by the Department of Health and Human Services to prepare the nation's public health system to respond to such a bioterrorism event.2 An effective public health response would need to be timely because there is only a short window to provide prophylaxis or implement other control measures that are designed to minimize the number of casualties.3 To enhance local and state preparedness, the CDC funded cooperative agreements with states and several large cities that focused on preparedness activities.4 Five areas were emphasized during the first 3 years of this program ( ): preparedness planning and readiness assessment, surveillance and epidemiology capacity, biological laboratory capacity, chemical laboratory capacity, and health alert network and training. The recent mail-borne attacks using spores of Bacillus anthracis have made bioterrorism a reality in the United States.5,6 These attacks demonstrated the 2 types of scenarios characteristic of bioterrorism events: covert (unannounced) and overt (announced). An unannounced release of anthrax spores would likely go unnoticed, as happened with the index case, Mr. Stevens.7 Others co exposed in the Stevens attack left the area long before the act of terrorism became evident. The first signs that anthrax spores had been released did not become apparent until days later, when 2 individuals became ill and sought medical care. An astute clinician and laboratorian provided public health officials with initial clues that an unannounced attack had occurred. However, increased physician awareness of unusual signs and symptoms that may be associated with a bioterrorist attack is needed because 7 individuals with cutaneous or inhalational anthrax sought medical treatment in New York, New Jersey, and Florida before the index case was recognized.8 Because of their terrorism response training, traditional "first responders" (e.g., firefighters or law enforcement) are the most likely to respond to an announced attack, such as the letter received by Senator Tom Daschle's office,6 or to the numerous hoaxes that occurred during the same period. Therefore, the initial recognition of bioterrorism, whether unannounced or announced, would be at the local level and state level and would result in a comprehensive public health response involving epidemiological investigation, medical treatment and prophylaxis for affected persons, and initiation of disease prevention activities. The success of this response depends on, to a large extent, physician recognition and rapid and accurate identification of the threat agent. In this issue of Mayo Clinic Proceedings, articles by Varkey et al,9 Espy et al,10 and Uhl et al 11 highlight the need for physician awareness and laboratory preparedness. Many biological agents can cause illness in humans, but not all are capable of affecting public health and medical infrastructure on a large scale. To focus on public health preparedness activities, the CDC convened a meeting of national experts to review potential criteria for selecting the biological agents that posed the greatest threat to civilian populations.12 The list of "Critical Agents"2 was prioritized based on considerations such as (1) ability of the agent to cause mass casualties, (2) ability of the agent to be disseminated widely, (3) ability of the agent to be transmitted from person to person, (4) public perception associated with the intentional release of the agent, and (5) special public health preparedness needs based on stockpile requirements, enhanced surveillance, or diagnostic needs. As currently defined, Category A agents are those most likely to cause mass casualties if deliberately disseminated and require broad-based public health preparedness efforts. Such agents are responsible for anthrax, smallpox, tularemia, plague, botulism, and viral hemorrhagic fevers. In the United States, both clinical and laboratory experience are limited for Category A agents. Physician education is of paramount importance for early recognition of covert releases of biothreat agents. Several Web sites (e.g., ) and articles such as that by Varkey et al 9 provide practicing physicians with knowledge of the clinical presentation, diagnosis, and management of diseases caused by Category A agents. Physicians also need to be aware that biological agents can be introduced into a civilian population in several ways (e.g., aerosol, food contamination, water, animal vectors) and that clinical presentation can vary depending on the route of entry. Rapid laboratory identification of the biothreat agent is necessary to minimize the impact of a bioterrorist attack. However, as previously mentioned, laboratory experience with Category A agents is limited. Before October 2001, the low number of infections caused by these agents in the United States was given as a major reason why the commercial sector did not develop and manufacture specific Food and Drug Administration-approved diagnostic tests in this country (i.e., a small market). In addition, many of these agents pose risks to laboratory workers and must be handled at Biosafety Level 3 or 4.12 This situation has created the need for development and restricted distribution of biodetection assays and specialized reagents, regardless of the size of the market. Such market-independent actions are necessary to support the emergency public health response infrastructure and to meet the security interests of the United States. These assays and reagents are employed within the Laboratory Response Network (LRN) for Bioterrorism, which was developed by the CDC in concert with the Association of Public Health Laboratories and with collaboration from the Federal Bureau of Investigation, US Army Medical Research Institute of Infectious Diseases, Naval Medical Research Center, and Lawrence Livermore National Laboratory to detect and respond to agents that are released by a bioterrorist and those that occur naturally. This is particularly important because the dispersal mechanism (i.e., intentional vs natural) will generally not be known at the time of initial detection. The LRN is a multilevel system that will ultimately link state and some local public health laboratories with military, veterinary, agricultural, water, and food-testing laboratories. Currently, the LRN operates as a network of laboratories (laboratory levels designated A through D) with progressively stringent levels of safety, containment, and technical proficiency necessary to perform the essential rule-out, rule-in, and referral functions required for agent identification. Level A laboratories are, for the most part, the hospital and other clinical laboratories with certified biological safety cabinets that participate in the LRN by ruling out or referring the critical agents that they encounter in their routine work to the nearest Level B or C laboratory. However, before the clinical laboratory can fulfill this role it must address several issues: (1) knowledge of the current biosafety level within the laboratory; (2) familiarity with Level A protocols, which are available on the Internet at either or ; (3) knowledge of current guidelines to ensure safe handling and shipment of biological agents 13; (4) familiarity with protocols related to chain of custody, collection, preservation, and shipment of specimens; and (5) location of the nearest LRN Level B or C laboratory. Until rapid and reliable detection systems and diagnostic tests are available for the Level A laboratory, it must rely on conventional methods to obtain as much information in the least amount of time to rule out specific agents effectively. The article by Uhl et al 11 describes the use and advantages of real-time polymerase chain reaction (PCR) assays and their potential use in clinical laboratories, in an attempt to rapidly rule in or rule out infection by a likely bioterrorism infectious agent. Most clinical laboratories are incapable of processing specimens for the diagnosis of smallpox. The article by Espy et al 10 describes the observation that proper autoclaving of specimens can eliminate hazards associated with infectivity while the DNA of the microorganism remains detectable by PCR. Nucleic acid amplification assays, such as those described by Espy et al, have high analytic sensitivity and do not distinguish between viable and nonviable agents. Thus, care should be taken to avoid cross-contamination of negative, nonviable specimens during autoclaving. Laboratory Response Network Level B, C, and D laboratories have access to the biodetection assays and specialized reagents that are used in validated protocols for the confirmation of critical agents. Level B laboratories are primarily state and local public health laboratories that have Biosafety Level 2 facilities where Biosafety Level 3 practices are observed; Level C laboratories are primarily public health laboratories with Biosafety Level 3 facilities or certified animal facilities, which are necessary for performing the mouse toxicity assay for botulinum toxin. Level C laboratories can perform all Level B tests and additional tests requiring Biosafety Level 3 containment, such as those that involve the handling of powders suspected of containing anthrax spores. Level D laboratories are federal laboratories (i.e., CDC and US Army Medical Research Institute of Infectious Diseases) with the Biosafety Level 4 capacity to handle agents (e.g., Ebola and variola major) that other laboratories cannot handle. Level D laboratories have the capacity to perform all the Level B and C procedures. In addition, they can identify agents in specimens that have been referred by Level B or C laboratories, identify recombinant microorganisms that may not be recognizable with conventional isolation and identification methods, and maintain extensive culture collection of critical agents against which the isolate(s) from a bioterrorist event may be compared to determine its origin. The LRN became operational in August Since then, considerable effort has been expended to develop and validate rapid assays. Robust real-time PCR assays using TaqMan (Roche Diagnostics Corporation, Indianapolis, Ind) chemistry have been developed and optimized for several commercially available platforms including the LightCycler (Roche Applied Science, Indianapolis, Ind), SmartCycler (Cepheid, Sunnyvale, Calif), and the GeneAmp 5700 and PRISM 7700 (Applied Biosystems, Foster City, Calif). The effort expended in the development of these assays ensures that they will have the accuracy, reproducibility, sensitivity, and specificity that are required for use in the LRN. High-confidence real-time PCR assay development starts with the identification of primer pairs or signatures that are able to recognize unique regions of the target microorganism. These primer pairs should detect nucleic acid derived from any strain of the target organism but not react with nucleic acid from phylogenetically related organisms or organisms that constitute the culture or sample background and could be copurified from the sample. The selected primer pair-probe combinations, referred to as specific signatures, are used as a panel to identify the target agent. For agent identification, a specific algorithm must be used based on positive or negative reactions of the signature panel. The robustness of these assays has been evaluated through a multicenter validation study involving an average of 10 laboratories. Having confidence in the test results also requires well-trained laboratorians. The CDC recently completed six 1-week courses during which 1 person from each state public health laboratory and other selected LRN laboratories received training in real-time PCR assays. In summary, an effective public health response to a bioterrorism event will depend on a few key factors: the ability of medical professionals to rapidly recognize the clinical signs and symptoms of disease caused by a biothreat agent; the capability of laboratory professionals to rapidly detect and confirm the identity of the agent; an epidemiological investigation to determine the source of the infection; and, most importantly, communication and coordination among all the responders involved in the event. Richard F. Meyer, PhD Rapid Response and Advanced Technology Laboratory; Bioterrorism Preparedness and Response Program; National Center for Infectious Diseases Stephen A. Morse, MSPH, PhD Bioterrorism Preparedness and Response Program; National Center for Infectious Diseases References Tucker JB. Historical trends related to bioterrorism: an empirical analysis. Emerg Infect Dis. 1999;5: 2. Centers for Disease Control and Prevention. Biological and chemical terrorism: strategic plan for preparedness and response: recommendations of the CDC Strategic Planning Workgroup. MMWR Recomm Rep. 2000;49(RR-4):1-14. 3. Kaufmann AF, Meltzer MI, Schmid GP. The economic impact of a bioterrorist attack: are prevention and post attack intervention programs justifiable? Emerg Infect Dis. 1997;3:83-94. 4. Khan AS, Morse S, Lillibridge S. Public-health preparedness for biological terrorism in the USA. Lancet. 2000;356: 5. Centers for Disease Control and Prevention. Update: investigation of anthrax associated with intentional exposure and interim public health guidelines, October MMWR Morb Mortal Wkly Rep. 2001;50: 6. Centers for Disease Control and Prevention. Update: investigation of bioterrorism-related anthrax and interim guidelines for exposure management and antimicrobial therapy, October 2001 [published correction appears in MMWR Morb Mortal Wkly Rep. 2001;50:962]. MMWR Morb Mortal Wkly Rep. 2001;50: 7. Jernigan JA, Stephens DS, Ashford DA, et al. Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States. Emerg Infect Dis. 2001;7: 8. Lipton E, Johnson K. A nation challenged: the anthrax trail; tracking bioterror's tangled course. New York Times. December 26, 2001; sect A:1. 9. Varkey P, Poland GA, Cockerill FR III, Smith TF, Hagen PT. Confronting bioterrorism: physicians on the front line. Mayo Clin Proc. 2002;77: 10. Espy MJ, Uhl JR, Sloan LM, Rosenblatt JE, Cockerill FR III, Smith TF. Detection of vaccinia virus, herpes simplex virus, varicellazoster virus, and Bacillus anthracis DNA by LightCycler polymerase chain reaction after autoclaving: implications for biosafety of bioterrorism agents. Mayo Clin Proc. 2002;77: 11. Uhl JR, Bell CA, Sloan LM, et al. Application of rapid-cycle real-time polymerase chain reaction for the detection of microbial pathogens: the Mayo-Roche Rapid Anthrax Test. Mayo Clin Proc. 2002;77: 12. Rotz LD, Khan AS, Lillibridge SR, Ostroff SM, Hughes JM. Public health assessment of potential biological terrorism agents. Emerg Infect Dis. 2002;8: 13. Centers for Disease Control and Prevention, National Institutes of Health. Biosafety in Microbiological and Biomedical Laboratories, 4th ed. Washington, DC: US Government Printing Office; Available at: . Accessibility verified May 29, 2002.

42 Laboratory Workers Decontamination and Disinfection
Effective sporicidal solutions: Commercially-available bleach diluted to 0.5% Sodium hypochlorite (1 part household bleach to 9 parts water) Rinse off concentrated bleach to avoid caustic effects Approved sporicidal agents CORE SLIDE These are CDC Recommendations: As mentioned in the last slide, the need for effective decontamination and disinfection procedures are essential when working with biological warfare agents. The most cost effective and readily available sporicidal solution is commercially available bleach at a 1:10 dilution. This is made by simply mixing one part of household bleach with 9 parts of water (0.5% sodium hypochlorite solution). This can be used as a sporicidal and disinfecting agent for all surfaces. If dilute bleach is used on skin, it should be thoroughly rinsed off to avoid any caustic effects. Full strength bleach (5.0% sodium hypochlorite) can be used for decontaminating equipment. There are also numerous other sporicidal agents available that can be used with equal efficacy.

43 Section 4 Plague as a Biological Weapon
Objectives: To be able to describe the pathophysiology and epidemiology of plague. To be able to recognize and treat the different clinical forms of plague. To be able to control the secondary transmission of plague

44 Plague History 200,000,000 deaths Biblical (I Sam.) BC, Philistines Major Pandemics 541 - Plague of Justinian ‘Black Death’ Modern Pandemic CORE SLIDE Plague has been a part of human history for thousands of years. It has been estimated that over time plague has been responsible for in excess of 200 million deaths and is referenced as far back as 1320 B.C. There have been several major pandemics, including the plague of Justinian in 541, the first great pandemic of Europe, the Black Death in We are currently in what would be considered the modern pandemic that began in 1894.

45 Plague Distribution 1894 - Began in China 1898 - Southwest to India
South to Vietnam Trans-Pacific to United States CORE SLIDE This map represents an estimation of the present day distribution of plague in the world. As mentioned in the previous slide, plague is suspected to have originated in Central Asia, subsequently moved into Europe, and Africa. However, in the 1890s with the advent of steamships being able to cross the ocean at a much higher rate of speed, plague was introduced into the North American continent. In 1894, the present day pandemic spread, via overland routes, to Southwest India, Indochina, and the sub-continent of Asia. It was brought to the American west coast in 1900 via an infected Chinese sailor who made port in San Francisco. He did introduce plague to the city, however aggressive measures were taken and no large-scale epidemic ever developed. Plague continues to make its way eastward today. . Map reference: Known worldwide foci of human plague infection: Human plague in WHO Weekly Epidemiological Record. 1 Nov 1991;44:321–324. Human plague in WHO Weekly Epidemiological Record. 17 Feb 1995;7:45–48. Barkway J. World Health Organization, Geneva, Switzerland. Personal communication, February 1996.

46 Plague Epidemiology Vector: fleas, >80 species
Xenopsylla cheopis (Oriental rat flea) Fleas feed on plague-infected mammal Bacteria multiply in gut Coagulum blocks gut Plague organisms are regurgitated into bite wound with next feeding CORE SLIDE Photomicrograph: Ken Gage, Ph.D., Centers for Disease Control and Prevention, Fort Collins, CO A short discussion of the natural epidemiology of plague is helpful to understand plague it as a biological weapon. Plague is a vector-borne (which means it is transmitted by a biting insect) illness with fleas as the primary vector. Plague is a zoonotic (usually infects animals) disease that is maintained in an animal reservoir including rats, cats, prairie dogs. The Oriental rat flea, among many other flea species and other insects, is the number one vector between the animal reservoir and the human population. The oriental rat flea (Xenopsylla cheopis) has historically been most responsible for the spread of plague to humans. This flea has a proventriculus (equivalent to a human’s upper GI tract (esophagus and stomach)) blocked with a mass of blood and Yersinia pestis bacteria. Simply put, when the flea feeds on a plague-infected mammal, such as a rat, the flea ingests plague bacteria with their blood meal. These plague bacteria subsequently multiply in the gut of the flea creating a fibrinoid mass of blood and bacteria. If there ever was anything like as a ravenous flea, this flea would be it. A flea’s mouth has two functions: 1) Squirting saliva and partly digested blood into the bite, and 2) sucking up blood from the host. When the flea bites its next host, it regurgitates thousands of bacteria into the bite wound, thereby inoculating that animal with plague bacillus (Yersinis pestis). In this way the flea vector mechanically transmits the plague organisms between the animals within the mammalian reservoir, including humans. Photo: Ken Gage, Ph.D., CDC, Fort Collins, CO

47 Plague Epidemiology Reservoir: mammals, >200 species.
Rattus rattus (Black rat) Ground squirrels, prairie dogs, cats CORE SLIDE The mammal reservoir for plague is quite large, including an excess of 200 species. The most well known of these would be the rat. The rat is an important reservoir animal because it typically lives in close proximity to humans. The rat population of cities may often exceed the human population of that city. In the U.S. there have been numerous cases of ground squirrels, prairie dogs, as well as cats becoming reservoirs for the plague bacterium. In the state of Colorado there are numerous prairie dog colonies infested with plague.

48 Plague Pathogenesis Yersinia pestis - a Gram negative, nonmotile, nonsporulating bacteria Size: 0.5–0.8 × 1.5–2.0 µm Normally a disease of rodents Virulence Factors: antiphagocytic fraction 1 capsule, pH 6 antigen, antiphagocytic Yops H and E, V antigens, Yop M, and plasminogen activator CORE SLIDE The causative organism of plague is the Yersinia pestis bacterium. Yersinia pestis is a gram negative, non-motile, bipolar staining bacteria. This organism does not form a spore like anthrax. This very important fact makes it a much more difficult organism to weaponize. Nonetheless, this is what we would also call a zoonotic disease, like that of anthrax. Zoonotic meaning that it is normally a disease of animals, however, can also be a disease of humans. The plague bacillus has several virulence factors. These virulence factors include a capsules (layers that envelope the bacterium and protect it from the body’s immune system) as well as antigens (protein molecules that are attached to the outside of the organism) that prevent the organism from being killed by the host’s immune system. It also secretes other exotoxins, such as plasminogen activator that causes bleeding, increasing the severity of plague as an illness.

49 Plague Pathophysiology
Inoculation or inhalation (1-10 organisms) (100-20,000 organisms) Macrophage Lymphatics Lung Meninges Liver Spleen CORE SLIDE In this slide we see a simple schematic showing the typical pathophysiology of plague. It is important to note that plague can either be in the bubonic form or the pneumonic form. The bubonic form is the form of plague that is acquired from the bite of a plague-carrying vector such as the flea. The bubonic form is much more common than the pneumonic form. Bubonic plague is still endemic to a number of areas of the world including Asia and Africa. The pneumonic, or inhalational, form of plague is acquired via inhalation of aerosolized plague organisms. This is a naturally occurring form of plague but is also the likely form that will result from an aerosolized plague biological weapon. Upon inoculation, or introduction of the plague organism into the human host, these plague bacterium are phagocytized (engulfed) by macrophages. We discussed macrophages in the anthrax section of this learning module. Macrophages are white blood cells that circulate in tissue (e.g. lung, skin, abdominal organs), engulf foreign organisms, such as bacteria and viruses, and transport them to regional lymph nodes, where they can be acted upon by the immune system. Plague is no different from anthrax in this sense. Whether in tissue (in the case of bubonic plague), or in the lungs (in the case of pneumonic plague) macrophages will bring these organisms back to the lymph nodes via the lymphatic drainage. Once in the regional lymph nodes, Yersinia pestis undergo multiplication and initiate a localized infection. These enlarged lymph are usually in the groin or axilla (as flea bites are most often on the arm and legs), extremely tender, may reach several inches in diameter, and are called buboes. The infection then spreads hematogenously (via the blood) from these buboes to the rest of the body. An important thing to note here: when this hematogenous seeding reaches the lungs, the plague organism will grow in the sputum, and/or develop into a pneumonia. This is essentially the same as a case of primary pneumonic plague. Therefore, when this patient coughs, he can aerosolize plague and spread the infection. This bubonic to pneumonic to aerosolization route that plague can follow is why it is extremely contagious and can create epidemics involving millions. The infectious dose of plague can vary widely. The inoculation necessary for a bubonic form of plague may be as few as 10 organisms. The inoculation required for the pneumonic form is much higher, anywhere from 100 organisms to thousands of organisms. Again, the required inoculum (dose) to establish an infection varies with the virulence of the plague organisms itself, the efficiency of the delivery method and weaponization process, as well as the underlying health of the patient. In the United States, most patients (85%–90%) with human plague present clinically with the bubonic form, 10% to 15% with the primary septicemic form, and 1% with the pneumonic form. Secondary septicemic plague occurs in 23% of patients who present with bubonic plague, and secondary pneumonic plague occurs in 9%. If Y pestis were used as a biological warfare agent, the clinical manifestations of plague would be: epidemic pneumonia with blood-tinged sputum if aerosolized bacteria were used or (b) bubonic or septicemic plague, or both, if fleas were used as carriers. As few as 1 to 10 Y pestis organisms are sufficient to infect rodents and primates via the oral, intradermal, subcutaneous, and intravenous routes. Estimates of infectivity by the respiratory route for nonhuman primates vary from 100 to 20,000 organisms. Regional lymph nodes Blood

50 Bubonic Plague Clinical Presentation
Incubation 1-8 days (mode 3-5 days) Sudden onset of flu-like syndrome (Fever, rigors, malaise, myalgias, nausea) Bubo formation - within 24 hours Swollen, infected lymph node (very painful!) Cutaneous findings in 25% of cases Mortality: Untreated 60% Treated <5% CORE SLIDE Let’s discuss the clinical presentation of the bubonic form of plague for a moment. One may ask why would we be considering bubonic plague a form of bioterrorism as it is requires a flea vector to cause illness. There is an historical precedence for this. During WWII, the Japanese established Unit 731, under General Shiro Ishii, in Manchuria China. At a biological weapon development facility and prison, called Pinfan, countless Chinese civilian and prisoners-of-war were used to test biological weapons. These victims were infected with any pathogenic organism imaginable, and then subjected to study, including vivisection, by the Japanese military researchers. Under General Ishii’s direction they developed a biological weapon using plague delivered by infected fleas. Their weaponization techniques were rudimentary at best, and they could not weaponize a pure form of the Yersinia pestis that it could be delivered effectively. Therefore, they resorted to employing the natural vector of plague, the flea. On a number of occasions they released plague-infected fleas from low flying aircraft over villages in China, creating localized outbreaks of bubonic plague. After of these epidemics development, the Japanese would enter the area, purportedly to offer medical assistance, and would perform tests on the victims, or bring them back to Pingfan. Therefore, even though we tend to concentrate our discussion about aerosol biological weapons, terrorist may still use the natural vector(s) of a pathogenic organism to carry the bioweapon to its target and victims. The typical incubation period for bubonic plague ranges from 1-8 days, more typically within 3-5 days. After the initial flea bite there is a small area of infection localized to the site of the bite. However, it may be a relatively minor lesion. This is followed by the onset of a flu-like syndrome including fever, rigors, malaise, nausea, and muscle aches. Again, I want to emphasize that the early symptoms of many various biological agents are painfully nonspecific and may be difficult to differentiate between various bioweapons, or a naturally occurring disease. As mentioned earlier, once the organism is introduced via the fleabite, it is engulfed by macrophages and transported to the local lymph nodes via the lymphatic system. Subsequently these lymph nodes become extremely swollen and painful and are are referred to as buboes. These typically develop within 1 or 2 days following the initial bite. Subsequently these buboes may become quite enlarged, anywhere from a couple centimeters to 5-6 centimeters in diameter. If a person is bitten on the leg by a plague-infected flea these buboes will develop in the inguinal lymph nodes of that limb. Likewise, if they are bitten on the upper extremity they are likely to develop axillary lymphadenitis (buboes). Left untreated bubonic plague has a mortality rate of approximately 60%. This mortality rate can be reduced to <5% with appropriate antibiotic treatment. Keep in mind that these mortality rates and recovery rates are based upon naturally occurring plague and do not take into account potential molecular biological engineering that may increase the virulence of the plague organism (e.g. antibiotic resistant).

51 Bubonic Plague CORE SLIDE Photographs: Ken Gage, Ph.D., Centers for Disease Control and Prevention, Fort Collins, Colo. In this slide we see two examples of bubonic plague. The child on the left has a large inflamed lymph node in his right axilla, which is a buboe. The erythematous, eroded, crusting, necrotic ulcer on the child’s left upper quadrant is located at the presumed primary inoculation site, which occurs in anywhere from 4-10% of bubonic plague patients, is likely to be the primary inoculation site.   In the picture on the right we see a patient with an enlarged right inguinal lymph node. This patient was likely bitten and infected on his right lower extremity. Photographs: Ken Gage, Ph.D., Centers for Disease Control and Prevention, Fort Collins, CO

52 Pneumonic Plague Clinical Presentation
2 to 3 day incubation period followed by high fever, muscle aches, chills, headache Cough with bloody sputum within 24 hours pneumonia progresses rapidly – shortness of breath, stridor, cyanosis, difficulty breathing, chest pain respiratory failure, shock, bleeding In contrast to anthrax, Plague pneumonia and sepsis develop acutely and may be fulminant Patchy lung infiltrates or consolidation seen on chest x-ray CORE SLIDE Pneumonic plague may occur primarily, from inhalation of aerosols, or secondarily, from hematogenous dissemination. The clinical presentation of pneumonic plague is somewhat more rapid in onset compared to the bubonic form. The incubation period is somewhat shorter, approximately 2-3 days, as opposed to up to 8 days for bubonic plague. Typically this incubation period is followed by high fever, muscle aches, chills, sweats, and headache. Again, these are relatively non-specific symptoms. Due to its inhalational route of infection pneumonic plague presents very with early respiratory symptoms. Productive cough, often of bloody sputum, will develop within the first 24 hours. A frank pneumonia subsequently develops, associated with shortness of breath, stridor, chest pain, and cyanosis. Pneumonic plague progresses relatively rapidly, and may quickly be followed by respiratory failure, septic shock (low blood pressure due to overwhelming infection) and coagulopathy (abnormal blood clotting – usually leading to abnormal bleeding and bruising) leading to a bleeding diathesis. What this all means is that the pneumonic form of plague is a much more toxic form of plague, given that it is an infection of the chest and respiratory system. Patients typically become sicker, quicker, and as a result, have a much higher mortality rate. The untreated pneumonic plague victim faces approximately 100% mortality. In contrast to inhalational anthrax, the pneumonia that is see with pneumonic plague develops rather fulminantly with patients becoming ill extremely quickly and may actually progress straight to respiratory failure and death. Although pneumonia and pulmonary infiltrates can be seen with inhalational anthrax, they are not the most prominent feature of inhalational anthrax. However, pneumonia is the primary feature of pneumonic plague.

53 Pneumonic Plague CORE SLIDE This chest roentgenogram shows right middle-and lower-lobe involvement in a patient with pneumonic plague. Photograph: Ken Gage, Ph.D., Centers for Disease Control and Prevention, Fort Collins, CO. The findings on chest roentgenography may be variable, but bilateral alveolar infiltrates appear to be the most common finding in pneumonic plague. Photograph by Ken Gage, Ph.D., Centers of Disease Control and Prevention, Fort Collins, CO.

Plague Transmission Fleas (active or dormant) Aerosol PNEUMONIC Surface contact CORE SLIDE This slide is an important representation of plague transmission. Anthrax, although a deadly biological weapon, it is not communicable between patients. This is a very critical distinction between plague and anthrax. Conversely, plague is extremely contagious between patients. This is one of the primary reasons that it has created some of the great pandemics of history, killing millions of people at a time. On the left of this diagram we see a person who has been bitten by a flea. This person will develop bubonic plague. Once the infection spreads hematogenously (via the blood system) he will develop the septicemic (diffuse infection throughout the blood and organs of the entire body) form of plague. This means that the plague bacteria will travel through the entire blood supply of the person’s body, seeding various organs on the way. When the patient’s lungs are seeded, he will produce sputum that will contain the plague organism. Therefore, septicemic bubonic plague effectively creates the pneumonic form, albeit somewhat delayed from the time of the initial fleabite. Nonetheless, when this patient coughs, he will be aerosolizing plague in very small droplets. Should a nearby person inhale these droplets (middle person in diagram), that person will be exposed to these aerosolized plague organisms. As a result this second person will develop pneumonic plague. He can go on to cough and spread the organism as well (to right side person in diagram). This form of transmission will repeat itself as many times as there are susceptible people around the infected patient. It is via this route that plague is an extremely contagious infection and can become a catastrophic public health emergency. As mentioned above plague is different from anthrax. A single person can become infected with plague as a result of a biological weapons attack and go on to propagate that infection to many other individuals. Therefore the public health measures needed to contain plague are much more important. Any worker that has suspicion of a plague outbreak ANYWHERE should notify the appropriate medical and public health authorities immediately. Rodent BUBONIC and SEPTICEMIC SECONDARY PNEUMONIC and OROPHARYNGEAL

55 Plague Diagnosis Gram stain and culture of lymph node aspirates, sputum, or CSF samples Bipolar staining “Safety Pin” may be present Immunoassays are also available CORE SLIDE Photomicrograph: Ken Gage, Ph.D., Centers for Disease Control and Prevention, Fort Collins, CO. Further diagnosis of plague is usually done via laboratory studies. This is considered a highly contagious agent and should be dealt in a Biosafety Level 3 lab (BSL-3). One can gram stain and/or culture aspirates of any body fluid samples to grow the plague organism. Due to plague’s highly contagious nature, it is very important to notify one’s coworkers in the laboratory that if Y. pestis specimens are being sent to them for processing. Wright-Giemsa stain of body fluids with plague can demonstrate some characteristic staining features of the plague organism, Y.pestis. In this slide we see a picture of a gram stain of a peripheral blood smear from a patient with septicemic plague. Please look closely at the staining pattern of the small organisms seen on the slide. The larger blue and purple cells are white blood cells and the more orange appearing cells are red blood cells. The smallest appearing staining objects on the slide are Y. pestis organisms. Note that the staining is more pronounced at the ends of each bacterium. This is referred to as bipolar staining or a “safety pin” pattern. However, keep in mind that Y. pestis will not always demonstrate this staining pattern, and there are other organisms that may also demonstrate this staining pattern. Finally, there are numerous immunoassays and other laboratory studies that can be done to identify plague in a biological sample. This Wright-Giemsa stain of a peripheral blood smear from a patient with septicemic plague demonstrates the bipolar, safety-pin staining of Yersinia pestis. Gram’s and Wayson’s stains can also demonstrate this pattern. Photomicrograph: Ken Gage, Ph.D., Centers for Disease Control and Prevention, Fort Collins, CO.

56 Plague - Treatment Antibiotic Therapy:
Streptomycin (choice)15-30 mg/kg IM bid x 10 days Gentamicin - 2 mg/kg IV then mg/kg q8h or 5 mg/kg IV q24h x 10 days Doxycycline mg IV then mg bid x days Ciprofloxacin mg IV q12h x 10 days CORE SLIDE The treatment of choice for all forms of plague would be streptomycin. However, as a general rule streptomycin is no longer readily available. Therefore, other options include gentamicin IV as well as doxycycline and ciprofloxacin. Please recall that doxycycline as well as ciprofloxacin were recommended treatments for anthrax as well. This is not by accident as studies have been done to identify the most useful antibiotic to have on hand to treat multiple potential biological agents.

57 Plague Control of Secondary Transmission
Secondary transmission is possible and likely Standard, contact, and aerosol precautions for at least 48 hrs until sputum cultures are negative or pneumonic plague is excluded CORE SLIDE As mentioned earlier, plague is an extremely contagious and highly communicable disease. It is highly transmissible via aerosolization of Y. pestis by coughing. Stringent universal precautions (gloves, N95 face mask, eye protection) must be adhered to. A patient that presents with suspected pneumonic plague needs to be placed in respiratory isolation for a minimum of 48 hours of antibiotic treatment or until sputum cultures are negative for plague. In the interim all healthcare providers should adhere to strict universal precautions, which would include an appropriate respirator. The minimum recommended respirator would be a N95 OSHA-approved respirator. Recall that these respirators need to be fitted to the user and need to be kept on at all times. Furthermore strict body surface isolation should be adhered to with gowns, eye protection as well as gloves. It is important to emphasize that even though an individual patient may have an advanced form of pneumonic plague and treatment of that patient may indeed be futile, it is of paramount public health importance to prevent secondary cases of plague.

58 Section 5 Smallpox as a Biological Weapon
Objectives: To be able to describe the epidemiology and microbiology of smallpox To be able to recognize clinical smallpox To be able control the secondary transmission of smallpox To describe treatment and vaccination options for smallpox.

59 Smallpox The world’s first eradicated disease
1977- last endemic case in Somalia 1978- two laboratory cases in Britain 1980- WHO declares global eradication of smallpox CORE SLIDE Up until now we have been discussing anthrax and plague as biological weapons, both of which are bacteria. However, a number of viruses have also been weaponized as biological agents as well. The World Health Organization (WHO) declared small pox eradicated in This was after many decades of a concerted worldwide effort to immunize the world’s population against small pox. Contrary to the previous two agents discussed (anthrax and plague) small pox is not a zoonotic disease. With the exception of a few animal models that have been developed to study the virus in the laboratory, small pox is exclusively a disease of humans, and has no known animal reservoir. Nonetheless, the last endemic, or naturally occurring, cases of small pox were in Somalia in There have been no other cases of small pox reported since laboratory-associated (Note: these were occupational cases) cases in Subsequently, when the WHO declared small pox an eradicated disease in 1980, immunization programs were stopped. The U.S. stopped their general population immunizations in the 1972 and subsequently stopped immunizing the military in the late 1980s. Small pox was weaponized in the Biopreparat Biological Weapons Program of the former Soviet Union. Smallpox was chosen because it had characteristics that made it an effective biological weapon (highly contagious, infectious via the aerosolized route, no effective treatment) and, with the halting of large-scale immunization, the world’s populations were now susceptible. However, much like the 1979 Sverdlovsk anthrax outbreak, an accidental release, or poorly planned open-release test, of smallpox on Vozrozhdeniye island in the Aral sea lead to a small smallpox outbreak in Aralsk, in See below for additional discussion regarding this smallpox outbreak.

60 Smallpox Variola (Var-ï-óla) virus: an Orthopox virus, both minor and major forms of smallpox exist Structure is a large DNA virus Declared eradicated in 1980 and the U.S. stopped its civilian vaccination in 1981, U.S. military stopped in 1985 CORE SLIDE Smallpox is also known as the Variola virus. The variola virus is a member of the family Poxviridae, subfamily choropoxvirinae, and the genus orthopoxvirus. Smallpox is a large DNA virus; it is not as easily weaponized as anthrax, and requires additional measures to afford the virus the stability in the environment. Smallpox virus causes both minor and major forms of the disease smallpox. The poxviruses have a double stranded DNA genome.

61 Smallpox as a Bioweapon
1763- French & Indian War Fort Ticonderoga Lord Jeffrey Amherst World War II Unit 731 experiments in China Cold War USSR arsenal CORE SLIDE A brief review of the history of variola as a bioweapon would be useful here. Smallpox has been reported throughout the course of human history, causing episodic outbreaks, killing both rich and poor (many heads of states and noted figures of history died from smallpox). Its use as a biological weapon has been well documented. In 1763, Lord Jeffrey Amherst of the British Army utilized smallpox as a biological weapon against the Native American population of the New England area. They brought smallpox contaminated blankets from their smallpox infirmaries and gave them to the Native American tribes that had sided with the French during the French and Indian war. Needless to say this introduced smallpox into these communities, and wiping out whole Native American tribes. During World War II, the previously mentioned Unit 731 also performed experiments on the weaponization and use of smallpox as a biological weapon. In more recent history the former Soviet Union Biopreparat’s program spent a great deal of time weaponizing smallpox. In 1971 the Soviet Union was performing open-air experiments on smallpox as a biological weapon on Vozrozhdeniye Island. Vozrozhdeniye Island (also known as Rebirth island) is in the Aral Sea, lying between Kazakhstan and Uzbekistan. It was the Soviet Union’s equivalent of the American biological weapons program’s Dugway Proven Grounds in the desert of Utah. They performed open air testing of numerous biological pathogens on here including plague, anthrax, Q-fever, smallpox, tularemia, Venezuelan equine encephalitis, and botulin toxin. Typically, these agents would be aerosolized with test animals placed downwind. These experiments determined the effectiveness of the different bioweapons. As a result, there was a documented outbreak of smallpox as a result of open air testing of smallpox. As was learned from the anthrax release from Compound 19 at Sverdlovsk, the down wind range of an aerosolized biological weapon can be surprising long. Indeed, there was an open-air release of smallpox in 1971 that resulted in an outbreak of smallpox in the city of Aralsk, Kazakhstan. Between August and October of 1971 a total of 10 cases of smallpox were recorded, three of them being fatal. Given the fact that endemic smallpox had been eradicated from the Soviet Union in the 1930s and the last reported case of “imported” smallpox had occurred approximately 10 years earlier, it seems likely that these infections and deaths were the results of an open air biological weapons test by the Soviet Union gone awry. Reference: The 1971 smallpox epidemic in Aralsk, Kazakkstan, and the Soviet biological warfare program. Occasional paperno. 9, Jonathan B. Tucker and Raymond A. Zilinskas. Chemical and Biological Weapons Nonproliferation Program Center for Nonproliferation Studies Monterey Institute of International Studies 460 Pierce Street Monterey, California USA Tel: Fax: CNS Occasional Papers are available online at: Vozrozhdeniye Island is now somewhat of a worrisome legacy of the biological warfare era. Due to a number of factors the Aral Sea that surrounds Vozrozhdeniye Island has been shrinking. This has led the gradual development of a land bridge between the island and the mainland. This introduces the possibility that a biological warfare agent(s) tested there many years ago could be viable that be carried to the local region by animals or human visitation. It also introduces the possibility of terrorists gaining access to this island and acquiring samples of discarded stocks of biological weapons. It is well known that the Soviet Union has disposed of literally tons of anthrax spores on Vozrozhdeniye Island. Although decontamination procedures were done, and the island has seen a significant increase in security, the risk of a biological agent remaining viable and being vulnerable to theft by terrorists is significant. The risk is similar to the risk of biowarfare expertise, and possibly biological samples, being made available to terrorist organizations by former employees of the Biopreparat program who emigrated after the breakup of the U.S.S.R. and the demise of Biopreparat.

62 Why would smallpox Make A Good Biological Weapon?
Infectious via aerosol Vaccination discontinued Decreased potency of vaccine stocks Severe morbidity and mortality Transmissible Clinical inexperience “Brand-name” recognition CORE SLIDE Why would smallpox make a viable bioweapons today? It is infectious as an aerosol, the Soviet Union has shown that it can be effectively weaponized, and be deadly, as in the Aralsk smallpox outbreak of This is also in addition to the fact that Variola is still very infectious in the natural form of smallpox. A terrorist who can carry out his bioterrorist attack by infecting himself with Variola virus, and simply be traveling, working, or living among his victims spreading smallpox. Secondly, after decades of worldwide immunization, the program was discontinued approximately 30 years ago, leaving a large portion of the population unimmunized. Furthermore, it has been found that a large percentage of those who had been immunized against smallpox in their youth no longer have competent immunity against a smallpox challenge. This means that the entire general population is susceptible to smallpox. The U.S. has initiated a program to acquire several hundred million doses of smallpox vaccine in an effort to protect the entire American population. Smallpox as an illness carries with it severe morbidity and a high rate of mortality, making it an effective and very lethal weapon. It is extremely transmissible via an aerosol route. We often talk of weaponization of these agents into aerosols. As mentioned above, in the age of suicide bombers, a terrorist could infect himself with smallpox and then circulate among thousands of potential infectious contacts (e.g. commercial air travel, attending large events, visiting shopping malls) that may subsequently infect others, leading to a large epidemic. Furthermore, due to the fact that smallpox has not been seen for many years, most clinicians are relatively inexperienced in recognizing the disease, treating it, and managing the public health concern. Finally, when we talk of terrorist agents we often have to remember that terrorism is indeed a means of frightening and/or “terrorizing” an entire population. A good example of this is the fact that the anthrax attacks that took place in the U.S. in 2001 infected a total of 23 patients and caused five deaths. However, as many media poles showed at the time, a significant percentage of people were quite frightened by the possibility of becoming infected. Smallpox is at least as well known as anthrax, has a history of causing large epidemics, and is without a known cure. These factors, and others, combine to make smallpox a devastating bioweapon, as well as an effective terrorist weapon.

63 Clinical Smallpox Prodrome
Incubation 7-17 days (mean 12) Infection of respiratory mucosa Minor viremia: seeding of liver, spleen Major viremia: seeding of skin Acute onset fever, rigors, headache, vomiting Virus cultured from blood CORE SLIDE Smallpox, like other infectious bioweapons, requires an incubation period. During the incubation period of smallpox there is no viral shedding. During this time period the patient looks and feels relatively healthy and cannot infect others. The prodrome (symptomatic period after the incubation period) of smallpox is typically going to be a viral-syndrome-like febrile illness. Patients typically have fever with rigors and/or sweats, headache, back pain, nausea, vomiting, and severe malaise. The incubation period ranges between 7 and 17 days, with a mean of approximately 12 days. The 17 days, as we will learn later, is extremely critical when isolating potentially exposed patients. Infectious contacts of smallpox cases require strict isolation for a minimum of 17 days to insure that they will not develop smallpox and are not a risk to infect others. Smallpox begins as an infection of the respiratory mucosa by the variola virus. Patients will then develop a minor viremia. During this time there is rapid replication of the smallpox virus, with hematogenous seeding (minor viremia) of the organs, including the liver and spleen. This is similar to the period of bacteria replication with plague and/or anthrax infections. Within 2-3 days the temperature will fall and the patient will feel somewhat improved. A major viremia, marked by the development of the characteristic rash of smallpox, will then develop. The rash eruption is synchronous (all lesions appear at once). As mentioned above, viral seeding during the prodrome occurs hematogenously. Therefore, there should be a good viral load in the blood at this point and we can culture virus from a patient’s blood. However, a low viral load may yield a falsely negative viral culture making it difficult rule out smallpox early in the disease course. Isolation for 17 days and observation for the development of rash and fever are still recommended before “ruling out” variola infection.

64 Clinical Smallpox Enanthem Exanthem begins on face, hands, forearms
spreads to lower extremities centrifugal distribution Macules  papules  vesicles  pustules  scabs/crusts  scars CORE SLIDE An enanthem (rash on mucus membranes) primarily in the oropharynx including the mouth, tongue, and throat, begins towards the end of the prodrome phase. This typically precedes the exanthem (skin rash) by approximately a day or two. The smallpox rash typically begins as small reddish macules (flat red spots), with a diameter of approximately 2-3 mm, over the period of 1-2 days. They develop in a centrifugal (away from the center) distribution primarily on the head and neck, face, hands and forearms. Subsequently, they will include the lower extremities and the trunk. They remain sparser on the trunk compared to the extremities, head and neck. After several more days these initial papules will become vesicles (a small, raised, well-circumscribed fluid-filled blister) increasing in diameter to 2-5 mm. Approximately 4-7 days after the onset of the rash the rash will progress to slightly larger pustules (a small, raised, well-circumscribed pus-containing vesicle). As the rash progresses, these pustules will become very tense, and will be tender and painful. They will go umbilication (pustules develop a central depression resembling an umbilicus [belly button]) and crusting. Finally, after crusting the rash will undergo desquamation (shedding) of the dried up lesions. Approximately 65-80% of the survivors will have permanent pitted scars at the site of their pockmarks. Note: There are some variants of the smallpox rash that will be discussed later.

65 Breman & Henderson, NEJM, 346(17), April, 2002
CORE SLIDE Clinical Manifestations and Pathogenesis of Smallpox and the Immune Response. In this slide we see an extremely helpful chart, published in an excellent review on the recognition and management of smallpox by Breman and Henderson, outlining the onset of fever, the progression of the rash, the development of viremia, as well as the infectiousness over the time course of a smallpox illness. The interested reader is referred to the reference for this paper to fully review the clinical and pathophysiological features as well as management of clinical smallpox. However, in summary, this chart can be interpreted as to the following: Panel A at the top demonstrates the progression of the incubation infection and prodromal phases. Subsequently it shows photographs documenting the progression of the rash. Your attention is drawn to the red line through the pictures that shows the temperature of the patient. Note that it is highest and acutely so during the prodromal phase. At the bottom of panel A there is a relative time course demonstrating the length of each phase. Panel B of this diagram we see an artist’s illustration demonstrating the development of viremia after inhalational exposure. The first picture we see the inhalational exposure followed by the seeding of the respiratory mucosa and the infection of the regional lymph nodes. This is subsequently followed by a minor viremia with seeding of the spleen, liver, and reticuloendothelial system. Finally we see a major viremia develop coinciding with the prodromal phase causing the subsequent rash. On the far right we see both a photograph of the skin showing the characteristic lesions of smallpox, as well as a micrograph showing the pathological changes at the level of the dermis. Panel C is an illustration of the body’s immune response to smallpox. More importantly, however, we also see the infectiousness illustrated by the increased amount of oropharyngeal secretions beginning at approximately a few days before the prodromal phase. As you will note the oropharyngeal secretions are most significant within a few days of the prodromal phase and a few days of the development of the rash. Reference: JOEL G. BREMAN, M.D., D.T.P.H., D.A. HENDERSON, M.D., M.P.H. Diagnosis and Management of Smallpox. New England Journal of Medicine, Vol. 346, No. 17, pages , April 25, 2002. Panel A shows the initial phases of infection and the clinical manifestations, which include temperature spikes and progressive skin lesions (photographs of lesions courtesy of Dr. David Heymann, World Health Organization). Panel B shows the pathogenesis of the infection. The photographs at the right-hand side of the panel show the characteristic features of the vesicles caused by smallpox. (Photo reference: Strano AJ. Smallpox. In: Binford CH, Conner DH, eds. Pathology of tropical and extraordinary diseases: an atlas. Vol. 1. Washington, D.C.: Armed Forces Institute of pathology, 1976:65-7. Panel C shows the immune response to smallpox and the period of infectiousness. HI denotes hemagglutination inhibition, and CF complement fixation. Breman & Henderson, NEJM, 346(17), April, 2002

66 Smallpox Day 3 of Rash CORE SLIDE In the next three slides we see photographic documentation of the progression of the rash. All of these photos are from the WHO publication entitled “Smallpox and Its Eradication”. This publication is available online via the communicable disease surveillance and response page of the WHO web site. The web address is: The interested reader is referred to this web site for the complete text that documents the worldwide eradication of smallpox as well as numerous other sources of information. The interested reader is also referred to the WHO web site: The discussion of these slides is relatively self-evident. On day 3 we see the development of the rash in a very minor form on this child. On day 5 we see progression of this rash from the papule lesions to the pustular lesions. Finally by day 7 we see that the lesions have become umbilicated (pustules develop a central depression resembling an umbilicus). Also note that these photos demonstrate the centrifugal distribution of this rash, primarily on the head, neck, and extremities of this child. Although the trunk is not prominently featured in these pictures it is evident, especially on day 7 of the rash, that the lesions are more densely distributed to the face and arms of this child as opposed to the chest. This series of photographs illustrates the evolution of skin lesions in an unvaccinated infant with the classic form of variola major. (a) The third day of rash shows synchronous eruption of skin lesions; some are becoming vesiculated. (b) On the fifth day of rash, almost all papules are vesicular or pustular. (c) On the seventh day of rash, many lesions are umbilicated, and all lesions are in the same general stage of development. Photographs: From Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and Its Eradication. Geneva, Switzerland: World Health Organization; 1988: 10–14. Photographs by I. Arita. From: Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and Its Eradication. Geneva, Switzerland: World Health Organization; 1988: 10–14. Photographs by I. Arita.

67 Smallpox Day 5 of Rash CORE SLIDE From: Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and Its Eradication. Geneva, Switzerland: World Health Organization; 1988: 10–14. Photographs by I. Arita.

68 Smallpox Day 7 of Rash CORE SLIDE From: Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and Its Eradication. Geneva, Switzerland: World Health Organization; 1988: 10–14. Photographs by I. Arita.

69 Smallpox CORE SLIDE In this we see an adult with smallpox. This rash is in the advanced stages of the pustular phase. The important thing to note on this photo is again the centrifugal distribution of the rash. Also note that all the lesions are of the same general appearance. This is an important means to differentiate them from other illnesses that cause a vesicular rash such as chicken pox. Chicken pox lesions appear and develop in crops, so that there will be several “ages” of lesions on a patient at a given point. Also note the centrifugal distribution of the rash.

70 Smallpox Clinical Forms
Variola Major 30% fatal in unvaccinateds 3% fatal in vaccinateds Variola Minor Flat-Type Smallpox Hemorrhagic Smallpox Modified-Type Smallpox Variola Sine Eruptione CORE SLIDE As mentioned earlier, there are several forms of clinical smallpox. Thus far we have only discussed the variola major form. There is a “rule of threes” that is conventionally taught when referring to smallpox. Approximately 1/3 of the patients exposed to smallpox will acquire the illness and approximately 1/3 of those who acquired the illness will die. Bear in mind that nearly all of our experience with smallpox is via the natural route of infection, which is by aerosolized oropharyngeal secretions from an infected individual, and do not take into account the possible weaponization and mass aerosolization of a smallpox weapon. The rule of threes has been traditionally taught but there is some thought that the fatality rate may indeed be higher and will certainly vary with the underlying health and age of the patient. Unfortunately patients who have been vaccinated do not enjoy 100% protection. Even though the 1/3 fatality rate drops to approximately 3% in patients who have been vaccinated against smallpox, they still have a risk of death. The other forms of smallpox include variola minor, which has a similar rash, although much less significant rash and a much less toxic illness. Flat type smallpox and hemorrhagic, are mentioned above. Modified type smallpox variola sine eruptions are thought to occur in patients with partial immunity. Although most patients will usually acquire the variola major form, still a significant number may have one of these variants. The variola minor smallpox is considered to be a more mild strain of smallpox. This underscores the importance of the strain of the biological agent used. Consistent with this milder strain is a relatively low mortality in the 1% range. The flat type and hemorrhagic smallpox have fatality rates approaches 100%. However it is unclear as to who are susceptible for these variants. Finally, the modified type and sine eruption variants are thought to occur in partially immune individuals and are rarely fatal.

71 Variola Minor CORE SLIDE Variola minor, or alastrim, was distinguished by milder systemic toxicity and more diminutive pox lesions In this photo we see a patient with variola minor. The important thing to note on this photograph is the relatively sparse distribution of lesions on this patient’s face. Furthermore she is not a terribly ill appearing patient, with much milder systemic toxicity. This patient, although certainly infected with smallpox, may be more likely to travel and spread smallpox. From: Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and Its Eradication. Geneva, Switzerland: World Health Organization; 1988: 10–14. Photographs by I. Arita.

72 Flat-type Smallpox CORE SLIDE Here we see a photo showing flat type smallpox in an unvaccinated woman on the sixth day of rash. Although relatively rare, occurring in less than 5% of patients, it is accompanied by severe systemic toxicity and a relatively atypical rash. The initial rash progresses to these soft, flattened focal lesions, often confluent (distributed on the skin in a very dense pattern) instead of the vesicular (blister) phase. However, the rash typically does not complete the entire course of its evolution, as it is typically fatal before enough time passes for this progression to take place. Flat-type smallpox, noted in 2% to 5% of patients, was typified by (a) severe systemic toxicity and (b) the slow evolution of flat, soft, focal skin lesions Source: Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and Its Eradication. Geneva, Switzerland: World Health Organization; 1988: 33. Photographs by F. Dekking. From Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and Its Eradication. Geneva, Switzerland: World Health Organization; 1988: 33. Photograph by F. Dekking

73 Hemorrhagic Smallpox CORE SLIDE Here we see a photo of hemorrhagic smallpox lesions. Again, this is a relatively rare form of smallpox occurring in fewer than 3% of patients. However, important signs of the development of the hemorrhagic form of smallpox include the appearance of petechia (bleeding in the skin) as well as mucosal hemorrhages. The patient typically is extremely ill and usually dies, again, before the usual progression of the smallpox rash through to the pustular and desquamation phases. Hemorrhagic-type smallpox, seen in fewer than 3% of patients, was heralded by the appearance of extensive petechiae, mucosal hemorrhage, and intense toxemia; death usually intervened before the development of typical pox lesions. Early hemorrhagic-type smallpox with cutaneous signs of hemorrhagic diathesis. Death usually intervened before the complete evolution of pox lesions. Reference: Herrlich A, Mayr A, Munz E, Rodenwaldt E. Die pocken; Erreger, Epidemiologie und klinisches Bild. 2nd ed. Stuttgart, Germany: Thieme; In: Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and Its Eradication. Geneva, Switzerland: World Health Organization; 1988: 35. From Herrlich A, Mayr A, Munz E, Rodenwaldt E. Die pocken; Erreger, Epidemiologie und klinisches Bild. 2nd ed. Stuttgart, Germany: Thieme; 1967.

74 Smallpox vs. Chickenpox
Variola Varicella Incubation 7-17 days 14-21 days Prodrome 2-4 days minimal Distribution centrifugal centripetal Evolution synchronous asynchronous Scabs Form 10-14 days 4-7 days Scabs Separate 14-28 days <14 days Infectivity separation scabbing CORE SLIDE This is a comparison between the characteristic rashes of smallpox and chickenpox. This is a valuable comparison because, despite the other potential viral agents out there, chickenpox is still the most common, and therefore most likely disease, which would present in a fashion similar to smallpox. As we see with smallpox the incubation period as mentioned earlier is 7-17 days, where chickenpox is somewhat longer, up to 3 weeks. Smallpox’s prodrome is much more significant (sicker patient overall) than that of chickenpox. A very important characteristic is the distribution of the rash, being centrifugal (more on the extremities and head and neck) versus centripetal (more concentrated on the trunk) with varicella. The next important characteristic that can distinguish these two rashes is the fact that the smallpox lesions all appear synchronously. That is, they all appear at the same time and they go through the progression at the same pace, such that they all appear relatively uniform. In contrast, chickenpox lesions appear in what we would term “crops” because they develop asynchronously, with new lesions appearing over time. On examination you would notice a large variability in the ages and appearance of chickenpox lesions. Finally, the scabs typically form after 10 days to approximately 2 weeks with chickenpox, whereas with smallpox they typically form within a week. Likewise, the scabs separate between 2-4 weeks with smallpox and usually are done separating within 2 weeks with chickenpox. The most important distinction here is that the patient smallpox remains infectious until the separation of the scabs of the healing lesions. A smallpox patient will no longer be infectious once all of their scab lesions separate. In contrast, chickenpox lesions are infectious until they have scabbed. The patient with chickenpox is no longer infectious and may still have lesions that are scabbed over on their skin. For smallpox, the person is infectious from the onset of their enanthem until all scabs separate. Chickenpox is infectious from one day before the eruption of the rash until all vesicles scab.

75 Smallpox Management of Contacts
Immediate vaccination or boosting VIG 0.6 ml/kg Pregnant patients Dermatoses patients ?? Normal hosts Limited data: Vaccine + VIG better than vaccine alone? STRICT quarantine x 17 days CORE SLIDE As mentioned above, any case of smallpox represents a public health emergency. Management of all contacts (persons exposed to a person with smallpox) is important. Immediate vaccination, or boosting of a previous vaccination, is indicated. As mentioned earlier, the use of vaccinia immunoglobulin is also indicated especially for patients who have cannot safely be vaccinated, as pregnant patients, patients with a history of dermatoses, or patients with immunosuppression. Ultimately, the most important public health measure for the treatment of contacts is strict quarantine for a minimum of 17 days. This will not treat smallpox should they develop it, but it will limit the further spread of the disease and limit the outbreak and/or epidemic.

76 Vaccination Employs Vaccinia virus Given by scarification
One dose protective for 5-10 years Must keep vaccinia immunoglobulin (VIG) on hand to treat complications of vaccination CORE SLIDE Finally, vaccinia virus, another member of the orthopox genus of the poxvirus family, was used for vaccination. Indeed, the term vaccination comes from the name of this virus, vaccinia. It is related to the cowpox virus that was used to afford some immunity to smallpox. Vaccinia vaccination is given by a process of scarification. Scarification is a process of introducing the vaccinia virus into the superficial layers of skin via small punctures. Although it initially was thought that vaccination against smallpox would afford lifelong immunity, it has been found that immunity after a single vaccination wanes after approximately 10 years. Vaccination is not a risk free procedure, as we will discuss shortly. Vaccinia immunoglobulin is essentially a vaccinia anti-serum with antibodies against vaccinia virus. It provides “passive immunity” (the ability for an immediate immune response against a virus). It is used to treat patients with complications of immunization.

77 Complications of Vaccination
Normal host Inadvertent Autoinoculation (skin, eye) Generalized vaccinia Erythema multiforme Encephalitis Pregnancy - fetal vaccinia Dermatoses/Burns - eczema vaccinatum Immunocompromised - vaccinia necrosum CORE SLIDE However, vaccination with vaccinia virus is not without complications. In a normal healthy host there can be inadvertent autoinoculation (transferring the vaccinia virus from the vaccination site to another body site such as the eye), generalized vaccinia (overwhelming vaccinia infection), erythema multiforme, and vaccinia encephalitis. Populations with increased risk include pregnancy, which may lead to fetal vaccinia (the vaccinia infection of the fetus); patients with a history of dermatoses such as eczema and widespread burns can develop eczema vaccinatum; and immunocompromised patients can develop vaccinia necrosum (severe localized reaction to vaccinia inoculation causing invasive tissue reaction and tissue necrosis). Approximately one-third of vaccinations complication occur in contacts of vaccinated persons (e.g. family members).

78 Ocular Vaccinia CORE SLIDE This complication can cause corneal scarring and hence visual impairment. This was due to the inadvertent autoinoculation of that eye with vaccinia virus from an immunization site. Ocular vaccinia should be treated aggressively with a topical antiviral drug under close ophthalmological supervision. Untreated, this can lead to corneal scarring and therefore visual impairment. Reference: Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and Its Eradication. Geneva, Switzerland: World Health Organization; 1988: 298. Photograph by C. H. Kempe. From Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and Its Eradication. Geneva, Switzerland: World Health Organization; 1988: 298. Photograph by C. H. Kempe

79 Vaccinia Necrosum CORE SLIDE In this slide we see a child with vaccinia necrosum (severe reaction to vaccinia inoculation causing an invasive infection and localized tissue necrosis). Unfortunately, this child had an unrecognized congenital defect in cell-mediated immunity and underwent vaccination. The vaccinia vaccination went unchecked by the patient’s impaired immune system, developed into an overwhelming vaccinia infection with destruction of tissue at the inoculation site. Unfortunately, with the case fatality rate in excess of 75%, any patient with a history of any kind of cell-mediated immunodeficiency (e.g. AIDS, HIV infection) could not be safely vaccinated. Reference: Progressive vaccinia (vaccinia gangrenosum), which was fatal, in a child with a congenital defect in cell-mediated immunity. Progressive vaccinia or vaccinia necrosum. As seen in this child, progressive viral replication at the inoculation site in an immunocompromised individual leads to inexorable local tissue destruction. This complication occurred almost exclusively in persons with cellular immunodeficiency,1 with a case fatality rate of higher than 75%.2 1 - Fulginiti VA, Kempe CH, Hathaway WE, et al. Progressive vaccinia in immunologically deficient individuals. Birth Defects. 1968;4:129–145. 2- Freed ER, Richard JD, Escobar MR. Vaccinia necrosum and its relationship to impaired immunologic responsiveness. Am J Med. 1972;52:411–420. From: Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and Its Eradication. Geneva, Switzerland: World Health Organization; 1988: 298. Photograph by C. H. Kempe. From Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and Its Eradication. Geneva, Switzerland: World Health Organization; 1988: 298. Photograph by C. H. Kempe

80 Eczema Vaccinatum N Engl J Med, Vol. 346, No. 17, April 25, 2002
CORE SLIDE Here we see an example of eczema vaccinatum. In this patient we see a 27-year-old gentleman who was approximately 10 days post vaccinated against smallpox. He developed severe disseminated rash with severe facial lesions consistent with disseminated vaccinia. Subsequently, the patient did well after being treated with vaccinia and immunoglobulin. See attached reference. Reference: Eczema Vaccinatum — A Timely Reminder Ten days after being vaccinated against smallpox, a 27-year-old man was hospitalized with a high fever, facial edema, and an umbilicated, vesicular, crusting rash on his face, neck, upper chest, and hands (Panel A). He had a history of atopic dermatitis. Eczema vaccinatum was diagnosed. As the vesicular rash became disseminated, supraglottic edema with shortness of breath developed. The patient was transferred to the intensive care unit and received vaccinia immune globulin and supportive care. Over the next two weeks extensive crusting of the facial lesions occurred (Panel B). Cultures of vesicular fluid grew vaccinia virus, and skin biopsy showed necrotic epidermal cells with intranuclear inclusions compatible with the diagnosis of eczema vaccinatum. Three weeks after admission, the patient was discharged, with deep facial and chest scars. Eczema vaccinatum is a rare complication of smallpox vaccination. It develops in patients with a history of eczema who are given the vaccine or who are in close contact with vaccinated persons. ALLONE. MOSES, M.D. RONIT COHEN-PORADOSU, M.D. Hadassah University Medical Center Copyright © 2002 Massachusetts Medical Society. Jerusalem 91120, Israel New England Journal of Medicine, Vol. 346, No. 17, April 25, 2002 This article was published at on March 28, 2002. N Engl J Med, Vol. 346, No. 17, April 25, 2002

81 Smallpox Therapy Public health emergency Supportive care
Vaccinia Immunoglobulin Strict quarantine until scabs off At least 17 days Codofovir CORE SLIDE Smallpox therapy is not limited to purely vaccination. The first and most important order of business would be to declare a public health emergency to limit the spread of the disease. As mentioned earlier, smallpox is an extremely dangerous and communicable disease and therefore public health measures must be instituted EARLY to be effective. However, the care of the individual smallpox patient is primarily supportive and essentially intensive care unit (ICU) care. The use of vaccinia immunoglobulin is essential in all patients with complications of vaccination. As mentioned in the last slide, the patient was treated with vaccinia immunoglobulin to treat eczema vaccinatum. As mentioned earlier, the public health situation is extremely important, and whatever care the patient receives must be done in strict quarantine until he is well and no longer infectious. Cidofovir has been investigated as a potential medication for the treatment of smallpox. It is an antiviral agent, active against DNA viruses by inhibiting viral DNA replication. It has received FDA approval for treatment of cytomegalovirus retinitis in persons with AIDS. Its toxicities include nephrotoxicity (kidney damage), and neutropenia (decreased white blood cell count). Additional Information: Cidofovir is Active Against DNA Viruses •Nucleotide analogue, inhibits viral DNA polymerase by competitive inhibition of dCTP •Phosphorylated to active diphosphateform by host cellular enzymes •Cidofovir diphosphate-choline adduct –T½ = 87 hours –intracellular reservoir –weekly administration •FDA approved for treatment of CMV retinitis in persons with AIDS Cidofovir Dosing and Toxicity •Usual Dose = 5 mg/kg, intravenously, once weekly •Cleared in kidneys •Toxicities: –Nephrotoxicity, with proteinuria and↑creatinine - Neutropenia Reference: Lea AP, Bryson HM. Cidofovir. Drugs 1996;52:

82 Section 6 Other Viruses as Biological Weapons
Objectives: To become familiar with viral hemorrhagic fever viruses (VHFs) and Venezuelan equine encephalitis virus pathophysiology To be familiar with necessary PPE to able to limit the secondary spread of VHF To be able to treat victims of these biological agents

83 Viral Hemorrhagic Fevers Microbiology
RNA viruses causing high fevers and generalized vascular damage Filoviruses (Ebola, Marburg) Human infections by insect bites or by contact with blood and body fluids CORE SLIDE In this slide in the upper photograph we see a photomicrograph of the Marburg virus. The viral hemorrhagic fevers (VHFs) is a family of RNA viruses (viruses that use RNA, instead of DNA, to store their genetic information) that that have a predilection for the vascular system. There are many viral families, such as the filoviruses, which include the Ebola and Marburg viruses. In their natural form these viruses are contracted from an insect bite, or by contact with blood or body fluids from an infected individual. Lower Photograph: Source: Textbook of Military Medicine, Chapter 29, Viral Hemorrhagic Fevers, page 595: The picture of the arm is shows massive cutaneous ecchymosis associated with late-stage Crimean-Congo hemorrhagic fever virus infection, 7 to 10 days after clinical onset. Ecchymosis is indicative of multiple abnormalities in the coagulation system, coupled with loss of vascular integrity. Epistaxis and profuse bleeding from puncture sites, hematemesis, melena, and hematuria often accompany spreading ecchymosis, which may occur anywhere on the body as a result of needlesticks or other minor trauma. The sharply demarcated proximal border of this patient’s lesion is not explained. Photograph: Courtesy of Robert Swanepoel, PhD, DTVM, MRCVS, National Institute of Virology, Sandringham, South Africa. Additional Reference: Textbook of Military Medicine, Chapter 29, Viral Hemorrhagic Fevers, page 594: The Filoviridae includes the causative agents of Ebola and Marburg hemorrhagic fevers. These filoviruses have an exotic, threadlike appearance when observed via electron microscopy. Marburg virus was first recognized in 1967 when a lethal epidemic of VHF occurred in Marburg, Germany, among laboratory workers exposed to the blood and tissues of African green monkeys that had been imported from Uganda; secondary transmission to medical personnel and family members also occurred.10 In all, 31 patients became infected, 9 of whom died. Subsequently, Marburg virus has been associated with sporadic, isolated, usually fatal cases among residents and travelers in southeast Africa.11      Ebola viruses are taxonomically related to Marburg viruses; they were first recognized in association with explosive outbreaks that occurred almost simultaneously in 1976 in small communities in Zaire12 and Sudan.13 Significant secondary transmission occurred through reuse of unsterilized needles and syringes and nosocomial contacts. These independent outbreaks involved serologically distinct viral strains. The Ebola–Zaire outbreak involved 277 cases and 257 deaths (92% mortality), while the Ebola–Sudan outbreak involved 280 cases and 148 deaths (53% mortality). Sporadic cases occurred subsequently. In 1989, a third strain of Ebola virus appeared in Reston, Virginia, in association with an outbreak of VHF among cynomolgus monkeys imported to the United States from the Philippines.14 Hundreds of monkeys were infected (with high mortality) but no human cases occurred, although four animal caretakers seroconverted without overt disease. Recently, small outbreaks involving new strains of Ebola virus occurred in human populations in Côte d’Ivorie in 1994 and Gabon in 1995; a larger outbreak involving the Ebola-Zaire strain involved more than 300 people, with 75% mortality, in Zaire in Very little is known about the natural history of any of the filoviruses. Animal reservoirs and arthropod vectors have been aggressively sought without success. Photograph: Robert Swanepoel, PhD, DTVM, MRCVS, National Institute of Virology, Sandringham, South Africa.

84 Viral Hemorrhagic Fevers (VHFs)
RNA viruses causing high fevers and generalized vascular damage May be spread by aerosol, on fomites, and by oral secretions and eye drainage in animals Human infections by contact with blood and body fluids CORE SLIDE As mentioned earlier, viral hemorrhagic fever viruses are typically RNA viruses. They are associated with a febrile illness, and, as most viruses have a predilection for a certain cell type, viral hemorrhagic viruses have a predilection for the vasculature of a patient (i.e., the VHF viruses preferentially infect cells found in the blood and blood vessels, causing bleeding). They are highly communicable (contagious) and may be spread by aerosol (such as when a person coughs bloody sputum) or on fomites (furniture or contaminated medical equipment). Also they can be spread by bites from animals. Although most human infections are typically due to contact with the blood or body fluids of patients with this illness, the precise reservoir in the natural environment that causing natural outbreaks is unknown.

85 VHF Pathogenesis Fever, muscle aches, prostration
Cases evolve into shock and generalized mucous membrane hemorrhage Conjunctival injection, petechial hemorrhage, and hypotension Abnormal kidney and liver function tests  poor prognosis Mortality varies; % Ebola Zaire Disease severity and survival depends on various host factors; target organ is the blood vessel system. CORE SLIDE As mentioned above VHF infection begins with fevers, muscle aches, and rapid onset of a prostration and toxic illness. The cases rapidly evolve to have significant hypotension (shock), mucous membrane hemorrhage, as well as conjunctival injection and hemorrhage (bleeding of the eye tissue) and hemorrhage. The skin is notable for a maculopapular rash associated with petechial hemorrhages (very small spots of bleeding into the skin) and confluent ecchymoses (bruises). Full-blown cases will evolve into shock and generalized mucous membrane hemorrhage with involvement of the respiratory, bone marrow, and central nervous systems. Patients who develop evidence of significant liver damage or renal failure have a very poor prognosis. Overall, mortality varies widely between these different VHF illnesses, and even within an illness, with approximately 80% fatalities. Mortality rates range between 50 and 80 percent for strains of Ebola. Mortality rate and survival depends heavily on the underlying health of the host, as well as the availability of critical care medicine and supportive care such as blood products and transfusions. Additional Reference: Textbook of Military Medicine, Chapter 29, Viral Hemorrhagic Fevers, page : CLINICAL FEATURES OF THE VIRAL HEMORRHAGIC FEVER SYNDROME      The VHF syndrome develops to varying degrees in patients infected with these viruses. The exact nature of the disease depends on viral virulence and strain characteristics, routes of exposure, dose, and host factors. For example, dengue hemorrhagic fever is typically seen only in patients previously exposed to heterologous dengue serotypes.19 The target organ in the VHF syndrome is the vascular bed; correspondingly, the dominant clinical features are usually a consequence of microvascular damage and changes in vascular permeability.20 Common presenting complaints are fever, myalgia, and prostration; clinical examination may reveal only conjunctival injection, mild hypotension, flushing, and petechial hemorrhages. Full-blown VHF typically evolves to shock and generalized bleeding from the mucous membranes, and often is accompanied by evidence of neurological, hematopoietic, or pulmonary involvement. Hepatic involvement is common, but a clinical picture dominated by jaundice and other evidence of hepatic failure is seen in only a small percentage patients with Rift Valley fever, Crimean-Congo hemorrhagic fever, Marburg hemorrhagic fever, Ebola hemorrhagic fever, and yellow fever. Renal failure is proportional to cardiovascular compromise, except in HFRS caused by hantaviruses, where it is an integral part of the disease process; oliguria is a prominent feature of the acutely ill patient.8 VHF mortality may be substantial, ranging from 5% to 20% or higher in recognized cases. Ebola outbreaks in Africa have had particularly high case fatality rates, from 50% up to 90%.12,13      The clinical characteristics of the various VHFs are somewhat variable. For Lassa fever patients, hemorrhagic manifestations are not pronounced, and neurological complications are infrequent, occurring only late and in only the most severely ill group. Deafness is a frequent sequela of severe Lassa fever. For the South American arenaviruses, (Argentine and Bolivian hemorrhagic fevers), neurological and hemorrhagic manifestations are much more prominent. RVF virus is primarily hepatotropic; hemorrhagic disease is seen in only a small proportion of cases. In recent outbreaks in Egypt, retinitis was a frequently reported component of Rift Valley fever.21      Unlike Rift Valley fever, where hemorrhage is not prominent, Crimean-Congo hemorrhagic fever infection is usually associated with profound disseminated intravascular coagulation (DIC) (Figure 29-1). Patients with Crimean-Congo hemorrhagic fever may bleed profusely; and since this occurs during the acute, viremic phase, contact with the blood of an infected patient is a special concern: a number of nosocomial outbreaks have been associated with C-CHV virus.      The picture for diseases caused by hantaviruses is evolving, especially now in the context of HPS syndrome. The pathogenesis of HFRS may be somewhat different; immunopathological events seem to be a major factor. When patients present with HFRS, they are typically oliguric. Surprisingly, the oliguria occurs while the patient’s viremia is resolving and they are mounting a demonstrable antibody response. This has practical significance in that renal dialysis can be started with relative safety.      For the diseases caused by filoviruses, little clinical data from human outbreaks exist. Although mortality is high, outbreaks are rare and sporadic. Marburg and Ebola viruses produce prominent maculopapular rashes, and DIC is a major factor in their pathogenesis. Therefore, treatment of the DIC should be considered, if practicable, for these patients.      Among the flaviviruses, yellow fever virus is, of course, hepatotropic: black vomit caused by hematemesis has been associated with this disease. Patients with yellow fever develop clinical jaundice and die with something comparable to hepatorenal syndrome. Dengue hemorrhagic fever and shock are uncommon, life-threatening complications of dengue, and are thought—especially in children—to result from an immunopathological mechanism triggered by sequential infections with different dengue viral serotypes.19 Although this is the general epidemiological pattern, dengue virus may also rarely cause hemorrhagic fever in adults and in primary infections.22 DIAGNOSIS      The natural distribution and circulation of VHF agents are geographically restricted and mechanistically linked with the ecology of the reservoir species and vectors. Therefore, a high index of suspicion and elicitation of a detailed travel history are critical in making the diagnosis of VHF. Patients with arenaviral or hantaviral infections often recall having seen rodents during the presumed incubation period, but, since the viruses are spread to humans by aerosolized excreta or environmental contamination, actual contact with the reservoir is not necessary. Large mosquito populations are common during the seasons when RVF virus and the flaviviruses are transmitted, but a history of mosquito bite is sufficiently common to be of little assistance in making a diagnosis, whereas tick bites or nosocomial exposure are of some significance when Crimean-Congo hemorrhagic fever is suspected. History of exposure to animals in slaughterhouses should raise suspicions of Rift Valley fever and Crimean-Congo hemorrhagic fever in a patient with VHF. When large numbers of military personnel present with VHF manifestations in the same geographical area over a short period of time, medical personnel should suspect either a natural outbreak (in an endemic setting) or possibly a biowarfare attack (particularly if the virus causing the VHF is not endemic to the area).      VHF should be suspected in any patient presenting with a severe febrile illness and evidence of vascular involvement (subnormal blood pressure, postural hypotension, petechiae, hemorrhagic diathesis, flushing of the face and chest, nondependent edema) who has traveled to an area where the etiologic virus is known to occur, or where intelligence suggests a biological warfare threat. Signs and symptoms suggesting additional organ system involvement are common (headache, photophobia, pharyngitis, cough, nausea or vomiting, diarrhea, constipation, abdominal pain, hyperesthesia, dizziness, confusion, tremor), but they rarely dominate the picture. A macular eruption occurs in most patients who have Marburg and Ebola hemorrhagic fevers; this clinical manifestation is of diagnostic importance.      Laboratory findings can be helpful, although they vary from disease to disease and summarization is difficult. Leukopenia may be suggestive, but in some patients, white blood cell counts may be normal or even elevated. Thrombocytopenia is a component of most VHF diseases, but to a varying extent. In some, platelet counts may be near normal, and platelet function tests are required to explain the bleeding diathesis. A positive tourniquet test has been particularly useful in diagnosing dengue hemorrhagic fever, but this sign may be associated with other hemorrhagic fevers as well. Proteinuria or hematuria or both are common in VHF, and their absence virtually rules out Argentine hemorrhagic fever, Bolivian hemorrhagic fever, and hantaviral infections. Hematocrits are usually normal, and if there is sufficient loss of vascular integrity perhaps mixed with dehydration, hematocrits may be increased. Liver enzymes such as aspartate aminotransferase (AST) are frequently elevated. VHF viruses are not primarily hepatotropic, but livers are involved and an elevated AST may help to distinguish VHF from a simple febrile disease.    For much of the world, the major differential diagnosis is malaria. It must be borne in mind that parasitemia in patients partially immune to malaria does not prove that symptoms are due to malaria. Typhoid fever and rickettsial and leptospiral diseases are major confounding infections; nontyphoidal salmonellosis, shigellosis, relapsing fever, fulminant hepatitis, and meningococcemia are some of the other important diagnoses to exclude. Ascertaining the etiology of DIC is usually surrounded by confusion. Any condition leading to DIC could be mistaken for diseases such as acute leukemia, lupus erythematosus, idiopathic or thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome.      Definitive diagnosis in an individual case rests on specific virological diagnosis. Most patients have readily detectable viremia at presentation (the exception is those with hantaviral infections). Infectious virus and viral antigens can be detected and identified by a number of assays using fresh or frozen serum or plasma samples. Likewise, early immunoglobulin (Ig M) antibody responses to the VHF-causing agents can be detected by enzyme-linked immunosorbent assays (ELISA), often during the acute illness. Diagnosis by viral cultivation and identification requires 3 to 10 days for most (longer for the hantaviruses); and, with the exception of dengue, specialized microbiologic containment is required for safe handling of these viruses.23 Appropriate precautions should be observed in collection, handling, shipping, and processing of diagnostic samples.24 Both the Centers for Disease Control and Prevention (CDC, Atlanta, Georgia.) and the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID, Fort Detrick, Frederick, Maryland.) have diagnostic laboratories operating at the maximum Biosafety Level (BL-4; see Chapter 19, The U.S. Biological Warfare and Biological Defense Programs, for further discussion of BLs). Viral isolation should not be attempted without BL-4 containment.      In contrast, most antigen-capture and antibody-detection ELISAs for these agents can be performed with samples that have been inactivated by treatment with b-propiolactone (BPL).25 Likewise, diagnostic tests based on reverse transcriptase polymerase chain reaction (RT-PCR) technology are safely performed on samples following RNA extraction using chloroform and methanol. RT-PCR has been successfully applied to the real-time diagnosis of most of the VHF agents.26,27 When isolation of the infectious virus is difficult or impractical, RT-PCR has proven to be extremely valuable; for example, with HPS, where the agent was recognized by PCR months before it was finally isolated in culture.9      When the identity of a VHF agent is totally unknown, isolation in cell culture and direct visualization by electron microscopy, followed by immunological identification by immunohistochemical techniques is often successful.14 Immunohistochemical techniques are also useful for retrospective diagnosis using formalin-fixed tissues, where viral antigens can be detected and identified using batteries of specific immune sera and monoclonal antibodies. Although intensive efforts are being directed toward the development of simple, qualitative tests for rapid diagnosis in the field, definitive diagnosis for these diseases today requires, at a minimum, an ELISA capability coupled with specialized immunological reagents, supplemented (ideally) with an RT-PCR capability. Ebola and Marburg Virus Genomic Structure, Comparative and Molecular Biology Provided by John Crowley (B.S.) and Ted Crusberg (Ph.D.) Dept. of Biology & Biotechnology, Worcester Polytechnic Institute, Worcester MA 01609 Ebola is a member of the negative-stranded RNA virus family Filoviridae. These filoviruses (Ebola, Marburg and Reston) are very similar in morphology, density and sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) profile (Klenk, 1994). The particles are pleomorphic, meaning they can exist in many shapes. Their basic structure is long and filamentious, essentially bacilliform, but the viruses often takes on a "U" shape, and the particles can be up to 14,000 nm in length and average 80 nm in diameter. The virus consists of a nucleocapsid, surrounded by a cross-striated helical capsid. There is an axial channel in the nucleocapsid, and the whole virion is surrounded by a lipoprotein unit derived from the host cell. In addition, there are 7 nm spikes placed 10 nm apart visible on the surface of the virion. The genome consists of a single negative strand of RNA that is non-infectious itself, non-polyadenylated, with a linear arrangement of genes, with some occurrence of overlap. The order is: 3'-untranslated region nucleoprotein viral structural protein VP35 VP40 glycoprotein VP30 VP24 Polymerase (L) 5'-untranslated region Once inside the cell (mechanism not yet known) the virus transcribes its RNA and replicates in the cytoplasm of the infected cell. Replication is mediated by the synthesis of an antisense positive RNA strand what will serve as template for additional viral genomes. As the infection progresses the cytoplasm of the infected cell develops "prominent inclusion bodies" that contain the viral nucleocapsid, which will become highly structured. The virus then assembles, and buds off the host cell, attaining its lipoprotein coat from the infected cell's outer membrane. The transcriptional start site was determined to be at base 54 (3'-UACUCCUUCUAAUU-). The stop site was identified due to its sequence homology with the Sendai virus polyadentlation (transcriptional signaling) site and its position after the open reading frame for the nucleoprotein gene: (3'-UAAUUCUUUUUU). The location of these sequences determined that there are long non-coding sequences within the nucleoprotein gene itself, 417 bp at the 5'-end and 341 bp at the 3'-end. The coding region begins with two AUG codons and ends with a UGA stop codon. The first protein is predicted to have 739 amino acids, and 83.3 KDa molecular weight, lower than that observed by PAGE. A Kyte-Doolittle analysis of the predicted amino acid sequence yields a definitive hydrophobic N-terminal region, and a hydrophilic C-terminal end. Transcripts from the cloned gene run against natural viral mRNA on acid-urea-agarose gels were identical. Translated proteins from wild type and cloned genes were also identical on SDS-PAGE. Sanchez et al (1993) has published the sequence of the complete genome of Ebola virus and determined the gene order to be 3'-NP-VP35-VP40-GP-VP30-VP24-L. Three areas of overlap occur in the genome, that average 18 bp in length. The first overlap is between the VP35 and VP40 genes, the second between GP and VP30 and the third between VP24 and L. These overlaps are limited to the conserved sequences determined for the transcriptional signals. In addition there are three non-coding sequences between VP30 and VP24. Except for the start site the L gene (RNA dependent RNA polymerase) has yet to be completely sequenced. For the Ebola (+) leader RNA sequence a potential stem-loop structure is possible, and may play a role in gene expression (perhaps by altering ribosome binding). For example the hairpin shape of the (+) leader strand may be conducive to nucleoprotein binding, and the subsequent conformational change may either promote replication of transcription. Northern blot analysis performed on all transcripts yielded appropriate length mRNAs except for the glycoprotein gene (GP). It was found that the GP gene produces both a full-length transcript and a shorter mRNA derived from an atypical transcriptional stop sequence located in the middle coding region of the gene. The protein produced by this transcript would appear to have no particular purpose, and may not be tolerated within infected cells. Aligning the gene sequences of the Ebola and Marburg viruses along side one another and comparing protein products from the two viruses show similarities although no immunological cross reactivity exists: Ebola and Marburg genomes are both very large containing 3' and 5' non-coding regions. Both genomes contain overlaps that consist of the transcriptional start and stop signals, although Ebola has three and Marburg has only one. Their positions in relation to the intergenic regions also differ. The intergeneic regions are not conserved in either genome, and both contain one unusually lengthy region (>94 bp) All the polyadenylation sites contain (3'-UAAUU) except for the VP40 gene of the Marburg virus Both viruses produce mRNA that can form stem-loop structures Ebola GP gene produces two transcripts but Marburg produces only one The Marburg virus does not contain the polyadenylation sequence that is found in the Ebola GP gene. The proteins of Ebola and Marburg are likewise similar. The C-terminal end of the GP proteins of both viruses are 80.9% homologous and are comparable to the same protein found in oncogeneic retroviruses. This may provide some insight into the pathogenicity of this family of viruses, as synthetic peptides of this gene yield a highly compromised immune system, leading to an inhibition of blastogenesis of lymphocytes, a reduced chemotactiic ability of monocytes and macrophages and inhibition of natural killer (NK) cells. Other similarities in the GP protein include a variable hydrophilic central region flanked by hydrophobic regions that contain most of the cycfteins used in disulfide bridges. The sequences of the VP40 and VP24 proteins show that they are primarily hydrophobic, and may be membrane-associated. VP40 may be a matrix protein because of its net positive charge and similarity to like proteins from other viruses. VP30 is presumed to be a second nucleoprotein, base on its amino acid sequence and proposed structure. Elliott et al.,(1993) has reported on the amino acid composition of VP35 and VP40. VP35 is the second protein synthesized after the nucleoprotein so there is a distinct possibility that it may be a non-structural protein involved in transcriptional events. Unlike regulatory proteins VP35 is not phosphorylated. VP30 which on the other hand is phosphorylated is often found tightly bound to nucleoprotein, providing strong evidence that it too is in fact a nucleoprotein The Marburg virus GP is acylated by myristic and palmitic acids (Funke, et al., 1995) in an insect cell culture system, with cystein residues acting as the sites for acylation. In Marburg virus acylation appears to play a role in receptor binding and fusion activities. References Funke, C., et al., Acylation of the Marburg Virus Glycoprotein, Virology 208, (1995). Sanchez, A., et al. (1993), Sequence analysis of the Ebola virus genome: organization, genetic elements and comparison with the genome of Marburg virus, Virus Res. 29, (1993). Klenk, H.D., et al. (1994), Marburg and Ebola Viruses, Encyclopedia of Virology, Vol. 2, last modified May 18, 1995

86 Ebola Virus 1976 - First reported case in Sudan
Reston, VA health facility among imported monkeys April Ebola epidemic Kikwit, Zaire Ebola outbreak in Alice, TX - monkeys Gabon patient infection transferred to Johannesburg clinic healthcare worker 50 to 80% mortality rate in humans - extensive hemorrhage, shock, and end organ failure 2002 – Gabon – most recent outbreak CORE SLIDE Ebola virus. I will take a minute to discuss Ebola virus as it has been one of the better-known viral hemorrhagic fever viruses. The first case of naturally occurring Ebola virus infection was in Sudan in There have been repeated outbreaks of Ebola in Africa over the years, including a highly publicized outbreak in Kikwit, Zaire in The Ebola outbreak in Kikwit is a good example of the importance of universal precautions to limit the progression of an outbreak such as this. This outbreak was essentially out of control until the WHO and CDC representatives went to the site and instituted strict universal precautions as well as other critical care medicine to halt the outbreak. The variability in virulence between strains is well illustrated in an outbreak in Reston, Virginia in Imported green monkeys from the Philippines were found to have an Ebola virus infection. However, they were not ill. In fact, animal handlers who were exposed to these monkeys did not develop any clinical manifestations of Ebola infection. Most recently there has been an outbreak of Ebola in Gabon, Africa this year. It progressed over the course of several months and finally was controlled.

87 VHF Treatment Blood pressure resuscitation and monitoring
Careful fluid management Use of colloids (e.g. plasma) Vasopressors and inotropes Cautious sedation and analgesia No anti-platelet drugs or IM injections Coagulation studies and replacement of clotting factors, platelet transfusions CORE SLIDE The treatment of a patient with viral hemorrhagic fevers is essentially critical care medicine. In the prehospital, or out-of-hospital setting, limiting any procedures (like intravenous lines and other injections) and minimizing movement will limit bleeding. Encourage oral fluid intake if patient is able to drink and not vomiting. Supportive critical care medicine includes volume resuscitation (intravenous fluids to prevent dehydration) and blood products (such as blood and platelet transfusions and clotting factors) as needed. Medications to increase blood pressure (vasopressors and inotropes) can be used. Additional treatment includes pain medication and sedation, both to make the patient comfortable, as well as to limit movement and potential hemorrhages. Common sense interventions include minimizing, or avoiding, any kind of injections, or moving of the patient. Overall, the treatment goal for a viral hemorrhagic fever victim is to limit any further bleeding, ensure adequate fluid resuscitation, with transfusions as necessary. Additional Reference Material From: CDC: Management of patients with suspected viral hemorrhagic fever. MMWR 37(Supplement 3):1-16, Treatment is largely supportive and typically requires intensive care monitoring to avoid fluid overload (pulmonary edema) while maintaining hemodynamic stability and providing appropriate comfort measures (sedation, pain medication). Systemic coagulopathy should be treated in manner similar to DIC. Ribavirin is an antiviral (available only on an IND basis) medication that has been used in therapy and prophylaxis for Lassa fever, Hemorrhagic Fever with Renal Syndrome, and CCHF. The only available vaccine is against yellow fever. Other vaccines are currently under investigation. Additional Reference: Textbook of Military Medicine, Chapter 29, Viral Hemorrhagic Fevers, page : MEDICAL MANAGEMENT      Patients with VHF syndrome require close supervision, and some will require intensive care. Since the pathogenesis of VHF is not entirely understood and availability of specific antiviral drugs is limited, treatment is largely supportive. This care is essentially the same as the conventional care provided to patients with other causes of multisystem failure. The challenge is to provide this support while minimizing the risk of infection to other patients and medical personnel. Supportive Care      Patients with VHF syndrome generally benefit from rapid, nontraumatic hospitalization to prevent unnecessary damage to the fragile capillary bed. Transportation of these patients, especially by air, is usually contraindicated because of the effects of drastic changes in ambient pressure on lung water balance. Restlessness, confusion, myalgia, and hyperesthesia occur frequently and should be managed by reassurance and other supportive measures, including the judicious use of sedative, pain-relieving, and amnestic medications. Aspirin and other antiplatelet or anticlotting-factor drugs should be avoided.      Secondary infections are common and should be sought and aggressively treated. Concomitant malaria should be treated aggressively with a regimen known to be effective against the geographical strain of the parasite; however, the presence of malaria, particularly in immune individuals, should not preclude management of the patient for VHF syndrome if such is clinically indicated.      Intravenous lines, catheters, and other invasive techniques should be avoided unless they are clearly indicated for appropriate management of the patient. Attention should be given to pulmonary toilet, the usual measures to prevent super infection, and the provision of supplemental oxygen. Immunosuppression with steroids or other agents has no empirical and little theoretical basis, and is contraindicated except possibly for HFRS.      The diffuse nature of the vascular pathological process may lead to a requirement for support of several organ systems. Myocardial lesions detected at autopsy reflect cardiac insufficiency antemortem. Pulmonary insufficiency may develop, and, particularly with yellow fever, hepatorenal syndrome is prominent.16 Treatment of Bleeding     The management of bleeding is controversial. Uncontrolled clinical observations support vigorous administration of fresh frozen plasma, clotting factor concentrates, and platelets, as well as early use of heparin for prophylaxis of DIC. In the absence of definitive evidence, mild bleeding manifestations should not be treated at all. More-severe hemorrhage indicates that appropriate replacement therapy is needed. When definite laboratory evidence of DIC becomes available, heparin therapy should be employed if appropriate laboratory support is available. Treatment of Hypotension and Shock      Management of hypotension and shock is difficult. Patients often are modestly dehydrated from heat, fever, anorexia, vomiting, and diarrhea, in any combination. There are covert losses of intravascular volume through hemorrhage and increased vascular permeability.28 Nevertheless, these patients often respond poorly to fluid infusions and readily develop pulmonary edema, possibly due to myocardial impairment and increased pulmonary vascular permeability. Asanguineous fluids—either colloid or crystalloid solutions—should be given, but cautiously. Although it has never been evaluated critically for VHFs, dopamine would seem to be the agent of choice for patients with shock who are unresponsive to fluid replacement. a-Adrenergic vasoconstricting agents have not been clinically helpful except when emergent intervention to treat profound hypotension is necessary. Vasodilators have never been systematically evaluated. Pharmacological doses of corticosteroids (e.g., methylprednisolone 30 mg/kg) provide another possible but untested therapeutic modality in treating shock. Specific Antiviral Therapy      Ribavirin is a nonimmunosuppressive nucleoside analogue with broad antiviral properties,31 and is of proven value for some of the VHF agents. Ribavirin reduces mortality from Lassa fever in high-risk patients,32 and presumably decreases morbidity in all patients with Lassa fever, for whom current recommendations are to treat initially with ribavirin 30 mg/kg, administered intravenously, followed by 15 mg/kg every 6 hours for 4 days, and then 7.5 mg/kg every 8 hours for an additional 6 days.30 Treatment is most effective if begun within 7 days of onset; lower intravenous doses or oral administration of 2 g followed by 1 g/d for 10 days also may be useful.      The only significant side effects have been anemia and hyperbilirubinemia related to a mild hemolysis and reversible block of erythropoiesis. The anemia did not require transfusions or cessation of therapy in the published Sierra Leone study32 or in subsequent unpublished limited trials in West Africa. Ribavirin is contraindicated in pregnant women, but, in the case of definite Lassa fever, the predictability of fetal death and the need to evacuate the uterus justify its use. Safety of ribavirin in infants and children has not been established.      A similar dose of ribavirin begun within 4 days of disease is efficacious in patients with HFRS.33 In Argentina, ribavirin has been shown to reduce virological parameters of Junin virus infection (i.e., Argentine hemorrhagic fever), and is now used routinely as an adjunct to immune plasma. However, ribavirin does not penetrate the brain and is expected to protect only against the visceral, not the neurological phase of Junin infection.      Small studies investigating the use of ribavirin in the treatment of Bolivian hemorrhagic fever and Crimean-Congo hemorrhagic fever have been promising, as have preclinical studies for Rift Valley fever.33 Conversely, ongoing studies conducted at USAMRMC predict that ribavirin will be ineffective against both the filoviruses and the flaviviruses. No other antiviral compounds are currently available for the VHF agents.      Interferon alpha has no role in therapy, with the possible exception of Rift Valley fever,34 where fatal hemorrhagic fever has been associated with low interferon responses in experimental animals. However, as an adjunct to ribavirin, exogenous interferon gamma holds promise in treatment of arenaviral infections.

88 Prevention of Secondary VHF Transmission
Animal studies indicate aerosol transmission possible Single room with adjoining anteroom as only entrance Handwashing station with decontamination solution Negative air pressure room if possible Strict barrier precautions (PPE): Gloves, gown, mask. shoe covers, protective eyeware/faceshield Consider HEPA respirator (e.g. N95) for severe hemorrhage, vomiting, diarrhea, cough CORE SLIDE Prevention of viral hemorrhagic fever transmission. As mentioned earlier, viral hemorrhagic fevers are transmitted by infected body fluids from patients with this illness. Therefore, extreme care should be taken to isolate patients from other patients. Patients are best treated in private rooms with anteroom, which essentially will serve as a “warm zone” between the contaminated patient area and the rest of the hospital. In an anteroom healthcare providers can both change in and out of their personal protective equipment. A use of chemical toilets and fastidious disinfection of all furniture is indicated as well. If available, a negative pressure room would be indicated, as aerosolization of the virus has been demonstrated, showing that airborne transmission of the virus is possible. Additional Reference Material From: Isolation and Containment CDC: Management of patients with suspected viral hemorrhagic fever. MMWR 37(Supplement 3):1-16, VHF may be transmitted by bodily fluids, but the exact mechanism is unknown. This disease does not appear to be readily transmitted by the airborne route. (This is not the case for Ebola among monkeys.) The highest risk of transmission is during the latter stages of the illness, which are characterized by vomiting, diarrhea, shock, and hemorrhage. Since most strains of VHF are known to spread in the hospital environment, universal precautions are essential. Patients suspected of having VHF should be isolated in a single room with an adjoining anteroom that serves as the only entrance. This anteroom should be stocked with personal protective gear (gloves, gowns, and masks) for staff. The patient’s room should have negative air pressure compared with the anteroom and the outside hall. Strict barrier-nursing techniques should be enforced. Patients should be cared for at the hospital where they were first seen, since transferring patients may increase the potential for secondary transmission. These viruses are easily inactivated with soaps, detergents, and routine disinfectant solutions. In previous outbreaks, simple barrier nursing was enough to reduce health care provider infection rate to virtually zero.      Patients with VHF syndrome generally have significant quantities of virus in their blood, and perhaps in other secretions as well (with the exceptions of dengue and classic hantaviral disease). Well-documented secondary infections among contacts and medical personnel not parenterally exposed have occurred. Thus, caution should be exercised in evaluating and treating patients with suspected VHF syndrome. Over-reaction on the part of medical personnel is inappropriate and detrimental to both patient and staff, but it is prudent to provide isolation measures as rigorous as feasible.30 At a minimum, these should include the following: stringent barrier nursing; mask, gown, glove, and needle precautions; hazard-labeling of specimens submitted to the clinical laboratory; restricted access to the patient; and autoclaving or liberal disinfection of contaminated materials, using hypochlorite or phenolic disinfectants.      For more intensive care, however, increased precautions are advisable. Members of the patient care team should be limited to a small number of selected, trained individuals, and special care should be directed toward eliminating all parenteral exposures. Use of endoscopy, respirators, arterial catheters, routine blood sampling, and extensive laboratory analysis increase opportunities for aerosol dissemination of infectious blood and body fluids. For medical personnel, the wearing of flexible plastic hoods equipped with battery-powered blowers provides excellent protection of the mucous membranes and airways.

89 Prevention of Secondary VHF Transmission
Chemical toilet All body fluids disinfected Disposable equipment/sharps into rigid containers and autoclaved/incinerated Double-bag refuse-outside bag disinfected Electronic/mechanical equipment must be disinfected CORE SLIDE

90 Venezuelan Equine Encephalitis (VEE)
Alphavirus spread by mosquitoes Endemic to Central and South America, Mexico, and Florida Highly infectious - 100% of exposed individuals develop symptoms Low mortality rate - 1% CORE SLIDE Venezuelan equine encephalitis (VEE) belongs to the group of viruses called alpha viruses. They are spread primarily by mosquitoes and are endemic to Central and South America. However, they have been found in southern continental United States, including Florida and on the border with Mexico. Venezuelan equine encephalitis is high infectious with essentially 100% of exposed individuals developing the illness (i.e. a 100% attack rate). Fortunately, its mortality rate is relatively low, with deaths usually due to central nervous system complications such as seizures. However, the low mortality rate is in naturally-occurring disease. The fatality rate in a bioterrorist attack may is unknown.

91 VEE Clinical Course 20% Children 4% Adult cases Febrile syndrome
lasting 3 days, º fever chills, headache, photophobia, sore throat ?? Inhalational Mosquito-borne 1 to 5 day incubation Mild CNS symptoms for 3 days More severe CNS signs Liver Damage Weakness for 1 - 2 weeks CORE SLIDE Here we see a schematic of the clinical course of Venezuelan equine encephalitis. In the naturally occurring disease there is a 1-5 day incubation period after a mosquito-borne inoculation. Keep in mind this is an incubation period based on the natural course of the illness and it is unclear as to how long the incubation would be if the exposure were an inhalational exposure to an aerosolized weaponized form of VEE. However, the illness will develop as a non-specific viral syndrome lasting several days. It will have neurological symptoms including headache, photophobia, meningismus, as well as sore throat, high fever, malaise, myalgias and arthralgias (sore muscle and joints). Subsequently, the disease will continue for approximately 1-2 weeks with the patient being extremely ill but usually leading to recovery. The overall mortality for all patients developing the illness remains at approximately 1%. Patients may develop CNS symptoms for several days following the onset of illness. They may also develop an associated hepatitis. A subset of these patients, approximately 10-37% will have mortality. Again, this is with the caveat that the mortality rates and complications rates are based on the natural course of the illness. Additional Reference: Textbook of Military Medicine, Chapter 28, Viral Encephalitides The IA, IB, and IC variants of VEE virus are pathogenic for equines and have the capacity for explosive epizootics with epidemic human disease. Epidemics of VEE affecting 20,000 to 30,000 people, or more, have been documented in Venezuela and Ecuador. In contrast to the other alphavirus encephalitides, EEE and WEE, epizootic strains of VEE are mainly amplified in equines, rather than birds, so that equine disease normally occurs prior to reports of human disease. Enzootic VEE strains (variants ID, IE, and IF and subtypes II, III, IV, V, and VI) are not recognized as virulent for equines, but disease has been documented with most of these variants in humans who reside in or move into enzootic foci, or after laboratory infections. The resulting syndromes appear to be similar, if not indistinguishable, from the syndrome produced by epizootic variants, which ranges from undifferentiated febrile illness to fatal encephalitis.      Following an incubation period that can be as short as 28 hours but is usually 2 to 6 days, patients typically develop a prostrating syndrome of chills, high fever (38°C–40.5°C), headache, and malaise. Photophobia, sore throat, myalgias, and vomiting are also common symptoms. Frequent signs noted on physical examination include conjunctival injection, erythematous pharynx, and muscle tenderness. Although essentially all human infections with VEE virus are symptomatic, only a small percentage manifest neurological involvement. In one epidemic, it was estimated that the ratio of encephalitis to infections is less than 0.5% in adults, although possibly as high as 4% in children. Mild CNS involvement is evidenced by lethargy, somnolence, or mild confusion, with or without nuchal rigidity. Seizures, ataxia, paralysis, or coma herald more severe CNS involvement. In children with overt encephalitis, case fatalities range as high as 35% compared with 10% for adults. However, for those who survive encephalitic involvement, neurological recovery is usually complete. School-age children are believed to be more susceptible to a fulminant form of disease, in which depletion of lymphoid tissues is prominent and which follows a lethal course over 48 to 72 hours.      In the first 3 days of illness, leukopenia and elevated serum glutamic-oxaloacetic transaminase (SGOT) are common. For those with CNS involvement, a lymphocytic pleocytosis of up to 500 cells per microliter will be observed in the CSF. The CSF pleocytosis may acutely be polymorphonuclear but soon becomes predominantly lymphocytic.      Specific diagnosis of VEE can be accomplished by virus isolation, serologic testing, or both. During the first 1 to 3 days of symptoms of nonspecific febrile illness, VEE virus may be recovered from either the serum or the nasopharynx. Despite the theoretical possibility of person-to-person transmission of virus present in the nasopharynx, no evidence of such occurrences has been reported. Identification of the VEE subtype of an isolate involved can be accomplished by cross-neutralization tests. HI, enzyme-linked immunosorbent assay (ELISA), or plaque reduction neutralization (PRN) antibodies appear as viremia diminishes. Complement-fixing (CF) antibodies make their appearance later during convalescence. VEE IgM antibodies are present in acute phase sera, and it has been reported that the VEE IgM tests do not react with sera from patients with EEE or WEE. Since patients with encephalitis typically come to evaluation later in the course of clinical illness, virus is recovered less often from them, and they usually have serum antibody by the time of clinical presentation.      Immunity after infection is probably lifelong to the homologous serotype, but cross-immunity is weak or nonexistent to heterologous serotypes. Thus, when viewed either as an endemic disease threat or as a potential biological warfare threat, adequate immunization will require polyvalent vaccines. % mortality Recovery

92 VEE Diagnosis & Treatment
Immunoassay Viral Culture Serologic Testing TREATMENT Supportive No antiviral medication exists CORE SLIDE The diagnosis of Venezuelan equine encephalitis is difficult. It is usually done on clinical grounds in endemic areas. Laboratory testing includes viral culture or viral antigen immunoassay. Serological testing (testing for the presence of antibodies against VEE) in patients after they have developed the illness. Needless to say, the individual patient with Venezuelan equine encephalitis will be difficult to diagnose on a clinical basis. Therefore, the diagnosis of Venezuelan equine encephalitis will likely be based on an epidemiological pattern of a large number of patients presenting with the illness in an area in which it is endemic. Treatment options of Venezuelan equine encephalitis are relatively limited. There is no effective antiviral medication. Therefore, the treatment is primarily supportive, including treatment of seizures if they occur.

93 Section 7 Toxin Weapons Objectives:
To be able to explain how each of the presented toxin weapons act To be able to recognize victims to toxin weapon poisoning To understand that toxin weapons are NOT infectious and CANNOT be secondarily spread

94 Botulinum Toxin Neurotoxin produced by Clostridium botulinum - Botulism Most lethal compound per weight (15,000 times more toxic than the nerve agent VX) Different toxicity if inhaled or ingested CORE SLIDE Clostridium botulinum is a spore-forming bacteria (like B. anthracis). C. botulinum spores are often found in the environment (e.g. soil). When the spores are exposed to an anaerobic (very little or no oxygen, e.g. canned vegetables) the sporulate into vegetative bacilli (like B. anthracis) and produce botulinum toxin. It is this toxin that is weaponized as a biological weapon. Therefore, botulinum toxin causes a poisoning, NOT an infection. There is a lag time between the time of exposure and the onset of symptoms. However, this is not an incubation time, this is simply the time necessary for the toxin to exert its effect. Botulinum toxin acts at the neuromuscular junction (the site that the nervous system interacts with muscles), where the motor neuron secretes the neurotransmitter acetylcholine from the presynaptic nerve ending (the end of the motor nerve where acetylcholine is released into the synapse (gap between nerve cell and muscle cell). Acetylcholine binds to receptors on the muscle cell making up the other half of the neuromuscular junction to trigger muscular contraction. Botulinum toxin blocks the release of acetylcholine effectively cutting communication between the nervous system and the muscles. This inhibition causes muscular paralysis. Botulinum toxin (Botox) is 15,000 times as toxic as the nerve agent VX, and 100,000 times more toxic than Sarin. However, note that this toxicity is via ingestion of the toxin. The inhalational route is thought to be 1,400 times less toxic than the ingestion dose. Botulinum toxin is the most lethal toxin yet discovered. Top picture: C. botulinum bacilli with visible spores within the bacteria. Bottom picture: A vial of purified botulinum toxin (Botox®) that is used in medicine to relax wrinkle lines on a patient’s face by injecting extremely small amounts of the toxin directly into certain facial muscles, and to treat muscular spasm, among other indications. A bioterrorist could potentially obtain SMALL amounts of botulinum toxin from medical facilities (enough to possibly cause several causalities). Additional reference material: Textbook of Military Medicine, Chapter 33, Botulinum toxins.      The seven serotypes of botulinum toxin produced by Clostridium botulinum are the most toxic substances known. They are associated with lethal food poisoning after the consumption of canned foods. This family of toxins was evaluated by the United States as a potential biological weapon in the 1960s and is believed to be an agent that could be used against our troops. Unlike other threat toxins, botulinum neurotoxin appears to cause the same disease after inhalation, oral ingestion, or injection. Death results from skeletal muscle paralysis and resultant ventilatory failure. Because of its extreme toxicity, the toxin typically cannot be identified in body fluids, other than nasal secretions, after inhalation of a lethal dose. The best diagnostic sample for immunologic identification of the toxin is from swabs taken from the nasal mucosa within 24 hours after inhalational exposure. Because of the small quantity of toxin protein needed to kill, botulinum toxin exposure does not typically induce an antibody response after exposure.      Prophylactic administration of a licensed pentavalent vaccine fully protects laboratory animals from all routes of challenge. Passive immunotherapy with investigational hyperimmune plasma also prevents illness if it is administered before the onset of clinical intoxication.

95 Botulinum Toxin Normal Muscle Contraction
NMJ Acetylcholine CORE SLIDE This cartoon shows the neuromuscular junction (NMJ). It is the site of interaction between the Motor Nerve and the Muscle. The motor neuron (on the left) carries impulses from the central nervous system to control skeletal muscle movement. At the NMJ, this electrical signal is changed into a chemical one. When the nerve impulse reaches the distal end of the motor nerve, it triggers a process that results in the release of the neurotransmitter acetylcholine from the end of the nerve. Acetylcholine jumps a very narrow gap (synapse) to the surface of a skeletal muscle triggering muscular contraction. In normal function, this communication between the nervous system and the muscles, via the neuromuscular junction, controls muscle movement. To the presenter: It is helpful to orient the listener to the content of the slide prior to using the animation: The NMJ is the space between the motor nerve and the muscle; it is the gap that acetylcholine crosses to bind to the muscle after it is released from the motor nerve. To Presenter: This slide is animated. When the slide is brought as part of a slide presentation, a mouse click, hitting the space bar or the “right arrow” key will start the animation. A “lightning bolt” (representing an electrical nerve impulse) will enter from the left of the slide (representing the nerve impulse traveling to the end of the motor nerve. When the impulse reaches the end of the nerve, green spheres will emerge (representing the release of acetylcholine from the nerve in “vesicles” (essentially subcellular-sized bags of acetylcholine molecules) into the synapse). They move across the synapse (left to right) and bind at their receptors to cause muscular contraction. The presenter can “reload” the animation by hitting the left arrow key or a right mouse click (select “Previous” from pull-down menu); then restart by left mouse click, hitting the space bar, or the “right” arrow key. Repeating this animation with repeated explanation of the slide can be helpful. Motor Nerve MUSCLE CONTRACTION Muscle

96 Botulinum Toxin Botulinum-Paralyzed Muscle
NMJ BOTOX CORE SLIDE This slide represents the effect of botulinum toxin at the NMJ. If botulinum toxin has bound to its intracellular targets (the cellular machinery that causes the release of acetylcholine), when the nerve impulse arrives, the acetylcholine (although present in the nerve cell) is not released into the NMJ synapse. Therefore, no acetylcholine can bind at the muscular receptors, causing paralysis of the muscle. To the presenter: It is helpful to orient the audience to the content of the slide prior to using the animation. BOTOX (in red) represents botulinum toxin that has bound to, and therefore inhibited, the secretion machinery of this motor nerve. To Presenter: This slide is animated. When the slide is brought as part of a slide presentation a mouse click, hitting the space bar or right arrow key will start the animation. A “lightning bolt” (representing an electrical nerve impulse) will enter from the left of the slide (representing the nerve impulse traveling to the end of the motor nerve. When the impulse reaches the end of the nerve, green spheres will emerge, representing the acetylcholine in distal end of the motor nerve. However, they only appear in this slide, and do no leave the cell (representing botulinum toxin inhibiting the release of acetylcholine into the synapse). The presenter can “reload” the animation by hitting the left arrow key or a right mouse click (select “Previous” from pull-down menu); then restart by left mouse click, hitting the space bar, or the “right” arrow key. Repeating this animation with repeated explanation of the slide can be helpful. Motor Nerve NO MUSCLE CONTRACTION Muscle

97 Botulism Signs & Symptoms
Descending paralysis Bulbar Palsies Blurred vision Dilated pupil Double vision Drooping eyelids Light intolerance Difficulty swallowing Difficulty speaking Respiratory failure CORE SLIDE The most important characteristic feature of Botulism is a descending paralysis (head to toe) and bulbar palsies (a bulbar palsy is a loss of function in the nerves that originates from the upper part of the spinal cord where it attaches to the brain). This leads to dysfunction of the cranial nerves that control all the muscles of the face, the eyes, and swallowing). Symptoms include: blurred vision, dilated pupils (mydriasis), double vision (diplopia), drooping eyelids (ptosis), light sensitivities (photophobia); difficulty swallowing (dysphagia); and problems speaking. Soon, the skeletal muscles become weak, starting in the upper body and progressing downwards in a symmetrical fashion until involvement of the diaphragm and accessory breathing muscles causes respiratory failure. Upper Photograph: bio_agents.html, Notice the lack of facial expression on this patient, and the need to manually open her eyes. This is due to the paralysis of her facial muscles by botulinum toxin. Lower Photograph: Floppy baby paralysis is a form of botulism seen in infants that results from the ingestion of C. botulinum spores or bacteria. Because the gastrointestinal tract of young infants is not populated with the normal gut flora (bacteria) one would see in adults, C. botulinum can grow in the gut, produce and secrete botulinum toxin, which is absorbed by the infant, causing botulism. Honey contaminated with C. botulinum has been implicated in this disease. “Floppy” baby flaccid paralysis

98 Botulism Diagnosis and Treatment
Clinical diagnosis: bulbar palsies with descending paralysis Mouse neutralization assay confirms diagnosis Treatment is supportive Long-term mechanical ventilation Antitoxins are available but must be administered early to be effective CDC vaccine protective for A,B and E toxins CORE SLIDE Botulism is usually a rapidly progressing illness, therefore early treatment is essential. First, making the diagnosis (which if usually missed on the first presentation) is the obvious priority. The descending paralysis is so characteristic of botulism, that any illness that includes such a pattern of paralysis should always raise the botulism as a possible diagnosis. Bulbar palsy - paralysis of the cranial nerves. These nerves control the muscles of facial expression and swallowing. Patients may also have dry mouth and throat (and may complain of sore throat), and dizziness. Onset of botulism is usually over 24 to 36, but may be longer if exposure is inhalational. Botulinum toxin is not dermally active and is not absorbed through the skin. Diagnosis canned be confirmed by laboratory studies. There are several immunoassays than test for the presence of botulinum toxin in a specimen or sample. However, the “gold standard” is the mouse neutralization assay. In very basic terms, in this assay, mice are injected with sample material that is thought to contain botulinum toxin, with or without a specific antitoxin to the different botulinum toxins (types A-G). Survival of all mice would rule out botulinum toxin presence; death of mice without antitoxin would indicate the sample was positive for the presence of botulinum toxin. Which mice died among the antitoxin-treated group would indicate which botulinum toxin type was present (A-G). Treatment of botulism includes the use of antitoxins. There are several preparations of antitoxin available, including a trivalent (A,B,E). However, antitoxin is of limited help unless given early in the course of the disease. Many patients become paralyzed requiring long-term mechanical ventilation. Typically, motor nerves require 2 to 3 months to regenerate the cellular machinery irreversibly inhibited by botulinum toxin. Botulism is a public health emergency! Whether the result of a bioterrorist attack or a naturally occurring case, efforts must be made to identify anyone who may have ingested the same food as the patient, and to notify the department of public health. Remember, once the patient is paralyzed, he/she will not be able to provide this history. Furthermore, the insidious onset of the illness, and poorly recognized presentation, means that most patients are diagnosed well into there course.

99 Ricin Potent toxin - a protein byproduct of castor bean processing for castor oil 5 times more toxic per weight than VX Blocks protein synthesis within the cell, causes cell death, and airway tissue death and swelling when inhaled CORE SLIDE Ricin is a toxin produced by the castor bean Ricinus communis. It is a protein toxin that accumulates in the castor bean. Approximately 5% (by dry weight) of castor bean mash (pulverized castor beans after they have been crushed to extract castor bean oil) is ricin. Although not nearly as toxic as botulinum toxin, it is still considered 5 times more toxic that the most lethal nerve agents. The toxin binds to a cell surface receptor, and then is internalized into the cell. Inside the cell, ricin toxin blocks cellular protein synthesis, leading to cell death and tissue necrosis. If ricin is inhaled, the patient will develop airway necrosis and bleeding, severe inflammation of the trachea, bronchi, and distal airways (alveoli). Pneumonia and pulmonary edema develop with widespread necrosis of lung tissue. If ingested, ricin produces predominantly gastrointestinal symptoms first, including bloody stools, vomiting blood, and abdominal pain. Finally, ricin can be injected, and has been used in this fashion for assassinations (the interested reader is referred to the story of Gorgi Markov’s assassination by ricin). However, all these routes of exposure can culminate in systemic toxicity and death.

100 Ricin Diagnosis & Treatment
Fever, chest tightness, cough. Shortness of breath, nausea, and joint pain. Ingestion causes severe diarrhea, hemorrhage, and necrosis of the liver, spleen, and kidneys - shock and death within 3 days Treatment is supportive, including airway management No antitoxin or vaccine available CORE SLIDE After inhalation, victims begin to experience fever, chest tightness, cough, and shortness of breath within 4 to 8 hours of exposure. Death may occur in 36 to 72 hours. If ricin is ingested, victims often develop rapid onset of nausea, vomiting, severe diarrhea, and gastrointestinal hemorrhage with necrosis of the liver, spleen, and kidneys. Shock typically ensues with death occurring in 3 days. Treatment is supportive, ensuring adequate oxygenation and hydration. No antitoxin or vaccine is currently available.

101 Staphylococcal Enterotoxin B (SEB)
Common cause of food poisoning in improperly handled foods 80% of exposed individuals develop symptoms Symptoms vary by route of exposure - can be aerosolized or introduced into food system CORE SLIDE SEB is another toxin produced by bacteria (like B. anthracis and C. botulinum). It causes a hyperstimulation of the immune system. It is toxic both by the ingestion and inhalational routes. This results in sever inflammation of the GI tract (if ingested) or the respiratory tract (if inhaled).

102 SEB Signs & Symptoms Sudden onset of high fever, headache, chills, muscle aches, and non-productive cough, and malaise. Inhalational: Severe shortness of breath & chest pain with larger doses Ingestion: Nausea, vomiting, and diarrhea CORE SLIDE SEB is a “pyrogenic toxin” which means it induces a fever. A person who has been exposed to inhalational SEB may likely present like someone with a severe pneumonia. After inhalation, symptoms develop within 3 to 12 hours and include sudden onset of fever, headache, chills, generalized muscle aching, and a non-productive cough. Inhalational Exposure: Severe shortness of breath and chest pain would develop with larger doses. Ingestion Exposure: Nausea, vomiting, and diarrhea develop and can be severe, often bloody. Both Routes: Patient’s presentation may be very SIMILAR to a patient in septic shock from an infection (severe low blood pressure due to severe infection). Remember: SEB is a toxin, not an infection. The comparison between its presentation and septic shock is only for comparison.

103 SEB Treatment Supportive Care: Oxygenation Hydration
Most victims will recover No vaccine available No antibiotic is effective CORE SLIDE Chance of recovery is largely determined by dose, obviously, the larger the dose (by any route of exposure) the less likely the patient will recover. No effective treatment is available, therefore treatment is primarily supportive care.

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