2004, Vol.22, Issues 3, Bioterrorism

Dermatol Clin 22 (2004) ix x
Preface
Bioterrorism
Boni E. Elewski, MD Guest Editor
Biological warfare has gained recent attention in the public and medical literature. This issue of the Dermatologic Clinics reviews the topic of bioterrorism as it pertains to dermatology. Cutaneous presentations are common with many biological warfare agents and may appear soon after a biological terrorist attack. It is conceivable to assume that in this situation dermatologists would become front-line responders. Prompt and accurate diagnosis would result in early medical intervention, which could reduce mortality. Therefore, knowledge of the clinical features of likely biological warfare agents is important for all dermatologists to become familiar with. For example, during the anthrax attacks in the fall of 2001, dermatologists made the initial diagnosis of cutaneous anthrax in the New York City cases, which brought to the public attention the unique expertise of dermatologists as the authorities in cutaneous disorders. Many dermatologists were soon called upon to educate the public and the medical communities on cutaneous manifestations of anthrax and other worrisome bioweapons. The first article in this issue extensively reviews the history of biological warfare and will include detailed discussions on the anthrax attacks of 2001. Although anthrax is caused by a bacterium, the cutaneous and systemic pathology are the result of a toxin. This is discussed in the article by Drs. Wenner
and Kenner, which emphasizes the cutaneous manifestations and diagnosis of anthrax. The next article, written by Dr. Henghold, reviews other biologic toxins that have potential as bioweapons, such as ricin. Following the terrorist attacks on the World Trade Center and the anthrax attacks on New York City and Washington, DC, there was much concern about the use of smallpox as a bioweapon. Consequently, a mass vaccination of the military and some civilians occurred. The article by Dr. Hanifen thoroughly reviews this virulent disease. Additionally, variola vaccine reactions have become another concern; this is addressed in the article by Dr. Fallon-Friedlander. The final three articles review other biological warfare agents, including the article by Drs. Baddley and Salvaggio on Ebola, hemorrhagic fever, and related diseases. Although plague occurs sporadically in nature it can be used as a biologic agent; this topic is reviewed by Drs. Cobbs and Chansolme. Finally, miscellaneous bioweapons, including tularemia, are discussed by Dr. Cronquist. This issue of the Dermatologic Clinics is designed to familiarize the reader with common cutaneous presentations and provide an overview of the pathogenesis of select biological warfare agents. Knowledge of their clinical changes and pathogenesis will likely hasten diagnosis and would hopefully mitigate the result of a biological attack. Although it is an
0733-8635/04/$ see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.det.2004.03.018
B.E. Elewski / Dermatol Clin 22 (2004) ixx
important area for dermatologists to master, the study of biological cutaneous manifestations of biological warfare agents has been somewhat neglected in residency training and received little attention until after the terrorist attacks in New York City. I hope you enjoy reading this interesting and informative edition of the Dermatologic Clinics.
Boni E. Elewski, MD Professor of Dermatology The University of Alabama at Birmingham 700 Eighteenth Street, South, EFN 414 Birmingham, AL 35233-0009, USA E-mail address: beelewski@aol.com
Dermatol Clin 22 (2004) 231 246
The history of biologic warfare and bioterrorism

Michael K. Jacobs, MD, MPH
Department of Dermatology, University of Alabama at Birmingham School of Medicine, 1530 3rd Avenue South, EFH 414, Birmingham, AL 35294, USA
The threat of biologic agent use against military and civilian populations is perhaps the greatest it has been at any point in our history [1]. It is estimated that at least 10 nations possess biologic weapons programs [1,2]. Despite signing the Biologic Weapons convention in 1972, the former Soviet Union maintained an extensive biologic weapons program as late as 1992, when Soviet President Boris Yeltsin promised to abandon the program [3]. However, the current extent of the program (now under Russian control) and the location of its former scientists are unknown [1,4]. After the Persian Gulf War in 1991, statements made by Iraqi officials and United Nations inspectors revealed that Iraq had developed several biologic weapons, some of which were reportedly deployed during the war [5]. Moreover, the biologic weapons threat is not limited to state-sponsored biologic warfare. As the anthrax letters of 2001 unfortunately underscore, terrorism with biologic agents is on the rise. Before the late 1990s, the Federal Bureau of Investigation (FBI) investigated about 12 cases per year involving weapons of mass destruction (WMD). The number of cases rose to 181 in 1998, 267 in 1999, and 257 in 2000, of which 112, 187, and 115 involved biologic agents threats, respectively. These numbers rose sharply after the anthrax letter attacks of Fall 2001; in a few weeks the FBI responded to over 7000 potential anthrax letters, over 900 other WMD threats, and over 29,000 telephone calls about suspicious packages [6,7]. Biologic weapons are unique in several ways. They are difficult to detect and relatively inexpensive and easy to reproduce. Biologic agents can spread beyond
E-mail address: mj@alumni.duke.edu
the initial delivery area, and can have devastating consequences with only small amounts of agent [8]. For example, the World Health Organization estimates that an attack on a city of 500,000 with 50 kg of aerosolized anthrax spores would result in 95,000 deaths and 125,000 illnesses [9]. Early victim recognition and subsequent containment is a central component of any disaster preparation plan for a bioweapons attack. Clinicians, therefore, are central to biologic weapons disaster preparedness. The diagnosis of initial cutaneous anthrax cases was delayed by up to a month in the anthrax letter attacks of 2001 [10]. There were just over 200 cases of cutaneous anthrax reported in the United States between 1944 and 1994; it is likely that the delay in diagnosis seen in 2001 was the result of a medical community that was unfamiliar and unsuspecting of cutaneous anthrax [11]. Similarly, the last endemic case of smallpox was reported in 1977. This lead the planners of the 2001 Dark Winter exercise (a senior-level government exercise simulating a covert smallpox attack on the United States) to assume that initial diagnoses of smallpox cases would be delayed, thus further promoting spread of disease [12]. Dermatologists, therefore, must be familiar with the effects of biologic weapons, especially those with prominent cutaneous findings such as anthrax and smallpox. In addition, consideration of the history of biologic warfare and bioterrorism will reveal to medical and public health professionals that the pursuit and use of biologic weapons has occurred since ancient times, and will likely persist into the future. The past usage of biologic weapons, however, does not necessarily predict future uses and their consequences (especially because a successful largescale aerosolized attack, which would have the most devastating effects, has never occurred) [11].
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Studying the history of biologic weapons use can be difficult. This is because allegations of biologic weapons use are difficult to confirm because often there is little historic, epidemiologic, or microbiologic data available regarding alleged attacks. Also, biologic weapons programs are conducted in secret, and allegations of biologic weapons attacks have been used as propaganda. Finally, in times of war, it is difficult to discern deliberate biologic weapons attacks from increased rates of naturally occurring infectious diseases often seen in wartime conditions [13].
Early biologic warfare The following early examples of biologic warfare are especially difficult to substantiate. Historic accounts reveal that the use of biologic agents has been part of military strategy since ancient times. Presentday knowledge of the epidemiology and pathophysiology of biologic agents may leave doubt as to the efficacy of specific early biowarfare methods, but, given prevailing ideas of disease at the time, perpetrators no doubt intended and expected to spread infection in these attacks [14]. Early use of infectious agents in war were based on three main ideas: (1) contamination of food or water supplies, (2) launching of potentially infective materials into enemy strongholds, and (3) dissemination of infected clothing or blankets. All were crude attempts using cadavers, dead animals, plant poisons, contaminated fabrics, and, in one case, snakes. Contamination Two of the earliest documented instances of biologic agents use occurred in the sixth century B.C. The Assyrians reportedly poisoned the wells of their enemies with rye ergot, a fungus whose toxins cause muscle cramps, vomiting, gangrene, and hallucinations [1]. Later, the Athenian politician Solon, during the siege of the city of Cirrha, dumped the herb purgative hellebore into the citys water supply, the River Pleistus. After drinking the tainted water, the guardians of Cirrha were gripped with intractable diarrhea and deserted their posts. Solon then captured the city from the Megarians [15]. The contamination of drinking water became a common military tactic. In 1155 A.D., during the Battle of Tortona, Barbarossa contaminated the enemys wells with the bodies of his dead soldiers [16]. Later, in 1171, Emperor Manuel of Ragusa seized and imprisoned some Venetian citizens. The Doge of Venice soon attacked Ragusa to reclaim his citizens.
Emperor Manuel opened negotiations with the Venetians, but then delayed them until the Venetians were forced to overwinter at nearby Chios, where drinking wells had previously been contaminated. Many of the Venetian forces subsequently became ill after drinking from the contaminated wells and were forced to return home without the prisoners [16]. In 1495, during the French invasion of Naples, Spanish forces gave French soldiers wine mixed with the blood from victims of leprosy. This attempt to infect the French forces was unsuccessful [16]. During the American Civil War, Confederate troops were accused of deliberately attempting to contaminate Union water supplies. General W.T. Sherman reported that during their retreat from Vicksburg in July of 1863, Confederate soldiers attempted to slow Union forces down by dragging farm animals into ponds and shooting them, so that the carcasses would have to be removed before the water could be used [15]. Projectile biologic weapons Biologic agents were first incorporated into projectile weapons as early as 400 B.C. when Scythian archers dipped their arrowheads in decaying cadavers or feces [17]. In 184 B.C., live animals were incorporated into a unique biologic weapons system. According to Justinus, Hannibal, at the Battle of Eurymedon, commanded his men to fill pots with serpents of every kind. During the battle, Hannibal launched these vessels onto the flagship of his enemy, King Eumenes of Pergamum. The subsequent turmoil the snakes created helped secure victory for Hannibal [15]. The development of the catapult and later the more capable trebuchet during the Middle Ages led to the common practice of launching potentially infected cadavers, animal carcasses, or human waste at enemies. This military technique persisted into the twentieth century [17]. According to the chronicler Jean Froissart, the Duke of Normandy used this tactic during the siege of the castle at Thun lEveque in 1340. The Duke used siege machines to cast in deed horses, and beestes stynking, wherby they within had great[er] dystress thane with any other thynge, for the ayre was hote as in the myddes of somer: the stynke and ayre was so abomynable, that they consydred howe that finally they coude nat long endure [18]. The psychologic impact of such an attack is evident in Froissarts account, although it is unclear if this incident was meant to spread disease or simply destroy morale. During the same time period, in what is presentday Ukraine, the Tatars created the first plague bio-
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logic weapon (as described by De Mussis). In 1346, the Tatars besieged the Genoese-controlled walled city of Caffa (now Feodosiya), an important seaport on the Eastern trade route. Caffa was essentially impregnable, and it withstood the Tatar siege for 3 years. After 3 years the plague erupted among the Tatar forces, killing thousands of their forces a day. Desperate, the Mongul leaders used their hurling machines to launch the bodies of plague victims into the city. Soon, the plague epidemic spread to the people of Caffa, forcing the retreat of the Genoese forces. The plague then spread to the places where these retreating forces landed: Sicily, Sardinia, Corsica, and Genoa [19]. It is likely that the plague spread to the city through a more common mechanismflea-infested ratsrather than the hurled corpses. However, the intention of the Monguls to spread the epidemic from which they were suffering to their enemies seems clear [13,17]. A similar incident occurred in 1422, when Corbut launched dead soldiers and cartloads of excrement into the enemy at Carolstein whereby a great number of the defenders fell victim to the fever which resulted from the stench [20]. Infected fabrics Attempts to deliberately spread smallpox through contaminated clothing and bedding represents the third form of early biologic warfare. In the fifteenth century, Pizarro, in his bloody conquest of South America, presented the native people with gifts of clothing contaminated with smallpox (variola virus) [21]. Later, the British used the same tactic during the French and Indian War (1754 1763). Fearing that an attack was imminent, Captain Ecuyer made gifts of blankets and a handkerchief from a smallpox hospital to the Native Americans camped around Fort Pitt (now Pittsburgh). Sir Jeffrey Amherst, the commander of British forces in North America, previously advocated this tactic in letters to his officers. Over the ensuing months, smallpox epidemics spread throughout the tribes of the Ohio River Valley [13,21]. Dr. Luke Blackburn, a Confederate surgeon who later became governor of Kentucky, resurrected the smallpox biologic weapon during the American Civil War. In 1863, Blackburn was arrested for importing clothing from smallpox and yellow fever patients and selling it to Union troops. Although yellow fever cannot be transmitted through clothing, at least one Union officers obituary claimed that he died of smallpox from Dr. Blackburns scheme [16,22]. It is difficult to determine if these attempts to use smallpox as a biologic weapon were successful. Smallpox (variola virus) is best transmitted from
person to person via a respiratory route, and transmission via contaminated fabrics has been shown to be infrequent. Moreover, smallpox outbreaks in the nonimmune Native Americans had occurred since the very first Old World settlers. Therefore, it is likely that other person-to-person contacts led to respiratory transmission between colonists and Native Peoples and later soldiers in the American Civil War [23,24].
World War I Modern microbiology was born in the late nineteenth century with the recognition that specific pathogens caused specific diseases. This observation, along with technical advances in the ability to isolate and culture specific pathogens, gave rise to the modern era of biologic warfare. During the World War I, evidence suggests that Germany took advantage of these advances in creating a well-developed biologic warfare program. Germanys program focused on plans to use the agents Bacillus anthracis (anthrax) and Burkholderia (Pseudomonas) mallei (glanders) against livestock destined for use by the Allied forces [16,22]. However, the use of biologic agents by German agents is difficult to prove, because glanders and anthrax occurred naturally in livestock in the early twentieth century [21]. At the beginning of the war, Major Franz von in Washington, Papen, a German military attache unsuccessfully attempted to contaminate feed for horses and cattle that were to be exported to England [16]. Anton Dilger, a German surgeon educated in the United States, was discharged from the German army after a nervous breakdown in 1915. With the United States still neutral in the war, he was subsequently sent to rest with his parents in Virginia. Secretly, Dilger carried with him cultures of Bacillus anthracis and Burkholderia mallei, with which he was to seed a biologic sabotage program against horses in the United States. Bacteria grown in Dilgers laboratory in Maryland were given to Captain Frederick Hinsch, who used them to inoculate horses in Baltimore before they were shipped to Allied forces in Europe [22]. In 1916, German agents attempted to infect sheep bound for Russia. The agents were caught, and cultures confiscated from them were identified as Bacillus anthracis and Burkholderia mallei by the Bucharest Institute of Bacteriology and Pathology. In 1917, a German saboteur was arrested for allegedly infecting 4500 mules with glanders [13]. In Argentina, some 200 mules died of anthrax and glanders infections thought to be the result of actions by German agents [16].
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Many other allegations were made, including attempts by German agents to infect French horses, to spread plague in St. Petersburg in 1915, and to cause infections in Romanian cities by dropping fruit, toys, and chocolate tainted with bacteria [22]. A League of Nations investigation after the war in 1924 concluded that Germanys bacteriological arm has not been employed in war. Furthermore, Germany denied all allegations that it conducted bacteriologic warfare. Regardless, it appears that Germanys biologic warfare program had little strategic effect [21,25]. 1925 Geneva Protocol The international community responded to the frightening use of chemical weapons and reports of biologic weapons during World War I by attempting to limit their proliferation through the 1925 Geneva Protocol for the Prohibition of the Use in War of Asphyxiating, Poisonous or Other Gases, and of Bacteriologic Methods of Warfare [13]. Although ratified by many nations, the Geneva Protocol only prohibited chemical and biologic weapons use. However, it did not forbid possession, research, or stockpiling of biologic weapons. Consequently, Belgium, Canada, France, Great Britain, Italy, The Netherlands, and Poland, all signatories of the Geneva Protocol, began biologic weapons research programs after World War I. Furthermore, the Geneva Protocol effectively became merely a no-first-use agreement when the signatories France, the Soviet Union, and Great Britain declared that they would not be bound by the Protocol if their enemies used biologic or chemical weapons first [8,26]. Although the United States initially signed the Geneva Protocol, it was not ratified by the senate until 1975 [13].
Post World War I through World War II United States Despite allegations of German bacteriologic sabotage during World War I, the United States, along with the newly formed League of Nations, initially felt that biologic warfare was impractical and likely ineffective in actual wartime use. Consequently, during the 1920s and 1930s, the United States did not pursue biologic weapons. However, this would change in the lead up to World War II [22]. In the late 1930s, there were increasing allegations of Japanese use of biologic weapons against Chinese troops. This suspected threat prompted United States
Secretary of War Harry L. Stimson to ask the National Academy of Sciences to form the Biologic Warfare Committee to direct research on biologic weapons in 1941, before the attack on Pearl Harbor. In August of 1942, with the recommendation of the Biologic Warfare Committee and under strict secrecy, President Franklin D. Roosevelt approved a plan to form the War Research Service under the leadership of George W. Merck, president of the Merck Company, a pharmaceutical manufacturer. The War Research Service was simply a coordinating committee, leaving the actual research to be done by existing government, university, and private research institutions. Additional information was gleaned from scientists at the National Research Council and the National Academy of Sciences [22]. However, Merck soon realized that a centralized, large-scale effort was needed to adequately research and develop biologic weapons [27]. Two years after its formation, the War Research Service was abolished, and biologic warfare research duties were transferred to the Special Projects Division of the War Departments Chemical Weapons Service (CWS). Merck remained affiliated as chairman of the Biologic Warfare Committee. Under this new arrangement, the US biologic weapons program flourished. In April 1943, the CWS established Camp Detrick (renamed Fort Detrick in 1956) in Maryland as the main biologic weapons research facility. Camp Detrick consisted mainly of four Pilot Plants (PP), along with several smaller research plants. PP1 was activated in October 1943, for the production of botulinum toxin. PP2 was established in March 1944, to produce spores of Bacillus anthracis and its simulant Bacillus globigii. PP3 and PP4, both completed in 1945, researched plant pathogens (such as brown spot rice fungus) and the pathogens of brucellosis and psittacosis, respectively [22]. Despite this explosion of biologic weapons research during World War II, the United States actually had little offensive or defensive biologic weapons capability during the war and did not deploy any biologic weapons. By June 1944, the United States had limited stockpiles of botulinum toxin and anthrax cattle cakes (developed with the United Kingdom) that could be used in retaliation (President Roosevelt had earlier stated that the United States would not use biologic weapons unless first used by the enemy) [8,16]. However, plans to produce biologic weapons were in place by the end of the war. In 1944, a biologic weapons production plant was established in Terra Haute, Indiana. This plant would take advantage of the biologic weapons programs key technologic advancement during the warthe development of small
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biologic agent particle sizes and systems capable of dispersing themto produce the SPD Mk I anthrax bomb. In fact, an order for one million such bombs was placed at the Indiana plant only to be cancelled at the wars end [22,27]. Japan The Japanese biologic weapons program was a surprisingly extensive effort that spanned the 15 years ending in 1945. Its full extent was not discovered until after the war. Lead by military scientists and doctors hoping to effect Japanese military supremacy, the program consisted of advanced methods of production and dissemination refined after considerable tests on prisoners of war and field tests against the Chinese in Manchuria. Much of the history of the Japanese biologic warfare program remains unknown, because after the war documents were either destroyed or not made public. Secret information gleaned the programs participants was protected by the United States to prevent other biologic weapons programs from benefiting from it [28]. Japans biologic weapons research effort began in 1930, and lasted until the end of World War II, first under the direction of Shiro Ishii (1930 1942) and then Kitano Misaji (1942 1945) [13]. Japanese interest in biologic weapons began with the flamboyant, self-promoting Doctor Ishii. In 1930, Ishii returned from a 2-year tour of international research laboratories, including laboratories in the United States. He reported to his superiors that the major powers were secretly conducting biologic weapons research, and that Japan would be vulnerable without biologic weapons. Ishiis efforts paid off in 1930, when the Japanese military established the Department of Immunology at the Tokyo Army Medical School with Ishii as chairman. At the Tokyo Army Medical School, Ishii began early biologic weapons research [29,30]. With further convincing, Ishii secured a dedicated, more secret, biologic weapons research facility in occupied Manchuria, initially at Harbin in 1932, and later moving to Beiyinhe. It was in Beiyinhe in 1933, where Ishii first began experiments on human subjects at the Zhong Ma camp. The facility then moved to Ping Fan in 1939, and grew to include 3000 staff, five satellite camps, and 150 buildings at its peak. In 1941, the camp was renamed Unit 731 [31]. The full Japanese biologic warfare research effort consisted of three additional units located in Manchuria. Unit 100, located in Changchun, prepared biologic weapons against plants, animals, and humans using biologic agents developed by Unit 731. Another unit,
located in Mukden, trained biologic weapons scientists under the direction of Kitano Misaji (who would later take over Unit 731). The final unit, Ei-1644, was located in a commandeered Chinese hospital in Nanking. This facility performed toxin research on human subjects [21,31]. These facilities in total employed a staff of more than 3000, including microbiologists, entomologists, and botanists, and technicians as well as at least 50 physicians [17]. This staff, under the direction of Ishii, performed grisly biologic weapon experiments on humans on a scale not seen before or since. Between 1932 and 1945, at least 10,000 prisoners died after experiments in which they were exposed to biologic agents such as Bacillus anthracis, Clostridium botulinum, Clostridium perfringens, Neisseria meningitides, Shigella spp., Vibrio cholera, and Yersinia pestis [13,17,21,32]. Those who survived the experiments were executed and analyzed to determine the pathogenesis of these agents. According to Japanese soldiers who worked at the facilities, not one prisoner was allowed to survive [8]. In addition, tens of thousands of Chinese civilians died as a result of epidemics (mostly plague) around the sites of Japanese biologic weapons facilities [30]. Ishii and his research units translated this gruesome research into large field trials of new biologic weapons. According to testimony from Japanese participants in Unit 731 who were captured by the Soviet Union, at least 12 large field tests of Japanese biologic weapons were conducted between 1939 and 1942 [29]. Most of these attacks targeted Chinese civilians, and it is estimated that thousands were hospitalized, and as many as 700 died as a result [17,21,29]. The Japanese contaminated river water with biologic weapons agents (including typhoid) in the Nomonhan incident, which occurred in 1939, during a battle with the Russians along the Manchurian/Siberian border. Soon after, the various biologic weapons units twice poisoned civilian wells in Harbin with Salmonella typhi, caused a cholera outbreak in Changchun, and poisoned wells in Nanking [31]. In 1941, plagueinfested fleas were sprayed over the cities of Ningpo and Chuhsien, resulting in 1000 casualties and 500 deaths in Ningpo, and 12 deaths in Chuhsien from plague epidemics [21,30,31]. Plague had not previously been seen in Chuhsien [21]. An aerial attack on Changteh, in 1941, reportedly resulted in 10,000 biologic weapons casualties, including the inadvertent deaths of 1700 Japanese troops, mostly of cholera [29]. In 1942, Ishiis Unit 731 gave Chinese children chocolates filled with Bacillus anthracis [8]. When Misaji assumed control of the Japanese biologic weapons program in 1942, field work was
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halted in favor of laboratory research [31]. This secret and deadly program ended in 1945, with the evacuation of Unit 731, but was not exposed until the Russian war crimes trial in Kharbarovsk in 1949 [16]. Much information from scientists who worked in the Japanese biologic weapons program (including Ishii and Misaji) was passed on to the US biologic weapons program at Camp Detrick in exchange for immunity from war crimes prosecution, and remains secret to this day. The United States feared that a public trial would make Japanese biologic weapons research available to the Soviet Union [8,29,30]. Germany Allied powers generally assumed that by the start of World War II, Germany had a well-developed biologic weapons program that began years earlier during the first World War. In reality, however, Germany had a limited biologic weapons program, such that it was not an offensive biologic weapons threat during World War II [13,32]. Germany did begin offensive biologic weapons research during the interwar period in 1933, with the Wickham Steed affair. As reported by the British journalist Henry Wickham Steed, in 1934, Germany allegedly conducted biologic weapons dispersion studies by dumping the simulant Serratia marcescens into Paris Metro ventilation shafts and around some French forts [16,22]. Additional efforts in 1936 and 1937 included antianimal experiments with foot-and-mouth disease at Luneburger Heide, anthrax studies at the German Military Bacteriological Institute in Berlin, and bacterial antiplant experiments at the German Agricultural Hochshule in Bonn [16]. However, these efforts were isolated, and not supported by central leadership, as evidence suggests that in 1939, Hitler banned offensive biologic weapons research [16,32]. Efforts after this ban concentrated on defensive measures against the perceived threat of Allied biologic weapons use [16]. These studies included experimental infections of prisoners of war during WWII with the pathogens Rickettsia prowazekii, Rickettsia mooseri, hepatitis A virus, and the parasites that cause malaria. However, these were studies of pathogenesis and treatment and not weapons development, and no German offensive biologic weapons program could be documented after the war [21]. France Most of the information concerning the French biologic warfare program was destroyed before the
German occupation. Those documents that remain show that Frances biologic weapons program expanded in during the period 1936 to 1940 in response to the Wickham Steed allegations. Francess program was largely concerned with viability studies of bacteria and viruses during storage and explosions. Further advances were limited by Frances defeat in the war [16,33]. Britain Like France, the Wickham Steed allegations in 1934 heightened British military officials concern about the threat of a biologic weapons attack, and in 1934, a committee was established to examine offensive and defensive biologic weapons. A biologic weapons facility was eventually established at Porton Down in 1940, after increasing intelligence reports of biologic weapons activities by Germany and Japan [16]. The Biology Department, Porton (BDP) mainly focused on anticrop and antianimal agents. BDP tested anthrax spores on sheep on Gruinard Island off the coast of Scotland, contaminating the island for 45 years. Having proved the utility of anthrax as a biologic weapon through these experiments, the BDP, like the US biologic weapons program, produced and stockpiled 5 million anthrax cattle cakes for retaliatory purposes. These were never used [8]. Canada Canadas biologic weapons research program began in 1939. Under the direction of Sir Frederick Banting, facilities at Connaught Laboratories and Suffield performed research on anthrax, botulinum toxin, plague, psittacosis, and the anticattle agent rinderpest in collaboration with the United States and Britain. Additionally, the United States, Britain, and Canada were jointly involved in a project to develop an anthrax cluster bomb, but the project did not reach the production stage before the end of World War II [16,27]. Russia Although little is known about the early Soviet biologic weapons program, it probably began in the late 1920s with the opening of a biologic weapons research facility north of the Caspian Sea [16]. Early efforts were crude: animals were made sick with pathogens of interest, then killed, dried, and ground into powder [6]. Later, in the 1930s, researchers at the Red Army Bacteriology Institute near Moscow con-
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ducted studies of gas gangrene, botulism, and plague, and of aircraft and artillery dispersal methods [27].
Post World War II and beyond United States After the Second World War, the United States continued its policy that it would not use biologic weapons unless they are used first by its enemies. However, the United States maintained a retaliatory biologic weapons capability through an extensive biologic weapons research programemploying nearly 4000 at its peakuntil 1969, when President Nixon ended the offensive program in favor of open defensive research [8,27]. Many of the details of the US biologic weapons program in this era have never been declassified [22]. During the early 1950s, the US biologic weapons research program, centered at Fort Detrick as a division of the Chemical Corps, focused its efforts on creating standardized protocols for biologic agents first researched during World War II and then developing weapons systems for those agents. The bulk of this initial research was directed toward antipersonnel agents, with anthrax taking highest priority [22]. During the late 1950s and 1960s, the program continued to expand, employing 3900 Army, Navy, and civilian personnel [8]. By 1960, Fort Detrick became the worlds largest consumer of guinea pigs while investigating numerous biologic agents, including Bacillus anthracis (anthrax), Francisella tularensis (tularemia), Brucella suis (brucellosis), Coxiella burnetti (Q fever), Venezuelan equine encephalitis (VEE) virus, yellow fever virus, botulinum toxin, staphylococcal enterotoxin [8,27]. Human tests, using military and civilian volunteers, were also conducted. Within the eight balla 1-million liter hollow metal spheresubjects were bombed with biologic weapons containing Francisella tularensis and Coxiella burnetti [13]. By 1960, the US military had standardized seven biologic agents [34]. As these biologic weapons agents were standardized at Fort Detrick, plants like the Production Development Laboratories, built in 1954 in Pine Bluff, Arkansas, developed means of mass producing them. In its first year, the facilities in Pine Bluff began production of Brucella suis, and eventually reached an output of 650 tons per month. The Production Development Laboratories could fill munitions within 4 days of receiving an order [22,27]. A separate production plant in Vigo, Indiana, had the capability to produce 100 tons of anthrax per month, if ordered [17]. In
1959, yellow fever virus, isolated from an outbreak in Trinidad in 1954, was standardized for use with the mosquito as a vector. Fort Detrick could produce 500,000 infected mosquitoes a month [22]. The Chemical Corps also standardized antiplant agents, the first of which was Puccinia graminis, which causes wheat stem rust, and could be used against cereal crops. Pyricularia oryzae, which causes rice blast, was also standardized before the program began to focus on chemical defoliants for use in Southeast Asia [22]. Despite investigating many biologic agents and dissemination systems, few biologic weapons were standardized. Among these was the M114 4-lb antipersonnel bomb filled Brucella suis. Over 100 of these 4-lb bombs were then used to fill the M33 500-lb cluster bomb. Antiplant biologic weapons included the M115 500-lb feather bomb, which carried the agent causing wheat stem rust as a dry particulate adhered to feathers, and the balloon bomb, which included a barometric trigger to release an agent at a designated altitude. The Corps also developed a unique naval biologic weapona covert submarine mine. Fired from a torpedo tube, the submarine mine would sink to the bottom for up to 2 hours, then rise to the surface to deliver up to 42 liters of a biologic agent, and finally scuttle itself [22]. The Chemical Corps supplemented its laboratory research at Fort Detrick and demonstrated US vulnerability to biologic weapons attack with several covert field tests using simulants of pathogenic agents and vectors. In September of 1950, agents released Serratia marcescens and Bacillus globigii organisms from ships into the waters of San Francisco Bay. Results of this test showed that in time, most of San Franciscos populace had inhaled substantial amounts of S marcescens [8,17]. In other covert tests in 1965 and 1966, the anthrax simulant Bacillus globigii was released at the National Airport and Greyhound Terminal in Washington, DC, and dropped from the street into subway stations in Manhattan. These experiments showed that disseminated at peak times, anthrax could infect large numbers of people in a similar real attack [22]. During the development of the yellow fever biologic weapon, uninfected mosquitoes were released from helicopters and planes over towns in Georgia and Florida. Within a day the mosquitoes had spread over several square miles and bitten numerous people, proving that infected mosquitoes could be an effective biologic weapons vector [22]. These field trials would eventually result in public distrust of military biologic weapons research [8]. Soon after the test release in the San Francisco Bay, an outbreak of S marcescens urinary tract infections
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occurred, resulting in one death. Although the strain released and the strain causing the infections were different, the wisdom of intentional S marcescens release was questioned [8,17]. Similarly, after Serratia species were aerosolized in tests over communities in Alabama and Florida, a record number of pneumonia cases were reported [17]. Although the United States denies ever employing actual biologic weapons in conflict, North Korea publicly made such accusations during the Korean War. On February 22, 1952, the North Korean Minister of Foreign Affairs accused the United States of numerous attacks with insects infected with plague and cholera [8]. China made similar allegations. Recent evidence showed that the allegations made by North Korea, China, and the Soviet Union were based on fabricated evidence [35]. These accusations, although ultimately shown to be false propaganda, resulted in loss of international goodwill toward the United States and its suspected biologic weapons program [13]. In the late 1960s, the international community became increasingly concerned over the use of biologic weapons. Great Britain, which had abandoned its own offensive biologic weapons research program in 1957, submitted a proposal to ban biologic weapons in 1969. A similar proposal was put forth by the Warsaw Pact nations later that year. Then, in November of 1969, President Nixon ended the US offensive biologic weapons program in an executive order renouncing future use by the United States of deadly or incapacitating biologic weapons and limiting future research to defensive strategies of vaccines and prevention and control measures. The order also mandated the destruction of all US biologic weapons stocks [13,21,22]. Between 1971 and 1973, the US biologic weapons stockpiles were destroyed at Pine Bluff Arsenal, Rocky Mountain Arsenal, and Fort Detrick. Small stocks of biologic weapons agents were preserved at Fort Detrick for use by the newly formed US Army Medical Research Institute of Infectious Diseases (USAMRIID) for defensive research in diagnostic testing, vaccinations, therapeutics, and other preventive measures against potential biologic weapons threats [13,22]. Ending offensive biologic weapons research in the United States was as much a practical decision as a moral one. Biologic weapons were largely untested in wartime conditions, and their use was considered risky and unpredictable, as in the case of the Japanese infecting their own troops in Manchuria. Additionally, prohibition and destruction of biologic weapons and related technologies helped prevent the proliferation of low-cost weapons of mass destruction and thus
preserved the United States and its allies advantage in conventional and nuclear weaponry [13]. 1972 Biological Weapons Convention Soon after the dismantling of the US biologic weapons program, the 1972 Convention on the Prohibition of the Development, Production, and Stockpiling of Bacteriological (Biologic) and Toxin Weapons and on Their Destruction (the Biological Weapons Convention or BWC) Treaty was created. This treaty was initially signed by over 100 countries, including the United States, the Soviet Union, and Iraq, and currently has over 140 signatories [27,36]. The BWC treaty made significant strides in limiting offensive biologic weapons proliferation by prohibiting the development, production, and stockpiling of biologic weapons agents and toxins as well as systems for disseminating them. Moreover, signatories were required to destroy all biologic agent stockpiles, weapons, and equipment after ratifying the treaty. Finally, the BWC treaty prohibits sharing biologic weapons technology with other countries [13]. However, the BWC treaty proved to have many weaknesses as a biologicl weapons control measure. Before ratification, all references to inspection, verification, and control procedures in the BWC treaty were deleted because they were unacceptable to the Soviet Union [27]. Thus, there is no mechanism by which signatories can verify another countrys compliance with the treaty [8]. The UN Security Council can initiate inspections, but these can be vetoed by Security Council members. Furthermore, the BWC treaty allowed for the possession of small quantities of biologic agents for research on prophylactic, protective or other peaceful purposes. Neither the quantities of biologic agents sufficient for defensive research nor the definition of defensive research are stipulated in the treaty [13]. As the Soviet Union and Iraq would illustrate, the BWC treaty proved an ineffective deterrent to biologic weapons proliferation, especially with recent advances in molecular biology and recombinant DNA techniques [8]. Soviet Union/Russia When Soviet diplomats signed the Biologic Weapons Convention in 1975, they amended the following: the Soviet Union does not possess any bacteriological agents and toxins, weapons, equipment or means of delivery [37]. However, almost immediately the Soviets continued biologic weapons research program as suspected by the United States [8]. From 1973 to 1974, the Soviet government created the Biopreparat
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(Chief Directorate for Biological Preparations), an ostensibly legal civilian biotechnology research outfit that was actually under state and military control, and tasked with carrying out the Soviet Unions biologic weapons research and production [38]. The world was alerted to the Soviet Unions secret biologic weapons program in April 1979, when an outbreak of respiratory anthrax occurred in Sverdlosk (now Ekaterinburg, Russia) [13]. The United States and Western intelligence believed that the epidemic was evidence of biologic weapons research, but the Soviets claimed that contaminated beef sold on the black market caused the outbreak [13]. In 1992, Russian President Boris Yeltsin admitted that the incident was the result of an accidental release of anthrax spores from a military research facility [39]. Up to 105 deaths occurred from the release of no more than 100 grams of spores [4]. Most of what is known about the former Soviet Unions biologic weapons program has been gathered from former high-level Soviet biologic weapons scientists who have defected to western nations. In 1992, Dr. Kenneth Alibek (formerly Kanatjan Alibekov) emigrated to the United States and subsequently described a Soviet bioweapons program so massive and advanced, it shocked US investigators during mutual inspections in the early 1990s [4]. Alibek was the former chief scientist and first deputy director of the Biopreparat complex, and worked in the program from 1975 to 1992 [4]. According to Alibek, in 1990, there were about 38 facilities at the Biopreparat alone, although a few performed legitimate pharmaceutical work to maintain cover appearances. The Biopreparat, the Ministry of Defense, and the Ministry of Agriculture employed 60,000 to 70,000 scientists, engineers, and technicians in creating a new class of genetically modified biologic weapons. Using new recombinant DNA technologies, the Soviets developed antibiotic-resistant strains of plague, anthrax, tularemia, and glanders that were stable as aerosols and persistent in the environment. The Biopreparat also created genetically altered smallpox, Venezuelan equine encephalomyelitis, and Q fever [4]. The Soviets were prepared to use these biologic agents as well. At full-surge capacity, the Biopreparat could produce hundreds of tons of pathogens per month at nine production sites [38]. Munitions could be filled within 2 to 3 days, and shipped to the front lines in a little over a week. These weapons, according to Soviet biologic weapons doctrine, would be used strategically alongside nuclear weapons in the event of total war where mutual destruction was possible. Alibek estimates that a single SS-18 inter-
continental ballistic missile equipped with multiple warheads filled with a strategic biologic agent [such as smallpox or plague] would be sufficient to cover a city the size of New York, killing at least 50 percent of the population [4]. At the same time that Yeltsin admitted the cause of the Sverdlosk outbreak, he asserted that he would end the Russian biologic weapons research effort [39]. However, because trilateral mutual inspections between the United States, the United Kingdom, and Russia ended in 1994, the current status of the Russian bioweapons program is unknown [4,27]. A former Soviet scientist and Western intelligence believe that Russia continues offensive biologic weapons research, particularly on genetic manipulations of vaccinia virus or monkeypox virus that are seemingly legitimate but could be quickly translated to smallpox bioweapons [4,27]. Also, after the breakup of the Soviet Union, the Biopreparat and related facilities were scaled back, and it is unclear where its former scientists and technicians have gone, and whether they took seed stocks of agents such as smallpox with them [4,36]. The Soviet Union also aided the communist Bulgarian government in at least two assassination attempts. In 1978, agents working for the Bulgarian government shot Bulgarian exiles Georgi Markov (in London) and Vladimar Kostov (in Paris) with tiny pellets fired from a gun disguised as an umbrella. The platinum/iridium pellets were drilled with holes that were filled with the deadly toxin ricin and capped with wax designed to melt at body temperature. Markov died from the attack, but apparently the wax on the pellet lodged in Kostovs subcutaneous tissue failed to melt. Kostovs life was saved when, on hearing of Markovs death, he saw a French doctor, who found and removed the pellet [8,31]. Iraq Although suspected of having a biologic weapons capability before the Persian Gulf War in 1991, Iraqs biologic weapons program was not fully revealed until after 4 years of postwar inspections by the United Nations Special Commission (UNSCOM). Iraqi defectors provided additional information [26]. Iraq began a sustained biologic weapons research program in 1985, with the Al-Muthanna research group at Salman Pak, and later at the Al-Hakam research facility [5,38]. At these sites, according to UNSCOM and Iraqi statements, Iraqis investigated and then produced 84,250 liters of Bacillus anthracis spores, 3400 liters of Clostridium perfringens spores, 380,000 liters of botulinum toxin, and 2200 liters of aflatoxin [5,38]. Additional work was performed on
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camel pox virus, enterovirus, rotavirus, ricin, and the antiplant agents wheat blunt and corn smut [5,38]. Iraq also used the agents produced to fill munitions, many of which were deployed and ready for use by the 1991 Persian Gulf War. Reportedly, Iraq had over 200 biologic weapons strategically deployed during the Persian Gulf War, including 200 R-400 400-lb. bombs and 25 modified SCUD missiles filled with botulinum toxin, anthrax, or aflatoxin [5,22]. Fortunately, for reasons that are unclear, Iraq did not use its biologic arsenal in 1991 [38]. Iraqi officials claimed that at the conclusion of the Persian Gulf War in 1991, they terminated all biologic weapons research and destroyed existing stockpiles [38,40]. However, Iraqs biologic weapons program may have been hidden in dual-use facilities. Spurred by new information from an Iraqi defector, UNSCOM investigators revisited the Al-Hakam facility, then producing a dry form of the biopesticide Bacillus thuringiensis, in 1994 and 1995. Sample testing revealed that the particle size of the B thuringiensis of less than 10 mm was better suited for aerosolized bioweapons applications than pesticide. Also, the fermentation process used at the site was capable of rapid conversion to anthrax production [26]. UNSCOMs continuous inspections of an often uncooperative Iraq ended in 1998 without the discovery of any biologic weapons. However, UNSCOM, as well as Western intelligence, believed that Iraqs biologic weapons program continued [38]. This belief that Iraq maintained biologic weapons of mass destruction (with the ability to mobilize biologic weapons in less than an hours time) was expressed often in building the case for the US-led coalition invasion of Iraq in March 2003 [41]. However, at the time of this writing, several months of continuing inspections have not revealed any stockpiles of weapons of mass destruction in Iraq, leading the former chief US weapons inspector to declare, were very unlikely to find large stockpiles of weapons . . . I dont think they exist [42]. The Iraq Survey Group did find evidence that Iraq maintained smaller, covert capabilities in bioweapons, such as a clandestine laboratory network, retained biologic agent cultures, and dual-use facilities, that could allow for surge production of biologic weapons agents [43].
are many, and include changes in motives, increased technologic sophistication, and increased resources of potential perpetrators [44,45]. Major motives for bioterrorism have changed from protesting governmental policies in the 1970s and 1980s to (in descending order of frequency) retaliation or revenge, furthering nationalist or separatist objectives, and apocalyptic prophecy, with the last often having mass casualties as the desired end [45]. Terrorist groups such as the Rajneeshees, with a state-certified medical laboratory, and the Aum Shinrikyo, with an estimated $1.5 billion in assets, are increasingly able to meet the technical and financial requirements to carry out acts of bioterrorism. Together, these trends may predict far deadlier future bioterrorist attacks, especially if groups can gain access to state biologic weapons programs (as may have happened in the US anthrax attacks of 2001) or achieve the technical expertise necessary to successfully aerosolize anthrax [10,44]. In a recent thorough review of bioterrorism and biocrimes from 1900 to 2000 (excluding, of course, the anthrax attacks of 2001), Carus defines bioterrorismthe threat or use of biological agents by [non-state] individuals or groups motivated by political, religious, ecological or other ideological objectives [44]. Biocrimes, however, have more traditional motives, such as revenge, financial gain, or psychologic pathology. Applying these definitions, Carus identified 54 cases of alleged bioterrorism, of which only 27 could be substantiated. Of these, only four terrorist groups actually used biologic agents, with one incident resulting in casualties [44]. Biocrimes were more numerous, with 56 confirmed cases of 82 potential incidents. Additionally, over 90 confirmed cases of anthrax hoax letters were identified that would likely be classified as criminal cases if more information about the perpetrators was known [44]. Like biologic warfare, cases of bioterrorism or biocrime are difficult to confirm. Events can only be studied if they are discoveredfor example, by law enforcement or news media. Many more events likely remain secret [45]. Of those events that are documented, many are not able to be substantiated through corroborating evidence, confirmation of agent possession, explanation of intent, or identification of a perpetrator [44,45].
Bioterrorism and biocrimes Bioterrorism As noted above, terrorist and criminal use of biologic agents is on the riseover 70% of substantiated cases of bioterrorism and biocrimes since 1900 occurred after 1990 [44]. The reasons for this increase Key cases in which possession of a biologic agent was confirmed (and in three cases used) are presented below.
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Aum Shinrikyo The Japanese Aum Shinrikyo cult, whose doctrine is dominated by apocalyptic prophecy, first came to international attention in March 1995, after it performed a well-coordinated attack using the nerve gas sarin on the Tokyo subway system, killing 12 and injuring 3800 people [46]. Subsequent investigations into the cult, including testimony of former members, revealed a heavy interest in biologic weapons [22,46]. With reported world-wide membership of 20,000 to 40,000 and assets of up the $1.5 billion, the cult financed two biologic weapons laboratories staffed with young recruited scientists and engineers who researched botulinum toxin, anthrax, cholera, and Q fever [46]. In 1993, the cults leader Shoko Ashahara, under the pretext of providing medical relief, led a team of 16 doctors and nurses to Zaire (now Democratic Republic of Congo) to attempt to collect Ebola virus [46]. Aum Shinrikyo members also performed several unsuccessful acts of bioterrorism in the early 1990s [46]. Twice cult members sprayed botulinum toxin from a specially equipped vehicle around targets in Tokyofirst government buildings in 1990, then near the wedding of the Crown Prince in 1993. Also, in 1993, cult members sprayed anthrax spores from the cults midrise Tokyo laboratory. After the attack, law enforcement authorities and the media reported brown steam, a foul smell, and some pet deaths. It is unclear why the attacks were unsuccessful [46]. After last-minute qualms, a cult member prevented a botulinum toxin subway attack in Tokyo by failing to load briefcase sprayers with the toxin [46]. Although most of Aum Shinrikyos leadership was arrested after the sarin attacks, the cult is once again recruiting new members and replenishing its coffers [46].
local county commission) grew antagonistic toward the cult and denied many of the permits the Rajneeshees needed to construct their commune [44]. Informant testimony later revealed that several commune leaders planned to clear the political way for construction of their community by making the electorate too sick to vote in the upcoming election for the Wasco County Court. They would then elect their own candidates by bringing thousands of homeless people into the commune and inducing them to vote [44]. In a trial run of the planned attack, cult members repeatedly and deliberately contaminated numerous local salad bars, restaurant coffee creamer, and grocery store produce with Salmonella typhimurium, resulting in the 1984 epidemic. In 1985, during a separate criminal investigation, the FBI confiscated a strain of S typhimurium identical to the outbreak strain from the compounds state-certified clinical laboratory. Additional testimony revealed that the group had plans to contaminate the municipal water supply [47]. Two members of the Rajneeshee cult were eventually convicted for their role in the first successful act of biologic terrorism on US soil [44].
Dark Harvest On October 10, 1981, a bucket of soil was found on the grounds of the Chemical Defense Establishment (the former site of Britains biologic weapons program) at Porton Down, Wiltshire, England. A group calling itself Dark Harvest claimed to have taken the soil from Gruinard Island, which was contaminated with anthrax during British biologic weapons testing during World War II, and returned the seeds of death in protest of the continued contamination of the island. Testing of the soil revealed low concentrations of anthrax spores. A second package of soil was placed at the site of a Conservative Party meeting a few days later, although no spores were found in this sample [44]. In 1986, 48 years after contaminating the island, the British government paid to have Gruinard Island decontaminated by spraying the island with 280 tons of formaldehyde mixed with 2000 tons of seawater [48].
Rajneeshees In September and October of 1984, an epidemic of Salmonella typhimurium caused 751 cases of gastroenteritis in The Dalles, Oregon [47]. Although an extensive outbreak investigation by the Oregon Health Division and the Centers for Disease Control and Prevention (CDC) linked the source of the outbreak to salad bars in local restaurants, it was not until a year later that officials discovered that the epidemic was the result of deliberate contamination. In 1981, followers of the Indian guru Bhagwan Shree Rajneesh bought land near The Dalles, Oregon, on which to build the cults international headquarters [47]. However, the community and the Wasco County Court (the
Minnesota Patriots Council In 1992, the FBI concluded a year-long investigation of a right-wing, antitax terrorist group in Minnesota when the wife of a member turned over a coffee can containing ricin and the solvent dimethyl sulfoxide (DMSO), incriminating her husband. Ac-
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cording to testimony, the group, known as the Patriots Council had plans to murder federal and local law enforcement agents by mixing the ricin with the DMSO to enable it to penetrate the skin. They also had plans to bomb a federal building. The group extracted the ricin from castor beans using instructions and beans they purchased by mail order [44]. Key members of the Patriots Council were later convicted and sentenced to jail for their role in the plot, becoming the first convictions under the Biological Weapons and Antiterrorism Act of 1989 [49]. R.I.S.E Another bioterrorism plot was thwarted in 1982, when Chicago police arrested two teenagers who were days away from contaminating Chicagos municipal water supply with Salmonella typhi [44]. The two teens created a white supremacist organization dubbed R.I.S.E, and planned to start a new master race by vaccinating the groups members against key pathogens so that they would survive the groups contamination of area water supplies [45]. The two had only begun to recruit and vaccinate a handful of members when two of the new recruits, alarmed by the plan, informed the police. Subsequent investigations revealed that the founders had stocks of S typhi. After their arrests, the two teens skipped bail and fled to Cuba, where one was later arrested by Cuban authorities for uncertain crimes [44]. Larry Wayne Harris Larry Wayne Harris was first arrested in 1995 for illegally obtaining stocks of Yersinia pestis. With $300 and false letterhead, Harris was able to order the pathogen from the American Type Culture Collection (ATCC) in Rockville, Maryland. Only when he complained about a delay in the shipment were employees at the ATCC suspicious enough to report him. A member of the white supremacy groups Aryan Nations and Christian Identity Church, Harris claimed that the cultures were part of his plan to find a cure for plague and protect citizens from the Iraqi biologic weapons threat. Harris was convicted of fraud in acquiring the cultures, and sentenced to 18 months probation [44]. Harris may have also been responsible for a disruptive explosion of anthrax hoax letters mailed in 1998 and 1999 [45]. Harris was arrested again in early 1998 for possession of anthrax. Although the anthrax strain proved to be a harmless veterinary vaccine strain, the case generated much publicity, and soon after, false anthrax claims became popular.
In both incidences, it is unclear if Harris had any terrorist intent [44].
Biocrimes Biocrimes, which are usually more limited in scope than bioterrorism, are often committed by one or two individuals (rather than groups) who are motivated by revenge, personal gain, and individual psychopathology rather than ideology. As in the case of biologic warfare, criminals began using pathogens to harass, manipulate, and murder almost as soon as the germs were recognized. Often, physicians or other medically trained personnel are responsible for these crimes [44]. Early criminals chose the unsubtle route of injecting their victims with pathogens and toxins. In 1911, Patrick OBrien de Lacy and Vladimir Pantchenko were convicted in Russia for the murder of de Lacys brother in law. De Lacy, a fallen captain of industry, solicited the aide of Pantchenko, a physician of suspect character, in a murderous plot to secure a large family fortune. Under the guise of administering anticholera serum, Pantchenko injected a lethal dose of diphtheria toxin to the unsuspecting brother-in-law [44]. In 1933, Benoyendra Chandra Pandey and Dr. Taranath Bhatacharya orchestrated a deadly attack using Yersinia pestis against Pandeys half-brother, ending a feud over the division of their fathers estate. Bhatacharya, another corrupt physician, obtained Y pestis cultures, and the two paid a stranger to bump into and inject the half-brother while he was standing on a train platform [44]. Contamination of food with bacteria and bacteriologic toxins was also a favorite tactic of wrongdoers. In 1921, a French insurance broker, Henri Gerard, was put on trial for poisoning several people in a series of fraudulent insurance schemes. Gerard convinced his victims to purchase life insurance policies naming him as the beneficiary. Then, he prepared meals laced with S typhi and poisonous mushrooms, killing two people [44]. During the early 1960s, a Japanese resident physician, frustrated by his subordinate role, infected up to 200 patients, colleagues, and family members with S typhi and Shigella dysenteriae. He may have also caused the outbreaks to further his thesis research in bacteriology [44,50]. More recently, a laboratory worker in a medical center in Texas was indicted for three charges of tampering with a food product after pastries she deliberately contaminated with S dysenteriae type 2 caused 12 coworkers to become ill, four seriously enough to be hospitalized. The lab worker laced blueberry muffins
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and donuts with the bacteria and placed them in a break room accessible only by key code. She then sent an anonymous e-mail inviting other lab employees to enjoy the treats [50].
Anthrax attacks of fall 2001 On October 4, 2001, about 1 month after the September 11 terrorist attacks on the World Trade Center towers in New York City and the Pentagon in Washington, DC, Florida health officials announced the first case of pulmonary anthrax seen in the United States in almost 25 years. The patient, a tabloid photo editor at American Media, Inc. (AMI), was initially thought to have contracted inhalational anthrax naturally, perhaps during a trip through North Carolina, in which he drank from a stream [10]. The photo editor died the next day, and soon anthrax spores were discovered on his office computer keyboard and in the nose of a mail clerk in the AMI building who had been hospitalized with pneumonia symptoms. After these findings, the AMI offices in Boca Raton, Florida, were closed, and a deliberate bioterrorism attack was suspected [10,51]. Over the next 3 weeks, additional cases of both inhalational and cutaneous anthrax were identified among employees of media outlets in New York City (the three major broadcast networks ABC, NBC, and CBS, as well as the New York Post). Most of these cases occurred in association with prior exposure to a powder-laden envelope, two of which were recovered, tested positive for anthrax, and traced to their origin in Trenton, New Jersey. Additional cases were confirmed in US Postal Service (USPS) workers who worked in the path of the letters [51]. A third anthrax letter was opened in mid-October in the office of Senator Tom Daschle (D-SD). USPS workers along the path of this letter developed inhalational anthrax, challenging the assumption that a securely sealed envelope could prevent the release of anthrax spores [10]. Cases among the Senate office workers were likely avoided by prophylactic antibiotic use [51]. A quarantine and search of all federal mail revealed an additional anthrax letter addressed to Senator Patrick Leahy (D-VT). These final two letters were also mailed from Trenton, New Jersey. In all, four anthrax letters were recovered, although as many as seven were suspected to have been mailed [10]. It was difficult to explain the mode of transmission in four anthrax victimstwo cutaneous cases (a mail carrier and a bookkeeper in New Jersey) and two inhalational cases (a hospital worker in New York City and a homebound elderly woman in Connecti-
cut). None of these patients were exposed to the involved government or media worksites or to the path of the contaminated letters, and only one of many environmental samples taken from their worksites and homes was positive for B anthracis. However, mail to these individuals did cross paths with the contaminated letters, suggesting that anthrax infections may be possible with the presumably very low inoculum carried to the victims on crosscontaminated letters [10,51]. Previous studies asserted that 8 to 10 thousand spores were necessary to cause inhalational anthrax [51]. As many as 5000 letters could have been crosscontaminated [10]. In all, the CDC identified a total of 22 cases of anthrax from October 2 to November 20, 2001. Of these, 11 cases were cutaneous anthrax (seven confirmed and four suspected) and 11 were confirmed inhalational anthrax, five of whom died. In addition, a lab worker contracted cutaneous anthrax through handling samples related to the attacks [51]. The perpetrator(s) of the attacks is still unknown. Initially, foreign terrorists were suspected in relation to the World Trade Center attacks. However, testing of the collected B anthracis samples revealed that the strain was genetically identical, except for some subtle, miniscule differences, to the Ames strain, which was developed as a biologic weapon by the United States. The strain is now maintained at Fort Detrick by the USAMRIID, and shared with a limited number of laboratories in the United States, Canada, and England [10,52]. With this revelation, the FBI suspected a domestic terrorist, and issued a profile of the perpetrator: a nonconfrontational loner adult male with a scientific background, access to labortory equipment, and a previous anthrax vaccine [10]. In the period after the anthrax attacks, anthrax hoaxes once again erupted. By December 2001, nearly 40 people faced federal charges in connection with anthrax hoaxes. The FBI investigated over 2300 suspected anthrax incidents, very few of which were substantiated. The USPS received over 15,800 anthrax reports [10]. In addition to the direct morbidity and mortality associated with the anthrax cases, the anthrax attacks had huge financial and psychologic effects. The Hart Office Building was quarantined for 96 days for decontamination at a cost of $42 million to the Environmental Protection Agency alone. Full cleanup costs for all contaminated buildings were in the range of hundreds of millions of dollars [10,53]. According to a poll taken at the height of the anthrax attacks, one in four people felt that they would be hurt or killed by a bioterrorist attack [54]. Roughly half of the respondents felt that the terrorist attacks (including the World
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M.K. Jacobs / Dermatol Clin 22 (2004) 231246 Motives for bioterrorism are changing to include
Trade Center attacks) would fundamentally change the way they would live their lives [54].
Summary Several conclusions can be drawn from this historic survey of biologic weapons:
Biologic weapons have been used throughout
history by states and individuals. This willingness to use biologic weapons will likely persist into the future. Compared with conventional weapons, biologic weapons are economical, effective, and relatively easy to procure. It is estimated that a major biologic weapon could be produced with just $10,000 and a room the size of a two-car garage [17]. A biocrime using contaminated food costs as little as the price of a box of donuts. Only 50 kg of anthrax spores could kill nearly 100,000 people in an aerosolized attack [17]. Many potential pathogens are easily found in the environment, reference laboratories, and even in our own bodies. These properties will ensure continued interest in biologic agents. Past uses of biologic weapons may not predict future uses. To date, attacks with biologic agents have not caused large-scale morbidity and mortality. However, a successful aerosolized attack with a biologic agent, such as Aum Shinrikyo attempted in 1993, would have much greater consequences. Effective dispersal systems for biologic agents appear difficult for small groups to develop, although this may change. Terrorists, criminals, and small countries represent the biggest biologic weapons threat. Wartime uses of biologic weapons have been mostly ineffective (although the former Soviet programs weapons were potentially very effective). Biologic agents and technical advances developed in former large-state biologic weapons programs may be transferred to terrorists or rogue states. The weaponized anthrax spores used in the 2001 anthrax letters likely originated from strains developed by the US biologic weapons program. Technical and financial barriers to successful bioweapons use by nonstate actors may be disappearing. Some groups, such as the Rajneeshees and Aum Shinrikyo, possessed sufficient funds and technical expertise to develop their own biologic weapons.
apocalyptic prophecy in which mass casualties are the desired result, not just disruption and fear. Through advances in genetic engineering, states may conduct covert biologic weapons research in related organisms that may quickly be translated to pathogens. Combined with dualuse facilities, countries may maintain a biologic weapons capability without weapons stockpiles or other direct evidence. Biologic weapons are resistant to antiproliferation attempts. The BWC treaty is not an enforceable control measure. For these reasons, biologic weapons will remain a formidable threat into the foreseeable future. As a result, a comprehensive defensive strategy against biologic weapons is necessary. Part of this strategy includes adequate education of health care professionals on the problem of biologic weapons.
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M.K. Jacobs / Dermatol Clin 22 (2004) 231246 attacks [working paper]. Washington (DC): Center for Counterproliferation Research, National Defense University; Nov 2001. Available at: http://www.ndu. edu/centercounter/ANTHRAX%20CHRONOLOGY. pdf. Accessed October 1, 2003. Inglesby TV, Henderson DA, Bartlett JG, Ascher MS, Eitzen EE, Friedlander AM, et al. Anthrax as a biological weapon: medical and public health management. JAMA 1999;281(18):1735 45. OToole T, Mair M, Inglesby TV. Shining light on Dark Winter. Clin Infect Dis 2002;34:972 83. Christopher GW, Cieslak TJ, Pavlin JA, Eitzen EM. Biological warfare: a historical perspective. JAMA 1997;278(5):412 7. Wheelis M. Biological warfare before 1914. In: Geissler E, van Courtland Moon JE, editors. Biological and toxin weapons: research, development and use from the Middle Ages to 1945. New York: Oxford University Press; 1999. p. 8 34. Rothschild JH. Tomorrows weapons: chemical and biological. New York: McGraw-Hill Book Company; 1964. p. 11 20. Robertson AG. From asps to allegations: biological warfare in history. Mil Med 1995;160(8):369 73. Lesho E, Dorsey D, Bunner D. Feces, dead horses, and fleas: evolution of the hostile use of biological agents. West J Med 1998;168:512 6. Froissart J. The chronicle of Froissart, translated out of French by Sir John Bourchier Lord Berners, Annis 1523 25, vol. 1. New York: AMS Press; 1967. p. 142 [Quoted in: Wheelis M Biological warfare before 1914. In: E Geissler, JE van Courtland Moon, editors. Biological and toxin weapons: research, development and use from the Middle Ages to 1945. New York: Oxford University Press; 1999. p. 8 34]. Derbes VJ. De Mussis and the great plague of 1348: a forgotten episode of bacteriological warfare. JAMA 1966;196(1):179 82. re sie de Viclef, Jean Hus, et Varillas. Histoire de lHe de Jerome de Prague. Lyon (France): Chez Iean Certe; 1682. p. 117 [Quoted in: VJ Derbes. De Mussis and the great plague of 1348: a forgotten episode of bacteriological warfare. JAMA 1966;196(1):179 82]. Eitzen EM, Takafuji ET. Historical overview of biological warfare. In: Sidell FR, Takafuji ET, Franz DR, editors. Medical aspects of chemical and biological warfare. Washington (DC): Borden Institute; 1997. p. 415 23. Smart JK. History of chemical and biological warfare: an American perspective. In: Sidell FR, Takafuji ET, Franz DR, editors. Medical aspects of chemical and biological warfare. Washington (DC): Borden Institute; 1997. p. 9 86. MacCallum FO, McDonald JR. Survival of variola virus in raw cotton. Bull World Health Organ 1957; 16:247 54. Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and its eradication. Geneva (Switzerland): World Health Organization; 1988.
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M.K. Jacobs / Dermatol Clin 22 (2004) 231246 of chemical and biological warfare. Washington (DC): Borden Institute; 1997. p. 451 66. CNN. Iraq war allies face tough questions on weapons. Available at: http://www.cnn.com/2003/WORLD/ meast/06/04/sprj.irq.main/index.html. Accessed January 26, 2004. CNN. Kay: No evidence Iraq stockpiled WMDs. Available at: http://www.cnn.com/2004/WORLD/meast/01/ 25/sprj.nirq.kay/index.html. Accessed January 26, 2004. Kay D. Statement before the House Permanent Select Committee on Intelligence, the House Committee on Appropriations, Subcommittee on Defense, and the Senate Select Committee on Intelligence on the interim progress report on the activities of the Iraq Survey Group (ISG). October, 2, 2003. Available at: http:// www.cia.gov/cia/public_affairs/speeches/2003/david_ kay_10022003.html. Accessed January 26, 2004. Carus WS. Bioterrorism and biocrimes: the illicit use of biological agents since 1900 [working paper]. Washington (DC): Center for Counterproliferation Research, National Defense University; Aug 1998, revised Feb 2001. Available at: http://www.ndu.edu/ centercounter/Full_Doc.pdf. Accessed October 1, 2003. Tucker JB. Historical trends related to bioterrorism: an empirical analysis. Emerg Infect Dis 1999;5(4): 498 504. Olson KB. Aum Shinrikyo: once and future threat? Emerg Infect Dis 1999;5(4):513 6. [47] To ro k TJ, Tauxe RV, Wise RP, Livengood JR, Sokolow R, Mauvais S, et al. A large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars. JAMA 1997;278(5):389 95. [48] Manchee RJ, Stewart R. The decontamination of Gruinard Island. Chem Br 1988;24:690 1. [49] Wangstad W. 2 Minnesota men first to be convicted under Biological Weapons Act. St. Paul Pioneer Press 1995;March 1:3B. [50] Kolavic SA, Kimura A, Simons SL, Slutsker L, Barth S, Haley CE. An outbreak of Shigella dysenteriae type 2 among laboratory workers due to intentional food contamination. JAMA 1997;278(5):396 8. [51] Jernigan DB, Raghunathan PL, Bell BP, Brechner R, Bresnitz EA, Butler JC, et al. Investigation of bioterrorism-related anthrax, United States, 2001: epidemiologic findings. Emerg Infect Dis 2002;8(10):1019 28. [52] Fainaru S, Warrick J. Ames strain of anthrax limited to few labs. Washington Post 2001;November 30:A1. [53] Shane S. Cleanup of anthrax will cost hundreds of millions of dollars. Baltimore Sun 2002;December 18. Available at: http://www.baltimoresun.com/bal-te. anthrax18dec18.story. Accessed January 26, 2004. [54] Pinkus S. Poll Analysis: Psychological effects of Sept. 11. Los Angeles Times 2001;December 21. Available at: http://www.latimes.com/news/custom/ timespoll/la-463pa4an,0,2583617.story?coll=la-newstimes_poll. Accessed January 26, 2004.
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Dermatol Clin 22 (2004) 247 256
Anthrax
Kimberly A. Wenner, MDa, Julie R. Kenner, MD, PhDb,c,*
a
Family Practice, Reynolds Army Community Hospital, 10 Briarcreek Drive, Fort Sill, OK 73505, USA b Department of Dermatology, Castle Medical Center, 640 Ulukahiki Street, Kailua, HI 96734, USA c Clinical Research, VaxGen, 1000 Marina Boulevard, Brisbane, CA 94005-1841, USA
Anthrax, a zoonotic disease that has become vanishingly rare in the industrialized world, tops the list of Category A (most lethal) biologic warfare and terroristic threats. Easily extracted from soil around the world, anthrax is cheap and readily grown in large quantities, and has long been part of the known bioweapons arsenal of several countries, most notably, the former Soviet Union and Iraq [1]. As a consequence, for many decades the standard education for military physicians has included training on the clinical recognition of this and other nonindigenous or exotic diseases. However, following the deadly anthrax mailings of 2001, it has become clear that the use of such agents is no longer confined to the battlefield. The World Health Organization has estimated that 50 kg of anthrax spores released upwind of a population center of 500,000 could result in up to 95,000 deaths, with a further 125,000 incapacitated [2]. These sobering facts compel every physician in the United States to familiarize themselves with the clinical presentation and management of anthrax. Dermatologists played a prominent role in the diagnosis of cutaneous anthrax in the recent terror attacks, and are expected to continue to play a critical role in this capacitynot only for anthrax, but also for other bioweapons that prominently affect the skin, such as smallpox, plaque, and tularemia.
History Anthrax is one of the oldest recorded diseases of humans and animals, with Egyptian and Greek references dating back several millennia [3]. The disease was known as the Black Bane in the Middle Ages, and major outbreaks (plagues) of anthrax are described in the Bible [4]. In the late 1870s, Robert Koch identified anthrax spores with a microscope, and, using experiments to answer a series of logical scientific postulates, proved to the medical community that microorganisms (germs) are responsible for human disease. Shortly thereafter, anthrax again made medical history when Louis Pasteur developed the first effective live bacterial vaccine using an attenuated strain of anthrax [5].
Anthrax as a weapon of war and terror Before October 2001, the role of anthrax as a weapon had been explored by numerous nations. During World War II, the Japanese military experimented with anthrax on prisoners of war held captive in Manchuria [6]. The British studied anthrax in that same war as a possible offensive agent to be used against the Germans, and exploded several bombs laden with anthrax spores on an island off Scotland [6]. The United States had an offensive biologic weapons program during the Cold War, and produced large amounts of anthrax before the termination of this program in 1969 [6]. The United States efforts appear to have been dwarfed by the former Soviet Union biologic weapons program, which focused heavily on anthrax as an offensive agent [7]. The
* Corresponding author. Department of Dermatology, Castle Medical Center, 640 Ulukahiki Street, Kailua, HI 96734. E-mail address: jkenner480@pol.net (J.R. Kenner).
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accidental release of anthrax spores from a military biologic facility near the city of Sverdlovsk, Russia, in 1979, resulted in the largest epidemic of inhalational anthrax to have ever occurred in humans [8]. At least 66 people in a 4-km radius of the facility died. Details regarding the epidemiologic and pathologic nature of these deaths now make up the basis of our understanding of this clinical form of anthrax, which occurs only rarely in nature [8,9]. In 1995, Iraq admitted to filling warheads with biologic agents, among them anthrax spores [10]. That same year, the religious cult Aum Shinrikyo was found to have released anthrax multiple times in Tokyo, Japan, although no cases of human infection resulted from these attacks [11]. Worldwide, it is thought that at least 13 countries have active bioweapons programs despite the Biological Weapons Treaty [11]. In October of 2001, the United States experienced an outbreak of 22 anthrax cases, after at least four and probably five letters were mailed to media companies and US senators [12]. The letters contained anthrax spores apparently specifically designed to aerosolize upon opening. In actuality, many spores were aerosolized from sealed letters that were processed through high-speed mail sorters, a fact that dramatically increased the radius of exposure. All together, 22 cases of anthrax occurred, 20 of which were in postal workers, media employees, or others known to have been in proximity with buildings contaminated by the anthrax-laced letters. Five people eventually died of inhalational anthrax, a number that could have been considerably higher had it not been for the prompt prophylactic treatment of those with possible exposure to the letters [13]. Nevertheless, two cases of anthrax occurred in individuals with no known link to the letters. Like the perpetrator, these cases remain a mystery [12].
cattle the major species infected. Humans contract the disease incidentally, through contact with infected animals or meat, or hides or animal fur containing anthrax spores. Naturally occurring anthrax can be found in agricultural regions throughout the world, especially in Africa, Greece, Turkey, Central Asia, and the Middle East [14]. Worldwide, it has been estimated that as many as 20,000 to 100,000 human cases of anthrax (almost exclusively cutaneous) develop each year, generally in underdeveloped regions of the world where livestock are not vaccinated [4]. Before 2001, only 236 cases of naturally occurring anthrax had been reported in the United States in the last 50 years, with less than one infection per year since 1980 [4,15]. The vast majority of cases were cutaneous anthrax, the most benign form of the disease. The 18 cases of inhalational anthrax that have occurred in the past century in the United States were predominantly associated with exposure to wool or animal hair (woolsorter disease) [16]. Gastrointestinal (GI) anthrax has never been reported in the United States [17].
Pathogenesis Clinical manifestations of anthrax occur when spores gain access to the body and germinate within macrophages. With cutaneous anthrax, germination generally stays localized at the site of infection, with edema and necrosis the result of toxins and hyperinflammatory responses [1]. However, in 5% to 20% of untreated cutaneous anthrax cases, the spores are carried to regional lymph nodes by the macrophages. There, the spores germinate, and the vegetative bacteria multiply rapidly and disseminate through the blood and lymphatic systems, resulting in hemorrhagic lymphadenitis, and possible death from massive septicemia and toxemia. Inhalational anthrax occurs when spores are inhaled and deposited in the lungs. The diameter of spores must be between 1.5 and 5 mm for disease to occur [14]. Particles larger than this cannot access the alveoli, and particles smaller than this are apparently too small to settle, and are exhaled [14]. The number of spores needed to cause infection or death in humans is not precisely known, but animal studies suggest the median lethal dose is from 2500 to 55,000 inhaled spores [18]. Epidemiologic findings from the October 2001 anthrax attacks suggest this number could be much lower in some individuals. Spore germination varies from 1 to 60 days or longer, as suggested by a primate death, which is
Epidemiology Anthrax is caused by Bacillus anthracis, an aerobic Gram-positive rod. In nature, the bacterium exists in soil in the form of spores, which are metabolically dormant, and resistant to light, heat, dessication, and many disinfectants [1]. This state may remain stable for many decades. It is only after the spores become introduced into an animal or human through abrasion, inhalation, or ingestion that the bacterium transforms into the vegitative state, becomes metabolically active, multiplies, expresses virulence factors, and produces clinical disease. Because the spores can be ingested in contaminated grasses, the disease is largely one of herbivore livestock, with sheep, goats, and
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reported to have occurred 98 days after experimental anthrax exposure [11,18]. From the lung, macrophages take up the spores and carry them to regional lymph nodes where they germinate and proliferate. Once germination occurs, bacteria rapidly proliferate leading to overwhelming septicemia, shock, and death [1]. The two major virulence factors of anthrax, the capsule and two toxins, are produced by plasmids, which are only activated in the vegetative form of the bacterium [1]. The polyglutamyl capsule of B anthracis, is coded for by genes on a plasmid called X02. This capsule protects the vegetative form of the bacilli from phagocytosis and allows for proliferation of the bacteria to proceed unchecked. The two powerful toxins of anthrax are produced from three exotoxin components expressed by the X01 plasmid [1]. These binary toxins are the major cause of morbidity and mortality associated with the disease. The protective antigen (PA) component is common to both toxins, and functions to enable the transport of the toxins across the cellular membrane [11]. Edema toxin, is made up of edema factor, and PA, and, as the name implies, is responsible for massive edema. Edema factor is an adenylate cyclase that converts adenosine triphosphate to cyclic adenosine monophosphate. Increased cellular levels of cyclic adenosine monophosphate leads to the massive exodus of water from cells, causing the characteristic edema of anthrax infections [1]. The toxin also inhibits neutrophil function, which likely augments the propagation of the infection [1]. Lethal toxin is comprised of lethal factor and PA. Lethal factor is a zinc metalloprotease, and inactivates mitogen-activated protein kinase kinase, which results in a hyperinflammatory state within the macrophages [1]. When injected into experimental animals, lethal toxin mimics the clinical and laboratory manifestations of systemic anthrax disease [19]. Two theories exist regarding the etiology of the lethality of this toxin. The long-standing theory based on in vitro modeling and nonclinical studies has been that lethal toxin causes lysis of the macrophages, which leads to a massive release of cytokines such as tumor necrosis factor alpha and (IL-1) [20]. The ensuing inflammation, septicemia, and toxemia with DIC lead to rapid deterioration and death from profound shock [14]. A more recent theory however, stipulates that the effects of lethal toxin are independent of tumor necrosis factor alpha release [19,21]. Instead of a cytokinemediated collapse with DIC, according to this theory lethal factor causes hypoxia-mediated tissue damage, which subsequently leads to shock and death [19,21]. Further studies are ongoing to clarify the exact mecha-
nisms of lethal toxins actions on tissues and how this might be related to new therapeutics.
Clinical disease Cutaneous anthrax The name anthrax is derived from the Greek word for coal, which describes the characteristic black eschar that develops in cutaneous anthrax [1]. The majority of cutaneous anthrax cases occur in people exposed to anthrax-infected animals such as farmers or veterinarians, or laboratory workers with occupational contact to the bacterium. Initially, the disease starts as a painless, often pruritic papule resembling an insect bite that occurs 1 to 12 days after spore exposure [22]. Spores may enter through small cuts, insect bites, or abrasions, most commonly on exposed skin of the head, neck, or upper extremity. The papule enlarges and develops a central vesicle or bulla within 1 day, with impressive surrounding edema due to toxin production. In areas with loose skin, such as the neck or periorbital region, the edema can become massive. Subsequently, the vesicle becomes hemorrhagic, cloudy, and depressed, with necrosis and ulcer formation. A black eschar of 1 to 5 cm forms over the ulcer, with many centimeters of surrounding edema and erythema. Satellite vesicles and ulcers may occur. This characteristic painless ulcer with black eschar and surrounding edema is the hallmark of cutaneous anthrax, and serves to differentiate it from other ulcerforming diseases, such as Brown Recluse spider bites, or tularemia [23,24]. During the peak of the lesion, those affected may experience fever, headache, malaise, and regional lymphadenopathy, possibly due to systemic dissemination of the bacillus [11]. This clinical pattern broadens the differential to include plague, rickettsial infection, and staphylococcal disease, among others. Fever in the setting of a painful purulent ulcer suggests secondary bacterial infection, most likely with staphylococcus or streptococcus. Over the next 1 to 2 weeks, the eschar loosens and falls off, and healing occurs often without leaving a permanent scar [1]. Spontaneous self-limited healing is the rule, with up to 90% of cases healing without complication. Nevertheless, rare complications can occur, such as malignant edema of the head and neck interfering with respiration or toxic shock due to overwhelming septicemia [1]. Mortality in untreated cases of cutaneous anthrax is estimated to be 5 to 20% [25]. Antibiotics are advocated early in the course of disease. However, this treatment has no affect on symptoms of the disease caused by toxin production.
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Antimicrobials, supportive care, and systemic steroids (if needed to treat malignant edema) together have reduced the mortality rate of cutaneous anthrax to less than 1% [4,18]. Half of the anthrax cases involved in 2001 terror attack (11 of 22) were cutaneous forms of the disease [12]. Most cases presented as individual lesions on the face, arms, or chest that demonstrated the characteristic features of anthrax. Two of the victims displayed multiple lesions, and one infant, initially diagnosed with a Brown recluse spider bite, became systemically ill with microangiopathic hemolytic anemia and coagulopathy [25]. This particular complication of cutaneous anthrax has only been described once before [26].
multitude of symptoms [9]. Because the disease is predominantly toxin-mediated, person-to-person spread has not been documented. In the majority of inhalational anthrax cases, chest X-rays are characteristically abnormal, showing mediastinal widening with infiltrates, or pleural effusions. In the inhalational anthrax cases of October 2001, 10 of the 11 cases demonstrated these distinctive radiographic findings [11,28].
Gastrointestinal anthrax GI anthrax occurs 2 to 5 days after spores are ingested, usually from raw or undercooked meat of infected animals [11]. Because anthrax is more common in the developing world where reporting is not common, the true incidence of GI anthrax is unknown. Likewise, the mortality rate is not precisely known, although rates have been estimated at 4% to 50% [5,23,29]. In the few community-based studies that exist, the clinical spectrum of GI anthrax ranges from gastroenteritis to an acute abdomen with bloody diarrhea, ascities, and shock [29]. Two different forms of GI anthrax are thought to occur. Oropharyngeal anthrax is the result of spores lodging in the oropharynx, which produce typical anthrax-like nodules or ulcers on the tonsils, pharynx, or hard palate. The resulting toxin-mediated swelling results in pain, dysphonia, dysphagia, and airway compromise [29]. The oral lesions undergo necrosis and ulceration, and often have a white to gray pseudomembranous surface [29]. Intestinal anthrax is the result of spores gaining access to the intestinal mucosa, with lesions occurring on the mid-jejunum, terminal ilieum, or cecum [23]. GI ulcers can be solitary or closely grouped, and are associated with regional lymphadenopathy. Massive enlargement of the lymph nodes and extensive ulceration can result in GI obstruction, bleeding, perforationm and acites [1,23]. Death occurs from hemorrhage, shock, or sepsis. Survivors (4 50%), typically take several days to weeks to recover completely [1].
Inhalational anthrax Before the recent anthrax terrorist attack, the last known case of inhalational anthrax in the United States was reported in 1976 [12]. Of the 18 total US cases reported in the last 100 years, 16 were associated with textile workers and 2 with laboratory workers. Before 2001, the case fatality rate of inhalational anthrax was generally quoted as 80% or more [16]. This rate was considerably less in the recent outbreak, with the death rate of 45% likely attributable to early identification of illness, aggressive supportive care, and possibly the use of more than one antibiotic in treatment [11,12]. Early diagnosis of inhalational anthrax can be difficult if there is no exposure history. The rarity of the naturally occurring disease, combined with the nonspecific nature of the systemic complains, often make for late diagnoses. Patients typically complain of an influenza-like illness with fever, malaise, and cough [27]. In the recent terror attack, the average time from exposure to symptoms was 4 days [27]. However, presumably because viable anthrax spores may remain in the lungs for a prolonged period before germination, there have been reports of late onset of symptoms (up to 6 weeks) following exposure to anthrax spores [8]. Inhalational anthrax is not a true pneumonia as the bronchoalveolar lung tissue is not primarily affected. The pulmonary system becomes involved through the invasion of the peribronchial and mediastinal lymph nodes by germinating spores, which release large amounts of lethal toxin, leading to hemorrhagic thoracic lymphadenitis, hemorrhagic mediastinatis, and pleural effusions [5,12]. Subsequently, the disease can disseminate widely, with involvement of the central nervous system and GI systems producing a
Diagnosis The diagnostic work up for suspected cutaneous anthrax involves first alerting the local Department of Health (directory at http://www.cdc.phpo), and obtaining specific instructions about collecting lab samples [22]. National reference laboratories have rapid diagnostic tests for anthrax including enzyme linked
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immunosorbent assay and polymerase chain reaction (PCR), but it may take several days to obtain definitive results [18]. When handling specimens, glove and gown precautions should be followed, but a mask is not necessary. All areas that may be exposed to lesional fluid should be thoroughly cleaned with standard hospital disinfectant, and instruments autoclaved. Vegetative anthrax bacilli are readily killed with normal hospital disinfectant containing hypochlorite. Spores, although resistant to short term exposure, are generally killed after an hour or more of exposure [30]. B anthracis is best identified when cutaneous lesions are in the vesicular or pustular stages, as fluid from the lesion contains numerous organisms that can easily be seen on Gram stain [22,23]. Sterile dacron or rayon swabs should be used for obtaining cultures, and placed in standard bacterial transport media. (sheeps blood agar, chocolate agar) [31]. If an eschar is present, swabs should be inserted under the eschar and rotated. An ulcer without an eschar may be sampled with a moist swab. Full-thickness punch biopsies should be obtained for histology, PCR studies, and immunohistochemistry studies. If the lesion is still in the vesicular/pustular stage, the biopsy is best taken from the border of the central lesion and surrounding erythematous skin. If an eschar is present, biopsies should be taken from the center of the eschar as well as the erythematous skin adjacent to the eschar [22]. Whenever anthrax is suspected, blood should be drawn for culture and sensitivities before antibiotic administration if possible. The laboratory should be notified, both to maintain adequate precautions when handling the specimens and to properly speciate any bacillus that results. Anthrax may be diagnosed retrospectively from acute and 6-week convalescent blood by assaying for anticapsule or anti-PA antibodies [23]. The 11 cutaneous anthrax cases that resulted from the 2001 terrorist attacks were diagnosed using a variety of techniques. The anthrax organism was cultured from skin lesions in two cases, found by immunohistochemical stains or PCR of skin biopsies in six cases, and identified serologically by anti-PA IGg titers in three cases [12]. Inhalational anthrax can be diagnosed by blood cultures, provided blood was drawn before antibiotic administration. Because of the massive amount of circulating bacteria, most cultures will be positive in the first 24 hours [12]. PCR of blood and pleural fluids is also helpful, and can be done at laboratories in the Laboratory Response Network [22]. If antibiotics have already been used, immunohistochemical examination of pleural fluid or bronchial biopsy can identify
antibodies to the capsule or cell wall of anthrax [27]. Serologic testing can be done at time of evaluation and 6 weeks postinfection [12]. Of the 11 inhalational anthrax cases identified in October 2001, seven were diagnosed by blood cultures, one by cerebral spinal fluid and blood cultures, one by PCR of the blood and pleural fluid, and two with positive immunohistochemistry testing of transbronchial and pleural biopsies [12,32]. The diagnosis of GI anthrax requires a high index of suspicion. In addition to primary infection, GI anthrax can occur secondarily from inhalational anthrax [9,12]. Laboratory diagnosis may be obtained from stool, blood, or peritoneal cultures, tissue samples, or serologic evidence of infection [30]. PCR can be used on any of the above fluids to help make the diagnosis [30].
Differential diagnosis Most commonly, the initial pruritic papule of cutaneous anthrax is thought to be an insect bite [11]. When the characteristic eschar forms, the differential diagnosis is broad, and can include Brown Recluse Spider bites, cutaneous leishmaniasis, plague, tularemia, and tyhus, among others (Box 1). In patients with systemic symptoms there is also a broad differential diagnosis including cat scratch disease, glanders, plague, and tularemia [23].
Box 1. Differential diagnosis of cutaneous anthrax with eschar formation Brown recluse spider bite Orf Cutaneous leishmaniasis Tuleremia Typhus Staphyloccal lymphadenitis Plague Glanders Ecthyma grangrenosum Rickettsial pox Rat-bite fever Aspergillosis Cutaneous tuberculosis Leprosy Mucomycosis See Refs. [1,22,23].
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Inhalational anthrax often presents as an influenzalike illness with cough, malaise, and fevers. A recent review of the anthrax cases from October 2001, and detailed clinical reports of other inhalational anthrax cases indicate that symptoms such as dizziness, confusion, nausea, and emesis along with the fever and cough can distinguish inhalational anthrax from influenza-like syndromes [32]. Currently, screening protocols are being studied to aide in differentiating patients with influenza-like disease verses inhalational anthrax who present for medical care [32]. Chest X-ray findings can be invaluable, with the presence of a widened mediastinum a characteristic diagnostic clue. Nevertheless, this finding is not pathognomonic, as pulmonary tularemia can also present with fever and medisatinal lymphadenopathy [23]. The differential diagnosis for GI anthrax ranges from viral gastroenteritis to any agent causing bloody diarrhea, bleeding ulcer, acute abdomen, GI obstruction, or ascites [28]. GI anthrax secondary to inhalational anthrax would present with a widened mediastinum on chest X ray [11].
Treatment In addition to the rare spontaneous penicillin and tetracycline resistant strains of B anthracis, attenuated strains of antibiotic resistant anthrax may have been developed for malicious purposes [33]. As such, the Centers for Disease and Prevention recommends ciprofloxacin as the drug of choice for treating anthrax until culture and sensitivities are available [27]. The usual treatment for uncomplicated, localized cutaneous anthrax is ciprofloxacin 500 mg twice a day. For those allergic to ciprofloxacin, doxycycline 100 mg twice a day is an alternative treatment. For naturally acquired cutaneous anthrax, the recommended treatment duration is 7 to 10 days. Disease resulting from a potential terrorist attack should be treated for at least 60 days to protect against late germinating spores potentially causing systemic anthrax [25]. The larger the suspected exposure inoculum, the longer the antibiotic prophylaxis is recommended, as lingering unphagocytosed spores are resistant to antibiotics and can cause fatal disease if antibiotic therapy is withdrawn prematurely [27]. If there are signs of extensive edema, systemic disease, or head and neck lesions, the patient should be hospitalized and treated with intravenous (i.v.) ciprofloxacin along with an additional antibiotic. Once the culture sensitivities are known and the patient is stabilized, treatment may be switched to a single oral antibiotic [27]. Especially in facial and neck lesions, corticosteroid treatment
may be necessary until the toxins are cleared to decrease life-threatening edema [27]. Inhalational or GI anthrax is treated with i.v. ciprofloxacin 400 mg (or doxycycline 100 mg) i.v. every 12 hours plus one or two other antimicrobial agents. Additional antibiotics may include rifampin, vancomycin, clindamycin, and imipenum among others [25]. Once stable, patients may be switched from i.v. to oral antibiotics. Total duration of treatment should be at least 60 days, for the reasons outlined above. Adjunctive treatments such as corticosteroids may be used for systemic and central nervous system symptoms as necessary. Aggressive correction of electrolytes, use of vasopressors and mechanical ventilation should also be used for supportive care as the toxins can cause rapid progression to septicemia and shock. Clindamycin is often used in other toxin mediated illness such as group A strep and staphylococcus, and may help limit toxin-mediated damage [27]. Drainage of hemorrhagic pleural effusions by chest tubes was also successful in stabilizing patients with inhalational anthrax during the recent terrorist attacks [34]. Fluoroquinolones are generally contraindicated in children and pregnant women due to concerns about arthropathy and cartilage growth in research animals [35]. Likewise, tetracyclines are contraindicated in children less than 9 years old due to the effects on growth and staining of teeth if given before permanent teeth develop [35]. Nevertheless, in the setting of inhalational anthrax or exposure thereof, the risks related to anthrax disease outweigh the risks of the medication side effects, and pregnant women and children should receive either ciprofloxacin or doxycycline until drug susceptibilities are known [36].
Prophylaxis After the terrorist attacks in October 2001, the Department of Health and Human Services announced additional options for persons possibly exposed to high concentrations of anthrax spores [27]. The options were 60 days of antimicrobial prophylaxis, 100 days of antimicrobial prophylaxis, or 100 days of antimicrobial prophylaxis plus three doses of the anthrax vaccine at 0, 2, and 4 weeks, under an investigational new drug protocol [27]. The options were created secondary to studies that show the persistence of spores can occur past 60 days [27], and disease can occur after discontinuation of therapy [37]. A model was recently developed to ascertain the optimum duration of antibiotic usage
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after an exposure. The anthrax attacks of 2001 were a low exposure event so 60 days of antibiotics was sufficient prophylaxis. A large exposure could require as long as 4 months of antibiotics until all the spores are cleared [38]. Studies have shown that after administering the anthrax vaccine at 0, 2, and 4 weeks antibiotics may be able to be discontinued [39]. This is the time it takes for > 95% of vaccines to develop a fourfold antibody response [39], but this has never been tested in humans, and was not recommended for the anthrax attacks of 2001. The Centers for Disease and Prevention currently recommends the anthrax vaccine be administered for preexposure prophylaxis only to those at risk for repeated exposures, such as military troops, laboratory workers, and veterinarians. Civilian first responders are not currently recommended to receive the vaccine because of the dosing schedule, supply issues, and the availability of effective postexposure prophylaxis. Although nasal swabs were used to screen people for anthrax spores deposited in the nose, the decision to treat should be based on history of exposure and not on the results of screening tests, which are used for epidemiological purposes [25]. As anthrax can take days or weeks to develop, postexposure prophylaxis is now the best method of protecting people after an exposure.
Vaccine Because antibiotics must be given early in the course of systemic anthrax, and even then are frequently not effective, vaccination against anthrax has been advocated in certain high-risk populations. In humans, the Anthrax Vaccine Absorbed (AVA) is the only Federal Drug Administration-approved vaccine for anthrax in the United States [39]. Licensed in 1970, it is an alum-adsorbed cell-free filtrate of a nonencapsulated attenuated strain with increased concentration of PA that stimulates the immune response in humans [40]. In monkeys, it was found to be 100% effective in preventing inhalational disease when spores were inhaled 8 weeks after initiation of immunization and 88% effective 2 years later [11]. In humans, a 95% seroconversion rate after three doses of vaccine is reported; however, the relationship between antibody titer and protection from anthrax are not known [39]. The AVA is produced by Bioport, and supplies are limited by the production capacity and the many doses that are required to produce immunity. Because anthrax used in the formation of the vaccine forms
spores, AVA production requires dedicated facilities, and production cannot be easily or rapidly expanded [41]. The vaccination schedule of 0, 2, and 4 weeks and 6, 12, and 18 months, with yearly boosters makes it difficult to maintain supplies and vaccinate the civilian population if needed. Developed before advances in molecular biology, the vaccine is also not highly purified, and is comprised of a poorly defined mixture of bacterial components and proteins, the most important of which is generally accepted to be PA, the nontoxic component of the nontoxic component of the lethal and edema toxins of anthrax [42]. The quantity of PA in AVA, however, varies from lot to lot [42]. Previous efficacy studies in animal models have demonstrated that PA must be present in nonliving anthrax vaccines, and to date, all human vaccines developed against anthrax are comprised largely of PA [41,43 45]. Nonclinical studies have demonstrated a strong positive correlation between serum anti-PA titers and protection from anthrax aerosol challenge in several animal models. Although safe and effective in humans with low systemic adverse effects, there was considerable controversy when the military started mandatory vaccination of service members with AVA in the 1990s [42]. Several studies have demonstrated a low incidence of systemic adverse effects but as high as 30% incidence in local reactions, mostly erythema, pain, and subcutaneous nodules [46]. These local site reactions are more commonly reported in women than men [39,47]. A recent study comparing local reactogenicity in individuals receiving AVA by the licensed subcutaneous route verses the intramuscular route showed a decrease in local reactions when the vaccine is given intramuscular versus subqutaneous without any significant change in anti-PA antibody titer [48]. Larger studies are ongoing to confirm the benefit of changing from subcutaneous to intramuscular injection site. Studies using the AVA with an increased dosing interval are also ongoing. One small study showed a statistically similar rise in anti-PA IgG concentration when two doses of the anthrax vaccine were given 4 weeks apart instead of the currently licensed three doses with 2 weeks interval regimen [48]. Another retrospective study showed that increasing the interval between doses to 4 weeks enhanced the antibody response in humans [49]. Larger studies are now ongoing to further determine the most advantageous vaccination schedule using AVA. There has been no evidence of long-term health effects due to the anthrax vaccine [42]. The vaccine has not been studied in humans less than 18 years or
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older than 65, and the safety and efficacay in these populations are not known. Contraindications include prior infection with anthrax, anaphylactic reaction to prior vaccination with AVA, pregnancy, and active acute infections. New vaccines are also currently being developed. Recombinant DNA technology was used to produce purified PA, which is the key component of the newest anthrax vaccine candidate. This vaccine, called rPA102, was initially produced by the Army, and has undergone multiple studies in rodent and nonhuman primate models with promising results. This vaccine is currently being developed by VaxGen, Inc (Brisbane, California), which has just completed a phase 1 rPA102 dose finding trial in humans. Early results from this study suggest this vaccine is safe, with significantly fewer local adverse events than AVA, while maintaining comparable elicited immunity at the higher doses [50,51]. Further studies are underway to target a single formulation of the rPA102 vaccine that can be used to replace AVA for both general use and postexposure prophylaxis to anthrax. Following the deliberate exposure of anthrax to the American public in 2001, the need became urgent for the development of a safe and rapidly immunogenic and effective anthrax vaccine to protect the population in advance of, or after, exposure to intentionally released anthrax spores. Finding a safe, effective, and rapidly producible two-dose vaccine is a priority in research, and would greatly aid efforts in protecting the population if another anthrax outbreak occurred.
References
[1] Dixon T, Meselson M, Guillemin J, Hanna P. Anthrax. N Engl J Med 1999;341(11):815 25. [2] Huxsoll D, Parrott C, Patrick W. Medicine in defense against biological warfare. JAMA 1989;262:677 9. [3] Witkowski J, Parish L. The story of anthrax from antiquity to the present: a biological weapon of nature and humans. Clin Dermatol 2002;20:336 42. [4] Pile J, Malone J, Eitzen E, Friedlander A. Anthrax as a potential biological warfare agent. Arch Intern Med 1998;158:429 34. [5] Friedlander AM, Anthrax. In: Sidell FR, Takafuji ET, Franz DR, editors. Textbook of military medicine: medical aspects of chemical and biological warfare, part 1. Washington (DC): Walter Reed Army Medical Center; 1997. p. 467 78. [6] Noah D, Huebner K, Darling R, Waeckerle J. The history and threat of biological warfare and terrorism. Emerg Med Clin North Am 2002;20(2):255 71. [7] Davis D, Johnson-Winegar A. The anthrax terror DODs number-one biological threat. Aerospace Power J. Winter 2000. Available at: http://www.airpower. maxwell.af.mil.airchronicles/apj/apj00/win00/davis. htm). Accessed October 12, 2003. [8] Meselson M, Guillemin J, Hugh-Jones M, Langmuir A, Popova I, Shelokov A, et al. The Sverdlovsk anthrax outbreak of 1979. Science 1994;226:1202 8. [9] Abramova F, Grinberg L, Yampolskaya O, Walker D. Pathology of inhalational anthrax in 42 cases from the Sverdlovsk outbreak of 1979. Proc Natl Acad Sci USA 1993;90:2291 4. [10] Zilinskas RA. Iraqs biological weapons. The past as future? JAMA 1997;278:418 24. [11] Inglesby T, OToole T, Henderson D, Bartlett J, Ascher M, Eitzen E, et al. Anthrax as a biological weapon, 2002. JAMA 2002;287(17):2236 52. [12] Jernigan DB, Raghunathan PL, Bell BP, Brechner R, Bresnitz EA, Butler JC, et al. Investigation of bioterrorism-related anthrax, United States, 2001: epidemiologic findings. Emerg Infect Dis [serial online] 2002; 8(10). Available at: http://www.cdc.gov/ncidod/EID/ vol8no10/02-0353.htm. Accessed September 3, 2003. [13] Bonn D. Anthrax update. Lancet Infect Dis 2002; 2(3):129. [14] Hart CA, Beeching NJ. A spotlight on anthrax. Clin Dermatol 2002;20:365 75. [15] Bales M, Dannenberg A, Brachman P, Kaufmann A, Klatsky P, Ashford D. Epidemiologic response to anthrax outbreaks: field investigations, 1950 2001. Emerg Infect Dis [serial online] 2002;8(10). Available at: http://www.cdc.gov/ncidod/EID/vol8no10/ 02-0223.htm. Accessed September 3, 2003. [16] Plotkin S, Brachman P, Utell M, Bumford F, Atchison M. An epidemic of inhalation anthrax, the first in the twentieth century. Am J Med 1960;29:992 1001. [17] Brachman PS. Inhalation anthrax. Ann NY Acad Sci 1980;353:83 93. [18] Inglesby T, Henderson D, Bartlett J, Ascher M, Eitzen
Summary Anthrax is an ancient disease that has become a modern threat in the face of bioterrorism. Even though there are treatments that can decrease mortality, the mechanisms of how anthrax kills and evades the immune system are still unknown. Given the mass panic and strain on the health care infrastructure a small release of anthrax caused in October of 2001, the potentially devastating effects of a larger release are apparent. Future research focusing on the pathogenesis of the disease, early identification of ill and exposed persons, adjuvant treatments including antitoxin therapy, and an improved and widely available vaccine are critical. Early recognition by astute clinicians remains the cornerstone of our defense and cannot be overstated. The three clinical syndromes cutaneous, inhalational, and GIare distinct syndromes, and should be considered, especially if presenting in someone with risk factors for the disease.
K.A. Wenner, J.R. Kenner / Dermatol Clin 22 (2004) 247256 E, Friedlander A, et al. Anthrax as a biological weapon. JAMA 1999;281(18):1735 45. Moayeri M, Haines D, Young HA, Leppla SH. Bacillus anthracis lethal toxin induces TNF-alpha-independent hypoxia-mediated toxicity in mice. J Clin Invest 2003; 112(5):670 82. Hanna PC, Acosta D, Collier RJ. On the role of macrophages in anthrax. Proc Natl Acad Sci USA 1993;90: 10198 201. Prince AS. The host response to anthrax lethal toxin: unexpected observations. J Clin Invest 2003;112(5): 656 8. Carucci J, McGovern T, Norton S, et al. Cutaneous anthrax management algorithm. J Am Acad Dermatol 2002;47:766 9. Swartz M. Recognition and manangement of anthrax an update. N Engl J Med 2001;345(22):1621 6. Turnbull PC. Bacillus. Medmicro, Chapter 15. Available at: http://gsbs.utmb.edu/microbook/ch015.htm. Accessed September 23, 2003. CDC. Update: investigation of bioterrorism-related anthrax and interim guidelines for exposure management and antimicrobial therapy; October 2001. MMWR [serial online] 2001;50(42):909 19. Available at: http:// www.cdc.gov/mmwr/preview/mmwrhtml/mm5042a1. htm. Accessed August 13, 2003. Freedman A, Afonja O, Chang M, Mostashari F, Blaser M, Perez-Perez G. Cutaneous anthrax associated with microangiopathic hemolytic anemia and coagulopathy in a 7-month-old infant. JAMA 2002;287(7): 869 74. Bell DM, Kozarsky PE, Stephens DS. CDC conference summary: clinical issues in the prophylaxis, diagnosis and treatment of anthrax. Emerg Infect Dis [serial online] February 2002. Available at: http://www. cdc.gov/ncid/EID/vol8no2/01-0521.htm. Accessed November 5, 2003. Jernigan J, Stephens D, Ashford D, Omenaca C, Topiel M, Galbraith M, et al. Bioterrorism-related inhalational anthrax: The first 10 cases reported in the United States. Emerg Infect Dis [serial online] 2001;7(6) Available from: URL: http://www.cdc.gov/ncidod/EID/vol7no6/ Jernigan.htm. Accessed September 23, 2003. Sirisanthana T, Brown A. Anthrax of the gastrointestinal tract. Emerg Infect Dis [serial online] 2002;8(7). Available at: http://www.cdc.gov.ncidod/EID/vol8no7/ 02-0062.htm. Accessed November 5, 2003. Spotts Whitney E, Beatty M, Taylor T, Weyant R, Sobel J, Arduino M, et al. Inactivation of Bacillus anthracis spores. Emerg Infect Dis [serial online] 2003;9(5) Available from: URL: http://www.cdc.gov/ ncidod/EID/vol8no7/02-0157.htm. Accessed November 5, 2003. CDC. Level A laboratory procedures for identification of Bacillus anthracis, last revised Mar 2003. Available from: URL: http://www.bt.cdc.gov/agent/anthrax/ LevelAProtocol/anthraxlabprotocol.pdf. Accessed September 25, 2003. Hupert N, Bearman GM, Mushlin AI, Callahan MA.
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Accuracy of screening for inhalatinal anthrax after a bioterrorist attack. Ann Intern Med 2003;139:337 45. Darling RG, Catlett CL, Huebner KD, Jarrett DG. Threats in bioterrorism I: CDC category A agents. Emerg Med Clin 2002;20(2):273 309. CDC. Anthrax page. Available from: http://www.bt. cdc.gov/Agent/anthrax/PreventiveTreatment12212001. asp. Accessed November 5, 2003. American Hospital Formulary Service. AHFS drug information. Bethesda (MD): Author; 2003. CDC. Notice to readers: update: interim recommendations for antimicrobial prophylxis for children and breastfeeding mothers and treatment of children with anthrax. MMWR 2001;50:1014 6. Friedlander AM, Welkos SL, Pitt ML, Ezzell JW, Worsham PL, Rose KJ, et al. Postexposure prophylaxis against inhalation anthrax. J Infect Dis 1993;167: 1239 42. Brookmeyer R, Johnson E, Bollinger R. Modeling the optimum duration of antibiotic prophylaxis in an anthrax outbreak. Proc Natl Acad Sci USA 2003;100: 10129 32. CDC. Use of anthrax vaccine in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR 2000;49:341 5. Available from: URL: http://www.cdc.gov/mmwr/ preview/mmwrhtml/rr4915a1.htm. Accessed September 3, 2003. Turnbull PC. Anthrax vaccines: past, present and future. Vaccine 1991;9:533 9. Russell P. Vaccines in civilian defense against bioterrorism. Emerg Infect Dis [serial online] 1999 Jul Aug. Available from: URL: http://www.cdc.gov/ ncidod/EID/vol5no4/russell.htm. Accessed September 20, 2003. The anthrax vaccine. Is it safe? Does it work? Institute of Medicine, Washington, DC: National Academy Press; 2002. Hambleton P, Turnbull PCB. Anthrax vaccine development: a continuing story. In: Bacterial vaccines. New York: Alan R. Liss, Inc.; 1990. p. 105 22. Turnbull PCB, Leppla SH, Broster MG, Melling J. Antibodies to anthrax toxin in humans and guinea pigs and their relevance to protective immunity. Med Microbiol Immunol 1988;177:293 303. Ivins BE, Fellows PF, Pitt MLM, Estep JE, Welkos SL, Worsham PL, et al. Efficacy of a standard human anthrax vaccine against Bacillus anthracis aerosol spore challenge in rhesus monkeys. Salisbury Med Bull 1996;87:125 6. Bioport Corporation labeling. Anthrax vaccine adsorbed [package insert]. Lansing (MI): Bioport Corporation; January 2002. Wasserman G, Grabenstein J, Pittman P, Rubertone M, Gibbs P, Wang L, et al. Analysis of adverse events after anthrax immunization in us army medical personnel. J Occup Environ Med 2003;45:222 32. Pittman PR, Kim-Ahn G, Pifat DY, Coonan K, Gibbs P, Little S, et al. Anthrax vaccine: immunogenicity and
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K.A. Wenner, J.R. Kenner / Dermatol Clin 22 (2004) 247256 safety and immunogenicity. Presented at the 2004 ASM Biodefense Research Meeting. Baltimore, MD, March 9, 2004. [51] Keyserling HL, Gorse GJ, Keitel W, et al. Ascending dose safety and immunogenicity study of a recombinant protective antigen (PA) anthrax vaccine (rPA102). The International Conference on Emerging Infectious Diseases (ICEID). Atlanta, GA, March 2, 2004.
safety of a dose-reduction, route-change comparison study in humans. Vaccine 2002;20:1412 20. [49] Pittman P, Mangiafico J, Rossi C, Cannon T, Gibbs P, Parker G, et al. Anthrax vaccine: increasing intervals between the first two doses enhances antibody response in humans. Vaccine 2001;19:213 6. [50] Taylor DN, Gorse G, Keitel W, Keyserling HL, Longhi M, Hirsch A, et al. Phase 1 study of a recombinant protective antigen anthrax vaccine (rPA102):
Dermatol Clin 22 (2004) 257 262
Other biologic toxin bioweapons: ricin, staphylococcal enterotoxin B, and trichothecene mycotoxins
William B. Henghold II, MD, MAJ(P), MC, USAR
Dermatology Service, Tripler Army Medical Center Hawaii, 1 Jarrett White Road, HI 96859-5000, USA
A biologic toxin is a poisonous substance having a protein structure and produced by a living organism. Pound for pound, biologic toxins are among the most deadly substances known to exist, with botulinum toxin reported as the most lethal of all [1]. Many organisms (animal, plant, bacterium) produce substances toxic to other organisms, but few of these harmful substances are suitable for use as bioweapons. Those that are potential biologic warfare (BW) agents have been identified as such due largely to their relative ubiquity in nature and ease of production and dispersal. This article deals with three toxins (ricin, staphylococcal enterotoxin B, and trichothecene mycotoxins), which have been reported as potential threat agents for use against military and civilian targets. Of note, only the trichothecene mycotoxins produce cutaneous manifestations as part of their clinical spectrum of disease.
Ricin History and significance Ricin is an extremely potent toxin produced by the castor plant, Ricinus communis. The entire plant is poisonous, but the toxin reaches the greatest concentration in the seeds. Ricin does not produce cutaneous disease in the exposed individual, but the origin of its name begs special attention from the dermatologist. Ricinus is the Latin word for tick (as dermatologists well know, ticks are the vectors for several diseases with cutaneous manifestations), and the plant
E-mail address: William.Henghold@amedd.army.mil 0733-8635/04/$ see front matter. Published by Elsevier Inc. doi:10.1016/j.det.2004.03.004
takes its name due to the resemblance of its seeds to engorged ticks [2]. The plant, cultivated in several varieties worldwide for it oil, is native to tropical Africa, but is now so common as to be considered a weed in certain parts of the United States. It is perennial in the tropics and an annual in subtropical and temperate areas, reaching a size of up to 15 feet and recognizable by its large, typically eight-lobed, palmate leaves [3]. Castor oil is produced in large quantities worldwide and has many industrial and commercial uses, many of which have been around for centuries. It is perhaps best known for its use as a lubricant for engines (Castrol-R racing motor oil) and as a medicinal. Once the oil is extracted from the plant, the aqueous residue, or mash, is 5% ricin by weight. The toxin can then be separated from the mash in a relatively simple procedure using undergraduate chemistry techniques [4]. The toxic nature of the plant and its seeds has also been well known for centuries, primarily through its harm to livestock. By weight, the toxin is one of the most deadly naturally occurring substances known [5]. As mentioned, the seeds contain the highest concentration of ricin, and if chewed, one seed may be fatal to a child. Ricin is a relatively stable protein, and can be dispersed as an aerosol, by injection, or as a food and water contaminant. Ricin is much more potent if inhaled or injected. There have been numerous reports concerning the actual or potential use of ricin as a weapon [5 7], including a recent (October 2003) incident of a package containing ricin, which was sent, along with an accompanying threatening note, to a South Carolina post office [8]. One of the most often-cited instances (due to the James Bond-esque nature of
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the event) deals with the assassination in London in 1978 of Georgi Markov, a Bulgarian dissident, who died shortly after being injected with a ricin-containing pellet via a weapon disguised as an umbrella [9]. Some of the more recent news reports are perhaps the most troubling, given their links to terrorist groups openly hostile to the US and its allies:
A recipe for the production of ricin was found
Clinical presentation The spectrum of disease in humans and other animals varies with the route of exposure and dose received. There are no cutaneous manifestations of ricin toxicity. The toxin can be manufactured as a liquid or powder, and subsequently administered via injection, aerosol, or used to contaminate food or water. It can also be delivered percutaneously if dissolved in a solvent such as dimethyl sulfoxide. This method of delivery was planned in a bizarre assassination plot involving an extremist group in Minnesota [14]. Ricin is most toxic when delivered as an aerosol or injection, with a lethal dose for 50% of the exposed population (LD50) estimated at 3.0 mg/kg. It is less toxic when ingested, likely due to enzymatic degradation. When inhaled, pathologic changes consisting of necrosis of respiratory epithelium can be seen as early as 8 hours after exposure. Death from respiratory failure due to a capillary leak-like syndrome may eventuate in 36 to 72 hours. If a significant dose is ingested, the exposed individual will experience, usually within a few hours, severe gastrointestinal distress brought on by mucosal ulceration and hemorrhage. Vascular collapse and death can be expected in several days [6,14].
on the body of a Chechen rebel who was killed by Russian troops. It is felt that some Chechen militants have connections to terrorist groups, notably al Qaeda [10]. Plans to produce ricin were found in Kabul, Afghanistan, in November 2001, and linked to al Qaeda [7]. More than 10 individuals, most of who are from North Africa, have been arrested in connection with the discovery of ricin in a London apartment in January 2003 [11]. Ricin is much less poisonous by weight compared with other toxins, such as botulinum (lethal dose) and Staphylococcal enterotoxin B (SEB) (incapacitating dose), and so it is thought to be less of a potential threat to our armed forces; however, its abundance in nature and cheap manufacture make it an ideal weapon in the hands of terrorist organizations who may only wish to incite panic and disrupt daily life.
Differential diagnosis The differential diagnosis includes any biologic agent that can produce acute respiratory disease such as SEB, mycotoxins, anthrax, tularemia, plague, and a host of other bacterial and viral pathogens [15]. Some chemical warfare agents also need to be considered. A complete discussion is beyond the scope of this article. Certainly, the important point for the dermatologist is the absence of cutaneous findings in ricin poisoning. Diagnosis can be confirmed through the use of enzyme-linked immunosorbent assays (ELISA) of blood or other body fluids or by tissue immunohistochemistry [6]. Ricin is a potent immunogen, and individuals who survive intoxication will likely have high circulating antibody levels several weeks after exposure.
Pathogenicity The toxic and immunologic properties of ricin have been studied extensively for over a century. The toxin acts at the cellular level through a complex series of steps resulting in an enzymatic inhibition of protein synthesis [12,13].The glycoprotein ricin is a 66-kilodalton (kDa) globular heterodimer that consists of a 32-kDa A-chain and a 32-kDa B-chain, linked together by a disulfide bond. Just one ricin molecule can inactivate over 1500 ribosomes per minute and kill the cell, but both chains are required to produce the cytotoxic effect. The B-chain has lectin (agglutination) properties that facilitate binding to cell surface molecules, thereby triggering endocytosis of the toxin. It is then transported through the Golgi complex to the cytoplasm where the A-chain enzymatically alters the 28S ribosomal subunit, effectively shutting down protein synthesis. For this reason, ricin is also referred to as a ribosome-inactivating protein or RIP (a rather appropriate acronym when one thinks about it).
Management There is no treatment for acute ricin intoxication other than symptomatic, supportive care. Use of a protective mask is effective in preventing aerosol exposure. Once exposed, individuals cannot transmit
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the agent to others, as would be the case with an infectious agent. No antitoxin is available, and no human vaccine is currently under development, although ricins potent immunogenicity provides hope that an effective vaccine will be available soon. Recent animal studies are encouraging [16].
Staphylococcal enterotoxin B History and significance Exotoxins are proteins that exert their poisonous effect outside the organism that excreted them. The bacterium Staphylococcus aureus produces a number of exotoxins, one of which is SEB. The dermatologist is readily familiar with at least two of the other toxins produced by strains of S aureus: exfoliative toxin A (which causes bullous impetigo and staphylococcal scalded skin syndrome) and toxic shock syndrome toxin. An enterotoxin is so named as it primarily affects the gastrointestinal tract, and SEB is one of the most common causes of food poisoning in the United States; however, the toxin causes a markedly different and more severe clinical syndrome when inhaled than it characteristically produces when ingested [17,18]. During the days when the United States had an active offensive biologic warfare program, it was realized that SEB could be a particularly effective weapon when released as an aerosol. The toxin remains stable when aerosolized, and the dose required to incapacitate an enemy is much lower than that required to kill him. It was estimated that an effective attack could render 80% or more of exposed clinical personnel incapable of performing their mission for up to 2 weeks. For this reason, its intended purpose was as an incapacitating agent, and SEB was one of the seven biologic agents stockpiled by the United States [14]. To date, no claims of its use in war or terrorist actions has been documented, but an accidental exposure to the aerosolized toxin has been reported [17].
of cytokines (eg, tumor necrosis factor [TNF]-a, interleukin [IL]-6, interferon [IFN]-g) and driving an inflammatory cascade that damages tissue. For this reason SEB is termed a superantigen [19]. A corollary of this brisk inflammatory reaction is manifested in the acute flare experienced by atopic dermatitis patients who are secondarily infected with S aureus [20]. Cutaneous disease is not part of the clinical presentation in a biologic attack with aerosolized SEB given the small doses to which any one individual would be exposed; however, SEB has been demonstrated to induce dermatitis in normal as well as atopic patients when applied to the skin [21].
Clinical presentation Symptoms of SEB poisoning depend on the route of exposure, but are usually seen within 3 to 12 hours after either inhalation or ingestion of the toxin. Flulike symptoms predominate early on, followed by chest pain, cough, and difficulty breathing in inhalational exposure, and classic food poisoning symptoms with oral intoxication. Gastrointestinal disease may occur with aerosol exposure due to swallowing of the toxin after mucocilliary clearance. No cutaneous disease is seen. If death occurs, it is usually due to respiratory failure. Although SEB is not considered highly lethal, it may incapacitate for several weeks those exposed many miles downwind from the toxin release point [14,17].
Differential diagnosis The differential diagnosis of SEB exposure is similar to that of ricin intoxication, and rests largely on clinical and epidemiologic grounds, with large numbers of patients presenting over a very short period of time, probably within a 24-hour period. As with ricin exposure, no cutaneous disease is manifest. Diagnosis can be confirmed with ELISA of tissue, body fluids, or environmental samples [14,15,17].
Pathogenicity SEB is one of at least seven antigenically distinct enterotoxins that have been identified [18]. It is a moderately heat-stable 28-kDa protein that exerts its pathologic effect largely due to its ability to bind to the class II major histocompatibility complex of antigenpresenting cells, independent of the normal control of T-cell activation. This stimulates a much greater number of T cells than usual, leading to a massive release Management At this time there are no vaccines or therapeutic agents available to protect against or treat SEB intoxication, and so treatment is limited to symptomatic, supportive care. After several weeks of illness, the vast majority of patients would be expected to make a full recovery. Vaccine testing in primates is
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proving successful, and so a human vaccine is possibly just around the corner [22].
terfering with peptidyl transferase activity during mRNA translation, effectively shutting down protein production [25,26,30].
Trichothecene mycotoxins History and significance A mycotoxin, as the name indicates, is a poison produced by a fungus. The trichothecene mycotoxins are especially potent variants produced by species of Alternaria, Aspergillus, Claviceps, Fusarium, and Penicillium [23]. Of the suspected BW toxins, only the trichothecenes produce cutaneous reactions in exposed individuals, and so knowledge of this group is especially important to the dermatologist [24]. T-2 mycotoxin is perhaps the most extensively studied of the trichothecenes due to its extreme toxicity and relative abundance in nature. Numerous reports implicate mycotoxins in the illness and death of humans and other animals, usually through the accidental ingestion of moldy agricultural products [25,26]. A syndrome named alimentary toxic aleukia, which killed over 10% of the population in Orenburg, Russia, was later linked to T-2 toxin [27]. The discovery that a naturally existing toxin could produce such devastation on a population almost certainly led to its investigation as a BW agent. The trichothecenes are extremely stable proteins, resistant to autoclaving and ultraviolet light inactivation. They can be delivered as dusts, droplets, aerosols, or smoke from a variety of dispersal systems and exploding munitions. The toxin is highly soluble in a number of organic solvents, and after extraction from culture, a yellow, greasy, crystalline substance is obtained. This is the material the Soviet Union and its allies were believed to have used in the yellow rain incidents in the mid- to late 1970s and early 1980s, killing thousands in Southeast Asia and Afghanistan [28]. Iraq is also known to have produced and stockpiled mycotoxins for use as weapons [29]. Clinical presentation The route of exposure may be topical, oral, or inhalational, and symptoms may begin within minutes to hours, given the lipophilic nature of the toxin. As mentioned, it is the only biotoxin that produces cutaneous disease, and although less lethal by weight than either ricin or SEB, it is many times more potent than the chemical weapons that are dermally active, such as the alkylating agents (eg, mustard gas) [27]. Cutaneous signs and symptoms include erythema, edema, pain, and pruritis, with blisters peaking at 48 hours, and ultimate necrosis and sloughing of large areas of skin. Severe ocular irritation with corneal injury may also be seen. Airway and intestinal necrosis may occur, and the final pathway for all routes of exposure to a sufficient dose is hemorrhage, shock, and death in 24 to 48 hours. In sublethal intentional exposures, or in small, accidental exposures, severe cutaneous irritation lasting several weeks may occur. Likewise, the effects on the ocular, gastrointestinal, and respiratory systems are less profound than in a lethal dose, but still produce severe morbidity. A late effect of systemic absorption is pancytopenia, predisposing to bleeding and sepsis [14,27].
Differential diagnosis A diagnosis of trichothecene exposure is difficult at best, but clinical and epidemiologic clues are helpful. In the alleged yellow rain incidents, cutaneous, ocular, respiratory, and gastrointestinal signs and symptoms coexisted. A patient presenting with red, burning, itching skin with eye irritation and a runny nose, sore throat, cough, difficulty breathing, and vomiting and diarrhea minutes to hours after being exposed to a yellow (or other pigmented) oily liquid is highly suggestive of a chemical or biologic attack. Exposure to SEB or ricin aerosols can cause severe respiratory symptoms, but no cutaneous signs will be seen. Mustard and other blistering agents must be considered in such a patient presentation, and they can be rapidly detected by a field chemical test. At the present time, a rapid diagnostic test for mycotoxins is not commercially available. Blood and urine samples should be collected and sent to a reference lab for antigen detection. A diagnosis can be made up
Pathogenicity Trichothecenes are low molecular weight (250 500 Da) potent inhibitors of protein synthesis, with secondary effects on DNA and RNA synthesis. They are most toxic to rapidly dividing cells, and are referred to as radiomimetic agents, as they mimic the signs and symptoms of radiation sickness [14,27]. Although the mechanisms of action of these cytotoxins on eukaryotic cells are diverse and not entirely elucidated, they are thought to act primarily by in-
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to a month after patient exposure as the toxin metabolites persist that long [14,27,31].
Management In a suspected chemical or biologic attack, the examiner must always avoid becoming a casualty as well by first donning a protective mask (equipped to filter particles 3 mm or larger) and clothing, as the patients contaminated garments can serve as a reservoir for further toxin exposure. All of the patients clothes must be removed and decontaminated. Although care for the patient is essentially supportive, some treatments in laboratory animals have demonstrated a survival benefit (eg, thorough washing of the skin with soap and water to reduce systemic absorption, administration of activated charcoal, and high-dose systemic steroids) [27,32,33]. No specific antitoxin is available, and no human vaccine appears close to development, although studies are ongoing in animal models.
[7]
[8]
[9]
[10]
[11]
Summary Biologic toxins are highly poisonous compounds synthesized by living organisms. They are widely abundant in nature, relatively cheap to manufacture and weaponize in a variety of forms, and have no definitive treatment or prophylaxis. In many respects, they are the perfect weapons for terrorists. Of the suspected biologic toxin bioweapons, only the trichothecene mycotoxins produce cutaneous findings in those exposed.
[12]
[13] [14]
[15]
References
[16] [1] Gill MD. Bacterial toxins: a table of lethal amounts. Microbiol Rev 1982;46:86 94. [2] Armstrong WP. The castor bean: a plant named after a tick. Noteworthy plants for March 1999. Available at: http://waynesword.palomar.edu/plmar99.htm. Accessed August 16, 2003. [3] Cornell University Poisonous Plants Informational Database. Available at: http://www.ansci.cornell.edu/ plants. Accessed August 16, 2003. [4] Darby SM, Miller ML, Allen RO. Forensic determination of ricin and the alkaloid marker ricinine from castor bean extracts. J Forensic Sci 2001;46(5): 1033 42. [5] Balint GA. Ricin: the toxic protein of castor oil seeds. Toxicology 1974;2(1):77 102. [6] Franz DR, Jaax NK. Ricin toxin. In: Sidell FR, Taka-
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fuji ET, Franz DR, editors. Medical aspects of chemical and biological warfare. Textbook of military medicine. Part 1. Warfare, weaponry, and the casualty, Vol. 3. Washington (DC): Office of the Surgeon General, Dept of the Army; 1997. p. 631 42. Anonymous. Ricin as a weapon. CNN.com. 23 Oct 2003. Available at: http://cnn.com/2003/WORLD/ europe/01/07/terror.poison.extremists/index.html. Accessed November 3, 2003. Smith T, Paras A. Weeklong delay on ricin threat draws scrutiny. GreenvilleOnline.com. 24 Oct 2003. Available at: http://greenvilleonline.com/news/2003/10/24/ 2003102417544.htm. Accessed November 3, 2003. Eitzen EM, Takafuji ET. Historical overview of biological warfare. In: Sidell FR, Takafuji ET, Franz DR, editors. Medical aspects of chemical and biological warfare. Textbook of military medicine. Part 1. Warfare, weaponry, and the casualty, Vol. 3. Washington (DC): Office of the Surgeon General, Dept of the Army; 1997. p. 420 1. Dougherty J. Moscow: ricin recipe found on Chechen fighter. CNN.com. 13 Jan 2003. Available at: http:// www.cnn.com/2003/WORLD/europe/01/13/russia. ricin/ index.html. Accessed November 3, 2003. Anonymous. UK ricin find: Six more arrested. CNN.com. 15 Jan 2003. Available at: http://www. cnn.com/2003/WORLD/europe/01/13/ricin.courts/ index.html. Accessed November 3, 2003. Endo Y, Mitsui K, Motizuki M, Tsurugi K. The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes: The site and the characteristics of the modification in 28S ribosomal RNA caused by the toxins. J Biol Chem 1987;262(12):5908 12. Robertus JD. The structure and action of ricin, a cytotoxic N-glycosidase. Sem Cell Biol 1991;2:23 30. US Army Medical Research Institute of Infectious Diseases. Medical management of biological casualties handbook. 4th edition. Fort Detrick, Frederick (MD): USAMRIID; 2001. Franz DR, Jahrling PB, Friedlander AM, et al. Clinical recognition and management of patients exposed to biologic warfare agents. JAMA 1997;278:399 411. Smallshaw JE, Firan A, Fulmer JR, Ruback SL, Ghetie V, Vitetta ES. A novel recombinant vaccine which protects mice against ricin intoxication. Vaccine 2002; 20(27 28):3422 7. Ulrich RG, Sheldon S, Taylor TJ, Wilhelmsen CL, Franz DR. Staphylococcal Enterotoxin B and related pyrogenic toxins. In: Sidell FR, Takafuji ET, Franz DR, editors. Medical aspects of chemical and biological warfare. Textbook of military medicine. Part 1. Warfare, weaponry, and the casualty, Vol. 3. Washington (DC): Office of the Surgeon General, Dept of the Army; 1997. p. 621 30. Marrack P, Kappler J. The staphylococcal enterotoxins and their relatives. Science 1990;248:705 11. Skov L, Baadsgaard O. Bacterial superantigens and inflammatory skin diseases. Clin Exp Dermatol 2000; 25:57 61.
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W.B. Henghold II / Dermatol Clin 22 (2004) 257262 [27] Wannemacher Jr RW, Wiener SL. Trichothecene mycotoxins. In: Sidell FR, Takafuji ET, Franz DR, editors. Medical aspects of chemical and biological warfare. Textbook of military medicine. Part 1. Warfare, weaponry, and the casualty, Vol. 3. Washington (DC): Office of the Surgeon General, Dept of the Army; 1997. p. 655 76. [28] Seely TD, Nowicke JW, Meselson M, Guillemin J, Akratanakkul P. Yellow rain. Sci Am 1985;253:128 37. [29] Zilinskas RA. Iraqs biological weapons. The past as future? JAMA 1997;278(5):418 24. [30] Pace JG, Watts MR, Canterbury WJ. T-2 mycotoxin inhibits mitochondrial protein synthesis. Toxicon 1988; 26(1):77 85. [31] Black RM, Clarke RJ, Read RW. Detection of trace levels of trichothecene mycotoxins in human urine by gas chromatography-mass spectrometry. J Chromatogr 1986;367(1):103 15. [32] Fricke RF, Jorge J. Assessment of efficacy of activated charcoal for treatment of acute T-2 toxin poisoning. J Toxicol Clin Toxicol 1990;28(4):421 31. [33] Shohami E, Wisotsky B, Kempski O, et al. Therapeutic effect of dexamethasone in T-2 toxicosis. Pharm Res 1987;4(6):527 30.
[20] Skov L, Olsen JV, Giorno R, Schlievert PM, Baadsgaard O, Leung DY. Application of staphylococcal enterotoxin B on normal and atopic skin induces up-regulation of T-cells by a superantigen-mediated mechanism. J Allergy Clin Immunol 2000;105(4): 820 6. [21] Strange P, Skov L, Lisby S, Nielsen PL, Baadsgaard O. Staphylococcal enterotoxin B applied to intact normal and intact atopic skin induces dermatitis. Arch Dermatol 1996;132:27 33. [22] Boles JW, Pitt ML, LeClaire RD, et al. Generation of protective immunity by inactivated recombinant staphylococcal enterotoxin B vaccine in nonhuman primates and identification of correlates of immunity. Clin Immunol 2003;108(1):51 9. [23] Steyn PS. Mcotoxins: general view, chemistry, and structure. Toxicol Lett 1995;82/83:843 51. [24] McGovern TW, Christopher GW, Eitzen EM. Cutaneous manifestations of biological warfare and related threat agents. Arch Dermatol 1999;135:311 22. [25] Hussein HS, Brasel JM. Toxicity, metabolism, and impact of mycotoxins on humans and animals. Toxicology 2001;167(2):101 34. [26] Ueno Y. Trichothecene mycotoxins: mycology, chemistry, and toxicology. Adv Nutr Res 1989;3:301 53.
Dermatol Clin 22 (2004) 263 274
Smallpox: the basics

Mark K. Slifka, PhDa, Jon M. Hanifin, MDb,*
Vaccine and Gene Therapy Institute, Oregon Health & Science University, 505 NW 185th Avenue, Beaverton, OR 97006-3448, USA b Department of Dermatology, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239-3098, USA
a
Variola major is the causative agent of smallpox, a severe disease that typically resulted in 20% to 30% mortality and was arguably one of the most serious human pathogens ever encountered in recorded history [1,2]. Humans are the only known reservoir of variola major; no known animal or insect reservoirs have been identified. Thus, after eradication of smallpox through a massive global immunization effort, this incredibly lethal scourge was eliminated from all corners of the globe, with the last naturally occurring case of smallpox reported in Somalia in 1977. Despite the total eradication of naturally occurring smallpox, there are still stockpiles of smallpox virus maintained in the United States and the former Soviet Union. Unfortunately, it is impossible to know if all smallpox stocks have been accounted for or whether unknown or unreported stocks of smallpox may still exist in these and other countries [2]. In addition, it is possible that other orthopoxviruses such as monkeypox or camelpox might be used as weapons of biowarfare. In the age of genetic engineering, these viruses could theoretically be modified to increase their virulence to the levels associated with smallpox itself [2]. Fears of genetically altered poxvirus-based biological weapons rose sharply [3] when an Australian group first published a study in which they made mousepox (ectromelia) far more deadly in mice by simply engineering the gene for interleukin-4 (IL-4) into the mousepox genome [4]. There has been great
* Corresponding author. E-mail address: hanifinj@ohsu.edu (J.M. Hanifin).
speculation that if smallpox was engineered to have the human version of the IL-4 gene, then it might be transformed into a superbug that would be much more virulent than before and possibly even resistant to the current smallpox vaccine. Although it is possible that a genetically altered strain of smallpox could be more virulent in humans, there is also no guarantee what the outcome might be. For instance, if antiviral antibody plays more of a role in protection against smallpox than T-cell mediated immunity, then the effects of recombinant virus-produced IL-4 on human disease might be considerably dampened compared with what was found with mousepox infection of mice. In other words, the increased mortality associated with mousepox strains that express IL-4 may be unique to this strain of virus and its special interactions with its murine host [3]. When mice were challenged with a closely related virus (vaccinia) that was also genetically engineered to express IL-4, the effects in vivo were relatively minor compared with the effects observed in mousepox [5 8]. Although one study found that the virusproduced IL-4 inhibited CD8+ T-cell proliferation/ expansion [8], another study indicated that there was virtually no effect on the size of the antiviral T-cell response [7]. Instead, they noted substantial skewing of cytolytic activity from being mainly perforin-mediated to being predominantly Fas-FasL mediated. These discrepancies indicate that there is still much to be learned about the underlying mechanisms of IL-4 enhanced disease in this model system. Interestingly, recombinant vaccinia virus expressing IL-4 elicits antibody responses that shift toward IgG1 (a Th-2-induced IgG subclass) rather than IgG2a (a Th-1-induced IgG subclass), but there appears to be
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no effect on the total level of antigen-specific antibody produced [6]. This suggests that humoral immune responses are largely left intact even in the presence of virus-produced IL-4. Although orthopoxviruses are closely related (see later discussion), there are many variations in the pathogenesis of each virus and its unique relationship to an individual host [9]so any genetic improvement in virulence would have to be made empirically. This is perhaps most problematic for smallpox, because this virus only infects humans and there is no animal model in which one could test genetically altered strains of virus for increased virulence. On the other hand, a bioterrorist could theoretically develop genetically altered strains of smallpox that carry more dangerous payloads than IL-4, such as biotoxins that are already known to be pathogenic in humans. Again, the success of such a venture would not be guaranteed; the toxin could act as an adjuvant and thereby interfere with the many host evasion genes of smallpox. Alternatively, if a recombinant toxin is overproduced or lethal to the smallpox-infected cell itself, then it may result in host cell death before the virus can effectively replicate to high titersthus reducing the biological fitness and virulence of the altered strain of virus. If an effective smallpox super virus were indeed produced, some experts believe that the result may paradoxically be a smaller, and possibly more controllable, smallpox outbreak instead of a catastrophic outbreak that would cause global destruction [10]. For instance, if a more lethal strain of smallpox were to be produced, then the incubation period would likely be shorter, thus reducing the period of time available for transmission. Moreover, the clinical symptoms would likely be more pronounced and more easily identified by the attending physician, thus aiding in the rate at which quarantine, postexposure vaccination, and therapeutic drugs/supportive care could be administered to immediate contacts. Although the potential use of genetically altered biological weapons of mass destruction should not be taken lightly, it should also not overshadow our current preparedness efforts for bioterrorist attacks that are more likely to employ biological agents in their native state.
The smallpox virus Variola major is the strain of smallpox that is most commonly described in the literature and was known for inducing a severe and often disfiguring disease with approximately 30% mortality. Smallpox repre-
sents a prototypical orthopoxvirus with an oval or brick-shaped virion containing a stable doublestranded DNA genome that is not prone to the mutation events that occur with most major RNA viruses. For instance, comparisons between two fully sequenced variola major viruses, VAR-Bangladesh (20% mortality in a 1975 outbreak) and VAR-India (30% mortality in a 1967 outbreak), revealed 99.3% homology, even though these strains were isolated 8 years apart in two neighboring countries [11]. Of approximately 200 potential genes analyzed, 122 were 100% identical, 42 showed a substitution of only one amino acid, 11 had only two amino acids that differed, and the rest were more divergent. If a 569-base pair and a 210-base pair deletion are removed from analysis, then the homology between these two strains of smallpox increases to 99.7%, thus demonstrating the well-conserved genomic structure that is maintained over both time and distance. In addition to variola major, another epidemiologically distinct strain of smallpox, variola minor, was identified by its far less virulent nature; variola minor outbreaks were associated with only approximately 0.8% mortality instead of the 20% to 30% mortality described in cases of variola major. Amazingly, approximately one third of the sequenced genes in variola minor (VAR-Garcia) are 100% identical to those found in variola major (VAR-India and VARBangladesh) and the remaining genes are 95% identical [12]. Overall, variola minor is 98% identical to variola major and yet induces far less morbidity and mortality. A better understanding of why these closely related viruses differ so dramatically in their clinical outcomes/mortality rates would be an important step forward in the development of future vaccines and antiviral therapies. Not only are different strains of smallpox quite similar to each other, they are also very similar to other orthopoxviruses [13]. Cowpox and vaccinia represent the first viruses used to vaccinate against smallpox and they have 147 genes and 159 genes, respectively, that are more than 90% homologous to genes found in smallpox. Other human pathogens such as monkeypox and camelpox also have approximately 144 and 176 genes, respectively, that are more than 90% homologous to smallpox. The high degree of genetic similarity between these viruses is the main reason for the incredible success of the smallpox eradication program and explains why there is extensive and reciprocal cross-protection between essentially all orthopoxviruses [1]. For example, immunization with vaccinia not only protects against smallpox, but also monkeypox, camelpox, and cowpox. In animal models, vaccinia infection also pro-
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tects against other closely related orthopoxviruses such as mousepox and rabbitpox [1]. In striking contrast, another common human pathogen, molluscum contagiosum, is a distantly related poxvirus (Molluscipoxvirus) with essentially the same size and genomic structure as the orthopoxviruses, but has just five genes with 70% to 80% homology with smallpox and no known antigenic cross-reactivity with orthopoxviruses [14]. This indicates that the orthopoxviruses are very closely related to each other, but have diverged significantly from other poxviruses that reside outside of this genus.
Immunology of smallpox infection Naturally occurring smallpox was eradicated before sophisticated techniques were developed for quantitating cellular immune responses. For this reason, historical data consist mainly of analysis of antiviral antibody responses, because these were the only techniques available to measure host immunity at the time. Thus the absolute requirements for full protective immunity against smallpox are not known [1] but are likely to involve a combination of innate immunity and adaptive immune responses, including both antibody production and T-cell mediated immunity. Mousepox infection of mice by footpad injection represents one of the most commonly used animal models of smallpox infection. Although this has been a very useful model in characterizing viral dissemination and mechanisms of protective immunity, there is also the possibility that the observations made in this model may not directly reflect the immunobiology and virology of aerosolized smallpox infection in humans. As stated before, each poxvirus has a unique niche with its primary host, which results in infections that may remain mainly localized (eg, molluscum contagiosum in humans) or result in rapid dissemination (eg, mousepox or cowpox in mice) and these interactions can be dramatically altered by the route of infection, dose and strain of virus used, or selection of the host species under study [9]. An example of historical significance is that respiratory smallpox infection of humans leads to approximately 30% mortality, whereas deliberate smallpox inoculation into the skin results in only 0.5% to l% mortality and yet provides full protection against later smallpox exposure. In terms of differences in host species specific immunity, one group analyzed the cellular immune responses mounted against vaccinia virus in both mice [15] and humans [16,17] by measuring direct ex vivo cytolytic activity against autologous vaccinia-infected target cells. In
mice, strong direct ex vivo lytic activity was mediated primarily by major histocompatibility complex (MHC)-restricted T-cells, whereas in humans, direct ex vivo lytic activity was not MHC-restricted, could not be demonstrated in purified T-cell fractions, and instead was found to be mediated almost entirely by natural killer cells through antibody-dependent cell cytotoxicity. Further characterization revealed that vaccinia-specific antibody was required for antibody-dependent cell cytotoxicity to occur, because addition of blocking antibodies against human IgG before analysis resulted in loss of vaccinia-infected target cell lysis. This reveals an important mechanism by which innate and adaptive immune responses work together in a complementary means of antiviral activity. Together, these experiments also indicate that even when the same virus is used for study, substantially different host-specific mechanisms may be involved with protective immunity. Although these early studies were unable to detect a strong virus-specific T-cell response following vaccinia infection [16,17], this is likely due to the relatively low frequency of virus-specific CD4+ and CD8+ T-cells that are mounted during the acute viral infection. Several studies have identified virus-specific T-cells after vaccinia infection using in vitro expansion of human cytolytic T-cell clones [18 20] or, most recently, by using highly sensitive and quantitative direct ex vivo techniquesincluding the ELISPOT (enzyme-linked immunosorbent) assayfor measuring virus-specific interferon (IFN)-g production [21 24], intracellular cytokine staining to measure both IFN-g and tumor necrosis factor a simultaneously [25], or MHC class I tetramer staining, which detects antiviral CD8+ T-cells based directly on their peptide specificity [24]. Although antiviral immunity (including T-cell memory) was believed to be lost within 3 to 5 years [2,26 30], recent studies have clearly refuted this opinion. Cross-sectional analysis of over 300 volunteers revealed that both CD4+ and CD8+ T-cell memory was maintained for decades after vaccinia infection, but declined slowly with a half-life of approximately 8 to 15 years [25]. Moreover, antiviral T-cell responses could be detected in a subpopulation of individuals for up to 75 years after a single vaccinia immunization, demonstrating that vaccinia-specific T-cell memory may, under certain circumstances, be maintained at a detectable level for life. Another study measured total IFN-g producing vaccinia-specific T-cells using ELISPOT assay and came to a similar conclusion: total T-cell memory (both CD4+ and CD8+ T-cell responses combined) is maintained for many years and declines with a half-life of ap-
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proximately 14 years overall [23]. The first study also identified an interesting dichotomy in terms of maintaining T-cell memoryonly about 50% of volunteers vaccinated 20 to 50 years previously maintained demonstrable CD8+ T-cell responses, whereas close to 90% of these individuals continued to maintain a substantial level of CD4+ T-cell memory [25]. The effect that this has on maintaining protective immunity against smallpox infection is not fully understood and represents an area in need of further investigation. In contrast to T-cell mediated immunity following vaccinia or smallpox infections, much more is known about antiviral antibody responses to these viruses. For instance, two early studies found that individuals who had high levels of neutralizing antibodies were more likely to be protected against smallpox infection [31,32]. One study showed that contacts of smallpox patients who had vacciniaspecific neutralizing titers of less than 1:32 were more likely to contact smallpox during an outbreak (3 of 15 or 20% of contacts infected) than contacts who had neutralizing titers above 1:32 (0 of 127 or < 1% of contacts became infected with smallpox) [31]. Similar results were obtained in a later independent study; 6 of 43 contacts of smallpox victims who had neutralizing titers of less than l:20 became infected with smallpox, whereas 0 of 13 contacts who had pre-existing antibody titers of greater than 1:20 became infected [32]. These results were further substantiated by a third study that monitored antibody titers during the acute stages of smallpox infection [33]. Here it was noted that previously vaccinated individuals who had high neutralizing antibody titers at the early stages of smallpox infection had a much higher survival rate than patients who had the lowest levels of neutralizing antibody. These three studies did not formally prove that high antiviral antibody levels were directly responsible for protection, because it was possible that a high antibody titer could simply have been a biomarker of high underlying antiviral T-cell memory as wella question that could not be addressed at the time of naturally occurring smallpox infections. With the tools and technology available today, one recent study has shed new light on this important question. Following smallpox vaccination, direct comparisons between the level of antiviral antibody production and the levels of either vaccinia-specific CD4+ T-cell memory or CD8+ T-cell memory revealed that there was no statistically significant correlation between these two arms of the adaptive immune response, regardless of whether or not the tests were performed at 1 month to 7 years, 14 to 40 years, or 41 to 75 years postvaccination [25]. Relating this information back
to the historical accounts of smallpox protection correlating with high antiviral antibody titers [31,32], these results imply that neutralizing antibody plays a far more important role in protection against this virulent pathogen than was previously expected. More than 90% of volunteers vaccinated 25 to 75 years previously maintained their antiviral antibody responses, and a large proportion of these individuals also demonstrated some form of T-cell memory. How do these immunologic parameters (T-cell memory and antiviral antibody production) relate to historical accounts of the true duration of immunity against smallpox? Several, often large, epidemiologic studies have found that smallpox vaccination protected 90% to 95% of vaccinees against lethal smallpox infection for many decades and in some cases even for life [34 38]. Perhaps the clearest example of this was a study of imported smallpox epidemics in Europe from 1950 to 1971 [38]. During these outbreaks, the mortality rate was 55% (22 of 40) in unvaccinated individuals. Of those individuals fortunate enough to have been vaccinated before smallpox exposure (excluding patients who may have been admitted for other clinical conditions but were inadvertently infected with smallpox), the mortality rate was 2% for those vaccinated fewer than 10 years previously, 6% at 11 to 20 years, and 7% for those vaccinated more than 20 years previously. This indicates that in hospital staff, their contacts, and the general public, vaccination resulted in 98% protection during the first 10 years after vaccination but faded only to 93% protection after 20 or more years had passed. This correlates well with a recently published mathematical model of long-term immunity [39] and several studies demonstrating prolonged and often lifelong antiviral T-cell or antibody mediated immunity [20,23,25,40 43]. Although immunity does indeed decline slowly over time, there is clearly no absolute loss of immunity during the previously assumed 3- to 5-year time frame. These calculations of protective immunity may also be considered conservative, because they only account for the individuals that contracted clinically apparent disease and dont take into account that most previously vaccinated individuals remain uninfected or show no signs of illness after exposure to smallpox. In a metaanalysis of 10 epidemiologic studies [1], it was noted that fewer than 4% of previously vaccinated household contacts contract a clinically apparent infection when exposed to smallpox. Taken together, a 7% mortality rate in the 4% of previously vaccinated individuals that show disease symptoms suggests that smallpox vaccination could provide as much as 99% protection overall.
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Pathology of smallpox The virus enters the body through respiratory and oral mucosa, then travels to regional lymph nodes. After several days of replication in lymphatic tissues, asymptomatic viremia (usually macrophage-associated) begins, seeding virus to skin and reticuloendothelial organs [1]. Ten to 14 days after infection, leukocytes containing viral particles pass from dermal or submucosal capillaries and the virus begins to enter epithelial cells. The earliest histologic changes occur in dermal vessels with endothelial swelling, vascular dilatation, and perivascular lymphocyte and dendritic cell infiltration. In the more severe, fulminant forms, dermal and submucosal hemorrhage occurs. With other types, epidermal changes predominate with formation of eosinophilic cytoplasmic inclusions (ie, Guarnieri bodies), along with swelling and vacuolation with ballooning degeneration of cells in the spinous layer. Proliferation and progressive swelling and degeneration of these cells produces thickening of the epidermis within which thin-roofed, multiloculated vesicles form. Pustulation follows as neutrophilic infiltration occurs. Sebaceous glands appear to be destroyed preferentially, which likely contributes to scarring [44 46]. Bras [44] suggested that the greater concentration of sebaceous glands on the face accounted for the large number of lesions and the severe facial scarring that occurs. Although the upper portions of hair follicles may also be destroyed, hair follicles and sweat glands were relatively unaffected compared with sebaceous glands [44,47]. Although the majority of histopathological studies have focused on the skin, virtually all organs are affected, especially in the more severe and fulminant forms. In general, mucosal lesions resemble those seen in skin, showing Guarnieri bodies and reticulate degeneration. The absence of a stratum corneum layer leads to rapid erosion rather than vesiculation. Consequently, early in the exanthem phase, oral and pharyngeal ulcerations occur, and pharyngeal shedding can be continuous, with virus detectable from 7 to 13 days after onset of fever in nonfatal cases [48]. Mucosal lesions are arrayed in descending frequency over pharynx, uvula, tongue, and the upper trachea and esophagus. Necrotic foci are sometimes seen in pharyngeal and tonsillar lymphoid tissue [47]. The reticuloendothelial system is particularly affected; endothelial cells lining sinusoids of the liver can be swollen and sometimes necrotic, and cloudy swelling of parenchymal cells is common. Splenic engorgement with increased lymphoid development is typical. Renal hemorrhages are often seen in the fulminant type of smallpox and are usually associated
with hematuria. Hemorrhage is often present also in gastric laryngeal and pharyngeal mucous membranes and in endocardium and myocardial tissue. Striking depletion of megakaryocytes accompany the thrombocytopenia in hemorrhagic smallpox. Encephalitis occurrs occasionally, accompanied by perivascular demyelination and lymphocytic infiltration. Testis are also affected at times, with small foci of necrosis in all areas [47].
Transmission Unless there is close contact with soiled bedding or other materials contaminated with highly infectious fomites, the spread of smallpox appears to be primarily transmitted by nasal or oral secretions rather than from cutaneous lesions. Studies using a mousepox model showed that infection occurred first in mucosal cells in the upper and lower respiratory tract and in alveolar macrophages [49]. In virologic studies, approximately 10% of household contacts of smallpox patients harbored virus in pharyngeal secretions, but only 4 of 34 such individuals developed smallpox themselves [48]. Smallpox can also occur by accidental inoculation in skin and, rarely, through the conjunctiva [1]. Inoculation smallpox, well-described from observations of variolation, tends to have an incubation period that is 2 to 3 days shorter than infections contracted through the respiratory route. Rare cases of transplacental spread have also been reported.
Clinical features The clinical presentation of smallpox can vary greatly, influenced by vaccination status, level of nutrition and immunity, viral dose and strain, and possibly ethnic differences. Clinical descriptions also vary according to authors experiences, medical specialties, and research interests. Historical recollections and old photographs are our only informational source, because the last endemic case of smallpox occurred in Somalia in 1977 and the last recorded infection was acquired in a laboratory in the United Kingdom in 1978 [50]. The incubation period is approximately 12 days in variola major and may be a few days longer in variola minor. Variola major has highly varied symptoms and morphology, ranging from a benign abortive type with short-lived fever and a few papules to the fulminant, hemorrhagic type with purpura, ecchymosis,
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and death within 2 days. Between these extremes is a panoply of features [45]. The onset of the prodromal phase is sudden, with high fever (39C 41C), headache, myalgia, nausea, cramping, vomiting, and diarrhea. Sometimes evanescent morbilliform or urticarial lesions were observed, more readily in fair-skinned persons. Occurring mainly in previously vaccinated persons, it was sometimes called an allergic rash and occurred around the vaccination site and in the axillae, the popliteals, and the groin [1]. Typically, the earliest viral lesions began as erythematous papules and erosions on oropharyngeal mucosa, followed quickly by skin lesions. The cutaneous phase begins as erythematous macules that become raised and indurated over the course of a few hours. During the next few days, they evolve as firm, pearly and vesicular lesions, often with umbilication. The early vesicles often had a faint erythematous halo. Although vesicular in gross and histologic appearance, the lesions have been described as shotty with hard, round bodies palpable in the epidermis [1]. During the second week, vesicles became pustular and confluent, with edema and gross facial distortion. By 2 weeks, lesions dry with thick crusts and gradual separation to reveal deep scars.
abdominal sparing is evident [51]. Another distributional feature mentioned is the tendency of lesion density to be greater in areas of trauma or inflammationcalled the garter effect. This was apparently a Koebner phenomenon in which vessels, dilated by inflammation, were prone to viral invasion and more rapid lesion development. This was commonly noted along recent scratches or areas of chemical irritation. In one case, a confluent eruption of smallpox lesions was observed on a patients forehead after using alcohol compresses to help ease headache [1].
Clinical classifications of smallpox There are also variations in clinical classification. Rook and colleagues 1968 description [45] relied heavily on Dixons [52] tabulation of nine types of smallpox, while a 2002 review uses a proposed World Health Organization classification developed by Rao [50,51] that encompasses five smallpox types based on an epidemic in India. Further classification complexity results from the recognition of variola major and variola minor during epidemics of severe and mild smallpox in the nineteenth century. Table 1 provides a rough comparison of the types and terminology used in Dixons and Raos reviews. We will follow Raos classification in subsequent descriptions. The clinical characteristics described above represent the classical form of variola major, now referred to as ordinary smallpox. Within this grouping are gradations of severity along a spectrum ranging from confluent to semiconfluent to discrete. The confluent subtype, with fatality rates of 62% in Raos series, spares little of the facial or extensor surfaces. The semiconfluent classification is arbitrarily designated when lesions are discrete on the body and extensors but confluent on the face. Raos series fatality rate was 37%, in contrast to the discrete group with 9%
Distribution of smallpox lesions Distribution is described as centrifugal. Lesions usually began on the forehead and facial prominences, presumably due to the susceptibility of sebaceous glands as noted above. Lesions then quickly spread to the scalp, forearms, hands, back, and upper chest, with the legs and abdomen affected last. Thus the most mature and concentrated lesions are present on the head and upper body and, by the seventh day, the characteristic centrifugal pattern with relative
Table 1 Principal clinical characteristics of the types of smallpox Dixon Variola sine eruptione Benign abortive Mild Benign discrete Benign semiconfluent Benign confluent Malignant semiconfluent Malignant confluent Fulminating (purpura variolosa) Mortality % 0 0 0 2 10 20 25 70 100 Rao
Mortality % (unvaccinated/vaccinated) 0/0 0/0 30/3 37/8 62/26 97/67 96/94
Variola sine eruptione Modified Ordinary discrete Ordinay semiconfluent Ordinary confluent Flat type Hemorrhagic
Data from Rao AR. Smallpox. Bombay (India): Kothari Book Depot; 1972; and Dixon CW. Smallpox. London: J. & A. Churchill; 1962.
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fatalities. The discrete ordinary smallpox was the most common, comprising 42% of unvaccinated and 58% of vaccinated individuals. In this form of the disease, pustules are separated by normal skin on all areas, and only 9% of cases were fatal. The modified type of smallpox is similar to the ordinary type but follows an accelerated course (ie, it peaks and resolves more rapidly) and occurs mainly in vaccinated people. Lesions are fewer in number, evolve quickly, and have been described as abortive. Papules remain small, with many failing to progress to vesicles and pustules [1]. Crusting is complete in 10 days, but the prodromal illness tends to be as severe as in ordinary smallpox [51]. Variola sine eruption is the term used for the febrile illness that occurred among vaccinated individuals who had contact with smallpox patients. Febrile onset is sudden and might be accompanied by backache and other symptoms. Serological studies and viral isolations from pharyngeal swabs confirmed the diagnosis in some cases. In some reports, conjunctivitis was the only associated feature; in a series reported by Kempe and colleagues [53], virus was isolated from conjunctival exudates in 12 of 21 cases. A striking case description of contact fever is reprinted here because it illustrates so well the dangers and the crucial role of vaccination for physicians who care for smallpox patients:
Variola major was introduced into Durban from India in 1943 and spread widely in South Africa. I was personally involved with one of the patients admitted to Baragwanath Hospital. The physician-in-charge phoned to say that a patient had developed a profuse rash which he felt was probably due to a virus infection. One look at the patient convinced me that she had virulent confluent smallpox. The patient coughed in my face as I was examining her. In spite of having been revaccinated many times, indeed each time I saw a patient with smallpox and again on this occasion and each time responding with an immune reaction, I developed a high fever 12 days later, beginning with chills, muscle pain, especially in the small of the back, and headache and photophobia. My throat became sore and intensely itchy and a white membrane formed on the tonsils and pharynx, presumably an outward sign of an immune reaction taking place at the virus-blood junction. Also of interest was a marked erythematous reaction which developed at the site of the inoculation of the vaccine, presumably an immunological reaction against the antigen deposited at the site in the skin. This reaction became apparent at the time of defervescence. At the same time two vesicles, one on my ankle and one on my wrist, appeared and went through the typical stages of vesicle, pustule and scab.
My infection seems to have been a case of contact fever, a condition which had been recognized as occurring in fully vaccinated individuals many years ago. One of the sisters and the physician attending this patient developed a similar illness also, in spite of revaccination immediately the diagnosis was made. [1] (referring to J.H.S. Gear, personal communication, 1983)
Flat type smallpox was the designation given to cases in which the lesions were barely raised, usually in children and unvaccinated people. Patients were extremely ill with toxic fever. Most died, often with hemorrhagic lesions and pneumonia. The course and appearance of the lesions seemed to reflect absence of cellular immunity [1]. The fulminant or hemorrhagic type of variola majoralthough rare (2.4% of Raos series)is important to recognize because of the likelihood of misdiagnosis. The patient becomes extremely ill with an exhausted appearance, though fever may not be as high. As in ordinary smallpox, death often occurrs before obvious cutaneous signs appear, though scattered petechiae are usually evident and, if the patient survives a few days, widespread painful, erythematous, petechial lesions and ecchymoses appear. A syndrome similar to disseminated intravascular coagulation with thrombocytopenia becomes evident and death is usually due to massive hemorrhage from mucosal surfaces [54,55].
Variola minor This milder form of smallpox became recognized only toward the end of the nineteenth century when some epidemics occurred. The first report, in 1904, described a mild form, called kaffir-pox, with a fatality rate of 1% occurring in South Africa [56]. A similar mild disease had been noted in North America beginning in 1896 and by then had spread to South America, Europe, and Australia. With various names in different countries, along with controversy about its relationship to smallpox, often called alastrim, as a distinct entity until the mid-1950s [57], the term variola minor came to be used for clinico-epidemiologic variants with low case-fatality rates. Surprisingly, despite dramatic differences in clinical outcome, genetic analysis shows that variola minor is 98% identical to variola major [12]. All cases would have been described as modified or as ordinary-discrete but indistinguishable from variola major in any given individual. It was only after assessing the overall severity and fatality rate in an outbreak that allowed its designation as variola minor [58]. While the pre-
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eruptive illness could be just as severe, lesions were smaller, less dense and nonumbilicated. The course of the eruption was shorter and patients were less toxemic and often ambulatory during the eruptive phase.
Box 1. Differential diagnosis of smallpox Monkeypox and other orthopox infections Varicella/Chickenpox Bullous Drug Eruption Widespread herpes simplex Immunobullous diseases (bullous pemphigoid, dermatitis herpetiformis) Orf and Milkers Nodules Molluscum contagiosum Bullous impetigo Scabies Erythema multiforme Enteroviruses Secondary syphilis Meningococcemia Disseminated intravascular coagulation syndromes A useful algorithm for diagnosis is available from the Centers for Disease Control and Prevention at http://www.bt.cdc.gov/ agent/smallpox/diagnosis/riskalgorithm/ index.asp.
Diagnosis Diagnosis of smallpox depends on clinical features, epidemiological context, and laboratory confirmation. Clinical diagnosis requires recognition of the following key features:
Pre-eruptive fever, severe symptoms, and
enanthem
Pattern of lesion appearance, first on the face,
then forearms, upper trunk, and lower extremities

Monomorphous lesions of similar maturity on
any one area of the body

Individual lesion evolution from papules to vesi-
cles to umbilicated pustules

Palpation of hard, round, shotty vesicles
It is important to recognize that previously vaccinated individuals (ie, most of those born before 1972) may present with atypical smallpox with modifications to the general criteria listed. Hemorrhagic type smallpox may also be difficult to recognize and may escape detection without epidemiologic alerts and good laboratory support. Generally, the most common and easily confused entity that must be distinguished from smallpox is varicella/chickenpox (Box 1). Potentially, any other of the vesisculobullous diseases could be confused with smallpox, but usually if the key features above are kept in mind, these can be ruled out. Recent publications have also alerted us to the sporadic outbreaks of monkeypox and cowpox/feline orthopox infections that may become more frequent with global transport and travel [59,60]. Symptoms and lesions of smallpox in vaccinated individuals may be accelerated, abortive, sparse, and atypical. Laboratory diagnosis can be difficult, given the wide array of diseases to be considered. Moreover, previously vaccinated individuals with residual immunity from childhood vaccinations [23,25,40 43] may present with few or none of the commonly described symptoms. If smallpox is suspected, an oropharyngeal smear may be the earliest specimen for demonstrating virus. On the skin, a Tzanck smear positive for multinucleated giant cells can substantially support the diagnosis of varicella or herpes simplex infections; alternatively, the presence of
Guanieri bodies will be highly suggestive of an orthopox lesion. Where available, direct fluorescent antivaricella antibody test on smears from vesicle bases can provide quick, sensitive, and specific diagnosis of varicella lesions. Viral cultures, while not helpful for varicella, can identify herpes simplex and other viruses relatively quickly. Skin biopsy with both routine and immunofluorescent antibody preparations can help distinguish many of the conditions that might be mistaken for smallpox. Also, electron microscopic preparations can be helpful for identifying viruses. Based on current methodology, it is clear that more rapid and highly sensitive diagnostic assays need to be developed to streamline and confirm the diagnosis of potential smallpox infections. With the monkeypox outbreak, infection could only be confirmed by PCRno other test was considered accurate enough for diagnostic confirmation of infection.
Treatment options for smallpox infection According to the most current information provided by the Centers for Disease Control and Pre-
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vention (CDC), smallpox can only be prevented by the use of the smallpox vaccine, and there is no proven treatment for smallpox [61]. The CDC Web site states that research to evaluate new antiviral agents is ongoing, and one such candidatecidofovirshould be used to treat smallpox only under the evaluation of experts at the CDC and the National Institutes of Health. In general, the CDC suggests that smallpox patients can benefit from supportive therapy and antibiotics to control any secondary bacterial infections should they occur. In agreement and in addition to these recommendations, analysis of recently identified or historically relevant therapies indicates that other treatment options should also be considered. These include: (1) postexposure vaccination, (2) the use of vaccinia immune globulin (VIG), and (3) administration of cidofovir alone or in combination with VIG. Unlike many vaccines, the smallpox vaccine still provides considerable protection against morbidity and mortality even after a person has already been exposed to the virus. It is generally believed that vaccination within 1 to 3 days postexposure will provide strong protection against smallpox, with the effectiveness of treatment diminishing rapidly as the period after smallpox exposure increases [61,62].
Previous studies A classic study of 75 patients published in 1913 [35], and an anonymous medical record of 28 patients discovered in the Central Public Health Laboratory (Colindale, London, United Kingdom) [63] directly addressed this issue and demonstrated strong protective immunity by postexposure vaccination, especially in individuals who were also vaccinated at some point before exposure to smallpox. Further evaluation of these datasets indicates that substantial protection against severe/lethal disease may occur even if postexposure vaccination is delayed by 6 to 8 days after smallpox exposure (M.K. Slifka, manuscript in preparation). In the event of a deliberate smallpox outbreak, rapid identification and postexposure vaccination of all known or potential contacts could play an important role in decreasing the size and severity of the first round of infections and greatly suppress secondary or tertiary rounds of smallpox infection, especially when used in combination with isolation and quarantine procedures. At this time, VIG is only being recommended for severe adverse events related to smallpox vaccination and is not currently recommended as a treatment option for smallpox. This is unfortunate, because his-
torical studies indicate that passive immunotherapy with either convalescent smallpox serum [64], hyperimmune antivaccinia gammaglobulin of animal origin [65], or VIG [66 68] can have a substantial impact on the outcome of smallpox infection and spread. One outstanding study published in 1941 presented an outbreak of variola major in which convalescent serum or whole blood (obtained approximately 10 days after lesions had scabbed over) was administered daily to smallpox patients [64]. There were three casualties in the first 10 cases of smallpox (ie, 30% mortality) that were not treated by passive immunotherapy. After that, all other smallpox patients were treated with convalescent serum or whole blood and, remarkably, none of the 250 patients died (<0.4% mortality). Another study published in 1940 came to a far different conclusion in that they were unable to identify any therapeutic effect when using convalescent serum or antivaccinia serum produced in animals [69]. However, one of the main reasons for the failure of demonstrable efficacy in this latter study is that the patients were first monitored for disease symptoms, and only the most severe cases (already at late stages of disease) were then treated. This delay in treatment is likely to be a major reason for the lack of therapeutic efficacy reported. It is well-known in a variety of other viral infections that passive immunotherapy must be provided at the earliest stages of disease to provide full protective benefits and is unlikely to provide protection against lethal infection if administered after severe disease symptoms have already manifested [70]. Because smallpox-specific convalescent serum is not available, the next best thing is VIG, and it, too, has shown therapeutic efficacy in large field studies involving smallpox outbreaks. For instance, in one study involving 705 smallpox contacts, the administration of a small volume of VIG (10 mL/adult intramuscularly) in addition to immediate smallpox vaccination reduced the number of smallpox cases by 70% over that achieved through postexposure vaccination alone [68]. Likewise, administration of a similar volume of hyperimmune antivaccinia antibody of animal origin (in addition to vaccination) resulted in 0 of 13 cases of smallpox in close contacts compared with 13 of 29 cases of smallpox in a control group of contacts that received postexposure vaccination alone [65]. This clearly demonstrates that even small doses of VIG, of either human or animal origin, given intramuscularly can have a significant effect on smallpox spread. The CDC has stockpiled new VIG formulations (Calgene) that are as potent as the older formulations (Baxter) but can be used for intravenous administration [71], which is likely to greatly increase
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its efficacy over previous VIG administration methods that required intramuscular injections. Cidofovir (Vistide) is an antiviral drug that has been approved by the US Food and Drug Administration and is licensed for the treatment of cytomegalovirus retinitis. This antiviral compound has demonstrated efficacy in blocking the in vitro replication of most major poxviruses, including smallpox, and has been shown to provide significant protection against lethal or systemic poxvirus infection in several animal models [72]. Administration of cidofovir as early as 4 days before cowpox infection of mice or up to 2 days after infection protected 80% to 100% of mice from lethal disease [73]. In a nonhuman primate model of monkeypox infection (closely mimicking human smallpox infection), cidofovir apparently protected against up to 1000 LD50 of aerosolized monkeypox, provided that administration was started within the first few days after exposure (J.W. Higgins, unpublished data). The main drawback to the use of this drug is its nephrotoxicity, which can be potentially severe at high doses. However, a small clinical trial consisting of eight immunocompromised bone marrow transplant patients [74] and a recent case study [75], all with adenovirus-associated hemorrhagic cystitis, enteritis, hepatitis, or renal dysfunction, showed that they were able to successfully treat these patients with low-dose cidofovir (1 mg/kg intravenously, once daily three times a week with probenecid given orally at 1.25 g/m2 in divided doses daily). In both studies, this regimen was well tolerated, no cidofovir-related side effects were observed, and the treatment resulted in elimination of the adenovirus infection and improved renal function [73]. In the noted case study, the patient already had renal dysfunction before cidofovir therapy which then improved following treatment; therefore, it is likely that this low-dose cidofovir regimen would be considered relatively safe in patients that do not have preexisting renal ailments and who do not have other predisposing contraindications for cidofovir/probenecid treatment. Moreover, the combined use of lowdose cidofovir with VIG is a therapeutic approach that has yet to be formally explored. Because these treatments work independently of each other (one blocks viral replication at the level of the DNA polymerase and the other neutralizes infectious viral particles once they have already formed), it is possible that doses of cidofovir and VIG that alone might only provide suboptimal protection may be far more effective when used in combination. Considering that cidofovir is likely to interfere with active immunization by blocking vaccinia replication, it should be given only to smallpox patients showing progres-
sion of disease symptoms in spite of VIG or postexposure vaccination.
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Smallpox: vaccine reactions and contraindications

Wynnis L. Tom, MDa, Julie R. Kenner, MD, PhDb, Sheila F. Friedlander, MDc,d,*
Department of Internal Medicine, San Diego School of Medicine, University of California, 200 West Arbor Drive, Mail Code 8422, San Diego, CA 92103, USA b Department of Dermatology, Castle Medical Center, 640 Ulukahiki Street, Kailua, HI 96734, USA c Division of Pediatric and Adolescent Dermatology, Childrens Hospital and Medical Center, 8010 Frost Street, Suite 602, San Diego, CA 92123, USA d Departments of Pediatrics and Medicine, San Diego School of Medicine, University of California, 200 West Arbor Drive, Mail Code 8420, San Diego, CA 92103, USA
a
Smallpox is caused by the variola virus, an orthopoxvirus that has a case fatality rate of up to 30% or more. There is no cure for the disease, but vaccination is an effective method of protection. Intensive vaccination efforts and tracking of cases in the 1960s and 1970s led to global eradication of the disease by 1980. With recent threats of the possible reintroduction of smallpox as a weapon of bioterrorism, smallpox vaccination has been reinstituted on a limited basis in the United States. This article reviews the historic background leading to the development of smallpox vaccines, the contraindications and adverse events associated with vaccination, the challenges encountered during the ongoing smallpox vaccination program, and the future direction of smallpox vaccines, which are being designed to provide effective immunity with fewer adverse effects.
Historic background As early as the tenth century, it was noted that those who contracted smallpox and survived thereafter enjoyed protection from further attacks. This observation spawned the process of variolation, the
* Corresponding author. Division of Pediatric and Adolescent Dermatology, Childrens Hospital and Medical Center, 8010 Frost Street, Suite 602, San Diego, CA 92123. E-mail address: sfriedlander@chsd.org (S.F. Friedlander).
technique by which variola virus was deliberately given to healthy individuals to protect them from more severe disease. The idea was to try to induce a mild form of the disease from which the individual would recover and gain lifelong immunity [1]. For centuries, variolation was practiced in various forms across the world. The Chinese used a method of inhalation of powder made from the crusts of a recovered patient who had suffered a mild case of smallpox. In other areas, such as India and Europe, the contents from either a vesicle or pustule were taken and inserted into a cut in the skin [2,3]. In England in the mid-eighteenth century, Robert Sutton began the practice of using a lancet to insert a minute amount of fluid from a vesicle obliquely into the skin [4]. Although a significant improvement from the natural disease, variolation was associated with a mortality rate of 1% to 2% [3]. Furthermore, those inoculated could spread their infection to contacts in an uncontrolled manner, often resulting in severe disease or outbreaks. These two factors significantly limited the utility of using variola virus for protection, and attention shifted to the use of heterologous orthopoxviruses to induce crossprotective immunity. This medical breakthrough was first discovered from observations of dairymaids. These maids, who had frequent exposure to cowpox, a smallpox-like disease of cattle which only minimally affected humans, seemed to be uniquely protected from contracting smallpox. Edward Jenner performed extensive studies and experimentation on the use of cowpox virus as a means of providing immunity to smallpox
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[4]. In 1796, he introduced material from a dairymaids cowpox lesion into the arm of an 8-year old boy. The boy did not develop smallpox when Jenner subsequently exposed him to the variola virus. He called the use of cowpox to provide crossprotection vaccination, vacca being the Latin word for cow, and this term continues to today [5]. Jenners goal was for vaccination to produce such mild disease that the vaccinee would gain immunity but would not be a source of infection to others. Arm-to-arm vaccination and occasionally, use of virus from the lesions on inoculated calves, was practiced until the development of lymph vaccine preparations. Since the time of Jenner, further modifications have been made in the technique of vaccination. During the nineteenth century, a switch was made to the vaccinia virus in lieu of cowpox virus for vaccination. It is thought that the vaccinia virus is a spontaneously evolved relative of cowpox virus [3]. In the first half of the twentieth century, commercial methods of freeze-drying allowed bulk production of vaccine that could be stored for years. In 1961, the bifurcated needle was invented, which simplified the administration of vaccine and allowed smaller doses to be used [4,6]. Vaccination turned a devastating disease into one unseen in the past 25 years. The last case of smallpox in the United States (US) was in 1949, and routine civilian vaccination was discontinued in the US in 1972 [3]. With intensified global surveillance and vaccination efforts, the last case of natural smallpox occurred in Somalia in 1977, and smallpox was
declared eradicated in 1980 by the World Health Organization. Vaccination of military personnel was discontinued in the US in 1990 [7]. Smallpox became the model example of how effective public health efforts can eradicate a disease. However, because of recent events involving terrorism, including the September 11, 2001, and postal anthrax attacks, smallpox has again become a public health concern. The majority of the population in the US today would be vulnerable in the event of a smallpox attack; over 40% of the population has never been vaccinated, and the rest have uncertain levels of immunity [8,9]. With the renewed threat of smallpox being used for bioterrorism and biologic warfare, smallpox vaccination was reinitiated in the US for selected groups. Vaccination of military personnel began in December 2002. Voluntary vaccination of public health and hospital-based health care workers who would respond in the case of a smallpox outbreak began in late January 2003 [10]. Unfortunately, a significant number of unexpected adverse cardiovascular events (detailed below) have occurred during military and civilian vaccination. These events, coupled with a perceived decrease in the potential for a bioterrorist event, have led to a marked slowing of the pace of vaccination (Fig. 1). Many experts now believe that a significant nationwide vaccination program should not be implemented unless a real exposure event occurs, or a safer vaccine is widely available for public use [11]. Next-generation safer and effective smallpox vaccines are currently undergoing accelerated development. The
Fig. 1. Number of civilian smallpox vaccinations administered since the reinstitution of vaccination January 2003. (From Centers for Disease Control and Prevention [CDC].)
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two most likely attenuated vaccine candidates (modified vaccinia Ankara and LC16m8) are currently in Phase 1 and 2 trials [10]. Therefore, widespread availability of a safer smallpox vaccine will most likely not occur for several years.
Smallpox vaccine and its administration The current vaccine being administered in the United States Smallpox Vaccination Program is a lyophilized formulation of the New York City Board of Health (NYCBOH) strain of vaccinia virus called Dryvax (Wyeth Pharmaceuticals, Marietta, Pennsylvania). This vaccine, prepared from the lymph of calves inoculated with vaccinia virus, has been in use in the US for decades [12]. Dryvax is 90% to 96% effective in preventing disease when given before exposure to variola virus, and can significantly attenuate or abort an infection if given within the first 3 to 4 days after exposure [3]. Unlike natural smallpox or variolation, vaccination does not render lifelong immunity to smallpox, and the duration of protection against infection is a subject of debate. There is evidence of some cell-mediated immunity and neutralizing antibodies being present at 30 or more years after primary vaccination, but how this correlates with protection against infection is unknown [9]. Most estimates suggest primary vaccination gives full protection for 3 to 5 years, with some but waning immunity at 10 years and thereafter. Revaccination probably provides substantial protection for at least 20 to 30 years [13]. Dryvax vaccine is administered via a bifurcated needle inserted perpendicularly into the skin over the insertion of the deltoid muscle or the posterior aspect of the arm over the triceps muscle. Skin preparation is not required unless the area is grossly contaminated, in which case soap and water are used. Alcohol or other chemical antiseptics can inactivate the vaccine virus and should not be used [12]. When the bifurcated needle is inserted into the vial of reconstituted vaccine, a 0.0025-mL droplet adheres between the prongs of the needle. Multiple punctures are then rapidly made at the insertion site. According to the product insert, two to three insertions are recommended for primary vaccination and 15 for revaccination. Punctures should be vigorous enough for a trace of blood to appear after 15 to 20 seconds. If no trace of blood is visible, three additional insertions should be made with the same needle without reinserting into the vaccine vial [14]. Smallpox vaccine administered simultaneously with either the oral polio vaccine, Bacille of Cal-
rin vaccine, yellow fever vaccine, measles mette-Gue vaccine, or the diphtheria and tetanus toxoids and whole-cell pertussis vaccine has been found to have the same level of safety and efficacy as the vaccines administered separately [15]. Although there is no data on simultaneous administration of smallpox vaccine with the other vaccines now routinely administered in the US, current recommendations allow for simultaneous administration of smallpox vaccine with other inactivated or live-virus vaccines, with the exception of the varicella vaccine. Because varicella vaccine virus lesions might be confused with vaccinia lesions, the two vaccines should be given at least 4 weeks apart [14]. Suppression of reactivity to the purified protein derivative skin test for tuberculosis screening has been observed after administration of smallpox and other parenteral live-virus vaccines [16]. The purified protein derivative test should not be given within 1 month of smallpox vaccination to prevent possible false-negative reactions [14].
Vaccine reactions In primary vaccinees, the smallpox vaccine produces a local reaction consisting of a papule at about 3 to 5 days after administration. The papule develops into a vesicle (Jennerian vesicle) and then into a pustule around day 8. The pustule subsequently crusts over (day 10 to 14) and scabs, with a scar left after the scab detaches at around day 17 to 21 (Fig. 2). This progression from papular to pustular lesion with a surrounding ring of erythema or induration signifies a take of the vaccine, indicating vaccinia growth and replication and the development of an immune response. Pain or pruritis at the site of vaccination are also common. Vaccination should be repeated if a take does not occur by 6 to 8 days after vaccination [17]. Revaccinated individuals tend to have a milder local reaction over an accelerated time course, depending on the duration since prior vaccination. Some may develop a pustule or scab at day 6 to 8, while others with substantial residual immunity may only have erythema at the vaccination site. Because it is difficult to distinguish the modified take reaction due to residual immunity from that reaction seen from improper vaccine administration, a repeat revaccination should be administered if the first attempt fails to produce the major (primary) take reaction and the vaccination status of the individual is not known [18]. Additional minor local reactions affect up to 6% of primary vaccinees. One to 10 satellite lesions often develop around and progress with the primary lesion (Fig. 3). Lymphadenopathy, lymphangitis, and intense
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Fig. 2. Normal progression of the major take reaction in a primary vaccinee. (From Centers for Disease Control and Prevention [CDC] Public Health Image Library [PHIL].)
surrounding erythema or edema are other common local reactions [19]. About 2% to 16% of primary vaccinees develop what is called a robust take, where the local reaction is 10 cm or more in diameter (Fig. 4). The area can be very painful or pruritic. A robust take may be mistaken for bacterial cellulitis (Fig. 5). Most cases of robust take occur at day 8 to 10 after vaccination and improve in 24 to 72 hours
without specific therapy. In contrast, secondary bacterial infections usually occur within 5 days of vaccination or 30 days after, and unless treated with antibiotics, continue to progress [20]. Systemic symptoms following vaccination include fever (100F to 102F, more common in children than adults), chills, nausea, malaise, myalgias, and headache [19]. These are considered normal reactions and they generally peak 8 to 10 days after vaccination and last 1 to 3 days. About 30% of vaccinees feel too
Fig. 3. Satellite lesions surrounding the primary lesion. (From Centers for Disease Control and Prevention [CDC] Public Health Image Library (PHIL) and contributed by M. Grossman, MD, of the California Emergency Preparedness Office, Immunization Branch.)
Fig. 4. Example of a robust take in a vaccinee. (From Centers for Disease Control and Prevention [CDC] Public Health Image Library [PHIL].)
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Fig. 5. A case of secondary staphylococcal infection at the smallpox vaccination site. (From Centers for Disease Control and Prevention [CDC] Public Health Image Library [PHIL], and contributed by A.W. Mathies, MD, of the California Emergency Preparedness Office, Immunization Branch.)
ill to participate in normal activities. Symptomatic treatment alone is needed for these self-limited symptoms [20].
rashes, broad roseola-like erythematous macules and patches, and urticarial rashes. They are thought to be immune-mediated reactions rather than the result of direct viral invasion. These rashes are not usually associated with fever, and typically self-resolve in 2 to 4 days. Hypersensitivity reactions may also occur [20]. Erythema multiforme can range from macules, papules, and urticaria to the typical targetoid lesions (Fig. 6). Oral antipruritics can be given if needed. Rarely, Stevens-Johnson syndrome (bullous erythema multiforme with mucous membrane involvement) can develop, and require hospitalization and supportive care [17,23]. Reactions specific to the vaccinia virus include accidental, or inadvertent, vaccinia, the autoinoculation of vaccinia virus from the vaccination site to other areas. This is the most common adverse reaction associated with smallpox vaccination, and accounts for about 50% of all adverse events [12]. Viable virus is present at the vaccination site from the time
Adverse reactions in the vaccinee Of concern in reinstituting smallpox vaccination are the serious adverse reactions associated with the current vaccine. Data from the 1960s (Table 1) estimated about 1254 complications per 1 million primary vaccinations [21]. Rates of adverse reactions were highest among persons less than 5 years of age, and were 10 times more common in primary vaccinees than those being revaccinated [22]. Nonspecific skin rashes can occur after the vaccine is given. These include fine maculopapular
Table 1 Rates of adverse reactions in a 1968 10-state survey Events per million primary vaccinees 164.6 529.2 241.5 38.5 1.5 12.3 1.5 Events per million revaccinees 10.0 42.1 9.0 3.0 3.0 2.0
Adverse event Erythema multiforme Accidental vaccinia Generalized vaccinia Eczema vaccinatum Progressive vaccinia Postvaccinial encephalitis Deaths
Data from Lane JM, Ruben FL, Neff JM, Millar JD. Complications of smallpox vaccination, 1968: results of 10 statewide surveys. J Infect Dis 1970;122:303 9.
Fig. 6. Erythema multiforme in a 1-year-old following vaccination. (From Centers for Disease Control and Prevention [CDC] Public Health Image Library [PHIL], and contributed by A.W. Mathies, MD, and J. Leedom, MD, of the California Emergency Preparedness Office, Immunization Branch.)
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the papule forms until scab separation. Virus can be transferred when the vaccinee touches or scratches the site and then subsequently touches or inoculates another area of the body, most commonly the eyelids, nose, mouth, genitalia, and anus. Lesions are usually seen 7 to 10 days after vaccination, and often follow the course of the primary lesion at the vaccination site [24,25]. If accidental inoculation occurs more than 5 days after vaccination, the lesions may be more attenuated due to the developing immune response. Uncomplicated cases do not require therapy, and usually resolve in 3 weeks. Extensive involvement can be treated with vaccinia immune globulin (VIG) to decrease spread and speed recovery. VIG is a sterile solution of the immunoglobulin fraction of plasma from persons vaccinated with vaccinia vaccine. It can provide passive immunity and is given by the Centers for Disease Control and Prevention (CDC) by Investigation New Drug protocol [20]. Also available by Investigation New Drug protocol in the case of failure to respond to VIG is cidofovir, a nucleotide analog approved for the treatment of cytomegalovirus infections. It has activity against vaccinia and certain other orthopoxviruses in cell-based in vitro and animal studies, but no clinical data is available [7,20]. Ocular vaccinia is the most common form of accidental inoculation. Autoinoculation to the eyes can be in form of conjunctivitis (Fig. 7), keratitis, or iritis. Vaccinia keratitis (about 6% of cases of ocular vaccinia [26]) causes central ulceration of the cornea that can lead to scarring and residual visual loss [27]. There are currently no approved medications for the treatment of ocular vaccinia. CDC treatment guidelines include the use of topical antiviral eye drops in moderate to severe cases [20]. Before 1972, when smallpox vaccination was still routine, vidarabine ointment was found to successfully treat cases of ocu-
Fig. 8. Generalized vaccinia in a child on day 10 following smallpox vaccination. (From Centers for Disease Control and Prevention [CDC] Public Health Image Library [PHIL], and contributed by M. Grossman, MD, of the California Emergency Preparedness Office, Immunization Branch.)
Fig. 7. Ocular vaccinia: conjunctivitis from autoinoculation of vaccinia virus. (From Centers for Disease Control and Prevention [CDC].)
lar vaccinia. It is no longer manufactured, although some supplies still exist. Trifluridine eye drops, indicated for treatment of conjunctivitis and keratitis associated with herpes simplex virus infection, may be administered for ocular vaccinia, although there is no supporting clinical trial data for this [28]. With the exception of topical trifluridine, which should not be given for more than 14 days, topical antivirals should be administered until all periocular or eylid lesions have healed and the scabs have fallen off. Overuse of trifluridine can lead to superficial punctate keratopathy, which resolves on discontinuation of the medication. VIG has not been found to improve the course of vaccinia keratitis, and sometimes appears to exacerbate the condition; therefore, it should not be given in cases of isolated keratitis. However, VIG should not be withheld if indicated by another comorbid condition (eg, eczema vaccinatum [EV] or progressive vaccinia [PV]), and should be considered for severe ocular disease without keratitis [20]. Generalized vaccinia (GV) is the dissemination of macular, papular, or occasionally, vesicular lesions on otherwise normal skin, without evidence of accidental autoinoculation. Lesions may be limited or numerous, and may occur anywhere on the body (Fig. 8). GV is thought to be due to spread of the virus through the bloodstream [10]. It usually occurs 6 to 9 days after a primary vaccination, and most commonly in children [22]. The lesions vesiculate and progress as would any vaccinial lesion. Vaccinees usually do not feel ill, although they may have a fever preceding the rash. GV lesions may contain vaccinia virus, and affected individuals should keep the lesions covered and avoid
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physical contact with others. In immunocompetent individuals, GV is self-limited. Supportive care, such as with nonsteroidal antiinflammatory agents and oral antipruritics, may be of help; VIG is not needed. However, in the setting of immunodeficiency, GV is often more severe, and these individuals might benefit from VIG, especially if systemically ill [20]. Potentially fatal reactions from smallpox vaccination include EV, PV, and postvaccinial central nervous system disease. In the era of routine vaccination, about one death occurred per 1 million primary vaccinations and 0.25 deaths per 1 million revaccinations [22]. EV is localized or generalized spread of vaccinia virus in individuals with atopic dermatitis (AD) or less frequently, other chronic dermatoses, such as Dariers disease (keratosis follicularis) [29]. Papules, pustules, or vesicles can occur anywhere, but have a predilection for areas with prior AD lesions (Fig. 9) [20]. The lesions usually develop at the same time or shortly after the appearance of lesions at the vaccination site. The affected individual is systemically ill, often with fever, malaise, and lymphadenopathy. Cases range from mild to severe, with hundreds of rapidly evolving lesions and such extensive involvement that there is substantial loss of the cutaneous barrier [10]. The most serious cases of EV are in primary vaccinees, and are independent of the sever-
Fig. 10. Progressive vaccinia in a female with chronic myelogenous leukemia 1 month after smallpox vaccination. (From Centers for Disease Control and Prevention [CDC] Public Health Image Library [PHIL], and contributed by A.W. Mathies, MD, and J. Leedom, MD, of the California Emergency Preparedness Office, Immunization Branch.)
Fig. 9. Eczema vaccinatum in a primary vaccinee 8 days after vaccination. (From Centers for Disease Control and Prevention [CDC] Public Health Image Library [PHIL] and contributed by the CDC/Arthur E. Kaye.)
ity or activity of the underlying skin disease [30]. Meticulous skin care as for burn victims along with volume and electrolyte repletion may be needed for disruption of the cutaneous barrier. Antibacterial and antifungal agents may be used to prevent secondary bacterial and fungal infections of the lesions. Early treatment with VIG has been shown to reduce mortality. One study found the fatality rate of 30% to 40% was reduced to 7% after the introduction of VIG [31]. Cidofovir may be considered a secondary treatment for severely ill patients, although this has never been tested [20]. Because vaccinia virus can be isolated from EV lesions, precautions should be used to prevent secondary transmission and nosocomial infection [14]. Introduction of vaccinia virus to individuals with severe impairment of the immune system leads to PV, also called prolonged vaccinia, vaccinia necrosum, or vaccinia gangrenosum [20]. Most cases have been in patients with defective cell-mediated immunity, but a few cases have been reported in patients with agammaglobulinemia and other humoral defects [32]. Unlike in immunocompetent vaccinees, there may be no signs of inflammation at the vaccination site and the primary lesion does not heal. Instead, it continues to enlarge and progresses to a painless ulcer, often with central necrosis (Fig. 10) [33]. Presumably because of immunodeficiency, viral replication is not halted and the vaccinia virus instead continues to spread. With viremia, similar metastatic lesions may develop at distant sites on the skin, bone, or viscera. PV should be suspected if the initial vaccination lesion progresses without healing for 15 or more days after vaccination. The degree of immunodeficiency prob-
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ably correlates with the risk for PV, although the precise protective level of cellular or humoral immunity is unknown [20]. Progressive vaccinia had a case fatality rate of 100% before the introduction of VIG and ICU care; the rate is now approximately 20% to 30% [17]. Topical or systemic cidofovir therapy may also be useful in these patients [6]. Central nervous system (CNS) complications with the smallpox vaccine include encephalopathy, encephalitis, and encephalomyelitis. They occur in apparently normal individuals with no predisposing conditions. Postvaccinial encephalopathy (PVE) most commonly affects infants less than 2 years of age [20]. Symptoms and signs may develop abruptly 6 to 10 days after vaccination, and include fever, headache, seizures, hemiplegia, Guillain-Barre syndrome, aphasia, and transient amnesia. There is generalized cerebral edema without inflammation. Postvaccinial encephalitis or encephalomyelitis (PVEM) may occur 11 to 15 days after vaccination with abrupt onset of fever, vomiting, malaise, headache, and anorexia, which can then progress to confusion, drowsiness, seizures, spinal cord signs, amnesia, and coma. Pathologic features are similar to that observed in other postinfectious encephalitides [34]. These CNS complications are thought to be autoimmune reactions, as very few affected individuals have vaccinia virus in sampled CNS tissue or cerebrospinal fluid [10]. No clinical criteria, radiographic findings, or laboratory tests are specific for PVE or PVEM; they are diagnoses of exclusion. Treatment is supportive care, as there is no evidence to indicate that VIG is an effective therapy [20]. Different strains of vaccine virus may have different rates of CNS complications, with higher rates in non-NYCBOH strains [3]. About 15% to 25% of recipients of the NYCBOH strain who develop PVE or PVEM die, 25% of survivors are left with residual neurologic sequelae (mental impairment, paralysis), and the remainder recover in about 2 weeks [35]. Pregnancy-related concerns with smallpox vaccination mainly relate to the fetus. About 20 cases of fetal vaccinia were reported between 1932 and 1974 [36]. Fetal vaccinia can manifest with lesions of the skin, mucous membranes, and placenta. The skin lesions in the neonate are similar to those of GV or PV, and may be confluent or extensive. Fetal vaccinia often results in fetal or neonatal death [37]. Smallpox vaccination of pregnant women has not been clearly associated with prematurity, low birth weight, or congenital malformations. Reported cases of fetal vaccinia include women vaccinated in all three trimesters, primary vaccinees, those being revaccinated, and nonvaccinated contacts of vaccinees. It is not
known whether the virus infects the fetus through the blood or by direct contact with infected amniotic fluid. No known reliable intrauterine diagnostic test is available to confirm fetal infection [10]. VIG might be considered for a viable infant born with vaccinial lesions, but the appropriate dosage or efficacy is not known. If a pregnant woman is inadvertently vaccinated or if a woman becomes pregnant within 4 weeks after vaccination, she should be counseled regarding potential complications. However, given the rarity of congenital vaccinia among live-born infants, vaccination during pregnancy is not ordinarily a reason for termination. No indication exists for prophylactic use of VIG for an inadvertently vaccinated pregnant woman. However, VIG should not be withheld if a pregnant woman has a condition where VIG is needed, such as EV [20].
Contact vaccinia Despite Jenners belief that vaccination would not lead to infection of contacts of vaccine recipients, secondary transmission does occur. Surveys in the 1960s found contact vaccinia to occur at a frequency of two to six cases per 100,000 primary vaccinations [30]. The majority of cases have been in children in contact with a recently vaccinated family member, although nosocomial cases have also been reported. Most fatal cases of contact vaccinia were in infants less than 1 year of age [31]. Contact eczema vaccinatum (Fig. 11) accounts for nearly all serious cases of contact vaccinia. EV resulting from secondary transmission usually involves skin eruptions 5 to 19 days after exposure [20]. All other reported cases
Fig. 11. Eczema vaccinatum in an 8-month old contact (sibling). (From Centers for Disease Control and Prevention [CDC] Public Health Image Library [PHIL] and contributed by the CDC/Arthur E. Kaye.)
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Fig. 12. Accidental vaccinia in a contact. (From Centers for Disease Control and Prevention [CDC] Public Health Image Library [PHIL] and contributed by the CDC/Dr. Robinson.)
of contact vaccinia are cases of accidental vaccinia in contacts (Fig. 12) and two cases of secondary transmission resulting in fetal vaccinia. No secondary cases of progressive vaccinia or postvaccinial CNS disease have been reported to date [38]. Secondary transmission can be prevented by keeping the vaccination site covered with gauze and avoidance of touching or applying any topicals to the area. Vaccinated health care workers should additionally have a semipermeable dressing over the gauze as an additional barrier to the vaccinia virus to prevent nosocomial transmission. Infection-control measures should be taken, including careful hand hygiene on touching the site or anything that has been in contact with the site [14].
Other associated adverse events Other less common adverse events have been reported to occur in temporal association with smallpox vaccination, but causality has not been established. These include osteomyelitis, neurologic complications such as transverse myelitis, paralysis, seizures, and polyneuritis, the precipitation of erythema nodosum leprosum or neuritis in patients with leprosy, and myocarditis and pericarditis [3]. Between December 13, 2002 and May 9, 2003, there were 37 reported cases of myocarditis or pericarditis among the approximately 449,198 military personnel who have received the smallpox vaccine [39]. The first 18 cases were all primary vaccinees (information not yet available on the other 19) [40]. Among civilian vaccinees,
there have been 22 reported cases (15 cases consistent with myocarditis, 6 with pericarditis, 1 unspecified). Age of the civilian cases ranged from 29 to 61 years (median age of 48). Onset of symptoms took place 1 to 42 days after vaccination (median of 12 days). Nineteen of the first 21 cases (90%) were revaccinees. There have been no case-fatalities, and all civilian cases have recovered [39]. Myopericarditis had been reported in Europe and Australia during the preeradication era, mainly with vaccinia strains more virulent than NYCBOH vaccinia, and was thought to be the cause of the rare cases of cardiac-associated deaths following smallpox vaccination [41,42]. At least one case of myocarditis has been reported previously in association with the NYCBOH strain [43,44]. When compared with the rate reported in an unvaccinated military population during 2002, the rate of myopericarditis among military vaccinees is substantially elevated (3.6-fold), suggesting, but not proving, causality [40]. In addition, because smallpox vaccination was reinitiated in late 2002, one case of fatal myocardial infarction (MI) in a military vaccinee and nine cases of ischemic cardiac events have been reported in civilians (age range of 46 to 65 years, six cases with evidence of MI, three with angina) [39]. For the civilian cases, eight were revaccinees (information unavailable for the ninth). Symptom onset ranged from the day of vaccination to 26 days later (median of 10 days). All but one case had either a prior history of MI, angina, or exertional chest pain or had known cardiac risk factors. Two of the six civilian cases of MI died (female patients age 55 and 57 years). These are the first reports of cardiac ischemic events in association with smallpox vaccination [39,42]. All cases of ischemic events occurred before the April 2003 establishment of exclusionary criteria (discussed below). Preliminary autopsy findings for the three patients with MI who died showed no evidence of disseminated vaccinia infection or myopericarditis. The number of reported deaths from cardiac disease among civilians and military personnel is consistent with expected background rates [39]. Two cases of nonischemic dilated cardiomyopathy (DCM) have also been reported for the first time in association with smallpox vaccination (two women, ages 53 and 55 years). Both were revaccinees who 3 months after revaccination, developed a new murmur, new left bundle branch block on EKG, and echocardiographic findings of diffuse hypokinesis with impaired systolic function (ejection fraction V35%) [45]. Whether these ischemic and dilated cardiomyopathy conditions are caused by smallpox vaccination or are coincidental occurrences is unclear.
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Studies to evaluate possible mechanisms for cardiac adverse events following smallpox vaccination are being considered.
Vaccine safety and current vaccine contraindications To limit the number of adverse events from smallpox vaccination in the current nonemergent setting (presmallpox outbreak), the Advisory Committee on Immunization Practices (ACIP) has delimited certain conditions as contraindications to vaccination (Box 1).
Box 1. Contraindications to smallpox vaccination in the preexposure setting (pre-smallpox outbreak) Contraindication Atopic dermatitis or eczema or household contacts of persons with these conditions Active acute, chronic, or exfoliative skin conditions or household contacts of persons with these conditions Inflammatory eye diseases requiring opthalmic steroid treatment Immunodeficiency or Immunosuppression or household contacts of persons with immunodeficiency/ suppression Severe autoimmune disease Women who are pregnant or intend to become pregnant within 4 weeks of vaccination or their household contacts Women who are breastfeeding Infants less than 1 year of age Serious allergy to any component of the vaccine or its diluent Known cardiac disease or ischemic cardiovascular disease Three or more cardiac risk factors: hypertension, hypercholesterolemia, diabetes mellitus, smoking, or a first-degree relative with heart disease before the age of 50 From Refs. [14,46,48,52].
Due to the increased risk of eczema vaccinatum, smallpox vaccine should not be administered to individuals with a history of eczema or atopic dermatitis or to their household contacts, irrespective of disease severity or activity [46]. Although not all types of eczema are at risk for EV, the majority of primary care providers do not routinely make the distinction between eczema and AD, particularly with regard to chronic exfoliative skin conditions in infants and children. There is also concern regarding accuracy of disease recall. A recent study found that only 59% of 94 adults with atopic dermatitis or eczema correctly self-reported having the skin disease, while 70% of 177 parents correctly recalled that their child had AD [47]. A history of Dariers disease is also a contraindication for smallpox vaccination because of reported cases of EV in affected individuals [29]. Individuals with active acute, chronic, or exfoliative skin conditions that disrupt the epidermis are at increased risk for severe accidental inoculation vaccinia. Examples include varicella-zoster, impetigo, psoriasis, contact dermatitis, wounds, and burns [14]. Vaccination should not be performed in either these individuals or their close contacts until the skin condition has resolved and the skin is fully reepithelialized, or in the case of chronic disorders, when the disorder is under good control. Individuals taking therapy that can cause severe xerosis or eczematous lesions (eg, oral isotretinoin) or erosions (eg, topical fluorouracil) should not be vaccinated until 72 hours after discontinuing the drug [46]. Those with inflammatory eye diseases requiring opthalmic steroid treatment should not be vaccinated until the condition resolves and therapy is complete to avoid vaccinia inoculation from touching or rubbing the eye [20]. Immunocompromise is another contraindication, because enhanced vaccinia viral replication at the vaccination site may occur in these individuals and lead to adverse reactions such as progressive vaccinia. Persons with congenital or acquired (eg, HIV, Acquired Immune Deficiency Syndrome) immunodeficiencies should not be vaccinated. Conditions associated with immunosuppression (eg, leukemia, lymphoma, generalized malignancies) are also contraindications. Household contacts of immunocompromised individuals should not receive the vaccine due to the risk of vaccinia transmission. Smallpox vaccination should be avoided or delayed in persons treated with immunosuppressive agents, such as solid organ transplantation recipients as well as certain hematopoietic stem cell transplantation recipients (those less than 24 months posttransplantation or
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those more than 24 months posttransplantion but with graft-versus-host-disease or disease relapse). The vaccine should not be administered to persons treated with systemic corticosteroids at immunosuppressive doses (2 mg/kg body weight or greater doses, or 20 mg/day or more of prednisone for 2 or more weeks) within 1 month of completing the therapy. Those treated with other immunosuppressive drugs (eg, alkylating agents, antimetabolites) within the previous 3 months should not be vaccinated [14]. Vaccination should be delayed until 72 hours after discontinuation of topical therapies which might disrupt immune defenses, such as topical immunomodulators (tacrolimus, pimecrolimus) and topical corticosteroids of any strength [46]. Persons with severe autoimmune diseases such as systemic lupus erythematosis may have significant suppression of their immune system from the disease. Although currently there is no data indicating that they are at risk from live-virus vaccines (in the absence of any immunosuppressive therapy), current recommendations are to avoid vaccinating these individuals [14]. Pregnancy or intended pregnancy within 4 weeks of vaccination are contraindications to vaccination, given the risk of fetal vaccinia. The vaccine also should not be given to women who are breastfeeding, as it is unknown whether vaccine virus is excreted in breast milk and the close physical contact during breastfeeding increases the risk of accidental transfer of virus to the infant [48]. The vaccine should not be given to infants less than 1 year of age, given that serious outcomes of any type were more frequent in this age group [49]. Before the eradication of smallpox, vaccination was routinely given during childhood. However, because of the increased risk for adverse effects in this age group, smallpox vaccination is no longer recommended for individuals less than age 18 in the nonemergent setting. The smallpox vaccine should not be given to those who have a serious allergy to any component of the vaccine or its diluent. According to the package insert, Dryvax vaccine contains trace amounts of polymyxin B, streptomycin, tetracycline, and neomycin, and the diluent contains glycerin and phenol [14]. Because the association between the smallpox vaccine and cardiac ischemic events is not yet clear, beginning March 28, 2003, the ACIP has added precautionary recommendations. Persons with known cardiac disease or ischemic cardiovascular disease should not receive the smallpox vaccine, regardless of presence or absence of symptoms. Such conditions include coronary artery disease, congestive heart failure, cardiomyopathy, stroke, or transient ischemic attack [50,51]. Persons with three or more of the
following cardiac risk factors should also not be vaccinated: hypertension, hypercholesterolemia, diabetes mellitus, smoking, or a first-degree relative with heart disease before the age of 50 [52]. In the event of a smallpox outbreak, the CDC recommends vaccination of anyone directly exposed to the variola virus, because the disease poses greater risk than the vaccine [14].
The US population in 2003 and adverse events to date Compared with the 1960s, the US now has a higher proportion of persons at increased risk of adverse reactions from smallpox vaccination. Atopic dermatitis has been increasing in prevalence over the last 30 years; currently, 6% to 22% of the population are estimated to be affected [49]. There are also more persons who are immunocompromised as a result of cancer, autoimmune conditions, radiation and immunosuppressive therapies, chemotherapy, and acquired immunodeficiencies, most notably HIV/AIDS [30]. It is estimated that about 25% of the population should not receive the smallpox vaccine based on these factors [53]. Reports from the recent vaccination program indicate that 102 of the first 276 military personnel screened (37%) were exempt from vaccination based on contraindications given before the start of the program in December 2002. About 50% of these persons were exempt because of a contraindication in a household contact [54]. Given the frequency of heart disease (particularly coronary artery disease) in the population, the additional cardiac contraindications introduced April 2003 will make an even larger proportion of the population ineligible for vaccination in the predisease outbreak setting. Between January 24 to August 8, 2003, 38,257 civilians have received the smallpox vaccine [55]. Adverse events reported to the Vaccine Adverse Event Reporting System are listed in Table 2. No cases of eczema vaccinatum, erythema multiforme major, or progressive vaccinia have been reported. Although over 100 women have been inadvertently vaccinated during pregnancy or became pregnant within 4 weeks of vaccination, no cases of fetal vaccinia have been reported [56]. One suspected case of postvaccinial encephalomyelitis has been reported. No cases of vaccine transmission from civilian vaccinees to their contacts have been reported. Sixteen cases of transmission from military personnel to civilian contacts have been reported. There have been no new cardiac ischemic events reported since the
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Table 2 Adverse reactions in civilians (n = 38,257) in the United States Smallpox Vaccination Program (January 24, 2003 to August 8, 2003) Adverse event Stevens-Johnson syndrome Accidental vaccinia (nonocular) Ocular vaccinia Generalized vaccinia Eczema vaccinatum Progressive vaccinia Postvaccinial central nervous system disease Fetal vaccinia Myo/pericarditis Cardiac ischemic events Pyogenic infection of vaccination site Other serious eventsa Nonserious eventsb Number of cases 0 21 3 3 0 0 1 0 22 9 0 77 653
Not all of these reported cases have been confirmed. a These include events resulting in hospitalization, permanent disability, life-threatening illness, or death, and are temporally but not necessarily causally associated with vaccination. b These include expected self-limited vaccine reactions (eg, local reaction, fever, rash, headache, pain, fatigue) and nonserious adverse events temporally but not necessarily causally associated with vaccination. Data from National Immunization Program, CDC. MMWR Morbidity and Mortality Weekly Report. 2003 Aug 29; 52(34):819 20.
ACIP recommended not vaccinating individuals with cardiac disease or cardiac risk factors in the current stage of the vaccination program (data as of August 8, 2003) [39,55].
Smallpox vaccines in development Current supplies of Dryvax are limited, and even if diluted, would be unable to meet the needs of the unvaccinated US population [57]. The development of new live smallpox vaccines has focused on two strategies: (1) the modernized production of the proven Dryvax vaccine, and (2) the development of attenuated, replication deficient strains of vaccinia [10]. Weltzin et al [58] recently published findings relating to ACAM1000, a clone of NYCBOH vaccinia virus derived from pooled vials of Dryvax and grown on a human diploid cell line. This clone was the most promising due to its comparability to Dryvax in mice, monkeys, and rabbits with regard to immunogenicity and virulence. In a small clinical trial, these investigators showed that ACAM1000 was able to produce
a take reaction in all those vaccinated. Immune responses were similar to that of Dryvax, although humoral responses were somewhat less and cellmediated immune responses slightly more. Dryvax and ACAM1000 showed similar safety profiles, with all study volunteers experiencing at least one mild to moderate adverse event following vaccination. No cardiac adverse events occurred during this study. Although the development of ACAM1000 and similar noncalf lymph-derived production strategies for increasing the supplies of smallpox vaccine may address some of the immediate needs of the country, these vaccines are not necessarily safer than the older Dryvax vaccine. Because ACAM1000 was chosen for its similarity in immunogenicity to Dryvax, adverse effects are likely to be similar, with perhaps decreased risk of CNS disease. Preclinical studies with ACAM1000 demonstrated a low rate of lethality when given by intracerebral inoculation to suckling mice and low rates of meningitis and nonlethal brain swelling when given intrathalamically to nonhuman primates [58]. Larger clinical trials are planned to further evaluate the safety of cell culture-derived NYCBOH strains of vaccinia. The second approach to smallpox vaccine development has focused on attenuated strains of vaccinia. The modified vaccinia Ankara (MVA) vaccine is an attenuated strain of vaccinia virus produced by passage in chick embryo fibroblasts 574 times. Because of a reduced capacity to replicate in mammalian cells, it is expected to be safer than Dryvax [59]. Specifically, it is hoped that MVA will be safe for use in special population groups normally not eligible for smallpox vaccination, such as persons with AD, immunocompromise, or other risk factors for vaccine-related adverse events. In the 1970s, MVA was given to over 150,000 people in Germany who were at high risk of side effects from smallpox vaccination. It was used to trigger a baseline immunity to prepare the body for the standard smallpox vaccine several months later. MVA did help people tolerate the standard vaccine, but efficacy against actual disease was never proven in vaccinees [60]. A two-dose smallpox vaccination strategy consisting of MVA followed by Dryvax may therefore allow vaccination of individuals who have contraindications to Dryvax. Unfortunately, the need for two doses separated in time may make it less useful in the setting of an actual outbreak. Recent studies in mice have found MVA equivalent to Dryvax in providing protection against pathogenic vaccinia virus [61]. Phase 1 clinical studies are currently underway [10]. A singular disadvantage of the MVA vaccine is the lack of a visible take reaction. Because MVA is given intramuscularly, it
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does not produce a lesion at the site of administration, which has thus far been the method of determining the take, or protective efficacy, of the smallpox vaccine. Of concern would be the difficulty in determining whether protection has been established [60]. Another live attenuated smallpox vaccine is LC16m8, derived from the Lister (Elstree) strain of vaccinia via repeated passage in rabbit kidney cells. It was initially developed based on its ability to produce smaller pock sizes on chorioallantoic membranes and for its markedly decreased neurovirulence in rodent and primate models compared with the parent Lister strain [62,63]. LC16m8 has been in use in Japan for over 30 years, and was licensed in that country in 1980. Prelicensing clinical trials in the 1960s and 1970s revealed this vaccine to have less local and systemic reactogenicity than conventional smallpox vaccines then in use (Ikeda, Lister, an so on), without compromising humoral or protective immunity (as inferred from Lister vaccinia challenge). It had an excellent safety profile in more than 50,000 children to whom it was administered in Japan [64]. Successful smallpox eradication efforts near the time of this vaccines original development precluded its use in a smallpox-endemic region of the world. LC16m8 is currently being developed for possible licensure in the US as a potentially safer alternative smallpox vaccine; phase 1 and 2 trials are underway [10].
[2]
[3] [4]
[5]
[6]
[7] [8] [9] [10]
[11] [12]
Summary The smallpox vaccine is an effective method of protection against a disease that kills nearly a third of its victims and leaves the rest scarred or blind. It is, however, not without its own complications. The increased prevalence of atopic dermatitis and immunocompromise today makes a greater proportion of the US population at risk for vaccine-associated adverse events. In the preexposure setting, careful screening of potential vaccinees is critical. Unfortunately, no screening program, no matter how well designed, can completely eliminate the risk of adverse reactions with the currently available vaccine. The risk-to-benefit ratio of any vaccine program must be considered before implementation, and in the absence of a known exposure risk, widescale vaccination must await the development and testing of safer versions of the vaccine.
[13]
[14]
[15] [16] [17]
[18] [19]
References
[20] [1] World Health Organization. Global Commission for the Certification of Smallpox Eradication. The global
eradication of smallpox: final report of the Global Commission for the Certification of Smallpox Eradication, Geneva, December 1979. Albany (NY): World Health Organization; 1980. Brock TD. Microorganisms: from smallpox to lyme disease: readings from Scientific American magazine. New York: Freeman; 1990. Fenner F. Smallpox and its eradication. Geneva: World Health Organization; 1988. Roddis LH. Edward Jenner and the discovery of smallpox vaccination. Menasha: George Banta Publishing Company; 1930. Bazin H, Jenner E. The eradication of smallpox: Edward Jenner and the first and only eradication of a human infectious disease. San Diego (CA): Academic Press; 2000. Bray M. Pathogenesis and potential antiviral therapy of complications of smallpox vaccination. Antiviral Res 2003;58:101 14. Smallpox vaccine. Med Lett Drugs Ther 2003;45:1 4. Bicknell WJ. The case for voluntary smallpox vaccination. N Engl J Med 2002;346:1323 5. Cohen J. Bioterrorism. Smallpox vaccinations: how much protection remains? Science 2001;294:985. Bartlett J, Borio L, Radonovich L, Mair JS, OToole T, Mair M, et al. Smallpox vaccination in 2003: key information for clinicians. Clin Infect Dis 2003;36: 883 902. Couzin J. Smallpox immunization. Panel urges caution over heart problems. Science 2003;300:2013 5. CDC. Vaccinia (smallpox) vaccine: recommendations of the Advisory Committee on Immunization Practices (ACIP), 2001. MMWR Recomm Rep 2001;50:1 25. Arita I. Duration of immunity after smallpox vaccination: a study on vaccination policy against smallpox bioterrorism in Japan. Jpn J Infect Dis 2002;55:112 6. Wharton M, Strikas RA, Harpaz R, Rotz LD, Schwartz B, Casey CG, et al. Recommendations for using smallpox vaccine in a pre-event vaccination program. Supplemental recommendations of the Advisory Committee on Immunization Practices (ACIP) and the Healthcare Infection Control Practices Advisory Committee (HICPAC). MMWR Recomm Rep 2003;52:1 16. Plotkin SA, Orenstein WA. Vaccines. 3rd edition. Philadelphia: W.B. Saunders Co.; 1999. Smithwick EM, Steiner M, Quick JD. Vaccinia virus and tuberculin reactivity. Pediatrics 1972;50:660 1. Goldstein JA, Neff JM, Lane JM, Koplan JP. Smallpox vaccination reactions, prophylaxis, and therapy of complications. Pediatrics 1975;55:342 7. World Health Organization. Expert Committee on Smallpox. First report. Geneva: WHO; 1964. Frey SE, Couch RB, Tacket CO, Treanor JJ, Wolff M, Newman FK, et al. Clinical responses to undiluted and diluted smallpox vaccine. N Engl J Med 2002; 346:1265 74. Cono J, Casey CG, Bell DM. Smallpox vaccination and adverse reactions. Guidance for clinicians. MMWR Recomm Rep 2003;52:1 28.
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W.L. Tom et al / Dermatol Clin 22 (2004) 275289 cinationUnited States, 2003. MMWR Morb Mortal Wkly Rep 2003;52:248 50. Finlay-Jones LR. Fatal myocarditis after vaccination against smallpox: report of a case. N Engl J Med 1964;270:41 2. Saurina G, Shirazi S, Lane JM, Daniel B, DiEugenia L. Myocarditis after smallpox vaccination: a case report. Clin Infect Dis 2003;37:145 6. CDC. Update. Cardiac and other adverse events following civilian smallpox vaccinationUnited States, 2003. MMWR Morb Mortal Wkly Rep 2003;52: 639 42. American Academy of Dermatology. AAD supplemental statement regarding contraindications to smallpox vaccination. 2003. Available at http:// www.aad.org/BioInfo/ Presidents%20Message_ statement2_10_03. pdf. Accessed 30 June 2003. Naleway AL, Belongia EA, Greenlee RT, Kieke BA, Chen RT, Shay DK. Eczematous skin disease and recall of past diagnoses: implications for smallpox vaccination. Ann Intern Med 2003;139:1 7. Centers for Disease Control and Prevention. Smallpox vaccination information for women who are pregnant or breastfeeding, 2003. Available at http://www.bt.cdc. gov/agent/smallpox/vaccination/preg-factsheet.asp. Accessed 30 June 2003. Engler RJ, Kenner J, Leung DY. Smallpox vaccination: risk considerations for patients with atopic dermatitis. J Allergy Clin Immunol 2002;110:357 65. Centers for Disease Control and Prevention. Fact sheet: smallpox vaccine and heart problems, 2003. Available at http://www.bt.cdc.gov/agent/smallpox/vaccination/ heartproblems.asp. Accessed 30 June 2003. CDC. Update. Adverse events following smallpox vaccinationUnited States, 2003. MMWR Morb Mortal Wkly Rep 2003;52:278 82. CDC. Notice to Readers. Supplemental recommendations on adverse events following smallpox vaccine in the pre-event vaccination program: recommendations of the Advisory Committee on Immunization Practices. MMWR Morb Mortal Wkly Rep 2003;52:282 4. Kemper AR, Davis MM, Freed GL. Expected adverse events in a mass smallpox vaccination campaign. Effective Clin Pract 2002;5:84 9. Available at http:// www.acponline.org/journals/acp/marapr02/kemper. htm. Accessed 30 June 2003. Press A. First vaccinations show 1 in 3 soldiers exempt. Newsday 2002;December 21:A10. CDC. Update. Adverse events following civilian smallpox vaccinationUnited States, 2003. MMWR Morb Mortal Wkly Rep 2003;52:819 20. CDC. Women with smallpox vaccine exposure during pregnancy reported to the National Smallpox Vaccine in Pregnancy RegistryUnited States, 2003. MMWR Morb Mortal Wkly Rep 2003;52:386 8. LeDuc JW, Becher J. Current status of smallpox vaccine. Emerg Infect Dis 1999;5:593 4. Weltzin R, Liu J, Pugachev KV, Myers GA, Coughlin
[21] Lane JM, Ruben FL, Neff JM, Millar JD. Complications of smallpox vaccination, 1968: results of ten statewide surveys. J Infect Dis 1970;122:303 9. [22] Lane JM, Ruben FL, Neff JM, Millar JD. Complications of smallpox vaccination, 1968. N Engl J Med 1969;281:1201 8. [23] Habif TP. Clinical dermatology: a color guide to diagnosis and therapy. 3rd edition. St. Louis: Mosby; 1996. [24] Neff JM, Levine RH, Lane JM, Ager EA, Moore H, Rosenstein BJ, et al. Complications of smallpox vaccination United States 1963. II. Results obtained by four statewide surveys. Pediatrics 1967;39:916 23. [25] Humphrey D. Localized accidental vaccinia of the vulva: report of 3 cases and a review of the world literature. Am J Obstet Gynecol 1963;86:460. [26] Ruben FL, Lane JM. Ocular vaccinia. An epidemiologic analysis of 348 cases. Arch Ophthalmol 1970;84: 45 8. [27] Semba RD. The ocular complications of smallpox and smallpox immunization. Arch Ophthalmol 2003;121: 715 9. [28] Vastag B. Experts weigh prevention, therapy for ocular vaccinia in smallpox vaccinees. JAMA 2003;289: 2198 9. [29] Carney JF, Caroline NL, Nankervis GA, Pomeranz JR. Eczema vaccinatum and eczema herpeticum in Darier disease. Arch Dermatol 1973;107:613 4. [30] Neff JM, Lane JM, Fulginiti VA, Henderson DA. Contact vacciniatransmission of vaccinia from smallpox vaccination. JAMA 2002;288:1901 5. [31] Kempe CH. Studies on smallpox and complications of smallpox vaccination. Pediatrics 1960;26:176 89. [32] Breman JG, Henderson DA. Diagnosis and management of smallpox. N Engl J Med 2002;346:1300 8. [33] Bray M, Wright ME. Progressive vaccinia. Clin Infect Dis 2003;36:766 74. [34] Cleri DJ, Villota FJ, Porwancher RB. Smallpox, bioterrorism, and the neurologist. Arch Neurol 2003;60: 489 94. [35] DeVries E. Postvaccinial perivenous encephalitis. Amsterdam: Elsevier; 1959. [36] Levine MM. Live-virus vaccines in pregnancy. Risks and recommendations. Lancet 1974;2:34 8. [37] Suarez VR, Hankins GD. Smallpox and pregnancy: from eradicated disease to bioterrorist threat. Obstet Gynecol 2002;100:87 93. [38] Sepkowitz KA. How contagious is vaccinia? N Engl J Med 2003;348:439 46. [39] CDC. Update. Cardiac-related events during the civilian smallpox vaccination programUnited States, 2003. MMWR Morb Mortal Wkly Rep 2003;52:492 6. [40] Halsell JS, Riddle JR, Atwood JE, Gardner P, Shope R, Poland GA, et al. Myopericarditis following smallpox vaccination among vaccinia-naive US military personnel. JAMA 2003;289:3283 9. [41] Feery BJ. Adverse reactions after smallpox vaccination. Med J Aust 1977;2:180 3. [42] CDC. Cardiac adverse events following smallpox vac-
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[62] Hashizume S. The bases of a new low-virulent vaccinia virus strain, LC16m8. Rinsho To Uirusu 1975;3: 229 35. [63] Morita M, Aoyama Y, Arita M, Amona H, Yoshizawa H, Hashizume S, et al. Comparative studies of several vaccinia virus strains by intrathalamic inoculation into cynomolgus monkeys. Arch Virol 1977;53:197 208. [64] Yamaguchi M, Kimura M, Hirayama M. Vaccination research group research report: Ministry of Health and Welfare special research: post-vaccination side effects and research regarding treatment of complications. Rinsho To Uirusu 1975;3:269 79.
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Other viral bioweapons: Ebola and Marburg hemorrhagic fever

Michelle R. Salvaggio, MDa, John W. Baddley, MDa,b,*
a
Division of Infectious Diseases, Department of Medicine, University of Alabama at Birmingham, 1900 University Boulevard, 229 Tinsley Harrison Tower, Birmingham, AL 35294, USA b Infectious Diseases, Birmingham Veterans Affairs Medical Center, 700 19th Street South, Birmingham, AL 35233, USA
Discussion in the private sector and the public health forum has increasingly focused on bioterrorism and preparedness for future acts of bioterrorism. Viral organisms identified as potential bioweapons include Variola major (smallpox) and some agents of viral hemorrhagic fevers: Lassa, Junin, CrimeanCongo hemorrhagic fever, Rift Valley fever, yellow fever, Ebola, and Marburg [1]. Viruses that cause hemorrhagic fevers (HFs) represent a particularly serious threat to the public as well as the medical field due to high morbidity and mortality and lack of familiarity with the hemorrhagic fever syndrome. The Centers for Disease Control Strategic Planning Workgroup has recently classified many hemorrhagic fever viruses (HFVs) in Category A, which include those pathogens for which preparedness is deemed to be of the highest priority [2]. The organisms in Category A are easily disseminated or transmitted person to person, could result in high mortality and thus greatly impact public heath, have potential to create public panic and lead to disruption of daily lives, and require special action for public health preparedness. HFVs have also been identified by the Working Group on
* Corresponding author. Division of Infectious Diseases, Department of Medicine, University of Alabama at Birmingham, 1900 University Boulevard, 229 Tinsley Harrison Tower, Birmingham, AL 35294. E-mail address: jbaddley@uab.edu (J.W. Baddley).
Civilian Biodefense as having further key characteristics that make them serious risks if used as bioweapons [3]. These viruses also (1) have low infectious doses and can be disseminated via aerosol; (2) have no effective vaccine currently available; (3) are available for procurement; (4) can be produced in large quantities; (5) are relatively stable in the environment; and (6) have been previously developed for use as a biological weapon [3,4]. The term viral hemorrhagic fever refers to a clinical syndrome characterized by acute onset of fever accompanied by nonspecific findings of malaise, prostration, diarrhea, and headache. Illness can progress to septic shock, often culminating in bleeding diatheses. More than 25 different viruses from four different families (Filoviridae, Flaviviridae, Bunyaviridae, and Arenaviridae) are known to cause viral hemorrhagic fevers [3,5] (Table 1). The majority of HFs are zoonotic diseases; humans contract disease from animals that serve as reservoirs or vectors. Distribution of HFVs is limited geographically by the natural habitat of the reservoir or vector. The clinical syndrome of hemorrhagic fever has been identified throughout the world. Interviews with researchers involved with the Soviet biologic weapons program indicate that HFVs had been extensively researched and weaponized before the dissolution of the former Soviet Union. The Soviet Union had produced Marburg, Ebola, Lassa, Junin, and Machupo viruses in large quantities for use in its biologic weapons program [6]. Animal studies performed with aerosolized Marburg and Ebola viruses established infection in nonhuman
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M.R. Salvaggio, J.W. Baddley / Dermatol Clin 22 (2004) 291302
Table 1 Selected characteristics of hemorrhagic fever viruses Family Arenaviridae Genus Arenavirus Virus Disease Lassa fever Bolivian hemorrhagic fever Argentine hemorrhagic fever Venezuelan hemorrhagic fever Brazilian hemorrhagic Fever Rift Valley Fever Crimean-Congo hemorrhagic fever Hemorrhagic fever with renal syndrome Ebola hemorrhagic fever Marburg hemorrhagic Fever Yellow fever Dengue fever Reservoir/vector Rodent Rodent Rodent Rodent Rodent Livestock/mosquito Vertebrate/tick Rodent Unknown Unknown Monkey/mosquito Human/mosquito Vertebrate/tick Rodent/tick Geographic distribution Africa South America South America South America South America Africa Europe, Asia, Africa Asia, Europe, Americas Africa Africa Africa, South America Asia, Africa, Americas India Central Asia
Lassa Machupo Junin Guanarito Sabia Bunyaviridae Phlebovirus Rift Valley fever Nairovirus Crimean-Congo hemorrhagic fever Hantavirus Hantaan and related virusesa Filoviridae Filovirus Ebola Marburg Flaviviridae Flavivirus Yellow fever Dengue
Kyasanur Forest disease Kyasanur Forest disease Omsk hemorrhagic fever Omsk hemorrhagic fever
a
Includes Hantaan as well as Seoul, Puumala, and Dobrova plus others.
primates with very low numbers of virions [3,4,6]. Our discussion will focus on hemorrhagic fevers caused by the Filoviruses, Ebola, and Marburg,
Historic background Over the past 35 years the discovery of the filoviruses and four distinct subtypes of Ebola have marked significant milestones within the field of investigation of emerging infectious diseases. A brief description of the outbreaks of Filoviruses is instructive in epidemiology and viral pathogenesis. Marburg, the first filovirus to be described, was identified after a cluster of hemorrhagic fever cases occurred in laboratory workers in Marburg, Germany, in 1967. All of the infected workers handled blood and tissue or cell cultures from African green monkeys originating from Uganda. A total of 32 cases, 26 primary and 6 secondary (contacts of primary patients) were documented, with overall mortality 23% [7,8]. Despite an intense epidemiologic investigation the origin of the virus was not discovered. The outbreak was controlled by quarantine of the involved animal facilities, and led to the initiation of quarantine rules for all imported animals intended for scientific research [9]. Two outbreaks of hemorrhagic fever occurring simultaneously in Democratic Republic of the Congo (DRC, formerly Zaire) and Sudan in 1976 brought filoviruses back to the attention of the global bio-
medical research community. The outbreaks, initially thought to represent Marburg infection, were caused by two different species of Ebola, named Ebola Zaire (EBO-Z) and Ebola Sudan (EBO-S) [10 14]. Each outbreak was associated with high mortality, especially among health care providers. Secondary transmission was propagated by the reuse of needles and syringes. The outbreak in the DRC involved 318 cases with 88% mortality; in Sudan, mortality was 53% among 284 total cases [10 14]. In 1989, Ebola was introduced to the United States. Cynomolgus monkeys imported from the Philippines began to exhibit signs of hemorrhagic fever while in mandatory quarantine in Reston, Virginia. Other infected cynomolgus monkeys originating from the Philippines were also found in Pennsylvania and Texas [15,16]. The filovirus found in the monkeys was determined to be a novel strain of Ebola, later named Reston (EBO-R) [17]. Over 500 humans who had varying amounts of contact with the animals were monitored for symptoms. No humans experienced symptomatic illness, yet serologic screening indicates four persons had asymptomatic seroconversion [16]. The most recent strain of Ebola to be identified was discovered in 1994. A researcher investigating several deaths among a chimpanzee troop in Ta Na te dIvoire became ill after pertional Park in Co forming necropsy on a chimpanzee. The procedure took place within 12 hours of the animals death. Virus te isolated from the researcher was named Ebola Co dIvoire (EBO-IC) [14,18]. The researcher recovered
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with supportive therapy. No other workers involved in the necropsy became ill, and no documented seroconversions were found among 74 contacts [18,19].
Virology The filoviruses are enveloped, negative sense RNA viruses. The family Filoviridae is divided into two genera, Ebola-like and Marburg-like vi te dIvoire are ruses. Reston, Sudan, Zaire, and Co the four species types included in the Ebola-like viruses. Marburg is the only Marburg-like virus yet to be identified [9,20]. The Filoviridae (phyllo- thread in Latin) are named for the unique thread-like morphology exhibited by the viruses within tissue culture as well as pathologic specimens (Fig. 1). Filament length varies among viruses, with Marburg virions being shorter on average than Ebola (800 nm and 1000 nm, respectively); the diameter of filoviruses approaches 80 nm [20,21]. Virions have been demonstrated in linear, circular, or U-shaped forms [7,10,21]. The genomes of Marburg and Ebola are approximately 19 kilobases in length. Sequencing analysis reveals 55% homology between Ebola and Marburg at the nucleotide level [22,23]. Homology between strains and subtypes within each species is high, especially for Marburg isolates [22 24]. Each negative sense RNA genome consists of seven genes. Ebola has three overlap areas (sequences that are part of two different transcriptional products) and Marburg has a single overlap area. Areas involved in overlap are highly conserved. Gene products between
the two species are similar and appear to have the similar functions [14,22,24]. The genomes of Marburg and Ebola viruses encode nine unique gene products [9]. Although much has been learned about the proteins of Ebola and Marburg, work continues to further our understanding of protein functioning. Discussion here is limited to a brief description of protein function. Two indepth reviews are recommended for more extensive information of filovirus protein structure and function [9,20]. Seven genes encode structural glycoproteins, four of which are associated with the nucleocapsid: NP, VP30, VP35, and L protein [25]. Both nucleoprotein (NP) and virion structural protein 30 (VP30) are believed to be involved in encapsidation of newly formed virions [9,20]. Virion protein 35 (VP35) and large protein (L) are involved in transcription. L is an RNA-dependent RNA polymerase and VP35 acts as a cofactor with L [20]. There is some evidence to support an immunomodulatory role in vivo for VP35 [26]. In an influenza virus mutant construct, VP35 antagonized interferon expression at the promoter level [26]. The remaining three structural proteins, glycoprotein (GP), VP24, and VP40, are associated with the membrane. GP, the only surface-bound protein found in filoviruses, is present in knob-like projections on the outer membrane [24]. GP possibly functions as a binding site for cellular receptors, and it may be involved with antigenicity [27 29]. Recent studies have implicated GP in vascular cell toxicity, which may be related to the bleeding diatheses seen in Ebola hemorrhagic fever [29,30]. Virion protein 40 (VP40)
Fig. 1. Electron micrograph of a thin section containing Ebola viral particles. (From Public Health Image Library. Content credit: Fred Murphy. Available at: http://phil.cdc.gov/phil/detail.asp?id=2738.)
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is the major component of the virion matrix. The function of virion protein 24 (VP24) is unknown at this time [9,20]. The two additional proteins, secreted glycoprotein (sGP) and delta protein, are secreted products [9,20,31]. Present only in Ebola-like viruses, sGP may function as an immunomodulator through neutrophil activation [29]. Delta protein has only recently been described. and its function has not been elucidated [9].
Epidemiology Transmission Transmission of filoviruses occurs through contact with infected persons or nonhuman primates. Persons at highest risk for infection include family members who have direct exposure to the sick family member, particularly during the latter stages of disease. This exposure is beyond casual contact, and usually consists of contact with bodily fluids or assisting with bathing [11,12,32,33]. Participants in the ritual cleansing of body in preparation for burial were at higher risk for Ebola disease than those who did not attend the funeral or participate in the ceremony [11,12, 32,33]. The finding of Ebola viral particles in skin of infected persons, especially in the sweat glands, is consistent with this mode of transmission [34]. Percutaneous inoculation is also an important mode of transmission. The earliest outbreaks of EBO-Z were propagated through the reuse of unsterilized needles and syringes in those seeking care at an outpatient facility [11,12]. Mortality was 100% for those inoculated by contaminated needles and syringes [11]. Both EBO-R and Marburg have been introduced to humans through needlestick injuries [16,35]. Although sexual transmission of Marburg has been demonstrated in at least one patient during the original Marburg outbreak of 1967 [8], there have been no clearly documented cases of Ebola transmission via sexual intercourse. During the EBO-Z outbreak in 1976, researchers were unable to differentiate those persons whose contact was of a sexual nature from those whose contact consisted of nursing their ill spouse [11]. In Kikwit, in 1995, convalescents and their household contacts were eligible for prospective evaluation of risk of potential sexual transmission [36]. Researchers were able to document that an asymptomatic woman who tested weakly positive for EBO IgM had unprotected sexual intercourse with her convalescing partner. Seminal fluid from the male
partner was positive for EBO RNA by reverse transcriptase polymerase chain reaction (RT-PCR). The initial serology was performed on the woman 52 days after exposure to her partner began. Subsequent testing on the female did not confirm seroconversion with expression of EBO IgG; thus, the elevated IgM may have been a false positive reaction [36]. Sexual transmission of Ebola is theoretically possible and most experts advocate use of condoms during sexual activity for at least 3 months after recovery [3,9]. Viable Ebola virus has been cultured from semen up to 82 days after illness onset [35,37]. Moreover, Ebola RNA has been detected by RT-PCR from vaginal secretions and semen 33 and 91 days after initiation of clinical syndrome, respectively [36,37]. Breast-feeding may represent another mode of transmission of filoviruses. Three infants breast fed by their ill mothers died of EBO-S in the Uganda outbreak in 2001 [33]. During a Marburg outbreak in the DRC in 1999, a mother and her breast-fed 8-month-old infant developed symptoms of hemorrhagic fever. Both mother and infant were found to be positive for Marburg by RT-PCR, and the mother was culture positive for Marburg from her blood. Both recovered fully, and 2 years later were found to be positive for Marburg IgG [38]. Route of transmission is unclear in this case. The mother had provided care to an infected relative, but the infant had not been in contact with the infected relative. Breast milk was not tested for virus, and direct transmission by other means cannot be excluded [38]. Filoviruses retain infectivity while at room temperature on environmental surfaces; thus, fomites may be sources of transmission [9]. In the Ebola outbreaks of 1976 and 2000, persons who shared a sleeping mat with an infected patient had a higher risk of becoming infected [11,12,33]. During the Ugandan outbreak of EBO-S, a blanket from a patient who died of Ebola may have been the source of infection for the patients brother [33]. The ability to disseminate disease through aerosol or airborne small-droplet nuclei would render filoviruses a potential biologic warfare threat. As previously mentioned, both Marburg and Ebola have been weaponized in this manner [3,4,6]. Aerosol transmission of Ebola has been demonstrated in experimental models involving nonhuman primates [3,9,39]. During an experiment to evaluate therapeutic benefit of interferon following infection with EBO-Z, two of three control rhesus monkeys (Macaca mulatto) became ill [40]. Control animals were caged 3 m from infected animals. Researchers were unable to document direct or percutaneous contact through injec-
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tions, and speculated inoculation to the control animals occurred via pulmonary, nasopharyngreal, oral, or conjunctival routes [40]. Definitive evidence that small-droplet nuclei pose a substantial transmission risk among humans during naturally occurring outbreaks is lacking [3,9,39]. The introduction of barrier precautions including gloves, gowns, and masks is credited with halting the Ebola outbreaks of 1976 and 1995 [11,12,41,42]. Early establishment of standard infection control precautions [43], with or without aerosol protection, even without definitive diagnosis of filovirus infection, has been sufficient in protecting health care worker from infection in a tertiary care setting [44]. Early institution of these precautions in a suspected outbreak of viral hemorrhagic fever is prudent.
and ecologic distribution of potential reservoirs [53]. Regions with the highest predicted Ebola disease density had high levels of precipitation and moderate-to-high temperatures. Predicted geographic distribution corroborated findings of phylogenetic analysis. For example, EBO-Z and EBO-IC occurred in areas that had similar conditions; but EBO-S, the most genetically disparate strain of Ebola, was predicted to occur under distinct ecologic conditions from other EBO strains. The ecologic conditions in African areas with high concentrations of EBO were similar to areas of the Phillipines [53]. Use of this technology may be useful to search for the reservoir(s) of Ebola and Marburg.
Clinical features Ecology Reservoir The reservoir(s) of the filoviruses has yet to be identified. The sporadic nature of outbreaks frequently takes local health care communities by surprise; therefore, extensive epidemiologic investigations are initiated retrospectively [3,9,45 47]. Despite transmission to humans of Ebola and Marburg from infected tissues of nonhuman primates it is unlikely that nonhuman primates are the reservoir because of high mortality among these animals [7,11, 12,16,19]. Experimental inoculation of Ebola in various plants and animals collected from Zaire after the 1995 outbreak resulted in viral replication in bats without apparent illness [48]. The possibility of an airborne vector that sheds virus in stool could explain the sporadic nature of the outbreaks; however, there have been no reports of isolation of Ebola or Marburg in bats collected in the wild to support this theory. Many other viruses that cause the syndrome of hemorrhagic fever use rodents as a reservoir or vector (Table 1). Decreasing human interaction with such rodents is a vital part of outbreak control for Hantavirus, Lassa, and Junin viruses [5,49]. No rodent vector or reservoir has been established for filoviruses. The location of outbreaks of Ebola and Marburg in Africa is indicative of a geographic distribution within the 10th North and South parallels centered on the Equator [20]. On the basis of phylogenetic analysis and a clear difference in pathogenic predilection, it has been suggested that EBO-R originated in Asia [17,50 52]. Recently, ecologic niche modeling was applied to recorded outbreaks of Ebola and Marburg in an attempt to delineate the geographic Filoviral hemorrhagic fever is characterized by the acute onset of fever accompanied by a multitude of nonspecific signs and symptoms [8,11,12,54,55]. Early complaints are similar to those experienced during other severe viral illnesses and include asthenia (64 95%), nonbloody diarrhea (66 90%), headache (52 100%), nausea or vomiting (59 74%), myalgias/ arthralgias (50 79%), abdominal pain (55 71%), lumbosacral back pain (12 26%), and chest pain (5 83%) [12,45,46,54]. The occurrence of hiccoughs has been associated with filoviral infections and occurs in 5% to 18% of patients [12,45,46,54]. Generally, patients are described as having expressionless faces that appear ghost-like [12]. There have been few documented cases of asymptomatic seroconversion reported with EBO-Z and Marburg [36,38,56]. Dermatologic manifestations of Ebola and Marburg infection are relatively common [3,9]. A maculopapular rash is distinct to filovirus infection, and may be observed within 5 days of illness [3,8,19,35, 55,57 59]. Although rash is more easily recognized in Caucasians, 52% of infected persons during the EBO-S outbreak in Sudan were noted to have a morbilliform rash or to desquamate in the later stages of disease [12]. Skin lesions have been described as occurring initially in patches, particularly on the extremities and trunk, and later coalescing to become confluent [12,35,57,59]. Desquamation is noted in many survivors and may be the first indication that a non-Caucasian has skin involvement [3,12,19,35,55, 57 59]. Complaints of unusual sensations on the skin such as burning and parasthesias are not infrequent; however, these sensations do not appear to correlate with subclinical rash or pending desquamation [54]. A prominent feature of viral hemorrhagic fever is bleeding disorders. Bleeding disorders occur in up to
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71% of patients with filovirus infection and manifest as petechiae, epistaxis, hematemesis, melena, bleeding gums, or bleeding from puncture sites [12,45,46, 54,55]. Patients can progress to fulminant disease with disseminated intravascular coagulation (DIC) [9,52,55]. Among patients from the Ebola outbreak in Kikwit in 1995, there was no significant difference in rate of bleeding complications between survivors and nonsurvivors [54]. Other complications of Ebola and Marburg hemorrhagic fever include increased vascular permeability, systemic inflammatory response syndrome manifested as adult respiratory distress syndrome, hepatitis, pancreatitis, renal impairment and coma, or other organ failure. If illness is to be fatal, most patients will die by the end of the first week. Patients may become normothermic just before death due to shock or massive hemorrhage [12,45,46,54,55]. There have been several reports of spontaneous abortion during acute illness with the filoviruses, and fetal infection and death can occur at all gestational stages [11,12,33,45]. During the Ebola outbreak in Zaire in 1977, all 11 children born to infected women died within 19 days of birth [11]. Evacuation of the fetus or handling of tissue or body fluids from the mother have been linked to transmission of the disease [11,12,33,45]. Long-term sequelae of Ebola infection have been well described [36,54]. Among some patients who survived infection with EBO-Z in Kikwit, arthralgias, uveitis, orchitis, and hearing loss persisted several months after illness [36,54]. Many patients were unable to go back to work or pursue activities of daily living even after an extended period of convalescence. Viral shedding of Ebola in urine and seminal fluid has been documented up to 3 months after infection [35,36]. It is unclear if shedding of Ebola is responsible for prolonged symptoms after resolution of infection. Evaluation of 18 survivors from the Marburg outbreak in 1967 revealed no long-term sequelae, and the majority of patients returned to work within 3 months of illness [8]. A patient who recovered from Marburg infection developed uveitis after 2 months of being asymptomatic, and Marburg was cultured from the anterior chamber of her eye [57]. The incubation period of Ebola and Marburg has been reported to be 3 to 14 days [11,12,19,35,38,44, 54,58], and is important for estimating potential exposure in the event of a biologic warfare act. Because HFs are usually detected retrospectively, it is difficult to pinpoint exact dates of potential exposure and infection. The most reliable data on incubation period is from health care providers who contracted illness from patients with unique travel
histories [44,57,58]. It is not understood if the incubation period is affected by route of transmission, load of inoculation, or species-related virulence.
Pathogenesis Investigation into the pathogenesis of filoviruses offers unique challenges to researchers, in part because human outbreaks are sporadic and geographically disparate. Moreover, selection of animal models is fraught with difficulties, stemming from differential lethality among Ebola subtypes for specific animals [60], and containment issues that require laboratory analysis to be performed in a level 4 biosafety facility [61]. Data on the pathogenicity and virulence of specific viruses or subtypes derived from outbreaks is limited and is described below. The Ebola subtypes have varying degrees of virulence among human and nonhuman primates. EBO-R, the agent of the first Ebola outbreak in the United States, appears to be highly pathogenic to cynomolgus monkeys. All monkeys infected with EBO-R became ill and eventually died or were euthanized [62]. In contrast, one human who was inoculated with EBO-R from a scalpel injury developed viremia without symptoms [55,63]. The strain EBO-IC, discovered in 1994, is lethal to chimpanzees [64], but only one of three humans involved in necropsy of chimpanzees during the outbreak became ill. Serologic screening of 74 contacts to the infected researcher identified no seroconversions [19]. High mortality among humans has been seen with infection from EBO-Z and EBO-S strains, with varied attack rates even within the same virus subtype. Overall mortality in the outbreaks of 1976 was 88% (EBO-Z) and 53% (EBO-S) [11,12]. Mortality associated with Marburg in the initial outbreak in Germany was 23% among 31 cases [7]. In later outbreaks in 1975, 1980, and 1987, involving a total of eight cases, mortality was 50% [8]. The largest outbreak of Marburg, occurring in 1998 in the DRC, had 61 deaths (84% mortality) among 73 cases [38,65]. Marburg outbreaks before 1998 showed evidence of decreased mortality among secondary cases when compared with primary cases [8,57,58]; however, this was not observed in the Marburg outbreak in DRC in 1998, and has not been observed during Ebola outbreaks [12,32,33,66]. A prominent area of research of the pathogenesis of filoviruses has focused on the mechanism by which filoviruses cause disease. The virus itself may exert direct destruction on tissues and cells [9,10,17,67]. Pathology specimens from nonhuman primates, chim-
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panzees, and humans reveal extensive hemorrhage and widespread necrosis in most examined organs, findings consistent among all species studied. Histologic findings of liver tissue are most impressive, with hepatocyte necrosis correlating with dense areas of filovirus antigens and viral inclusions [9,10,17,67]. In vitro and in vivo experiments have demonstrated that Ebola and Marburg preferentially infect endothelial cells, monocytes, and monocyte-derived dendritic cells [29,68 71]. Investigation is focused on the role of endothelial cells and monocytes in the production or propagation bleeding diatheses and sepsis, which is the essence of the viral hemorrhagic fever syndrome. In a nonhuman primate study performed with EBO-Z [70], low blood levels of protein C and elevated D-dimers were found soon after infection, consistent with DIC and sepsis syndrome. Extrinsic tissue factor, the predominate influence on the development of DIC, was upregulated in monocytes and found within tissues associated with splenic macrophages, polymorphonuclear cells, and endothelial cells [70]. Thrombi composed of fibrin and monocytes enmeshed in fibrin were demonstrated by transmission electron tomography and histopathology in vessels of all organs examined postmortem. Monocytes with Ebola viral antigens were frequently part of thrombi [70]. Bleeding disorders among patients with viral hemorrhagic fever may also be related to increased vascular permeability. Through a series of gene transfer experiments with endothelial cells, Ebola GP production was associated with cell death [30]. Further studies with human saphenous vein explants and porcine arteries infected with cells containing GP revealed increased permeability resulting from endothelial cell death and denudement of the basement membrane [30]. In addition, monocytes infected with Marburg produced tumor necrosis factor a (TNF-a) that resulted in increased permeability of endothelial cells [68]. The etiology of bleeding diatheses seen in patients with Ebola and Marburg infection is incompletely understood, but increased vascular permeability and DIC may be partly responsible. The inciting event of sepsis syndrome observed in patients may result from circulating substances such as TNF-a, interferon (IFN)-b, IFN-g, interleukin (IL)-2, and IL-10 expressed by activated monocytes and macrophages [9,67,68]. Once activated, monocytes and macrophages may have differential tropism for organs, possibly resulting in widespread release of more cytokine mediators and leading to the overall clinical picture of systemic inflammatory response [72]. Study of survivors and nonsurvivors from the
Ebola outbreaks in Kikwit [73] and Gabon [74,75] has highlighted the importance of cytokines in response to Ebola infections. For example, patients with fatal disease were noted to have high levels of TNF-a and IL-10 [73]. Patients who died in Kikwit were also noted to have high levels of IFN-a, IFN-g, and IL-2 [73]. As in other infectious processes, the host immune response is an important factor that directly affects the clinical manifestations of HFs. Survivors in Gabon after infection with EBO-Z experienced a rapid rise in IgG specific for viral NP and VP40, followed by clearing of viral antigens. In contrast, nonsurvivors had little Ebola specific-IgG production [74].
Diagnosis The diagnosis of viral hemorrhagic fever poses a difficult and potentially hazardous situation for health care workers. Tissue or fluids collected from infected patients require special handling to reduce the risk of transmission. Specific packaging and shipping guidelines are available and handling of specimens should be performed in a class 2 biologic safety cabinet according to biohazard safety level 3 practices [76,77]. In early outbreaks the diagnosis of Ebola was made by culture of virus in Vero cells; however, this method of identification does not differentiate the various strains of Ebola [7,10,78,79]. Recent work has used RT-PCR and sequencing technology to classify the virus and decrease time to identification [9,13,22,23]. Diagnostic testing that requires phlebotomy represents a potential exposure to health care workers, as evidenced by the high rate of percutaneous injuries and deaths described in Zaire in 1976 [11]. Serologic studies can be performed to assess antibody response or to detect antigen. Enzyme-linked immunosorbent assay (ELISA) for detection of antigen has been used with success in testing large numbers nonhuman primates during outbreaks of EBO-R [80]. Indirect fluorescent antibody testing, which can moderately differentiate between Ebola strains, has been performed in a small number of laboratories that are capable of handling specimens and reagents. A newly developed ELISA that detects IgM and IgG is more sensitive and specific in comparison to indirect fluorescent antibody testing and may be of use in seroprevalence epidemiologic studies [81]. A limitation of serologic testing is that patients in early stages of Ebola infection often die before developing detectable antibodies [74]. Recently, a novel method of immunohistochemical staining of skin samples for Ebola virus has been
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described [34]. Postmortem skin-snip biopsies were taken from the nape of the neck and axilla of known Ebola-infected persons during the Kikwit outbreak in 1995. Specimens were immediately fixed in formalin, and most were further prepared for electron microscopy with fixation in gluteraldehyde, effectively achieving viral inactivation. Polyclonal mouse and rabbit antibodies detected Ebola viral particles within skin biopsies, particularly associated with endothelial cells and concentrated around sweat glands. When immunohistochemical staining of skin biopsies were compared with testing by ELISA directed at antigen, IgM and IgG, and viral culture, all samples had concordant results [34].
administration of carbocyclic 3-deazaadenosine, an S-adenosylhomocysteine hydrolase inhibitor, protected against death when given on the day of infection or 1 day later. When treatment was delayed to day 2 or 3 following infection, survival decreased to 90% and 40%, respectively [84]. This drug has not been tested in humans, and it is unknown if it is safe or effective. Further investigation into the role of immunomodulators and novel antivirals and the treatment of DIC and sepsis syndrome observed during filovirus infection is warranted.
Prevention and control Infection control measures
Treatment The choice of therapy for a patient with potential viral hemorrhagic fever is difficult because presentation is nonspecific and may mimic many other infectious processes. A thorough travel history is important, and may help to narrow the differential diagnosis. Early infection with Ebola or Marburg may be confused with influenza and viral hepatitis. Other diseases in the same geographic distribution of the filoviruses to be considered include malaria, leptospirosis, rickettsial diseases, and dengue. Malaria parasitemia in patients from endemic areas can occur without associated illness; however, as malaria represents a possibly fatal yet treatable entity, antimalarial therapy should be initiated if blood smears are positive for malaria while awaiting further studies [3,9,52]. Currently, no specific therapy is recommended for filovirus infections [3,9,55]. Standard-of-care supportive measures are indicated, particularly as patients develop hemorrhage and shock. Conventional antivirals such as ribavirin and interferon are of no benefit in vitro or in vivo [9,10,82]. Convalescent serum with specific antibodies has been used successfully to treat Junin hemorrhagic fever [9]; however, antibody therapy is not beneficial in filovirus infections. Convalescent serum, nonspecific immunoglobulin, and polyclonal hyperimmune antibodies have been used as treatment in humans in noncontrolled studies and animal models without consistent success [9,11,35,55,57]. Whole blood transfusion, although used successfully among a small number of patients in Kikwit in 1995, has been studied in a noncontrolled manner and pathogenecity-related variables preclude generalizations regarding benefit [83]. Novel drugs that may prove to be beneficial in the treatment of filovirus infections are in early development [84]. In a mouse model of Ebola infection, Filoviruses have been transmitted through direct contact with skin, tissue, or fluids from infected individuals, percutaneous injury, and sexual intercourse, and can potentially be transmitted through aerosols and fomites. Infection control measures for management of infected persons have been published recently [3]. The guidelines support the use of aerosol and contact precautions for patients with Filovirus infection. Patients should be placed in negative pressure rooms, and persons providing direct patient care should protect themselves with the use of goggles, double gloves, and impermeable gowns. Laboratory workers who will come into contact with infectious fluids should also wear protective gear including gloves, face shields, and nonpermeable gowns. Routine laboratory work (complete blood counts and chemistry) should be kept to a minimum to reduce risk to personnel. If available, point-of-care analyzers should be used. However, if this capability is not available, laboratory tests should be performed on instruments dedicated to processing specimen from potential HF patients [3]. Standards for handling and shipping diagnostic specimen have been published [1,76,77]. Handling of dead patients with filovirus infection requires prompt burial or cremation to decrease potential for transmission. Every attempt should be made to decrease the frequency and magnitude of contact with the corpse, including autopsy and embalming [3]. Postexposure prophylaxis Currently, there are no interventions recommended for those who have been exposed to patients with active filovirus infection. The use of hyperimmune equine IgG was protective to guinea pigs when given early in the course of EBO-Z illness [82]. However,
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neither cynomolgous monkeys nor mice were protected from death. Among monkeys pretreated with IFN-a2b before Ebola infection, death was delayed for several days but not prevented [82]. Experimental agents such as carbocyclic 3-deazaadenosine may be of use pending further investigation in humans [84]. Vaccine Use of an effective filovirus vaccine may be of importance in halting an ongoing outbreak. Health care workers brought in from abroad could be vaccinated before deployment. Upon arrival, the international aid team could care for those ill as well as vaccinate locals who are uninfected. Filoviral genes or their products have been evaluated in vaccine models. Vaccines containing the genes of VP35, NP, VP40, and the secreted protein, sGP, have been ineffective [9]. However, a vaccine containing the GP gene protected cynomolgus monkeys from death when challenged with EBO-Z 4 weeks after vaccination [85]. In 2003, the National Institutes of Health began a phase I trial to investigate the safety of a human vaccine containing the GP gene from EBO-Z, but data are not yet available [86].
Summary The filoviruses Ebola and Marburg cause sporadic infection often associated with high morbidity and mortality. Important clinical features include fever and nonspecific symptoms such as malaise, prostration, diarrhea, and headache. Patients frequently develop rash, bleeding diatheses, and may progress to septic shock. No specific treatment exist other than supportive measures. Ebola, Marburg, and other agents of viral hemorrhagic fever are potential agents of biologic warfare because of severity of disease, ease of aerosol transmission, and the ability to manufacture mass quantities. Preparedness, coupled with a high index of suspicion, will be critical in limiting fatalities and calming public fears. Rapid institution of infection control practices will be essential in controlling an outbreak of Ebola or Marburg, whether naturally occurring or as a result of bioterorrism.
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A novel immunohistochemical assay for the detection of Ebola virus in the skin: implications for diagnosis, spread and surveillance of Ebola hemorrhagic fever. J Infect Dis 1999;179(Suppl 1): S36 47. Emond RTD, Evans B, Bowen ETW, Lloyd G. A case of Ebola infection. BMJ 1977;2:541 4. Rowe AK, Bertolli J, Kahn AS, Mukunu R, MuyembeTamfum JJ, Bressler D, et al. Clinical, virologic, and immunologic follow-up of convalescent Ebola hemorrhagic fever patients and their household contacts, Kikwit, Democratic Republic of the Congo. J Infect Dis 1999;179(Suppl 1):S28 35. Rodriguez LL, De Roo A, Guimard Y, Trappier SG, Sanchez A, Bressler D, et al. Persistence and genetic stability of Ebola virus during the outbreak in Kikwit, Democratic Republic of the Congo, 1995. J Iinfect Dis 1999;179(Suppl 1):S170 6. Borchert M, Muyembe-Tamfum JJ, Colebunder R, Libande M, Sabue M, Van der Stuyft P. A cluster of Marburg virus disease involving an infant. Trop Med Int Health 2002;7(10):902 6. Feldmann H, Czub M, Jones S, Dick D, Garbutt M, Grolla A, et al. Emerging and re-emerging infectious diseases. Med Microbiol Immunol 2002;191:63 74. Jaax N, Jarhling P, Geisbert T, Geisbert J, Steele K, McKee K. Transmission of Ebola virus (Zaire strain) to uninfected control monkeys in a biocontainment laboratory. Lancet 1995;346:1669 71. Update. Outbreak of Ebola viral hemorrhagic fever Zaire, 1995. MMWR 1995;44:468 9. Khan AS, Tshioki FK, Heymann DL, Le Guenno B, Nabeth P, Kerstie ns B, et al. The reemergence of Ebola hemorrhagic fever, Democratic Republic of the Congo, 1995. J Iinfect Dis 1999;179(Suppl 1):S76 86. Hospital Infection Control Practices Advisory Committee. Recommendations for isolation precautions in hospitals. Updated February 18, 1997. Available at http:// www.cdc.gov/ncidod/hip/isolat/isopart2.htm. Accessed September 23, 2003. Richards GA, Murphy S, Jobson R, Mer M, Zinman C, Taylor R, et al. Unexpected Ebola virus in a tertiary care setting: clinical and epidemiologic aspects. Crit Care Med 2000;28(1):240 4. Outbreak of Ebola hemorrhagic feverUganda, August 2000 January 2001. MMWR 2001;50:73 6. World Health Organization. Ebola hemorrhagic fever in the Republic of the CongoUpdate 12. May 7, 2003. Available at: http://www/who.int/csr/don/2003_05_07/ en/print.html. Accessed October 3, 2003. Leirs J, Mills JN, Krebs JW, Childs JE, Akaibe D, Woollen N, et al. Search for the Ebola virus reservoir in Kikwit, Democratic Republic of the Congo: reflections on a vertebrate collection. J Infect Dis 1999; 179(suppl 1):S155 63. Swanepoel R, Leman PA, Burt FJ, Zachariades NA, Braack LEO, Ksiazek TG, et al. Experimental inoculation of plants and animals with Ebola virus. Emerg Infect Dis 1996;2:321 5.
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[65] World Health Organization. Marburg fever, Democratic Republic of the Congo. May 14. WHO 1999; 74:145. [66] Bausch DG, Borchert M, Grein T, Roth C, Swanepeol R, Libande ML, et al. Risk factors for Marburg Hemorrhagic Fever, Democratic Republic of the Congo. Emerg Infect Dis 2003;9(12). Available at: http:// www.cdc.gov/ncidod/EID/vol9no12/03-0355.htm. Accessed December 5, 2003. [67] Zaki SR, Goldsmith CS. Pathologic features of filovirus infections in humans. Curr Top Microbiol Immunol 1999;235:97 116. [68] Feldmann H, Bugany H, Mahner F, Klenk H-D, Drenckhahn D, Schnittler H-J. Filovirus-induced endothelial leakage triggered by infected monocytes/ macrophages. J Virol 1996;70(4):2208 14. [69] Harcourt BH, Sanchez A, Offermann MK. Ebola virus selectively inhibits responses to interferons, but not to interleukin-1b, in endothelial cells. J Virol 1999;73(4): 3491 6. [70] Geisbert TW, Young HA, Jarhling PB, Davis KJ, Kagan E, Hensley LE. Mechanisms underlying coagulation abnormalities in Ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/ macrophages is a key event. J Infect Dis 2003;188: 1618 29. [71] Bosio CM, Aman J, Grogan C, Hogan R, Ruthel G, Negley D, et al. Ebola and Marburg viruses replicate in monocyte-derived dendritic cells without inducing the production of cytokines and full maturation. J Infect Dis 2003;188:1630 8. [72] Schnittler H-J, Feldmann H. Marburg and Ebola hemorrhagic fevers: does the primary course of infection depend on the accessibility of organ-specific macrophages? Clin Infect Dis 1998;27:404 6. [73] Villinger F, Rollin PE, Brar SS, Chikkala NF, Winter J, Sundstrom JB, et al. Markedly elevated levels of interferon (IFN)-g, IFN-a, Interleukin (IL)-2, IL-10, and tumor necrosis factor- a associated with fatal Ebola virus infection. J Infect Dis 1999;179(suppl 1): 188 91. [74] Baize S, Leroy EM, Georges-Courbot M-C, Capron M, Lansoud-Soukate J, Debre P, et al. Defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in Ebola virus-infected patients. Nat Med 1999;5(4):423 6. [75] Baize S, Leroy EM, Georges AJ, Georges-Courbot M-C, Capron M, Bedjabaga I. Inflammatory responses in Ebola virus-infected patients. Clin Exp Immunol 2002;128:163 8. [76] Centers for Disease Control. BMBL Section III. Laboratory Biosafety Level Criteria. Updated November 30, 2000. Available at: http://www.cdc.gov/od/ohs/biosfty/ bmbl4/bmbl4s3.htm. Accessed October 1, 2003. [77] Centers for Disease Control. BMBL Appendix A. Primary containment: biological safety cabinets. Updated June 17, 1999. Available at: http://www.cdc. gov/od/ohs/biosfty/bmbl4/b4aa.htm. Accessed October 1, 2003.
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[78] Bowen ETW, Lloyd G, Harris WJ, Platt GS, Baskerville A, Vella EE. Viral haemorrhagic fever in southern Sudan and northern Zaire. Lancet 1977;1:571 3. [79] Pattyn S, Van der Groen G, Jacob W, Piot P, Courteille G. Isolation of Marburg-like virus from a case of haemorrhagic fever in Zaire. Lancet 1977;1:573 4. [80] Ksiasek TG, Rollin PE, Jarhling PB, Johnson E, Dalgard DW, Peters CJ. Enzyme immunosorbent assay for Ebola virus antigens in tissues of infected primates. J Clin Microbiol 1992;30(4):947 50. [81] Ksiazek TG, West CP, Rollin PE, Jarhling PB, Peters CJ. ELISA for the detection of antibodies to Ebola viruses. J Infect Dis 1999;179(suppl 1):S192 8. [82] Jarhling PB, Geisbert TW, Geisbert JB, Swearengen JR, Bray M, Jaax NK, et al. Evaluation of immune globulin and recombinant interferon-a2b for treatment of experimental Ebola virus infection. J Infect Dis 1999;179(suppl 1):S224 34.
Dermatol Clin 22 (2004) 303 312
Plague
C. Glenn Cobbs, MD*, David H. Chansolme, MD
Division of Infectious Disases, Department of Medicine, School of Medicine, University of Alabama at Birmingham, THT 229, 1530 3rd Avenue South, Birmingham, AL 35294, USA
By present convention, plague refers to an infectious disorder caused by the bacterial pathogen Yersinia pestis. The name of the bacteria was changed approximately 20 years ago; before then, it had been designated Pasteurella pestis, after Louis Pasteur. Plague is of enormous historic importance with epidemics documented in manuscripts dating back as far as 500 B.C. [1]. Because of a lack of understanding of the pathogenesis of epidemic febrile illness until the last 150 years, it is unclear how many outbreaks of plague in fact represented the illness we designate with that term today. For example, the plague of Athens, which occurred around 450 B.C., has been speculated to be due to influenza viral disease complicated by staphylococcal pneumonia with staphylococcal toxic shock syndrome. Other disorders, such as epidemic typhus, typhoid fever, as well as smallpox and other viral infections, have all been confused with plague. Nevertheless, it is clear at the present time that there are a rather discrete group of infectious disorders that are caused by Y pestis. Several years ago a group in France performed an interesting study [1]. They obtained dental pulp from bodies of individuals buried at a site where European Black Death had occurred in 1347, 1590, and 1722 in Southern France. By extracting DNA from this material they were able to identify nucleic acid moieties consistent with the present constituents of Y pestis. They further speculated that their technique may allow us to identify the etiology of other ancient epidemics and distinguish yersinia disease from typhus, smallpox, and other infectious disorders.
The microorganism: Yersinia pestis Y pestis is a Gram-negative ovoid bacillus. It is a nonspore former, nonmotile, and characteristically stains in safety-pin fashion such that the microorganism demonstrates some Gram-positivity at either polar end similar to the case with some klebsiella strains. (Fig. 1) Microscopic examination of fresh culture may reveal a capsule. The bacteria grows most rapidly at 28C (82F), ferments glucose and mannitol, and is lactose, sucrose, and ramose negative [2]. It appears to be able to withstand dessication remarkably well in contrast to vegetative forms of many other bacteria, and may persist in dried secretions for as long as a week or more [3,4]. Important antigens that relate to its virulence include the Fraction I (F1) antigen, which is the component of the capsule that is antiphagocytic, and seems to be elaborated most optimally at 37C (98.6F) [5]. In contrast, some of the other antigens are synthesized more efficiently at 28C (82F). Other antigens are the V and W antigens, which are also antiphagocytic, and appear to be associated with virulence in an as-yet unknown manner [5]. The lipopolysaccharide endotoxin moiety of the cell wall is similar to the constituent found in other Gram-negative bacilli, and presumably affects the host similarly. Another virulence factor is the exotoxin coagulase, which appears to play an important role in the microorganisms survival in the flea vector and the transmission cycle [6,7]. It appears to block the proventricular alimentary organ of the flea, preventing the flea from completely ingesting the blood meal. This results in delivery of the infectious agent to the next host bitten by the flea. This coagulase has a temperature optimum of 28C (82F), which is presum-
* Corresponding author. E-mail address: gcobbs@uab.edu (C.G. Cobbs).
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C.G. Cobbs, D.H. Chansolme / Dermatol Clin 22 (2004) 303312
Fig. 1. Grams stain of peripheral blood smear of septicemic patient. Note the biploar staining of the bacteria and the safetypin morphology (arrows). (From Centers for Disease Control and Prevention: Laboratory Response Network. Level A laboratory procedures for identification of Yersinia pestis. Available at http://www.bt.cdc.gov/Agent/Plague/ype_la_cp_ 121301.pdf. Accessed November 29, 2003.)
ably more appropriate for elaboration in the insect [4]. Finally, there has been mention of a plasminogen activator protein, which results in virulence through activation of the clotting cascade with possible disseminated intravascular coagulation (DIC) resulting [5].
Relationships to other Yersinia A number of investigators have examined relationships among different Yersinia. Fig. 2 obtained from one report [8] describes the evolution of Y pes-
Fig. 2. Genealogy of Yersinia pestis: the evolution of different biovars of Y pestis and the epidemics associated with the variants. Note the geographic progression from North Africa to Western Europe and, finally, Asia.
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tis and the relationship of that microorganism to Yersinia pseudotuberculosis.
Pathogenesis of disease and clinical syndromes Bubonic plague There are three main clinical syndromes associated with human disease caused by Y pestis. Bubonic plague describes a disorder that follows the inoculation of the infectious agent into the skin or subcutaneous tissue of the host via the bite of a flea or by direct contact from an infected animal. The pathogenesis involves multiplication in skin and subcutaneous tissue followed by transmittal by lymphatics to regional lymph nodes. Intracellular persistence in phagocytic macrophages may play a role in virulence. These lymph nodes generally become inflamed, enlarged, and may be unable to contain the microorganism. In those instances where lymph node defenses are overwhelmed, the microorganism gains access to proximal lymphatic channels, thoracic duct, and blood stream. However, bacteremia does not occur in every patient [6]. Clearly, a few patients may recover from bubonic yersinia disease without specific antimicrobial therapy. However, untreated the estimated mortality approximates 50% [4]. In bubonic plague, illness generally follows an incubation period of 2 to 8 days after the bite of the infected flea, a bite that is often unapparent. Patients are characteristically afflicted by the sudden onset of fever, chills, weakness, headache, and prostration. Headache occurs in many febrile illness associated with the fever itself, and does not necessarily imply that microorganisms are multiplying in the central nervous system. Along with chills, fever, and profound malaise, enlarged, tender lymph nodes appear. Often the precise subcutaneous structure involved is unclear to the patient. In the case of plague following inoculation in the lower extremity, the nodes in the groin are prominent; in the case of upper extremity inoculation, epitrochlear nodes or axillary nodes are involved. Generally involved lymph nodes become greatly enlarged and often so exquisitely tender that the patient resists any active or passive motion. Involved nodes may evolve into diffusely swollen, tender masses. There may also be some mild discoloration of the overlying skin (see below). Occasionally, there may be frank suppuration with drainage although this is uncommon. Vital signs reveal high fever (as high as 41C) with tachycardia appropriate for the temperature elevation. Blood pressure may be normal or in some
occasions low, presumably as a result of the sepsis syndrome. Occasionally, patients will present with lymphadenopathy without associated systemic symptoms and signs. In this circumstance, it appears that for one reason or another, the host has been able to control the microorganisms at the initial lymphatic site. These patients have a more favorable outcome than those with generalized symptoms and signs. On rare occasions a diffuse rash may appear not precisely related to the buboes themselves, and occasionally, there may be a purpuric rash, presumably due to vasculitis or thrombocytopenia. It was this finding that led to the term Black Death during the middle ages (see Physical Examination below).
Septicemic plague As mentioned before, some patients with skin and lymph node involvement will become bacteremic. In one study, approximately 25% of untreated patients with bubonic plague had positive blood cultures for Y pestis [6]. Bacteremia is almost always a lethal complication. Rarely patients have been described with prostration and positive blood cultures without identifiable lymph node involvement, so-called septicemic plague. These patients have high-intensity bacteremia, and are presumably overwhelmed by the bacteremia before lymphadenitis develops. Patients with this variety of plague are frequently not as febrile as those with node disease, but hemorrhagic complications may be seen and in the absence of treatment death occurs rapidly. However, many patients with bubonic plague have demonstrable bacteremia as well, so the term septicemic plague is a poor one.
Pneumonic plague Another variety of Y pestis disease is pneumonic plague. In this disorder, patients inhale the infectious microorganism presumably from aerosolized droplet nuclei coughed out by patients with pulmonary involvement or possibly aerosolized rodent fecal material. Alternatively, pneumonia can develop as a result of hematogenous dissemination of pestis. The microorganism multiplies in lung parenchyma, and there is subsequent spread to mediastinal nodes and blood. In a patient with pneumonic plague, there is often characteristic sudden onset of cough, fever, and chest pain, occurring as briefly as 2 or 3 days after exposure. These symptoms and signs are rapidly followed by overwhelming pneumonia. Without treatment, mortality in pneumonic plague approaches 100%.
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Fig. 3. Map representing foci of plague activity. (From Centers for Disease Control. Available at http://www.cdc.gov/ncidod/ dvbid/plague/index.htm. Accessed November 12, 2003.)
Plague meningitis The striking occurrence of plague meningitis during the Vietnam War was noteworthy [9]. It appeared to be most common in patients who were inadequately treated for bubonic plague. Presumably there was localization of Y pestis in the subarachnoid space with relapse of the disease at that site after the discontinuation of (initially effective) therapy [5].
nental United States, particularly in the southwest where it is associated with prairie dogs, ground squirrels, and other rodents as well as cats [11]. There were an average of about a dozen cases per year reported in the United States from 1980 to 1999 [12]. Fig. 3 shows the endemic plague sites (note the Southwest United States).
Transmission Epidemiology The World Health Organization has established a Plague Manual, which describes the current incidence and prevalence of Y pestis disease as far as can be ascertained [10]. The latest report was published in 1999. During the time period 1954 to 1977, plague appeared in more than 35 countries afflicting an estimated 80,000 patients and causing 7000 deaths. Asia contributed the largest number of patients, approximately 60%, and the mortality rate on that continent was 54%. Prevalence of plague was substantial in Vietnam during the Vietnam War. Destruction of country side and dislocation of the ecosystem forced the plague vectors and rodent hosts into areas occupied by humans and led to the epidemic. For the period 1967 to 1971, Vietnam accounted for almost 90% of all plague cases reported worldwide [10]. Noteworthy for physicians in the United States is the continuing presence of plague within the contiPlague is generally transmitted from a rodent, the usual nonhuman animal host, to man via flea ectoparasites, particularly Xenopsylla cheopis and Ceratophyllus fasciatus. These fleas are the most important reservoirs of urban plague, that is, plague occurring in large populated areas. In contrast, sylvatic plague, which is the dominant variety in the United States, generally results from human contact with flea vectors, but 20% of cases are associated with direct contact with mammalian hosts such as ground squirrels, rock squirrels, prairie dogs, and other animals [11]. Man is, therefore, the accidental host in plague transmission, the primary host being the rodent. Humans are infected when bitten by a flea that has moved from an infected rodent. Because of blockage of the proventricular organ (see above), the flea regurgitates the previously ingested blood meal obtained from an infected rodent and this regurgitated infected blood serves as the transmission agent for disease in humans. Human-to-human spread appears
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unusual; the striking exception is pneumonic plague, where the illness is transmitted via aerosolized material man-to-man. Rarely, humans may become infected by handling infected tissue. Because of the pathogenesis involving primary disease in rodents, human bubonic plague is almost always associated with initial disease in the rodent population. Plague also appears to be more prevalent at certain times of year and in certain climates. Apparently the flea behaves differently when the weather is warm and moist compared with dry and hot or cold, and most outbreaks have occurred in warm humid conditions perhaps because the flea is able to survive longer between blood meals [5]. The temperature optimum for elaboration of the coagulase enzyme (28C [82F]) appears to be relevant because at lower temperatures the flea would presumably not be as likely to regurgitate the undigested blood meal, and higher temperatures, drier temperatures would not be ideal for elaboration of the enzyme [6]. In the circumstance of bioterrorism the presumed pathogenesis would include aerosolization of vegetative microorganisms. Contamination of water or soil would not be anticipated to result in a significant number of patients. On the other hand, if the organism could be appropriately prepared and maintained, depending on wind and climate, aerosolization might lead to pneumonic plague [13]. This is in contrast to the situation with anthrax, where the microorganism is a spore-forming bacillus, and inhalation of infectious spores is sufficient to cause anthrax. These spores are extremely hardy, and anthrax spores are able to germinate and invade normal skin. Y pestis would probably not be able to persist in fomites, for example, mail, the way anthrax can [14].
Peru would also be at risk. In the case of bioterrorism, cases might occur sporadically or en masse at any site in the United States or other developed or underdeveloped countries where terrorists might operate. Plague should be suspected as a possible etiology in the setting of unexplained fulminant pneumonia when there are a number of patients presenting with similar signs and symptoms [15]. Lymphadenitis and bubonic disease would be less likely as a result of a bioterrorist attack.
Physical examination The physical exam in plague will depend on the clinical variety that is encountered. However, in all patients, including those with bubonic, pneumonic, or septicemic plague, one anticipates markedly abnormal vital signs such as high fever, tachycardia, and general prostration. Lymphadenopathy is the hallmark of bubonic disease, and occurs in relationship to the site of inoculation. In the case of lower extremity infection, there is lymphadenopathy in the inguinal area; however, axillary or generalized adenopathy may occur. The buboes appear as ovoid masses with overlying stretched skin (Fig. 4). Typically, the involved nodes are firm, nonfluctuant, and extraordinarily tender to palpation. They usually appear as a single, smooth mass, but may present as a confluence of several lymph nodes with a more irregular appearance. Surrounding edema is also encountered, and erythema may extend along lymphatic channels [6]. A diffuse erythematous rash or purpura may be present when vasculitis or DIC occurs (Fig. 5). Examination
Diagnosis of plague As is the case in all serious medical disorders, the history, physical examination, laboratory studies, and special tests are all crucial in the early diagnosis of disease caused by Y pestis. In the United States, in the absence of a suspected bioterrorism attack, one would anticipate a history of exposure (presumably in the Southwestern area of the United States) to the rodents or small animal vectors, particularly prairie dogs, ground squirrels, and so on. Native Americans who live on reservations, hunters, and ranchers living in contact with prairie dog towns are at risk. Cases have also been reported in veterinarians and individuals in close contact with cats with signs and symptoms of Y pestis disease [11]. Travelers to endemic areas such as Southeast Asia, sub-Saharan Africa, Brazil, and
Fig. 4. Inguinal bubo in a patient with bubonic plague. The area has been prepared with iodine prior to aspiration. From Peterson PK, Dahl MV. Dermatologic manifestations of infectious diseases. Kalamazoo (MI): The Upjohn Company; 1982; with permission.)
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Dermatologic findings of plague Plague is well known in both medical and lay literature as The Black Death, a disorder that ravaged Europe in the fourteenth and fifteenth centuries, killing up to one third of the population. The origin of this moniker is the striking involvement of the skin, which may be encountered in severe cases. In one series from the 1970s among Vietnamese patients with culture-proven Y pestis infection, 9 of 40 (23%) patients presented with skin findings other than buboes [16]. The two most critically ill patients presented with lower extremity purpuric lesions, but other patients presented with a panoply of skin findings including erythema multiforme, petechiae, and diffuse erythematous pruritic papules [16]. Other lesions such as carbuncles and vesicles have been described at the site of inoculation. These lesions may represent small patches of gangrenous skin, and can develop into pustular lesions resembling smallpox lesions if the patient survives long enough for the lesions to evolve [3]. Pathologic examination of the purpuric lesions shows a process similar to that seen in other Gramnegative infections (eg, meningococcemia) represented by subepidermal hemorrhage, fibrin in capillaries and venules, and intraluminal thrombi. There may be embedded microorganisms visible. Other studies have demonstrated laboratory evidence of DIC despite a lack of overt bleeding [9]. This process presumably contributes to the purpuric lesions seen in critically ill patients with plague as indicated by histopathologic findings of involved dermal structures. Occasionally, this process can lead to gangrene of the affected area. Skin biopsies obtained from patients with diffuse erythematous popular lesions demonstrate perivascular infiltration by lymphocytes, eosinophils, and polymorphonuclear leukocytes [16].
Fig. 5. Purpura and gangrene of the extremities in a patient with septicemic plague. (Courtesy of the Centers for Disease Control.)
of the throat may occasionally reveal an intense inflammatory exudate involving the pharynx. Pharyngeal plague has been described in the rare epidemiologic circumstance of women grooming the hair of plague victims and having oral contact with fleas identified in the victims hair [6,10]. Patients with pneumonic disease exhibit respiratory distress, pleuritic chest pain, and cyanosis. In pneumonic plague, examination of the chest reveals findings consistent with consolidation including dullness to percussion, bronchophony, and whispered pectiroloquy. Cardiac findings with the exception of tachycardia are uncommon. The abdominal examination may reveal tender splenomegaly. In fact, one may think of the spleen as a large lymph node, and clearly, in bacteremic disease, it may become colonized by circulating microorganisms. Abdominal tenderness and vomiting are commonly seen in all varieties of Y pestis disease. In patients with meningitis, the symptoms and signs are similar to those in patients with other varieties of pyogenic meningitis such as pneumococcal and meningococcal diseases. In a study reported during the Vietnam War, the majority of patients with bubonic disease appeared to have no specific generalized skin lesions although pustules, vesicles, and papules near or associated with the bubo were described in some instances [16]. Pestis minor has been described in some areas as a variant of plague in which subclinical disease occurs, and medical attention is neither sought nor required [4]. Seroepidemiologic studies in endemic areas have shown higher than expected seropositivity, indicating the likelihood of subclincal disease in a number of patients who may not require medical care despite exposure to Y pestis [17].
Lab diagnosis Laboratory evaluation for diagnosis of plague consists of routine studies such as white blood cell count and differential count. The white blood cell count is almost always elevated and leftward shifted with band forms, Dohle bodies, and toxic granulations. Occasionally, the plague bacillus can be visualized in peripheral blood smears. It is noteworthy that in very few bacteremic disorders are the number of microorganisms sufficient to be identified in blood films. Most all microorgansims that gain access to the circulation are cleared by the reticuloendothelial system rapidly, and their numbers do not increase in
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blood. Normally 104 105 microorganisms per milliliter are required to be visible on blood smear, and this almost never occurs in the usual bacteremic disorders such as staphylococcal and streptococcal bacteremia, and so on. In contrast, Y pestis appears to be one of the few microorganisms that has the ability to actually multiply in the circulating blood. As noted above, the presence of purpura in some patients with plague suggests there is an aberration of the coagulation system with disseminated intravascular coagulation characterized by thrombocytopenia, elevated LDH, microangiopathic hemolytic anemia, and elevated fibrin split products. One study showed evidence of DIC in 19 of 22 (86%) of patients manifest with elevated levels of fibrin degradation products even though none of these patients had a clinically evident bleeding diathesis. Levels normalized after therapy was begun and patients improved [9]. Specimens submitted for microbiologic identification in suspected plague should be obtained from the respiratory tract in pneumonic disease, blood in septicemic, bubonic, or pneumonic disease, and from aspirates of buboes or other involved tissues (eg, liver, spleen, and so on), depending on the location of the abnormality [18]. These specimens can usually be processed in a laboratory using biological safety level 2 (BSL-2) practices, but BSL-3 precautions should be used when handling aerosols or potentially aerosolized material (animal carcasses, feces, and so on) [2]. Grams stain may reveal the classic bipolar (safety-pin) staining, but this phenomenon can be seen better on a Wright-Giemsa or a Waysons stain. Culture media for elaboration of Y pestis should be inoculated per standard protocol and taped shut to prevent inadvertent opening and aerosol spread. Cultures will demonstrate the fried-egg appearance typical of Y pestis colonies after 48 to 72 hours of incubation [2,5,18]. There have been a number of serologic tests used in the diagnosis of plague. Hemaglutination inhibition and enzyme-linked immunosorbent assay tests have been most popular in recent years [19 21]. Clinical and laboratory criteria for diagnosis of plague are shown in Table 1. Serologic criteria for a presumptive diagnosis include the presence of antiFraction I antigen antibody in a titer of greater than 1:10, along with a compatible clinical picture. A confirmed diagnosis requires a fourfold rise in antibody to this antigen by agglutination testing or a single titer of greater than 1:28 [22]. During the Vietnam War, Butler and colleagues performed studies on the serologic response in plague demonstrating the time course of antibody response during disease [23]. These observations described antibody titers begin-
Table 1 Laboratory test criteria for diagnosis of plaque Conditions for suspected plaque: 1. Clinical symptoms compatible with plaque such as fever and lymphadenopathy in a patient who lives near or who has traveled to a plaque-endemic area. 2. If small Gram-negative and/or bipolar-staining coccobaccilli are seen on a smear from affected tissues Conditions for presumptive plaque should include one of the following two items: 1. Immunoflourescence stain of material positive for Yersinia pestis F1 antigen 2. Single serum specimen with anti-F1 antigen titer of > 1:10 by hemagglutination inhibition test. Conditions for confirmed plaque require one of the following: 1. Isolated culture is lysed by a specific bacteriophage 2. Two serum specimens demonstrated fourfold anti-F1 antigen titer difference by hemagglutination inhibition testing. 3. A single serum specimen has an anti-F1 antigen titer of > 1:128 by hemagglutination inhibition testing in the absence of a history of vaccination or previous known plaque exposure Adapted from Centers for Disease Control: Division of Vector-Borne Infectious Diseases. Available at http://www.cdc. gov/ncidod/dvbid/plaque/lab-test-criteria.htm. Accessed November 15, 2003.
ning to rise on day 5 of illness and progressively increasing up to day 14, after which a plateau was noted [23]. More recently, polymerase chain reaction has been used in identification of Y pestis, and may prove to be an effective and rapid means of diagnosis with good sensitivity and specificity for Y pestis microorganisms [24,25].
Treatment In the United States, there are currently three Federal Drug Administration-approved antimicrobial agents for treatment of Y pestis disease. They include the historic agent streptomycin, as well as tetracycline hydrochloride, and doxycycline, a tetracycline derivative. Flouroquinolones might be effective in the management of plague because they are bactericidal against the microorganism in vitro, and studies in animals have suggested excellent activity in vivo [26,27]. However, no clinical trials have been reported up to now. The dose of streptomycin is 30 mg/kg per day divided in two doses. Streptomycin has been difficult
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to obtain in recent years. It is only rarely used for management of tuberculosis or Gram-negative aerobic microorganisms in developed countries. Gentamicin, a commonly used aminoglycoside, appears anecdotally to be as effective as streptomycin in treatment of plague, but has not been tested in large clinical trials. The World Health Organization recommends using 3 mg/kg/day of gentamicin divided in three doses for the treatment of plague in adult patients and higher doses (6.0 7.5 mg/kg/day) in children and infants [9,10]. Doxycycline should be used parenterally in a dose of 100 mg every 12 hours. This agent has a slightly better safety profile than tetracycline hydrochloride itself [13]. Chloramphenicol, 50 mg/kg/day divided in four doses, has been used as an oral regimen in combination with aminoglycosides with good success [10,13]. Because of good penetration of the subarachnoid space, chloramphenicol appears to be the most effective agent for treatment of meningeal disease. Beta-lactams have not been used as extensively as other agents, but animal models have shown good activity with third-generation cephalosporins comparable to flouroquinolone and aminoglycoside activity [26]. The duration of therapy is generally 10 days, and by this time patients will have begun to recover. In the past, Y pestis has been considered to be uniformly susceptible to the antimicrobials noted above, which have traditionally been used for therapy (ie, streptomycin and tetracycline), but recently, isolates have been recovered, which display resistance to these agents [28]. Supportive therapy of patients with plague includes volume resuscitation and management of sepsis syndrome. For patients with pneumonic involvement, ventilatory support is frequently necessary. Surgical drainage of suppurating nodes has been used, and although it is reasonable to anticipate that drainage of purulent, necrotic material might facilitate healing, there are no careful studies regarding the importance of this procedure. Y pestis is an easily transmissible microorganism, and suspected plague demands isolation procedures for patients [14]. This includes contact and respiratory isolation in a private negative pressure room if possible. All medical staff attending the patient should wear gowns, gloves, and masks, and use patient-dedicated examination equipment. Plague is a reportable disease, and any suspected or proven cases should be reported to the public health authorities at once to assist in case identification, contact tracing, and quarantine if necessary [15]. Public health measures directed against the rodent
host and the flea vector are appropriate as well, and would be instituted by the local authorities [3].
Immunization Several plague vaccines are available. The older vaccine is a product using the killed bacillus similar to the old typhoid vaccine [29]. This killed bacterial vaccine is unsatisfactory in a number of regards. First, there is a significant toxicity following inoculation, presumably due to the endotoxin moiety; second, the immunity is not solid, and does not seem to persist for longer than 6 months [30,31]. It affords only about 50% protection against bubonic plague, and is ineffective in preventing pneumonic disease [3]. A more recent vaccine prepared from the purified F1 antigen moiety appears to be as effective as the killed vaccine in terms of efficacy, and has fewer side effects [32,33]. Finally, there is a live bacterial vaccine prepared from the EV strain of Y pestis that was used in the 1930s. Oral administration of this agent was complicated in about half of the patients by pharyngeal inflammation [3]. Currently, vaccination with any of these products is recommended only for people working directly with Y pestis or those working in areas of known enzootic activity [5].
Plague as a bioterrorism agent Plague has been used in biologic warfare throughout history, and it remains a candidate bioweapon today [34]. Y pestis has been designated by the Centers for Disease Control and Prevention as a bioterrorism category A agent because it meets the following characteristics: (1) it can be easily disseminated; (2) causes high mortality; (3) causes public panic; and (4) requires special action for public health preparedness [35]. Only four bacteria are included in this designation: anthrax, tularemia, botulism, and plague. They represent the highest priorities in bioterrorism awareness. The World Health Organization has postulated that a 50-kg container of aerosolized Y pestis delivered over a city of 5 million people might result in 36,000 deaths and 80,000 to 100,000 hospitalizations [36]. Few municipalities are currently equipped to deal with an outbreak of this magnitude. An appropriate response should include quick recognition, a predetermined bioterrorism protocol, and the cooperation of the specific local, state, and federal authorities. As implied above, the most likely mode of delivering Y pestis in a bioterrorism setting would be as an aerosol form. Thus, one would expect to encounter
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pneumonic plague as the initial clinical syndrome after exposure to this agent. In addition to fever, chest pain, dyspnea, cough, and hemoptysis, patients might also present with gastrointestinal symptoms, hypotension, altered mental status, and oliguria. Because of the portal of entry (the respiratory tract), cervical buboes may occasionally be associated with disease; buboes at other sites would not be expected [36]. The initiation of appropriate antimicrobial therapy must not be delayed pending diagnosis if there is sufficient suspicion of a bioterrorist attack, as the mortality without therapy approaches 100% in those patients with pneumonic plague. Gram stain of the sputum may be the most expedient way to diagnose pneumonic plague, and could be employed to identify plague patients before definitive laboratory diagnosis. One must also consider the distribution of prophylactic therapy in those people who are deemed to be at high risk for infection due to common potential sources of exposure or direct contact with the patient. Appropriate agents for this purpose might include doxycycline, flouroquinolone, or chloramphenicol [14]. In the setting of suspected or proven pneumonic plague, caretakers must be aware of the potential for person-to-person spread through the aerosolized respiratory secretions of the patient, and appropriate isolation procedures should be undertaken immediately (see above). In addition to universal precautions, health care workers should wear N95 masks when in contact with the patient, and the patient should be placed in a negative-pressure airborne isolation environment. Great care should exercised in handling clinical specimens, and laboratory personnel should use biosafety level 2 protocols when processing blood, serum, sputum, urine, tissue, and other relevant samples [18]. When a case of pneumonic plague is suspected, one should notify the hospital-specific personnel (infection control practitioners, epidemiologists, infectious disease specialists, public affairs officials, and administrators), local and state health department officials, Centers for Disease Control and Prevention emergency response officials, and clinical microbiology laboratory personnel to ensure appropriate protocols are followed in the setting of a bioterrorism attack with a highly contagious agent [35].
Bubonic disease, pneumonia, and sepsis are seen in addition to a number of other less common manifestations. As an agent of bioterrorism, an aerosol Y pestis would pose an imminent threat if released in the appropriate environment. Diagnosis can be made with readily available techniques, but laboratory handling of specimens requires special care. Treatment should be instituted immediately when there is a strong suspicion of plague, as delaying therapy will result in increased morbidity and mortality.
Acknowledgments We would like to thank Susan Heath for her help in the preparation of this article.
References
[1] Drancourt M, Raoult D. Molecular insights into the history of plague. Microbes Infect 2002;4:105 9. [2] Aleksic S, Bockemu hl J. Yersinia and other Enterobacteriaceae. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, editors. Manual of clinical microbiology. 7th edition. Washington (DC): ASM Press; 1999. p. 483 96. [3] Plague and Meliodosis. In: Manson-Bahr PEC, Bell DR, editors. Mansons tropical diseases. 19th edition. ` re Tindall; 1987. p. 586 606. London: Ballie [4] Swartz MN. Yersinia, Francisella, Pasteurella, & Brucella. In: Davis B, Dulbecco R, Eisen HN, Ginsberg HS, editors. Microbiology. 4th edition. Philadelphia: JB Lippincott Co.; 1990. p. 601 14. [5] Yersinia. In: Joklik WK, Willett HP, Amos DB, Wilfert CM, editors. Zinsser Microbiology. 20th edition. Norwalk (CT): Appleton & Lange; 1992. p. 584 94. [6] Butler T. Plague. In: Strickland GT, editor. Hunters tropical medicine. 6th edition. Philadelphia: WB Saunders; 1984. p. 340 8. [7] Perry RD. A plague of fleas survival and transmission of Yersinia pestis. Am Soc Microbiol News 2003; 69(7):336 40. [8] Radnedge L, Agron PG, Worsham PL, Andersen GL. Genome plasticity in Yersinia pestis. Microbiology 2002;148:1687 98. [9] Butler T, Bell WR, Linh NN, Tiep ND, Arnold K. Yersinia pestis infection in Vietnam. I. Clinical and hematologic aspects. J Infect Dis 1974;129:S78 84. [10] World Health Organization. Plague manual: epidemiology, distribution, surveillance, and control. Available at http://www.who.int/emc-documents/plague/ whocdscsredc992c.html. Accessed October 12, 2003. [11] Gage KL, Dennis DT, Orloski KA, Ettestad P, Brown TL, Reynolds PJ, et al. Cases of cat-associated human plague in the Western US, 1977 98. Clin Infect Dis 2000;30:893 900.
Summary Plague is a disease that has been present for thousands of years and described since the earliest medical accounts. It is present today even in developed countries, and may present in a variety of forms.
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C.G. Cobbs, D.H. Chansolme / Dermatol Clin 22 (2004) 303312 by real-time PCR. J Clin Microbiol 2003;41(10): 4873 5. Engelthaler DM, Gage KL, Montenieri JA, Chu M, Carter LC. PCR detection of Yersinia pestis in fleas: comparison with mouse inoculation. J Clin Microbiol 1999;37(6):1980 4. Bonacarsi SP, Scavizzi MR, Guiyole A, Amouroux JH, Carniel E. Assessment of a flouroquinolone, three b-lactams, two aminoglycosides, and a cycline in treatment of murine Yersinia pestis infection. Antimicrob Agents Chemother 1994;38(3):481 6. Frean J, Klugman KP, Arntzen L, Bukofzer S. Susceptibility of Yersinia pestis to novel and conventional antimicrobial agents. J Antimicrob Chemother 2003; 52:294 6. Galimand M, Guiyole A, Gerbaud G, Rasoamanana B, Chanteau S, Carniel E, et al. Multidrug resistance in Yersinia pestis mediated by a transferable plasmid. N Engl J Med 1997;337(10):677 80. Cavanaugh DC, Elisberg BL, Llewellyn CH, Marshall Jr JD, Rust Jr JH, Williams JE, et al. Plague immunization. V. Indirect evidence for the efficacy of plague vaccine. J Infect Dis 1974;129:S37 40. Meyer KF, Smith G, Foster LE, Marshall Jr JD, Cavanaugh DC. Plague immunization. IV. clinical reactions and serologic response to inoculations of haffkine and freeze-dried plague vaccine. J Infect Dis 1974; 129:S30 6. Marshall Jr JS, Cavanaugh DC, Bartelloni PJ, Meyer KF. Plague immunization. III. Serologic response to multiple inoculations of vaccine. J Infect Dis 1974; 129:S26 9. Meyer KF, Hightower JA, McCrumb FR. Plague immunization. VI. Vaccination with the fraction I antigen of Yersinia pestis. J Infect Dis 1974;129:S41 5. Reddin KM, Easterbrook TJ, Eley SM, Russell P, Mobsby VA, Jones DH, et al. Comparison of the immunological and protective responses elicited by microencapsulated formulations of the FI antigen from Yersinia pestis. Vaccine 1998;16(8):761 7. Wheelis M. Biological warfare at the 1346 Siege of Caffa. Emerg Infect Dis 2002;8(9):971 5. Atlas RM. Bioterrorism: from threat to reality. Annu Rev Microbiol 2002;56:167 85. Tjaden JA, Lazarus AA, Martin GJ. Bacteria as agents of biowarfare: how to proceed when the worst is suspected. Postgrad Med 2002;112(2):57 70.
[12] Centers for Disease Control and Prevention. Summary of notifiable disease, United States, 2000. MMWR Morb Mortal Wkly Rep 2000;49(53):xvii. [13] Cunha BA. Anthrax, tularemia, plague, ebola or smallpox as agents of bioterrorism: recognition in the emergency room. Clin Microbiol Infect 2002;8:489 503. [14] Varkey P, Poland GA, Cockerill III FR, Smith TF, Hagen PT. Confronting bioterrorism: physicians on the front line. Mayo Clin Proc 2002;77:661 72. [15] Centers for Disease Control and Prevention. Recognition of illness associated with the intentional release of a biologic agent. MMWR Morb Mortal Wkly Rep 2001;50(41):893 7. [16] Butler T. A clinical study of bubonic plague: observations of the 1970 Vietnam epidemic with emphasis on coagulation studies, skin histology and electrocardiograms. Am J Med 1972;53:268 76. [17] Ratsitorahina M, Rabarijaona L, Chanteau S, Boisier P. Seroepidemiology of human plague in the Madagascar highlands. Trop Med Int Health 2000;5(2):94 8. [18] Centers for Disease Control and Prevention. Laboratory response network. Level A laboratory procedures for identification of Yersinia pestis. Available at http:// www.bt.cdc.gov/Agent/Plague/ype_la_cp_121301.pdf. Accessed November 29, 2003. [19] Williams JE, Arntzen L, Robinson DM, Cavanaugh DC, Isaa cson M. Comparison of passive haemagglutination and enzyme-linked immunosorbent assay for serodiagnosis of plague. Bull World Health Organ 1982;60(5):777 81. [20] William JE, Atas M, Cavanaugh DC. A comparison of serological tests for detecting antibody to plague. Bull World Health Organ 1976;54:232 3. [21] Williams JE, Gentry MK, Braden CA, Tyndal GL, Altieri PL, Berman S, et al. A monoclonal antibody for the specific diagnosis of plague. Bull World Health Organ 1988;66(1):77 82. [22] Centers for Disease Control and Prevention. Division of vector-borne infectious diseases. Laboratory test criteria for diagnosis of plague. Available at http://www. cdc.gov/ncidod/dvbid/plague/lab-test-criteria.htm. Accessed November 15, 2003. [23] Butler T, Hudson BW. The serological response to Yersinia pestis infection. Bull World Health Organ 1977;55:39 42. [24] Lo ez C, Herwegh S, Wallet F, Armand S, Guinet F, Courcul RJ. Detection of Yersinia pestis in sputum
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Dermatol Clin 22 (2004) 313 320
Tularemia: the disease and the weapon

Steven D. Cronquist, MD
Department of Dermatology, Naval Hospital Great Lakes, 3001A Sixth Street, Great Lakes, IL 60088, USA
The first scientific descriptions of tularemia were reported less than a century ago [1]. Most unimaginable in these early descriptions was the potential threat of tularemia as a weapon of biowarfare or bioterrorism. In the present day, this potential threat as a biologic weapon is plainly understood. Fortunately, there is no clearly established historic use of tularemia as a weapon, although tularemia has successfully been weaponized by select nations. Certain characteristics of tularemia categorize it as a high-risk threat. The present understanding of the cutaneous and systemic manifestations of tularemia is primarily derived from reports of naturally occurring cases.
The organism Tularemia is also known as rabbit fever and deerfly fever [2,3]. Francisella tularensis is an extremely virulent pleomorphic, aerobic, intracellular, Gramnegative coccobacillus. F tularensis does not form spores, but may survive for weeks in water, soil, animal carcasses, hides, and hay or straw. It may also survive in frozen meat [4,5]. Two main strains are described: F tularensis biovar tularensis (type A) and F tularensis biovar palaearctica (type B). Type A is significantly more virulent, and has a case fatality rate of 5% to 7% if untreated [6]. If inhaled or injected intradermally, as few as 50 type A organisms may cause disease. The inoculum required on oral chal-
The opinions stated in this article are those of the author and are not official policy of the Department of Defense or Navy. E-mail address: sdcronquist@NHGL.med.navy.mil
lenge is significantly higher, on the range of 108 organisms [3,7]. Type A disease has only rarely been reported outside of North America. Type B infection rarely results in death, and is found in Europe, Asia, and the United States [8 10]. Reports of tularemia are limited to the northern hemisphere. Tularemia is transmitted by arthropod bite, contact with blood or tissues of infected animals, ingestion of contaminated water or meat, or inhalation of aerosols [11]. Infective aerosols from lawn mowing and brush cutting have been suspected in at least one epidemic [12]. Aerosols generated from laboratory cultures are extremely dangerous to laboratory personnel. Contaminated drinking water has been associated with sporadic cases [13,14]. The large majority of all tularemia cases have occurred in rural areas. Reservoirs are several species of wild and domestic animals. Tularemia is most often linked with rabbits, including cottontail and jack rabbits in the United States. Squirrels, beavers, muskrats, meadow voles, rodents, and other small mammals have also been associated with the disease [8,11,15 17]. Animal bites, including those from cats and squirrels, have transmitted infection [3]. Arthropod vectors include ticks, mosquitoes, deerflies and fleas. In the United States, ticks are responsible for approximately 75% of cases. Over 10 species of ticks have been implicated. Amblyomma americanum (Lone Star tick), Dermacentor andersoni (American wood tick), and Dermacentor variabilis (American dog tick) are all reported as vectors in North America [18 20]. The organism is not found in the tick salivary gland, but rather in the tick feces [3]. Ticks also serve as a reservoir for F tularensis due to transovarian transmission [21]. Tularemia is a rare disease in the United States. Most recent cases of tularemia in the United States
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have been reported in the central and southwest states, but the disease has been reported in every state except Hawaii. The states of Arkansas, Missouri, South Dakota, and Oklahoma accounted for just over half of all reported cases from 1990 2000 [22]. The majority of cases during the 1990s were reported late spring and summer. Historically, most cases in spring and early summer are related to arthropod bites, whereas cases reported during the winter months are related to hunting or other direct contact with infected animals.
Biologic warfare and bioterrorism threat F tularensis is designated as a category A agent by the Centers for Disease Control and Prevention (CDC). Other category A agents include: Bacillus anthracis (anthrax), Yersinia pestis (plague), Botulanim toxin (botulism), Variola major (smallpox), and the viral hemorrhagic fevers. All suspected cases of these diseases require immediate reporting to public health authorities. Category A agents are given a high priority due to easy dissemination or person-to-person transmission, potential for major public health impact, ability to cause public panic and social disruption, and the requirement for additional public health preparedness [5]. Tularemia was maintained as a biologic weapon in the US arsenal until unilateral stockpile destruction in 1970. It was weaponized by freeze drying bacterialaden slurry and milling it into a fine powder for aerosol delivery [4,23]. It is known that the former Soviet Union also maintained stocks of weaponized tularemia, with reports of strains engineered for resistance to antibiotics and vaccines [24]. The Japanese studied tularemia as a potential biologic weapon during the World War II occupation of Manchuria [25]. It is not publicly known if any other countries have successfully weaponized tularemia. In his book Biohazard, former Soviet biologic weapons expert Dr. Kenneth Alibek (formerly known as Kanatjan Alibekov) alleged the former Soviet Union used tularemia against German troops during the battle for Stalingrad in 1942. A review by Croddy proposes that a naturally occurring outbreak, facilitated by conditions of war, was a more likely explanation for the epidemic [23]. The potential impact of an intentional release of tularemia was highlighted by a World Health Organization study in 1969. Expected casualties from an aerosol dispersal of 50 kg of F tularensis over a metropolitan area with 5 million inhabitants yielded estimates of 19,000 deaths and one quarter million
with incapacitating illness [4]. An aerosol with 1- to 10-mm particle size may remain suspended for hours in certain weather conditions, and may be delivered by relatively simple technology [26]. Some details have emerged of a largely secret air surveillance program within the United States aimed at minimizing the impact of a bioterrorist attack. This program, termed Biowatch, uses a network of outdoor airsampling sensors that is designed to warn officials within hours of any attempt by terrorists to release deadly microbes into the atmosphere. Of note, a sensor located in Texas reportedly detected small quantities of F tularensis that were naturally present in the atmosphere, and unrelated to an intentional release. There were no reports of associated illness in the community [27]. The potential use of tularemia as a biologic weapon led to reinstatement by the CDC on the list of nationally reportable diseases in 2000 [22]. Tularemia had been removed from the list in 1994 after the number of reported cases dwindled in the United States. When inhaled as an aerosol, very few organisms are required to cause human disease. Therefore, the pneumonic form of tularemia is considered to be the primary concern in the scenario of an intentional release of the microbe [28]. However, primary cutaneous manifestations of anthrax were seen with the bioterrorist postal attacks of 2001, and should be considered as a possible initial sign of weaponized tularemia exposure as well.
Clinical manifestations Clinical manifestations are largely dependent on the means of exposure to the microorganism. After an incubation period of 1 to 21 days (average 3 5 days), early infection is manifest with abrupt onset of fever, chills, myalgia, arthralgia, malaise, coryza, sore throat, and fatigue. In addition to these common features, six clinical syndromes characterize the illness: ulceroglandular, glandular, oculoglandular, oropharyngeal, typhoidal, and pneumonic (Table 1). Any of these forms of tularemia may result in hematogenous spread. In addition to the six classic clinical syndromes, hematogenous spread of tularemia has been associated with meningitis, pericarditis, hepatitis, peritonitis, endocarditis, ataxia, osteomyelitis, sepsis, and septic shock with rhabdomyolysis and acute renal failure [3,29,30]. Children infected with tularemia typically have a similar clinical presentation to adults, but have been reported to exhibit fever, pharyngitis, hepatosplenomegaly, and constitutional symptoms more often than affected adults [31].
S.D. Cronquist / Dermatol Clin 22 (2004) 313320 Table 1 Clinical presentations of tumaremia Presentation Ulceroglandular Findings Cutaneous ulcer at site of inoculation Proximal lymphadenopathy, may suppurate Majority of naturally occurring cases Lymphadenopathy, may suppurate No evidence of cutaneous involvement Ocular lesion, painful unilateral purulent conjunctivitis Cervical and preauricular lymphadenopathy Stomatitis, exudative, or membranous pharyngitis Cervical lymphadenopathy Pulmonary involvement +/ other systemic findings Considered most likely presentation if exposed to aerosol Systemic involvement without evidence of skin, mucosal, or lymphatic involvement
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described as secondary cutaneous manifestations of tularemia [20,32,33]. Sweets syndrome has also been reported in association with tularemia [34]. Glandular (5 10% of cases) Fever and tender lymphadenopathy are characteristic of this form. Skin manifestations are absent, but other systemic signs and symptoms may occur. Oculoglandular (1 2% of cases) Oculoglandular tularemia is characterized by painful purulent unilateral conjunctivitis with cervical and preauricular lymphadenopathy. Painful preauricular lymphadenopathy is unique for tularemia,and differentiates it from cat scratch disease, tuberculosis, sporotrichosis, and syphilis [3].The site of inoculation is typically the conjunctiva after contact with contaminated fingers or direct accidental inoculation with infected matter [35].Without treatment, corneal ulceration and perforation may occur [36]. Oropharyngeal and gastrointestinal
Glandular Oculoglandular
Oropharyngeal
Pneumonic
Typhoidal
Ulceroglandular (most common presentation in nature, 75 85% of cases) and other cutaneous manifestations of tularemia Two to 5 days after cutaneous exposure (range 1 10 days), a tender or pruritic inflammatory papule develops at the site of inoculation. The papule rapidly enlarges, and a primary ulcer usually appears with sharp demarcation and a thin, yellow exudate. Gradually, a tender necrotic base develops and a black eschar may form, often in phase with the onset of regional adenopathy. The most common sites of inoculation are the hands and distal extremities. The ulcer may persist for several months, and heals with scarring. Frequently, the bacteria spread through lymphatic vessels. Regional lymph nodes may enlarge and become tender with overlying erythematous skin. Involved lymph nodes may become fluctuant and spontaneously drain. Bacteremia may occur in ulceroglandular tularemia with spread to distant organs. Secondary skin eruptions (tularemids) are a common complication of tularemia, occurring in 3% to 25% of patients [32]. Tularemic exanthems appear most often during the second week of the disease, and may be seen in any clinical form of tularemia. Papular, macular, pustular, petechial, and papulovesicular exanthems have been described on extremities and face, with a lesser involvement of the trunk. Erythema nodosum (1 13% of cases) and erythema multiforme (1 2% of cases) have commonly been
Oropharyngeal tularemia presents with acute exudative or membranous pharyngitis, tonsillitis, or stomatitis with cervical adenopathy. Tonsils enlarge and may become covered in a yellow or white pseudomembrane. Gastrointestinal disease may include abdominal pain, nausea, vomiting, diarrhea, intestinal ulcerations, gastrointestinal bleeding, and mesenteric lymphadenopathy. Oropharyngeal tularemia usually follows ingestion of undercooked meat, contaminated water, or direct inoculation from contaminated hands after the handling of infected animal carcasses [3]. These forms of tularemia are extremely rare, in part due to the necessity of a much larger inoculum than that required for cutaneous or pulmonary disease. Pneumonic Pneumonia may be the sole manifestation of tularemia via the inhalation of aerosolized organisms. However, pneumonia is usually a secondary complication after hematogenous spread. Between 30% to 80% of typhoidal cases and 10% to 15% of ulceroglandular cases develop pneumonia [11]. Pneumonic tularemia will present as a variable infiltrate unresponsive to b-lactam antibiotics, and should be considered in the differential diagnosis of atypical pneumonias [3]. Symptoms include fever and nonproductive cough, with possible dyspnea or pleuritic
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chest pain. Laboratory workers exposed to tularemia cultures are at high risk for this form of the disease. Similarly, the use of tularemia as a weaponized aerosol would be expected to generate a large proportion of pulmonary cases. Typhoidal (5 15% historically, now considered rare in the United States) Inoculation by various means (cutaneous, respiratory, gastrointestinal) may result in the typhoidal form of tularemia. High continuous fever and other constitutional signs will occur in the absence of cutaneous, mucosal membrane, or lymphatic system involvement [3]. This form may present with sepsis or septic shock, and can be very difficult to diagnose. Blood cultures may rarely be positive.
Diagnosis The laboratory diagnosis of tularemia can be challenging (Table 2). Nonspecific findings include leukocytosis and mildly elevated hepatic transaminases. Hematocrit, hemoglobin, platelets, and differential counts are usually normal [5]. If pulmonary involvement occurs, chest radiograph findings are typically multifocal segmental or lobar infiltrates. Mediastinal lymphadenopathy, cavitary lesions, and
pleural effusions may also be found on chest imaging [37,38]. Serologic testing is the most frequent method of confirming the diagnosis of tularemia. Evidence of tularemia is established with detection of antibody response with the microagglutination test or enzymelinked imunosorbent assay. Agglutination titers with a fourfold increase (diagnostic) or a single titer greater of 1:160 (presumptive) are considered positive. The test is of confirmatory use only, as the antibody response usually takes 1 or more weeks to be detectable with current methods. Over 50% of cases demonstrate detectable titers in the second week of infection. Titers reach maximum level at 4 to 8 weeks, and may persist for years after infection [39]. The persistence of detectable antibodies may cause uncertainty in the patient with a remote history of tularemia exposure. For this reason, a fourfold rise in titer is more convincing as proof of acute infection. Both type A and type B strains react to the antigen used in the agglutination test. Serologic crossreactivity to Brucella species, Proteus Ox-19, and Yersinia species has been reported [2,3,8]. Fluorescent-labeled antibodies are available for microscopic examination of specimens such as tissue or smears. This test may provide rapid confirmation, and is available through reference laboratories [4]. Polymerase chain reaction testing may provide rapid and specific confirmation, including the acute phase
Table 2 Diagnostic methods for tularemia Methods Serum microagglutination test Findings Fourfold rise in convalescent titer (diagnostic) Single titer > 1:160 (presumptive) May be positive in tissue or smears May be positive in tissue, exudate, sputum, gastric washings, and rarely blood. Notify laboratory workersrequires special handling and media. Early lesions: neutrophilic response Late lesions: granulomatous response Nonspecific pulmonary infiltrates Hilar adenopathy, pleural effusions Consult reference laboratory, state health authorities, CDC
Fluorescent antibodies Culture
Histology
Chest radiograph
Other specialized tests: PCR Antigen detection assays Immunoblotting Pulsed-field gel electrophoresis
Abbreviations: CDC, Centers for Disease Control and Prevention; PCR, polymerase chain reaction.
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of disease [40,41]. Other techniques include antigen detection assays, immunoblotting, and pulsed-field gel electrophoresis. These techniques are largely confined to research or reference laboratories [2,4]. Direct culture is extremely hazardous to laboratory personnel, and requires careful handling and special media. Tissue, blood, exudate, sputum, and gastric washings may be cultured. Gram stain is of little value, but may reveal small, poorly staining, Gramnegative coccobacilli appearing as pink amorphous material. Cysteine is required for growth in culture, and potential media include chocolate, Thayer Martin, and buffered charcoal yeast extract [42]. Growth may not appear for 48 to 72 hours, and specimens should be held for at least 10 days before discarding. The laboratory must be notified of the clinical suspicion of tularemia so that protective measures may be exercised, as the organisms might aerosolize from culture plates. The plates must be sealed and handled using a Biosafety Level 2 (BSL-2) facility. If a BSL-2 facility presumptively identifies tularemia, further testing for additional study and susceptibility testing should be performed at a BSL-3 facility [4]. Histologic examination varies depending on the disease duration. Examination of a primary ulcer reveals a nonspecific pattern of epidermal necrosis that often extends into the upper dermis. Surrounding epidermis is often acanthotic with focal spongiosis. Chronic lesions demonstrate a more granulomatous pattern with necrotic foci. Involved lymph nodes usually show a greater degree of multiple granulomas with central necrosis. Immunofluorescent staining is required to clearly demonstrate organisms [43]. As with other potential agents of bioterrorism, temporally clustered cases occurring in large numbers should alert medical providers to possible mass exposure [44]. Due to the threat of bioterrorism, a Laboratory Response Network has been established, composed of both public and private laboratories, that will perform testing according to standard protocols developed by the CDC in partnership with the Association of Public Health Laboratories and American Society for Microbiology [42].
anthrax, scrub typhus, atypical mycobacterial infection, rat bite fever (Spirillum minus), and staphylococcal or streptococcal lymphadenitis [2,46]. With the exception of cat scratch disease, the majority of these infections do not result in same degree of lymphadenopathy that tularemia produces [3]. The differential diagnosis of pneumonic tularemia would include atypical pneumonias such as Chlamydia pneumoniae, Q fever, mycoplasma, psittacosis, histoplasmosis, and coccidiomycosis pneumonia. Typhoidal tularemia includes the differential diagnosis of typhoid fever, brucellosis, tuberculosis, malaria, Legionnaires disease, psittacosis, Q fever, sarcoidosis, and reticuloendothelial or hematologic malignancies [3,11]. Oropharyngeal tularemia should be differentiated from diphtheria and streptococcal pharyngitis [3]. The histologic differential diagnosis includes cat scratch disease, lymphogranuloma venereum, anthrax, tularemia, toxoplasmosis, sporotrichosis. and atypical mycobacteria.
Treatment The following treatment and guidelines were reached by consensus and recommended by the Working Group on Civilian Biodefense [4]. The treatment recommendations vary according to the relative number of cases, with different recommendations for mass exposure versus isolated or contained cases (Table 3). Streptomycin is the preferred treatment for isolated or contained cases. The recommended adult dose is 1 gram intramuscularly twice daily (or 7.5 10 mg/kg every 12 hours). This recommendation centers largely on historical success with streptomycin. Although currently recommended as a first-line agent, the limited availability of streptomycin may make alternatives more attractive. Of additional concern, a fully virulent streptomycin-resistant strain of F tularensis was developed during the 1950s, and is presumed to have been obtained by other countries [5]. This particular strain was sensitive to gentamicin.
Differential diagnosis The differential diagnosis of a necrotic skin ulcer includes anthrax, deep fungal infection, atypical mycobacterial infection, milkers nodule, orf, glanders, brown recluse spider bite, cutaneous leishmaniasis, or plague [45]. The diagnosis of glandular tularemia is more challenging, and the differential diagnosis would also include cat scratch disease, sporotrichosis,
Table 3 Current treatment recommendations for tularemia Isolated or contained cases Streptomycin 1 g im bid for 10 days Gentamicin 5 mg/kg iv/im daily for 10 days Mass casualty Ciprofloxacin 500 mg po bid for 10 days Doxycycline 100 mg po bid for 14 21 days
See text for pediatric guidelines and recommendations in pregnancy.
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The possibility of drug-resistant organisms must be a consideration in the setting of a bioterrorist incident. Gentamicin is an acceptable alternative to streptomycin for the treatment of isolated or contained cases, and is more readily available at most treatment facilities. A recommended adult dose of 5 mg/kg is administered daily via the intramuscular or intravenous route [4]. Treatment with aminoglycosides should be continued for 10 days. Tetracyclines (doxycycline 100 mg intravenously [iv] twice daily) and chloramphenicol (15 mg/kg iv every 6 hours) have been used to treat tularemia; however, treatment failures and higher relapse rates are reported. Treatment should be continued for 14 to 21 days to reduce the chance of relapse. Relapse is generally acknowledged to be secondary to survival of the organism in intracellular sites, and is more frequently associated with bacteriostatic agents (eg, tetracyclines and chloramphenicol) [2,47]. Fluoroquinolones have demonstrated efficacy in the treatment of tularemia, and ciprofloxacin may be an alternative at 400 mg iv twice daily for 10 days. Treatment reports with fluoroquinolones include success with levofloxacin [48]. The use of macrolide or b-lactam antibiotics is not recommended for the treatment of tularemia [4]. A Jarisch-Herxheimer-like reaction has been described after initiation of treatment for tularemia [3]. Children have been successfully treated with the same antibiotics recommended for adults. Streptomycin is the preferred treatment for isolated cases, and the recommended dose is 15 mg/kg intramuscularly every 12 hours. The pediatric dose should not exceed 2 g/day [4]. Alternatively, gentamicin may be used for the treatment of isolated cases. The recommended pediatric dose is 2.5 mg/kg intramuscularly or intravenously every 8 hours [49]. Pregnant patients should be treated at the same doses as other adults. There is scant evidence to support treatment recommendations in pregnant patients. Gentamicin has not been reported to produce fetal ototoxicity and fetal nephrotoxicity as have other aminoglycosides, and is therefore the preferred antimicrobial choice in pregnancy [4]. Mass casualty treatment recommendations for adults include the use of oral ciprofloxacin or doxycycline. The recommended adult ciprofloxacin dose is 500 mg orally twice daily for 10 days. Doxycycline is recommended at an adult dose of 100 mg orally twice daily. Treatment should be continued for 14 to 21 days [4]. The present antibiotic recommendations for pediatric cases in a mass casualty situation are the same selection as for adults. Pediatric dosing of doxycycline is 100 mg orally twice daily, if greater
than 45 kg body weight. If less than 45 kg, then the recommended dose of doxycycline is 2.2 mg/kg orally twice daily. Pediatric recommendations for ciprofloxacin are 15 mg/kg orally twice daily, with the maximum dose limited to 1 gram per day. In the setting of a mass casualty scenario, the benefits of treating pediatric patients with short courses of fluoroquinolones or doxycycline are generally accepted to outweigh the risks [4]. Oral ciprofloxacin has been reported to be very successful in treatment of pediatric cases without significant adverse reactions to the medication [50]. For pregnant patients in a mass casualty scenario, oral ciprofloxacin is considered the preferred treatment option [4]. Prophylaxis against tularemia is valuable if administered within 24 hours after airborne exposure. A 2-week course of ciprofloxacin or doxycycline is effective as postexposure prophylaxis when given within 24 hours of aerosol exposure [5,51]. As it is unlikely that intentional release of an infective aerosol would be detected within 24 hours, current recommendations are that those potentially exposed begin a fever watch. Standard treatment recommendations would then apply for those that subsequently develop symptoms within 14 days of presumed exposure [4]. A live-attenuated vaccine is available for those at high risk, such as veterinarians or laboratory workers routinely exposed to tularemia. This vaccine is derived from avirulent F tularensis biovar palaearctica (type B), and produces partial protection. Vaccineinduced protection could be overwhelmed by extremely high doses of the tularemia bacteria [5]. It is not recommended for postexposure prophylaxis [4]. Future development of tularemia vaccines is an ongoing effort, and previous endeavors have produced various degrees of success [52 54]. In the setting of an intentional release of an aerosol, persons receiving direct exposure should wash skin and clothing with soap and water. Decontamination of inanimate objects may be performed with a 10% bleach solution, which may be followed by a 70% solution of alcohol to further decontaminate and to decrease the corrosiveness of the bleach solution [4,5]. Quarantine or isolation is not necessary, as no known human-to-human transmission of tularemia has occurred. Therefore, antimicrobial prophylaxis of close contacts of tularemia patients is not recommended [4].
Prognosis In successfully treated patients, most become afebrile within 2 days, but lymph nodes and cutane-
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ous lesions may take an additional 1 to 2 weeks to heal. If treatment is delayed for several days or weeks, the response will take longer with fever possibly persisting greater than 48 to 72 hours after onset of treatment. Late lymph node suppuration may occur regardless of treatment received [3]. Mortality is less than 1% with appropriate treatment. Early treatment can prevent the development of significant clinical findings. If untreated, signs and symptoms typically last 1 to 4 weeks, but may persist for months. Mortality may be as high as 30% in untreated cases of pneumonic or typhoidal tularemia [3]. Lifelong immunity usually results after recovery from the illness. Immunity appears to be primarily cell mediated rather than humoral [3,55].
[8] [9]
[10]
[11] [12]
[13]
Summary
[14]
Tularemia is a bacterial infection usually transmitted via arthropod vectors or direct contact with infected animals. Naturally occurring cases are relatively rare, and can result in different clinical syndromes. Tularemia is also a potential agent of bioterrorism or biowarfare, and is categorized as a high-level threat. Effective antibiotic treatment is available, including the potential use of oral antibiotics in a mass casualty situation. An awareness of the clinical presentations of tularemia will facilitate timely intervention, appropriate diagnostic testing, and decreased morbidity in the event of a biologic attack with F tularensis.
[15]
[16]
[17]
[18]
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S.D. Cronquist / Dermatol Clin 22 (2004) 313320 Hoover DL, Bryne WR, et al. Clinical recognition and management of patients exposed to biological warfare agents. JAMA 1997;278(5):399 411. Mintz J. US provides a peek at air sensor program. Washington Post 2003;November 15:A03. McGovern TW, Christopher GW, Eitzen EM. Cutaneous manifestations of biological warfare and related threat agents. Arch Dermatol 1999;135(3):311 22. Rodgers BL, Duffield RP, Taylor T, Jacobs RF, Schutze GE. Tularemic meningitis. Pediatr Infect Dis J 1998; 17(5):439 41. LeDoux MS. Tularemia presenting with ataxia. Clin Infect Dis 2000;30(1):211 2. Jacobs RF, Condrey YM, Yamauchi T. Tularemia in adults and children: a changing presentation. Pediatrics 1985;76:818 22. Cerny Z. Skin manifestations of tularaemia. Int J Dermatol 1994;54:279. Peter R, Banyai T. Erythema nodosum revealing oculoglandular tularemia. Dermatology 2001;202:79 80. Ruiz AI, Gonzalez A, Miranda A, Torrero V, Gutierrez C, Garcia M. Sweets syndrome associated with francisella tularensis infection. Int J Dermatol 2001;40(12): 791 3. Guerrant RL, Humphries MK, Butler JE, Jackson RS. Tick-borne oculoglandular tularemia. Arch Intern Med 1976;136:811 3. Steinemann TL, Sheikholeslami MR, Brown HH, Bradsher RW. Oculoglandular tularemia. Arch Ophthalmol 1999;117(1):132 3. Ketai L, Alrahji AA, Hart B, Enria D, Mettler Jr F. Radiologic manifestations of potential bioterrorist agents of infection. Am J Roentgenol 2003;180(3): 565 75. Rubin SA. Radiographic spectrum of pleuropulmonary tularemia. Am J Roentgenol 1978;131:277 81. Bevanger L, Macland JA, Naess AI. Agglutinins and antibodies to Francisella tularensis outer membrane antigens in the early diagnosis of disease during an outbreak of tularemia. J Clin Microbiol 1988;26: 433 7. Karhukorpi EK, Karhukorpi J. Rapid laboratory diagnosis of ulceroglandular tularemia with polymerase chain reaction. Scand J Infect Dis 2001;33(5):383 5. Junhui Z, Ruifu Y, Jianchun L, Songle Z, Meiling C, Fengxiang C, et al. Detection of Francisella tularensis by the polymerase chain reaction. J Med Microbiol 1996;45(6):477 82. Robinson-Dunn B. The microbiology laboratorys role in response to bioterrorism. Arch Pathol Lab Med 2002; 126(3):291 4. Weedon D. Skin pathology. 2nd edition. London: Churchill Livingstone; 2002. p. 636. From the Centers for Disease Control and Prevention. Recognition of illness associated with the intentional release of a biologic agent. JAMA 2001;286(17): 2088 90. Werchniak AE, Herfort OP, Farrell TJ, Connolly KS, Baughman RD. Milkers nodule in a healthy young woman. J Am Acad Dermatol 2003;49(5):910 1. Kostman JR, DiNubile MJ. Nodular lymphangitis: a distinctive but often unrecognized syndrome. Ann Intern Med 1993;118:883 8. Russell P, Eley SM, Fulop MJ, Bell DL, Titball RW. The efficacy of ciprofloxacin and doxycycline against experimental tularaemia. J Antimicrob Chemother 1998;41(4):461 5. Aranda EA. Treatment of tularemia with levofloxacin. Clin Microbiol Infect 2001;7(3):167 8. Patt HA, Feigin RD. Diagnosis and management of suspected cases of bioterrorism: a pediatric perspective. Pediatrics 2002;109(4):685 92. Johansson A, Berglund L, Gothefors L, Sjostedt A, Tarnvik A. Ciprofloxacin for treatment of tularemia in children. Pediatr Infect Dis J 2000;19(5):449 53. Sawyer WD, Dangerfield HG, Hogge AL, Crozier D. Antibiotic prophylaxis and therapy of airborne tularemia. Bacteriol Rev 1966;30:542 8. Chen W, Shen H, Webb A, KuoLee R, Conlan JW. Tularemia in BALB/c and C57BL/6 mice vaccinated with Francisella tularensis LVS and challenged intradermally, or by aerosol with virulent isolates of the pathogen: protection varies depending on pathogen virulence, route of exposure, and host genetic background. Vaccine 2003;8:21(25 26):3690 700. Nersesyan AK. Vaccines against dangerous infections and cancer. Lancet Oncol 2002;3(6):331. Ellis J, Oyston PC, Green M, Titball RW. Tularemia. Clin Microbiol Rev 2002;15(4):631 46. Cox SK, Everett ED. Tularemia: an analysis of 25 cases. Mo Med 1981;78:70 4.
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Dermatol Clin 22 (2004) 321 324
A call to armsthe role of the dermatologist as front line responder

Toby Maurer, MD
Department of Dermatology, University of California San Francisco, San Francisco General Hospital, 1001 Potrero Avenue, Building 90, Ward 92, #224, San Francisco, CA 94110, USA
As the risk of bioterrorism becomes an increasingly significant issue in the United States and the world, dermatologists have found themselves as partners or front line participants in the effort to prepare for and respond to such a threat. In what was previously an unfamiliar part of practice for most dermatologists, bioterrorism has required that providers familiarize themselves with the pathophysiology, clinical presentation, diagnosis, and management of diseases from agents that could potentially be used. The threat of bioterrorism was realized when spores of Bacillus anthracis were intentionally distributed throughout the United States postal system in midSeptember 2001. A total of 22 persons developed anthrax and 5 persons died [1]. Dermatologists and other health care personnel aptly recognized the first cases and appropriately collected specimens and alerted public health officials, enabling rapid deployment of health and law enforcement personnel [2]. Medical personnel were also instrumental in providing prophylactic medications to persons who may have had exposure to anthrax [3]. During the response to anthrax, it became clear that other agents of bioterrorism also posed threats. Those biologic agents with primary cutaneous manifestations include smallpox, plague, tularemia, botulism, and hemorrhagic fever [4]. The threat of a smallpox event became the next area of concern. For dermatologists, smallpox held great historical interest; the dermatologic manifestations had been well-described and researched [5].
E-mail address: tmaurer@itsa.ucsf.edu
Very few practicing dermatologists and infectious disease specialists had ever seen a case of smallpox and were unaware of the disease to its fullest extent. The Centers for Disease Control (CDC) and other educational foundations developed Web sites that detailed the disease (www.cdc.gov/smallpox). Guidelines for specimen collection were widely distributed [6]. Every physician and medical institution in the United States received posters with pictures of all stages of smallpox and how to differentiate it from chickenpox. State health departments organized tabletop discussions in which disaster scenarios were played out [7,8]. Response teams were organized at every level of government and public works in the event that such a scenario occurred. Dermatologists and infectious disease specialists were called upon for their expertise in these matters. Not only were dermatologists asked to correctly identify disease, they were also expected to appropriately exclude diseases that were part of the differential diagnosis for bioterrorist-related skin diseases. Although rickettsialpox has always been endemic in New York City, 18 cases were diagnosed within a 20-month period after October 2001, representing a threefold increase in the number of cases compared with the previous 5 years [9]. Patients presented with fever, eschars, and papulovesicular eruptions. It was thought that after the anthrax attacks, clinicians and patients were more observant and concerned about such symptoms. Acute clinical judgment and rapid diagnostic tests reassured patients and stopped unneeded responses. With smallpox as an impending threat, the question arose as to how to vaccinate individuals once there was an event. The controversy was whether or
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not Americans should be vaccinated before an outbreak event versus organizing public health response teams that would be ready to engage in mass vaccination after an outbreak event had occured. On December 13, 2002, President Bush announced the Smallpox Vaccination Program, in which vaccine was recommended for smallpox response teams, health care workers, and emergency response teams. The plan for the initial phase was to involve approximately 500,000 civilians. At the same time, the United States military was preparing to enter into action in the Persian Gulf, where it was expected that personnel would have to be vaccinated against smallpox and anthrax. It was also expected that each state would formulate its own plan as to how mass vaccination would take place in the event of an outbreak; each state had to submit this plan to the CDC [10,11]. Before 1968, smallpox vaccine had been given nationwide on a regular basis, but once the risks of vaccine outweighed the threat of disease, the vaccine was stopped. The most frequent side effects were dermatologic and included inadvertent inoculation, eczema vaccinatum, generalized vaccinia, and progressive vaccinia. Because certain groups of dermatologic patients were at higher risk for developing these reactions, dermatologists were asked to aid in screening out these individuals for vaccination [12,13]. Dermatologists were also asked to define atopic dermatitis. They were asked to set guidelines that were easy to understand and that could be used by public health providers to screen out atopic individuals and others with skin disease that put them at risk for vaccination (American Academy of Dermatologys Supplemental Statement Regarding Contraindications to Smallpox Vaccination. Available at http://www.bt.cdc.gov/agent/smallpox/clinicians. asp#contra/skin%20conditions.). After vaccination, dermatologists were called upon to identify and help in the surveillance of side effects [14]. Over 100 Web sites sprang up to help providers understand the screening criteria as well as recognize the side effects [15]. Dermatologists volunteered at local and state levels to help with screening and surveillance. As part of this effort, dermatologistsespecially those who were part of a first response teamvolunteered to be vaccinated. Others found themselves in the center of the action as they were faced with questions from those who wanted to be vaccinated or from those who were experiencing a skin reaction after vaccination. Did the screening efforts work? As of February 2004, 581,000 persons in the US Department of Defense and 40,000 civilians have been vaccinated. There have been no cases of eczema vaccinatum or
progressive vaccinia. There have been eight probable cases and one confirmed case of generalized vaccinia, all mild and without sequelae [16]. Much of the success of the program has been attributed to excellent screening of high-risk individuals. Unexpected cardiac conditions have halted the expansion of phase 2 of the program, however [17,18]. Physicians have always understood risk and have provided health care for patients and the public, even when personal injury is at stake. Every provider should be able to recognize risk, identify events, and arrive at appropriate treatment decisions. In a national survey, few physicians thought they were well prepared for bioterrorism. Most noted that they would continue to care for patients in the event of a potentially deadly illness, though only a minority believed that it was their professional duty to treat patients through an epidemic [19]. A survey was sent out to 1000 randomly selected members of the American Academy of Dermatology (AAD) to assess how prepared they were to handle a bioterrorist event. Only 11% felt they were prepared to respond to an attack. Fifty-five percent said they would be able to recognize bioterrorist-related cutaneous manifestations, but 54% did not know where to report possible infections. Over 80% of responders were willing to participate in an AAD-sponsored online training program [20]. As of summer 2004, the volunteer smallpox program has virtually come to a halt and involved far fewer persons than was initially planned for phase 1. The CDC and other organizations are trying to understand why people declined vaccination. A survey to assess physician responses at Yale University with regard to smallpox vaccine noted that only 5% of responders had been or intended to be vaccinated. Of those who declined, 55% believed that the benefits did not outweigh the risks of vaccine, 18% considered the vaccination program to be unnecessary, 11% wanted to see what the side effects were, and 7% were concerned about compensation or liability. Ninety-four percent of responders who declined vaccination considered it a risk to themselves, their family, and their patients [21]. The University of Colorado Department of Dermatology is conducting a similar survey to assess dermatologists responses to smallpox vaccinations (R. Dellavalle, personal communication, 2003). What we do know from the CDC program is that there has been limited contact vaccinia between individuals outside intimate settings; the limited cases are usually due to breech of dressings. There has been no health provider to patient transmission of vaccinia. Although it had been recommended that health care
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personnel be kept away from immunosuppressed patients for 3 weeks postvaccination, in the military setting this was not done and there still was no transmission of vaccinia from health care personnel to patients [16,22]. There have been concerns about some of the implementation of these programs on the public health infrastructure; resources from traditional programs like routine immunizations have been pulled. A recent study showed that most centers that were involved in preparedness noted that there was more visibility and credibility for public health and that communication and coordination was improved compared with what was available previous to fall 2002 [23]. An example of this strengthened system is the rapid detection and reporting of monkeypox [24]. On May 24, 2004, a young girl was bitten by a pet prairie dog and developed a pustulovesicular lesion, fever, and chills. In the same state, another man had also been bitten by a prairie dog and developed similar symptoms. Astute clinical judgment and laboratory techniques that had been developed for smallpox preparation allowed for identification of monkeypox. Because state and federal agencies were set up to work with local agencies, rapid diagnosis and surveillance were possible. Alerts went out immediately, 72 cases were identified, and prairie dogs were no longer sold as pets. What could have become an epidemic quickly was stopped. What is the future for dermatologists in the field of bioterrorism? Because every state has an emergency plan in which they have partnered with local public health officials, the dermatologist can enter the system through local health departments. During states of emergency, dermatologists can volunteer with the National Medical Disaster System, which is administered by the Public Health Service and organizes federal and nonfederal agencies (www. ndms.dhbs.gov) to provided emergency care. The Medical Reserve Corps, led by the Office of the Surgeon General, also organizes emergency care for local communities (www.medicalreservecorps.gov). Although it appears that the fear of bioterrorist attacks has lessened, it has been noted that a threat remains and preparedness is an ongoing event [2]. The challenge is to creatively keep health care providers informed and to allow systems to exist whereby there can be rapid diagnosis and quick deployment of resources. Web sites that provide educational resources include the CDC at www.bt.cdc.gov and the AAD at www.aad.org. Vaccination continues, certainly amongst the military, but possibly also in the civilian population, as there is planning for revaccination of the currently
vaccinated core of individuals. Also, new vaccines have been developed and several clinical trials are ongoing [25]. These endeavors require dermatologic expertise. In the area of education and definitions, dermatologists continue to share their expertise in their field to allow for the discovery of new side effects of vaccines. They also ensure that diseases involving the skin are interpreted correctly and make sense from scientific and pathophysiologic perspectives. The Brighton Collaboration is an international effort to consolidate definitions of disease as a result of vaccination, and they are presently expanding into the field of dermatology. There is an ongoing need for dermatologists to remain engaged in the area of bioterrorism, to continue educating themselves and other professionals in infectious disease that affect the skin, and to continue to respond to the call to arms.
Acknowledgments I would like to thank Dr. Boris Lushniak for providing the contact information for volunteer dermatologists.
References
[1] Jernigan J, Stephens DS, Ashford DA, Omenaca C, Topiel MS, Galbraith M, et al. Bioterrorism-related inhalational anthrax. Emerg Infect Dis 2001;7:933 44. [2] Gerberding JL, Hughes JM, Koplan JP. Bioterrorism preparedness and response. JAMA 2002;287:898 900. [3] Update. Investigation of bioterrorism-related anthrax and adverse events from antimicrobial prophyslaxis. MMWR Morb Mortal Wkly Rep 2001;50:973 6. [4] Varkey P, Poland GA, Cockerill 3rd FR, Smith TF, Hagen PT. Confronting bioterrorism: physician on the front line. Mayo Clin Proc 2002;77:619 21. [5] Fenner F, Henderson DA, Jezek A, Landyl ID. Smallpox and its eradication. WHO; 1998. p. 1 68. [6] Medical treatment and response to suspected smallpox. Information for health care providers during biologic emergencies (draft). New York City Department of Health Bureau of Communicable Diseases, July 2000. [7] OToole T. Smallpox: an attack scenario. Emerg Infect Dis 1999;5:537 9. [8] Bardi J. Aftermath of a hypothetical smallpox disaster. Emerg Infect Dis 1999;5:547 51. [9] Koss T, Carter EL, Grossman ME, Silvers DN, Rabnowitz AD, Singleton Jr J, et al. Increased detection of rickettsialpox in a New York City hospital following the anthrax outbreak of 2001: use of immuno-
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T. Maurer / Dermatol Clin 22 (2004) 321324 histochemistry for the rapid confirmation of cases in an era of bioterrorism. Arch Dermatol 2003;139: 1545 52. Recommendations for using smallpox vaccine in a pre-event vaccination program (February 2003). Supplemental recommendations of the Advisory Committee on Immunization Practices (ACIP) and the Healthcare Infection Control Practices Advisory Committee (HICPAC). Recommendations for using smallpox vaccine in a pre-event vaccination program (April 2003). Supplemental recommendations of the Advisory Committee on Immunization Practices (ACIP) and the Healthcare Infection Control Practices Advisory Committee (HICPAC). Kempe C. Studies on smallpox and complications of smallpox vaccination. Pediatrics 1960;26:176 89. Lane J, Ruben F, Neff J, Miller J. Complications of smallpox, 1968: national surveillance in the United States. N Engl J Med 1969;281:1201 8. Cono J, Casey CG, Bell DM. Smallpox vaccination and adverse reactions. MMWR 2003;52:1 28. Ferguson NE, Steele L, Crawford CY, Huebner NL, Fonseka JC, Bonander JC, et al. Bioterrorism Web site resources for infectious disease clinicians and epidemiologists. Clin Infect Dis 2003;36:1458 76. Updates on smallpox vaccine adverse events among US civilians. MMWR 2003. [17] Cardiac adverse events following smallpox vaccinationUnited States, 2003. MMWR 2003;52:248 50. [18] Updates. Cardiac-related events during the civilian smallpox vaccination programUnited States, 2003. MMWR 2003;52:492 6. [19] Alexander GC, Wynia MK. Ready and willing? Physicians sense of preparedness for bioterrorism. Health Aff (Millwood) 2003;22:189 97. [20] Carroll C, Balkrishnan R, Khanna V, Feldman S. Bioterrorism preparedness in the dermatology community. Arch Dermatol 2003;139:1657 8. [21] Denin AL, Dembry L, Sharpiro ED, Holmboe ES. Reasons physicians accepted or declined smallpox vaccine, February through April, 2003. J Gen Intern Med 2004;19:85 9. [22] Secondary and tertiary transfer of vaccinia virus among US military personnelUnited States and worldwide, 2002 2004. MMWR 2004;53:103 5. [23] Staiti AB, Katz A, Hoadley JF. Has bioterrorism preparedness improved public health? Issue Brief Cent Stud Health Syst Change 2003;65:1 4. [24] Reed KD, Melski JW, Graham MD, Regnery RL, Sotir MJ, Wegner M, et al. The detection of monkeypox in humans in western hemisphere. N Engl J Med 2004; 350:324 7. [25] Rosenthal SR, Merchlinksky M, Kleppinger C, Goldenthal KL. Developing new smallpox vaccines. Emerg Infect Dis 2001;7:920 6.
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Current therapy
Herbal and complementary medicine in dermatology

Alan M. Dattner, MDa,b,c,*
Integrative Medicine and Dermatology, 17 Rodman Oval, New Rochelle, NY 10805, USA b HealthDataLink, 17 Rodman Oval, New Rochelle, NY 10805, USA c ImmunoClarity(SM) Research Associates, 17 Rodman Oval, New Rochelle, NY 10805, USA
a
Long before the era of modern purified pharmaceuticals, botanical products were used or extracted in their whole natural form. Carefully packaged packets of pressed herbs labeled Parke Davis and Co, which I keep in an antique tin in my study, attest to the fact that herbal use in medicine in this country was the norm until relatively recently. The majority of humans on our planet still depend on natural crude botanical preparations for their health treatment. The question on most physicians minds is why they should go back to using traditional crude botanical medicines when science has brought us powerful products that have been purified to contain just the active ingredients? This article will answer that question by putting the state of our scientific knowledge of the truth in perspective, and will introduce you to some principles and specifics of complementary medicine that you can use in your practice to enhance the efficacy of your therapeutic armamentarium. Complementary medicine is more than just using agents from a quaint pharmacopoeia to substitute for drugs in treating illnesses. It is the application of other diagnostic systems for evaluating the imbalance of a specific individual patient that has manifested in what we name as a disease. Often the principles of a system used will dictate a series of choices, from diet to therapeutic substance, and those choices will depend at least as much on the characteristics of the patient as the nature of the chief complaint or the disease in question. This can be a powerful tool to
split apart subgroups into those who respond to vastly different therapeutic approaches according to the genetics, exposure, history, and unique physiology that has led to a condition. As such, it can complement the powerful tools that we already have, and enable us to gain control of chronic diseases that have previously baffled us. Other systems have an observational basis that may extend over hundreds or even thousands of years. To extract the best information to integrate into dermatology, studies on treatments from other healing systems must be conducted from the perspective of that system from which it is derived. We are obliged to embrace other healing systems within the framework of dermatology because of the therapeutic efficacy they convey, if we are to continue to maintain respect for dermatologists as the ultimate healers of the skin. Many of our patients are using or seeking alternative treatments, so an understanding will help us counsel them wisely to get the best possible result with the combined treatment, or to stop them to eliminate untoward interactions.
Why use whole herb preparations or crude extractions First, there exists a long history and lore of traditional use for a wide variety of plants, which includes not only specificity of preparation, but when and how to harvest the plants. Indications for use are based not only on the disease process, but on specific characteristics of the individual patient, especially in Ayurvedic, Chinese, and certain Western schools of herbal prescribing. Pharmaceutical preparations gain potency by following the scientific wisdom of extracting the one
* Integrative Medicine and Dermatology, 17 Rodman Oval, New Rochelle, NY 10805. E-mail address: drdattner@yahoo.com
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component of a plant that has the most activity in producing the wanted effect. This is in direct contrast to herbal tradition, which uses whole plant extracts for their power. The plants have evolved to contain multiple constituents to support each crucial function, such as protection from oxidative damage from the sun. The products that make up the traditional crude extract of the plant may act synergistically to support the action of that prime constituent. British sailors discovered that eating limes and citrus fruit prevented the vasculitic disorder known as scurvy. Scientists later identified ascorbic acid, or Vitamin C, as the critical ingredient, and we now can buy pills of purified white crystalline vitamin C in the supermarket to supplement our diets. But the plant needs more than vitamin C as its own antioxidant protective against solar radiation, and contains a spectrum of flavinoids that accompany the vitamin C as protectants. Similarly, prescribing the whole orange, rose hip or acerola, rather than just the Vitamin C extract is more useful as an antioxidant or capillary protectant. A parallel story comes from the French explorer Jacques Cartier, whose men developed scurvy and were saved by a Native American who made them tea from pine bark and needles, which much later led to the French discovery of Proanthocyanadins [1,2]. These flavonoids have been well demonstrated to function in protecting the capillaries [3]. Many examples of different sort can be sited to support this. I have found that nearly half the patients with senile purpura I treat with bioflavinoids have an improvement of their condition. Bioflavinoids are found in the white of the skin in citrus fruit, in rose hips, acerola, grape seed extract, pacific maritime pine bark (called oligoproanthocyanidins). I give them with vitamin C 1500 to 3000 mg per day in three equal doses, at a dose of 1000 to 3000 mg per day of citrus bioflavinoids or 1 mg per pound body weight of oligoproanthocyanidins, which are much more expensive. Treatment of age-associated capillary leak syndrome, known as senile purpura, remains one of the major areas of nutritional undertreatment by dermatologists today. What specialty can better help patients with these preparations? In whose better hands can the parameters of the effects of this class of dietary antioxidants become observed and defined through the experience of clinical practice? Whether you use food or concentrated supplement, and follow the English observation of citrus bioflavonoids, the French experience of grape seed and pine product, or some other, it is time to make recommendations for your patients with senile purpura, and other benign
forms of capillaritis as well. The dark side of the scientific inquiry that has brought us so much progress in dermatology is the arrogance that decries observations before the scientific mechanisms are fully understood and double blind studies are complete. That alienates the patients who need to look beyond what medicine has to offer. With bioflavonoids, we can offer help for senile purpura, and other capillary disorders, now. I will mention a few other examples of the evolution that has led me to see new components that were inherent in the folk wisdom but not the original knowledge of the product. I originally assumed that cod liver oil was effective because of the Vitamin A functioning as an immune enhancer [4,5]. Now I understand the importance of the omega three unsaturated fatty acids like EPA and DHA [6] as critical ingredients contributing to calm down the immune hyper reactivity. These omegathree essential fatty acids from fish oil are antiinflammatory because they lead to the production of the antiinflammatory prostaglandin, PGE-3. They also inhibit clotting, and in occasional patients, will lead to an increased tendency toward bleeding. Some pregnant women deliver late term babies on high doses of fish oils.
My approach to complementary medicine After making a dermatologic diagnosis, I take a detailed history of the events and exposures preceding outbreaks. If I cannot find immune or other etiologic explanations to explore in connecting exposures with skin eruption, I explore other systems to hypothesize an etiologic diagnosis. For example, Jerome Litts extensive compilation of drug associations with skin disease [7] makes it easier to hypothesize specific pharmacologic agents as inciting skin diseases. The list of causes of lichenoid eruptions has grown considerably since the few items mentioned in Fitzpatrick [8] during my residency time. When an eruption does not clear after removing the drug, I consider herbal concepts of enhancing clearing by the liver, kidney, or whatever organs are known to act in metabolizing the drug. I use the principle of starting with the most gentle, least invasive treatments first, so I might well use dandelion leaf (kidney) [9] and root (liver), silymarin, or preparations containing these to enhance clearing in a chronic condition. Dandelion (Taraxacum officinale) contains the bitter glycoside taraxacerin as well as phytosterols, inulin, and saponins [10]. A water extract of dandelion was shown to slow down phase
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one hepatic detoxification of P450 E2 by 48% and speed up phase II detoxifying enzyme UDP-glucuronosyl transferase by 248% [11]. That would benefit a patient by making sure that the conversion of toxins to more reactive molecules for transport was slowed and the transport molecules were more available, to prevent the unfortunate situation of having an increase of toxins that are more reactive, water soluble, and not being removed from the body. Many healing systems see the skin as an organ of elimination, so treatment is aimed at enhancing the efficacy of other organs of elimination such as the bowel, kidneys, and liver. Silymarin is one of the major components of the milk thistle (Silybum marianum) seed, which has been shown to have numerous mechanisms by which it protects and helps regenerate the liver [12,13]. Well over 500 articles concerning the hepato-protective, antioxidant, anticarcinogenic, and other effects of silymarin are currently cataloged in the peer-reviewed scientific literature. Of particular interest to dermatologists is a study showing protection of cultured human hepatocytes from cytotoxicity induced by the combination of methotrexate and the cytochrome P450 2E1 inducers, ethanol or acetaminophen, by silymarin [14]. Burdock (Arctium lappa) is another mainstay used by herbalists as a detoxifier for most inflammatory skin conditions, which has hepatoprotective [15] and antiinflammatory effects [16] demonstrated. It can be used as a tea, a tincture, or prepared in food as a root. The Japanese experience of centuries of use of this herb (gobo) as a food argues for its safety. Herbal products have been in constant use since prerecorded history, and many are generally regarded as safe. The ratio of toxic to therapeutic dose is usually much lower for plant-derived products than pharmaceutical preparations. There are a number of therapeutic actions that can be obtained from herbal preparations for which there are no existing pharmaceutical preparations available. This is a most important complementary use of herbs that will be further expanded on in this article. Similarly, herbs may be substituted for drugs when the side effect is unacceptable, as in causing a skin rash, but there is no other drug available to get the same necessary clinical effect for the patient. Of course, on some occasions, such as when the hypertensive patient is breaking out from various classes of blood pressure medicines, it may take a lot of skillful diagnostic work and a total program of dietary and lifestyle changes besides the herbs, to bring that patients blood pressure within normal range. On the cautionary side, herbs are also weeds, and as such, can induce allergic reactions, especially
if the patient has sensitivities to products in the same family as the herb being considered. That is an important piece of history to obtain if you are recommending plant products. Plant products have multiple active components, and must be researched or observed carefully when combined with drugs, especially if the drugs dose range is critical, the patients condition is tenuous, or the herb is used for a long time. Some herbal products are adulterated with other herbs, and as such have caused toxic reactions. It is important to use a company that identifies the raw herb coming in by its characteristic properties (analeptic testing) as well as checking batches with high-pressure liquid chromatography to ascertain that proper peaks are present. I know the herbalists who run most of the herb companies whose products I use, and do not feel comfortable using products I dont know at all, any more than I like readings from a pathologist I dont know. I also tend to stick to herbs that are neither toxic, reserved for experts, nor tricky to use.
Antiinflammatory herbs and complementary techniques A large proportion of skin disorders are caused by inflammatory processes, accounting for the wide variety of corticosteroid preparations in our pharmacopoeia. These products produce dramatic results acutely, and can be practically curative in the case of self-limited processes from acute exposures to an allergen. In chronic cases, tachyphylaxis, plus the squellae of thinning and telangiectasia of the skin, as well as the chances of secondary infection, makes chronic use more problematic. As we choose from a wider palate of steroid strengths, so, too, should we be aware of herbal preparations with antiinflammatory properties. Chamomile (Matriarcha chamonilla) cream has been demonstrated to have antimicrobial action [17] antiinflammatory properties similar to that of .05% hydrocortisone [18]. Calendula (Marigold, Calendula officinalis) also has mild antiinflammatory effects [19], which I have written about previously, as well as positive effects on reepithelializtion, which may contribute to its wound healing effects [20]. Licorice (Glyceyrrhiza glabra), widely used in Chinese medicinal preparations, exerts a steroid sparing effect, likely due to slowing the reuptake and metabolism of cortisol by inhibiting 11B-hydroxysteroid dehydrogenase. Inhibition of the complement cascade centered around C 2 has also been demonstrated [21]. It can be given to enhance the effects of
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corticosteroids, and may have a role in patients care during the process of tapering corticosteroid oral or topical medication. It is a mineralocorticoid also, which can cause potassium depletion and sodium retention leading to hypertension, especially when used in doses above 5 g/day [22]. Identifying and eliminating the antigenic stimulus for an allergic reaction is another technique for treating inflammatory skin conditions. We are long familiar with the association of Group A streptococcus with acute guttate psoriasis. More recently, scientific consensus has identified psoriasis as an immune mediated disorder, and a host of new highly sophisticated agents are emerging from the pharmaceutical pipeline to specifically target and inhibit the immune sequence involved in the development of the psoriatic lesion. Although enthusiasm is high, based on patients who have been helped with with dramatic clearing, the specter of potential side effects of immune depression in the wrong subset of patients looms, as these drugs are deployed on a larger population.
Candida and inflammatory disease of the skin The yearly costs of these new silver bullets, the cost of lab monitoring, and the risks, may drive some dermatologists to follow the methods of Rosenberg and others in addressing chronic yeast and other problematic gut organisms as part of the solution to psoriasis [23]. For me, this involves not a 10-day course of fluconazole (Diflucan), but a slow progressive program starting with elimination of yeast, fungus, and their byproducts from the diet, as well as marked reduction in sugar and simple carbohydrates. This is followed by the addition of probiotic bacteria such as Lactobacillus acidophilus, bifidus, and Sacchromyces boulliardi with each meal, to favor growth of a more normal bacterial flora. Mild antiyeast products are then added, such as Caprylic acid, followed by stronger botanicals such Artemesia annua [24] or Grapefruit seed extract. Subsequently, if necessary, pharmaceutical agents, such as nystatin added, and this at increasing doses over a 2-week period building up to 6 million units per day given in three to four divided doses. Only then, when gut yeast populations are reduced, when their ecologic niche is constricted by removal of dietary enhancing factors, and when gut introduction of related yeasty foods that could induce high-dose immune tolerance to yeast have been eliminated, should one consider using medications like ketoconazole (Nizoral) or fluconazole (Diflucan) to further
treat the problem. To do so before reducing the yeast population and changing the ecologic yeast-favoring growth conditions in the gut could allow the growth of resistant strains such as Candida tropicalis to fill the niche. You would not be able to control this organism easily with anything in our current formulary. So, after their brief improvement, some of those chronic yeast patients treated acutely with big guns will have been done a distinct disservice. There is no question that this yeast elimination treatment is difficult, and a significant proportion of patients will not be able to maintain it for reasons of food addiction, lack of will power, or inconvenience with their busy schedules. Some patients even behave as if the organisms are sending neurohumoral signals to their brains to keep the sugar and simple carbohydrates coming to feed the yeast. As in the case of wheat elimination for dermatitis herpetiformis, gluten elimination is also difficult because of widespread inclusion in products, the difficulty of finding products that do not include it in convenience stores, and legal loopholes that allow a product containing gluten or yeast to be incorporated in another product without labeling its inclusion in the final product. This may be one of the reasons why investigators during the 1970s were able to demonstrate the success in Great Britain of using a glutenfree diet to treat dermatitis herpetiformis, while researchers in the United States could not. Also, it takes a practice or forum filled with highly sensitive patients to gluten or yeast to discover which products are gluten and yeast free, and which products have suddenly included them.
Reducing inflammation from food allergens Other methods for reducing antigen load to reduce causes of skin inflammation include identifying and eliminating food allergens, and improving the digestive process to better degrade antigenic substances in the food [25]. Much of what we see in dermatology is inflammatory reaction to unidentified antigenic stimuli, so it is important to address sources of antigen that incite skin inflammation by their crossreactive stimulus of inflammatory attack of the target. Identifying and removing other gut pathogens and parasites with the help of laboratories that specialize in this process, can reduce antigenic stimulation from those organisms. Removal of parasites will also reduce the inflammation of the gut wall, reducing the increased leakage of other intraluminal antigens into the Peyers patches and circulation to cause more reaction
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Topical and other preparations may be used to reduce itching My favorite herb for insect bites and stings is plantain. Fresh leaves are crushed or chewed and applied to the bite and held in place. The plant is found on lawns where herbicides have not been applied. I have been working on a preparation that can be packaged and still maintain the potency of the fresh plant ReLeaf [26], but until that is ready, I prefer the fresh plant to commercial preparations. Nettles (Urtica dioica) is well known for its antiallergic effects [27]. It can be used taken orally, as a tincture, combined with other herbs in a formula, or eaten cooked as a green. I have found that Hesperiden, a bioflavinoid from citrus fruit, can be useful as an antiinflammatory. Quercitin, taken orally, has antiinflammatory properties based on its inhibition of enzymes of prostaglandin formation [28], and its stabilization of mast cells and basophils, preventing histamine release [29]. Chickweed (Stellaria) is a common plant that has mild antiinflammatory properties. It can be applied as a poultice of crushed leaves, or as a cream containing this ingredient.
Immune stimulators Another complementary use of herbs is in stimulating the immune response. These herbs are a spectrum of preparations that predate and go beyond our limited formulary of immunostimulants such as imiquimod (Aldara), which is already showing new applications. Chinese formulas have addressed this process of general support of the immune system for centuries. A few of the critical ingredients of the fu zhen formulas include Astragalus root (Astragalus membranaceous) and Reishi mushroom (Ganoderma lucidum). Astragalus root is sold as tincture, or as tongue depressor-shaped root slices in Chinese herb shops. A few slices can be boiled in soups during the cold season, adding a slightly sweet taste, or chewed to extract the flavor. Studies by Mavligit at M.D. Anderson confirmed its immunostimulatory properties [30]. It would be useful whenever there was need to enhance cellular immunity, such as in chronic infection, debilitated patients, and possibly in enhancing rejection of warts or even tumors. Reishi mushroom (Ganoderma lucidum) is a woody mushroom that must either be broken down and extracted in alcohol, or finely shredded and boiled for an hour to extract immunostimulatory ingredients. It is also useful for enhancing cellular immunity, as with Astragalus, above. It also represents a class of immunostimulatory mushrooms including Maitake (Grifola frondosa) [31] (found in this country around the base of oak trees in fall as Hen of the Woods), Coreolus versicolor form China [32], and Shitake (Lentinula edodes), found in oriental cooking. A related preparation is MGN3, a modified arabinoxylane from rice bran, which has been found by one investigator to increase NK cells in cancer patients and to increase TNF-a production [33]. These preparations are all relatively safe except for the chance of sensitivity inherent in any plant product, and in some cases the expense or work of preparation. I trust that astute readers will incorporate them in their treatment regimens where they seem most appropriate, and further delineate their spheres of usefulness in dermatology.
Using herbs to complement pharmacologic therapy A number of medications used in dermatology have various degrees and likelihoods of inducing hepatotoxicity. These include methotrexate on the higher end, and ketoconazole and griseofulvin on the less likely pole. It would make a lot of sense to begin treatment of such patients with a benign, food grade herb like silymarin (milk thistle) and continue that herb throughout the treatment course to reduce the chances of liver damage. It might also be useful, in cases where liver damage was more likely, to do a functional evaluation of phase I and phase II liver detoxification in that patient. Great Smokies Diagnostic Laboratories can provide tests to help better know the status of phase I and II liver detoxification, which helps in know what cofactors to supply to the patient to restore ability to metabolize and excrete the drug being given. A concern after prolonged use of systemic corticosteroids is adrenal suppression. As mentioned before, licorice root can be used to aid the adrenal glands. Pantothenic acid (500 mg one to three times/day) and vitamin C can also be added for this purpose.
Homeopathics Homeopathy is a system that employs small (in the concentration range of 106 to 101000) doses of a substance to cause the body to complete the defensive reaction that would resolve its current
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symptomatology [34]. The purpose is to cause body to react with the opposite, or sometimes the same, effect as the substance itself produces. For example, small doses of the emetic Nux vomica may be employed to treat nausea. We are familiar with the concept of low doses used to immunize, but homeopathy sometimes involves dilutions so small that they exceed Avogadros number, and this concept of just the energetic presence of the substance remaining is a challenge to our scientific thinking. Nevertheless, the pharmacopoeia is made from a very carefully compiled set of observations that has grown over the past 150 years. It is a compilation of the associations between specific homeopathic remedies and the specific characteristics and symptoms of the patient, including and beyond the diagnosis and chief complaint. I have watched a colleague physician, Dr. William Shevin, use this system for 2 decades to help a loyal following of patients who were not adequately helped by conventional medicine alone. One of the homeopathic preparations I use most is Arnica montana, which is known for reducing pain and swelling and promoting healing after trauma. I give it to most of my patients postsurgery, and have found that they often did not need to use either Tylenol or codeine for pain after the procedure. I shared this with a few dermatologic surgeons, who seemed interested, but raised their eyebrows at the idea. I was pleasantly surprised later to hear a plug for use of Arnica at the American Academy of Dermatologys advanced plastic surgery course. I have never seen side effects, as the dose I usually use is 12 C (12 100-fold dilutions) which is right around Avogadros number, so there is likely only 1 molecule of Arnica present. Patients take two pills under their tongue after surgery, and a few every half hour that there is pain, or twice a day to promote healing. Another homeopathic I use is called Rhus tox, which you may recognize as extract of Rhus toxidendricorum or poison ivy. More classically used for arthritis by homeopaths, there is a small tradition of using low doses to prevent or even treat poison ivy and related Rhus dermatitis. I got a protocol from an elder homeopath in Washington State, and administered two tabs of Rhus tox 6 (six 10-fold dilutions of the Rhus) under the tongue one to two times per day to a group of people who had problems with poison ivy. This would presumably have some actual antigenic components, although in very low dosage. Some of those patients were convinced that they did not break out as severely upon contact as they had before, developing only a small localized rash instead of a progression of lesions that characterized their
past exposures. It is possible that the prevention information I gave at the same time contributed to the good result. It certainly did not work for everyone. I would still consider using it in patients who had a real seasonal problem with poison ivy or poison oak. One patient out of about 30 developed cold symptoms that could have been just a cold, or could have represented what as known as a proving of the remedy. The homeopathic repertoire is composed of the observations of many homeopaths recording symptoms or provings after taking a given remedy.
Melanoma Melanoma behaves as if it is influenced by immune, dietary, and mind body methodologies, covering the whole spectrum of complementary or holistic treatment. One of my first experiences with the power of immune enhancement with melanoma, in 1974, was injecting BCG into metastatic nodules of the forearm on a patient, under the tutelage of Dr. Mark A. Hardy, and watching the patient clear not only those lesions, but some distant metastases as well. This distant clearing does not occur in all patients, and those with already severely compromised immune systems are at risk for fatal BCGosis. In a psychoneuroimmunologic vein, I wrote a letter suggesting that the IRS not rake that patient over the coals for some back issue that only involved him by association. I have no doubt that the stress that they could have subjected him to would have tipped the scales of his precarious balance of immune control, keeping his metastatic melanoma from taking over. Another approach to melanoma is the Gerson Program, consisting of a low-sodium high-potassium lactovegetarian diet administered via fresh raw vegetable and fruit juice hourly, has anecdotal and some study evidence of value in Melanoma treatment [35]. It also requires coffee enemas, presumably to accelerate removal of toxic products from the biliary and enterohepatic circulation. A patient who visited me with persistent rectal problems from that treatment had a biopsy report of primary melanoma and a convincing story of clearing metastatic melanoma using the Gerson method. Most impressive was the fortitude and commitment of the patient and his wife, who hand squeezed a dozen glasses of vegetable juice for him every day for 22 months. There was a palpable sense of something far beyond diet in that couple who would not resign themselves to accept that there was a fatal diagnosis with no treatment. I assume that both the Gerson program and his marriage were critical to his apparent recovery.
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Summary A number of different systems, principles, and specific treatments have been touched upon in this article. I have emphasized that there are other ways that illness is understood by different healing traditions, and that we can look to see the value they may have, and incorporate their perspective to complement our treatment. Often, this involves steps to address earlier steps in the causal chain, which has lead to a skin disease. They may even be clearly understood when examined scientifically, especially as science evolves to answer the question on new levels. Several specific remedies are mentioned, such as the flavinoids for capillary fragility, which the dermatologist can use in his practice with great benefit and little risk, to have a primary experience of the complementary techniques discussed here. Observations made by the practicing physician with an understanding of dermatology, the underlying basic science, and the healing system from which herbal remedies come, will lead to new complementary treatment combinations tailored for individual patients who present with challenging skin disorders.
Acknowledgments Previous consultation was done for Abkit, Inc., distributor of Camocare cream, and a research study was done for Maitake Products, Inc.
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2004, Vol.22, Issues 3, Bioterrorism

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2004, Vol.22, Issues 3, Bioterrorism

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Dermatol Clin 22 (2004) ix x

Boni E. Elewski, MD Guest Editor

B.E. Elewski / Dermatol Clin 22 (2004) ixx

Dermatol Clin 22 (2004) 231 246

The history of biologic warfare and bioterrorism

E-mail address: mj@alumni.duke.edu

M.K. Jacobs / Dermatol Clin 22 (2004) 231246

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M.K. Jacobs / Dermatol Clin 22 (2004) 231246

M.K. Jacobs / Dermatol Clin 22 (2004) 231246

M.K. Jacobs / Dermatol Clin 22 (2004) 231246

In both incidences, it is unclear if Harris had any terrorist intent [44].

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Dermatol Clin 22 (2004) 247 256

K.A. Wenner, J.R. Kenner / Dermatol Clin 22 (2004) 247256

K.A. Wenner, J.R. Kenner / Dermatol Clin 22 (2004) 247256

K.A. Wenner, J.R. Kenner / Dermatol Clin 22 (2004) 247256

K.A. Wenner, J.R. Kenner / Dermatol Clin 22 (2004) 247256

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Dermatol Clin 22 (2004) 257 262

W.B. Henghold II / Dermatol Clin 22 (2004) 257262

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W.B. Henghold II / Dermatol Clin 22 (2004) 257262

Dermatol Clin 22 (2004) 263 274

Smallpox: the basics

* Corresponding author. E-mail address: hanifinj@ohsu.edu (J.M. Hanifin).

M.K. Slifka, J.M. Hanifin / Dermatol Clin 22 (2004) 263274

M.K. Slifka, J.M. Hanifin / Dermatol Clin 22 (2004) 263274

M.K. Slifka, J.M. Hanifin / Dermatol Clin 22 (2004) 263274

M.K. Slifka, J.M. Hanifin / Dermatol Clin 22 (2004) 263274

M.K. Slifka, J.M. Hanifin / Dermatol Clin 22 (2004) 263274

Mortality % (unvaccinated/vaccinated) 0/0 0/0 30/3 37/8 62/26 97/67 96/94

M.K. Slifka, J.M. Hanifin / Dermatol Clin 22 (2004) 263274

M.K. Slifka, J.M. Hanifin / Dermatol Clin 22 (2004) 263274

then forearms, upper trunk, and lower extremities

any one area of the body

cles to umbilicated pustules

M.K. Slifka, J.M. Hanifin / Dermatol Clin 22 (2004) 263274

M.K. Slifka, J.M. Hanifin / Dermatol Clin 22 (2004) 263274

sion of disease symptoms in spite of VIG or postexposure vaccination.

Dermatol Clin 22 (2004) 275 289

Smallpox: vaccine reactions and contraindications

W.L. Tom et al / Dermatol Clin 22 (2004) 275289

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