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Plague is a systemic zoonosis caused by Yersinia pestis. It predominantly affects small rodents in rural areas of Africa, Asia, and the Americas and is usually transmitted to humans by an arthropod vector (the flea). Less often, infection follows contact with animal tissues or respiratory droplets. Plague is an acute febrile illness that is treatable with antimicrobial agents, but mortality rates among untreated patients are high. Ancient DNA studies have confirmed that the fourteenth-century “Black Death” in Europe was Y. pestis infection. Patients can present with the bubonic, septicemic, or pneumonic form of the disease. Although there is concern among the general public about epidemic spread of plague by the respiratory route, this is not the usual route of plague transmission, and established infection-control measures for respiratory plague exist. However, the fatalities associated with plague and the capacity for infection via the respiratory tract mean that Y. pestis fits the profile of a potential agent of bioterrorism. Consequently, measures have been taken to restrict access to the organism, including legislation affecting diagnostic and research procedures in some countries (e.g., the United States).
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The genus Yersinia comprises gram-negative bacteria of the family Enterobacteriaceae (gamma proteobacteria). Overwhelming taxonomic evidence showing Y. pestis strains as a clonal group within Yersinia pseudotuberculosis suggests recent evolution from the latter organism—an enteric pathogen of mammals that is spread by the fecal-oral route and thus has a phenotype distinctly different from that of Y. pestis. When grown in vivo or at 37°C, Y. pestis forms an amorphous capsule made from a plasmid-specified fimbrial protein, Caf or fraction 1 (F1) antigen, which is an immunodiagnostic marker of infection.
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Human plague generally follows an outbreak in a host rodent population (epizootic). Mass deaths among the rodent primary hosts lead to a search by fleas for new hosts, with consequent incidental infection of other mammals. The precipitating cause for an epizootic may ultimately be related to climate or other environmental factors. The reservoir for Y. pestis causing enzootic plague in natural endemic foci between epizootics (i.e., when the organism may be difficult to detect in rodents or fleas) is a topic of ongoing research and may not be the same in all regions. The enzootic/epizootic pattern may be the result of complex dynamic interactions of host rodents that have different plague susceptibilities and different flea vectors; alternatively, an environmental reservoir may be important.
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In general, the enzootic areas for plague are lightly populated regions of Africa, Asia, and the Americas (Fig. 68-1). Between 2004 and 2009, 12,503 cases of plague, with a global case-fatality rate of 6.7%, were recorded by the World Health Organization (WHO); these figures were obtained by combining cases notified under the International Health Regulations with data from national surveillance programs and publications. More than 97% of these cases were in Africa; the majority of cases were reported from the Democratic Republic of the Congo and the island of Madagascar. The period covered spans a change in the International Health Regulations from a requirement for nations to notify the WHO of all cases of plague to a requirement to report pneumonic plague or any suspected case of plague in an area not known to be endemic for plague. In the past decade, outbreaks of pneumonic plague have been recorded in the Democratic Republic of the Congo, Uganda, Algeria, Madagascar, China, and Peru.
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Plague was introduced into North America via the port of San Francisco in 1900 as part of the Third Pandemic, which spread around the world from Hong Kong. The disease is presently enzootic on the western side of the continent from southwestern Canada to Mexico. Most human cases in the United States occur in two regions: “Four Corners” (the junction point of New Mexico, Arizona, Colorado, and Utah), especially northern New Mexico, northern Arizona, and southern Colorado; and further west in California, southern Oregon, and western Nevada (http://www.cdc.gov/plague/maps/index.html). From 1990 to 2011, 151 cases of plague were reported in the United States, a mean of seven cases per year. Most cases occurred from May to October—the time of year when people are outdoors and rodents and their fleas are most plentiful. The infection is most often acquired by fleabite in peridomestic environments; it can also be acquired through the handling of living or dead small mammals (e.g., rabbits, hares, and prairie dogs) or wild carnivores (e.g., wildcats, coyotes, or mountain lions). Dogs and cats may bring plague-infected fleas into the home, and infected cats may transmit plague directly to humans by the respiratory route. The last recorded case of person-to-person transmission in the United States occurred in 1925.
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Plague most often develops in areas with poor sanitary conditions and infestations of rats—in particular, the widely distributed roof rat Rattus rattus and the brown rat Rattus norvegicus (which serves as a laboratory model of plague). Rat control in warehouses and shipping facilities has been recognized as important in preventing the spread of plague since the early twentieth century and features in the current WHO International Health Regulations. Urban rodents acquire infection from wild rodents, and the proximity of the former to humans increases the risk of transmission. The oriental rat flea Xenopsylla cheopis is the most efficient vector for transmission of plague among rats and onward to humans in Asia, Africa, and South America.
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Worldwide, bubonic plague is the predominant form reported (80–95% of suspected cases), with mortality rates of 10–20%. The mortality rate is higher (22%) in the small proportion of patients (10–20%) with primary septicemic plague (i.e., systemic Y. pestis sepsis with no bubo; see “Clinical Manifestations,” below) and is highest with primary pulmonary plague; in this, the least common of the main plague presentations, the mortality rate approaches 100% without antimicrobial treatment and is >50% even with such treatment. Rare outbreaks of pharyngeal plague following consumption of raw or undercooked camel or goat meat have been reported.
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A total of 81 (76%) of the 107 plague cases reported in the United States from 1990 to 2005 were primary bubonic disease, 19 (18%) were primary septicemic disease, and 5 (5%) were primary pneumonic disease; 2 cases (2%) were not classified. Eleven cases (10%) were fatal.
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As mentioned earlier, genetic evidence suggests that Y. pestis is a clone derived from the enteric pathogen Y. pseudotuberculosis in the recent evolutionary past (9000–40,000 years ago). The change from infection by the fecal-oral route to a two-stage life cycle, with alternate parasitization of arthropod and mammalian hosts, followed the acquisition of two plasmids (pFra and pPst) and the inactivation of remarkably few Y. pseudotuberculosis genes in conjunction with preexisting properties of the Y. pseudotuberculosis ancestor (e.g., the presence of a third plasmid, pYV, and the capacity to cause septicemia). In the arthropod-parasitizing portion of its life cycle, Y. pestis multiplies and forms biofilm-embedded aggregates in the flea midgut after ingestion of a blood meal containing bacteria. In some fleas, biofilm-embedded bacteria eventually fill the proventriculus (a valve connecting the esophagus to the midgut) and block normal blood feeding. Both “blocked” fleas and those containing masses of biofilm-embedded Y. pestis without complete blockage inoculate Y. pestis into each bite site. The ability of Y. pestis to colonize and multiply in the flea requires phospholipase D encoded by the ymt gene on the pFra plasmid, and biofilm synthesis requires the chromosomal hms locus shared with Y. pseudotuberculosis. However, three Y. pseudotuberculosis genes inhibiting biofilm formation or promoting its degradation are inactivated in Y. pestis, together with urease, which causes acute flea gastrointestinal toxicity. Blockage takes days or weeks to come about after initial infection of the flea and is followed by the flea’s death. In addition, many flea vectors (including X. cheopis) are able to transmit plague in an early-phase unblocked state for up to 1 week after feeding, but 10 fleas in this state are required to infect a mammalian host (mass transmission).
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Y. pestis disseminates from the site of inoculation in the mammalian host in a process initially dependent on plasminogen activator Pla, which is encoded by the small pPst plasmid. This surface protease activates mammalian plasminogen, degrades complement, and adheres to the extracellular matrix component laminin. Pla is essential for the high-level virulence of Y. pestis in mice by subcutaneous or intradermal injection (laboratory proxies for fleabites) and for the development of primary pneumonic plague. When actual fleabite inoculation is used in mouse models, the fimbrial capsule-forming protein (Ca1 or fraction 1; F1 antigen) encoded on pFra increases the efficiency of transmission, and plasminogen activator is required for the formation of buboes. Because the antiphagocytic systems in Y. pestis are not fully operational at the time of inoculation into the mammalian host, the organism is taken up by macrophages from the inoculation site and transported to regional lymph nodes. After intracellular replication, Y. pestis switches to extracellular replication with full expression of its antiphagocytic systems: the type III secretion machines and their effectors encoded by pYV as well as the F1 capsule. Overproduction of the type III secretion substrate and translocation protein LcrV exerts an anti-inflammatory effect, reducing host immune responses. Likewise, Y. pestis lipopolysaccharide is modified to minimize stimulation of host Toll-like receptor 4, thereby reducing protective host inflammatory responses during peripheral infection and prolonging host survival with high-grade bacteremia—an effect that probably enhances the pathogen’s subsequent transmission by fleabite.
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Replication of Y. pestis in a regional lymph node results in the local swelling of the lymph node and periglandular region known as a bubo. On histology, the node is found to be hemorrhagic or necrotic, with thrombosed blood vessels, and the lymphoid cells and normal architecture are replaced by large numbers of bacteria and fibrin. Periglandular tissues are inflamed and also contain large numbers of bacteria in a serosanguineous, gelatinous exudate.
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Continued spread through the lymphatic vessels to contiguous lymph nodes produces second-order primary buboes. Infection is initially contained in the infected regional lymph nodes, although transient bacteremia can be detected. As the infection progresses, spread via efferent lymphatics to the thoracic duct produces high-grade bacteremia. Hematogenous spread to the spleen, liver, and secondary buboes follows, with subsequent uncontrolled septicemia, endotoxic shock, and disseminated intravascular coagulation leading to death. In some patients, this septicemic phase occurs without obvious prior bubo development or lung disease (septicemic plague). Hematogenous spread to the lungs results in secondary plague pneumonia, with bacteria initially more prominent in the interstitium than in the air spaces (the reverse being the case in primary plague pneumonia). Hematogenous spread to other organs, including the meninges, can occur.
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CLINICAL MANIFESTATIONS
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After an incubation period of 2–6 days, the onset of bubonic plague is sudden and is characterized by fever (>38°C), malaise, myalgia, dizziness, and increasing pain due to progressive lymphadenitis in the regional lymph nodes near the fleabite or other inoculation site. Lymphadenitis manifests as a tense, tender swelling (bubo) that, when palpated, has a boggy consistency with an underlying hard core. Generally, there is one painful and erythematous bubo with surrounding periganglionic edema. The bubo is most commonly inguinal but can also be crural, axillary (Fig. 68-2), cervical, or submaxillary, depending on the site of the bite. Abdominal pain from intraabdominal node involvement can occur without other visible signs. Children are most likely to present with cervical or axillary buboes.
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The differential diagnosis includes acute focal lymphadenopathy of other etiologies, such as streptococcal or staphylococcal infection, tularemia, cat-scratch disease, tick typhus, infectious mononucleosis, or lymphatic filariasis. These infections do not progress as rapidly, are not as painful, and are associated with visible cellulitis or ascending lymphangitis—both of which are absent in plague.
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Without treatment, Y. pestis dissemination occurs and causes serious illness, including pneumonia (secondary pneumonic plague) and meningitis. Secondary pneumonic plague can be the source of person-to-person transmission of respiratory infection by productive cough (droplet infection), with the consequent development of primary plague pneumonia. Appropriate treatment of bubonic plague results in fever resolution within 2–5 days, but buboes may remain enlarged for >1 week after initial treatment and can become fluctuant.
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Primary septicemic plague
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A minority (10–25%) of infections with Y. pestis present as gram-negative septicemia (hypotension, shock) without preceding lymphadenopathy. Septicemic plague occurs in all age groups, but persons older than age 40 years are at elevated risk. Some chronic conditions may predispose to septicemic plague: in 2009 in the United States, a fatal laboratory-acquired infection with an attenuated Y. pestis strain manifested as septicemic plague in a 60-year-old researcher with diabetes mellitus and undiagnosed hemochromatosis. These conditions also carry an increased risk of septicemia with other pathogenic Yersinia species. The term septicemic plague can be confusing since most patients with buboes have detectable bacteremia at some stage, with or without systemic signs of sepsis. In laboratory experiments, however, septicemic disease without histologic changes in lymph nodes is seen in a minority of mice infected via fleabites.
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Primary pneumonic plague results from inhalation of infectious bacteria in droplets expelled from another person or an animal with primary or secondary plague pneumonia. This syndrome has a short incubation period, averaging from a few hours to 2–3 days (range, 1–7 days), and is characterized by a sudden onset of fever, headache, myalgia, weakness, nausea, vomiting, and dizziness. Respiratory signs—cough, dyspnea, chest pain, and sputum production with hemoptysis—typically arise after 24 h. Progression of initial segmental pneumonitis to lobar pneumonia and then to bilateral lung involvement may occur (Fig. 68-3). The possible release of aerosolized Y. pestis bacteria in a bioterrorist attack, manifesting as an outbreak of primary pneumonic plague in nonendemic regions or in an urban setting where plague is rarely seen, has been a source of public health concern. Secondary pneumonic plague is a consequence of bacteremia occurring in ~10–15% of patients with bubonic plague. Bilateral alveolar infiltrates are seen on chest x-ray, and diffuse interstitial pneumonitis with scanty sputum production is typical.
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Meningeal plague is uncommon, occurring in ≤6% of plague cases reported in the United States. Presentation with headache and fever typically occurs >1 week after the onset of bubonic or septicemic plague and may be associated with suboptimal antimicrobial therapy (delayed therapy, penicillin administration, or low-dose tetracycline treatment) and cervical or axillary buboes.
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Symptomatic plague pharyngitis can follow the consumption of contaminated meat from an animal dying of plague or contact with persons or animals with pneumonic plague. This condition can resemble tonsillitis, with peritonsillar abscess and cervical lymphadenopathy. Asymptomatic pharyngeal carriage of Y. pestis can also occur in close contacts of patients with pneumonic plague.
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Because of the scarcity of laboratory facilities in regions where human Y. pestis infection is most common, and because of the potential significance of Y. pestis isolation in a nonendemic area or an area from which human plague has been absent for many years, the WHO recommends an initial presumptive diagnosis followed by reference laboratory confirmation (Table 68-1). In the United States, comprehensive national diagnostic facilities for plague have been in place since a federal Laboratory Response Network (LRN; www.bt.cdc.gov/lrn/) was set up in 1999 to detect possible use of biological terrorism agents, including Y. pestis. Routine diagnostic clinical microbiology laboratories that are included in this network as sentinel-level laboratories use joint protocols from the Centers for Disease Control and Prevention (CDC) and the American Society for Microbiology to identify suspected Y. pestis isolates and to refer these specimens to LRN reference laboratories for confirmatory tests (http://www.asm.org/index.php/issues/sentinel-laboratory-guidelines). Y. pestis is designated a “Tier 1 select agent” under the Public Health Security and Bioterrorism Preparedness and Response Act of 2002 and subsequent executive orders; the provisions of this act, the Patriot Act of 2001, and related executive orders apply to all U.S. laboratories and individuals working with Y. pestis. Details of the applicable regulations are available from the CDC.
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Yersinia species are gram-negative coccobacilli (short rods with rounded ends) 1–3 μm in length and 0.5–0.8 μm in diameter. Y. pestis in particular appears bipolar (with a “closed safety pin” appearance) and pleomorphic when stained with a polychromatic stain (Wayson or Wright-Giemsa; Fig. 68–4). Its lack of motility distinguishes Y. pestis from other Yersinia species, which are motile at 25°C and nonmotile at 37°C. Transport medium (e.g., Cary-Blair medium) preserves the viability of Y. pestis if transport is delayed.
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The appropriate specimens for diagnosis of bubonic, pneumonic, and septicemic plague are bubo aspirate, bronchoalveolar lavage fluid or sputum, and blood, respectively. Culture of postmortem organ biopsy samples can also be diagnostic. A bubo aspirate is obtained by injection of 1 mL of sterile normal saline into a bubo under local anesthetic and aspiration of a small amount of (usually blood-stained) fluid. Gram’s staining of these specimens may reveal gram-negative rods, which are shown by Wayson or Wright-Giemsa staining to be bipolar. These bacteria may even be visible in direct blood smears in septicemic plague (Fig. 68-4); this finding indicates very high numbers of circulating bacteria and a poor prognosis.
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Y. pestis grows on nutrient agar and other standard laboratory media but forms smaller colonies than do other Enterobacteriaceae. Specimens should be inoculated onto nutrient-rich media such as sheep blood agar (SBA), into nutrient-rich broth such as brain-heart infusion broth, and onto selective agar such as MacConkey or eosin methylene blue (EMB) agar. Yersinia-specific CIN (cefsulodin, triclosan [Irgasan], novobiocin) agar can be useful for culture of contaminated specimens, such as sputum. Blood should be cultured in a standard blood culture system. The optimal growth temperature is <37°C (25–29°C), with pinpoint colonies only on SBA at 24 h. Slower growth occurs at 37°C. Y. pestis is oxidase-negative, catalase-positive, urea-negative, indole-negative, and lactose-negative. Automated biochemical identification systems can misidentify Y. pestis as Y. pseudotuberculosis or other bacterial species.
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Reference laboratory tests for definitive identification of isolates include direct immunofluorescence for F1 antigen; specific polymerase chain reaction (PCR) for targets such as F1 antigen, the pesticin gene, and the plasminogen activator gene; and specific bacteriophage lysis. PCR can also be applied to diagnostic specimens, as can direct immunofluorescence for F1 antigen (produced in large amounts by Y. pestis) by slide microscopy. An immunochromatographic test strip for F1 antigen detection by monoclonal antibodies in clinical specimens has been devised in Madagascar. This method is effective for both laboratory and near-patient use and is now widely used in endemic countries. A similar test strip for Pla antigen has recently been developed and could be used to detect wild-type or engineered F1-negative virulent strains. Many other rapid diagnostic kits for possible bioterrorism pathogens, including Y. pestis, have been described in recent years, but none is widely used for primary or reference laboratory identification, and only one (a field real-time PCR for a range of potential bioterrorism agents) is approved by the U.S. Food and Drug Administration (FDA). Detailed phylogeographic DNA sequence data based on culture collections have been accumulated to trace plague evolution, and this system could be adapted in the future to determine real-time clinical plague epidemiology.
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In the absence of other positive laboratory diagnostic tests, a retrospective serologic diagnosis may be made on the basis of rising titers of hemagglutinating antibody to F1 antigen. Enzyme-linked immunosorbent assays (ELISAs) for IgG and IgM antibodies to F1 antigen are also available.
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The white blood cell (WBC) count is generally raised (to 10,000–20,000/μL) in plague, with neutrophilic leukocytosis and a left shift (numerous immature neutrophils); in some cases, however, the WBC count is normal or leukopenia develops. WBC counts are occasionally very high, especially in children (>100,000/μL). Levels of fibrinogen degradation products are elevated in a majority of patients, but platelet counts are usually normal or low-normal. However, disseminated intravascular coagulation, with low platelet counts, prolonged prothrombin times, reduced fibrinogen, and elevated fibrinogen degradation product levels, occurs in a significant minority of patients.
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TREATMENT Plague
Guidelines for the treatment of plague are given in Table 68-2. A 10-day course of antimicrobial therapy is recommended. Streptomycin has historically been the parenteral treatment of choice for plague and is approved for this indication by the FDA. Although not yet approved by the FDA for plague, gentamicin has proven safe and effective in clinical trials in Tanzania and Madagascar and in retrospectively reviewed cases in the United States. In view of streptomycin’s adverse-reaction profile and limited availability, some experts now recommend gentamicin over streptomycin. In 2012, the FDA approved levofloxacin for prophylaxis and treatment of plague (including septicemic and pneumonic disease), making it the first antibiotic approved for a new indication under a regulatory approach based on animal studies alone, known as the Animal Rule. An FDA decision on ciprofloxacin is pending. Levofloxacin is more efficacious than ciprofloxacin for postexposure prophylaxis of inhalational anthrax in animal models and also received FDA approval for this indication (Chap. 10); thus it is approved for multiagent prophylaxis in possible bioterrorism exposures.
While systemic chloramphenicol therapy is available in the resource-poor countries primarily affected by plague, it is less likely to be available or used in high-income countries because of its adverse effect profile. Tetracyclines are also effective and can be given by mouth but are not recommended for children under the age of 7 years because of tooth discoloration. Doxycycline is the tetracycline of choice; at an oral dosage of 100 mg twice daily, this drug was as effective as IM gentamicin (2.5 mg/kg twice daily) in a trial in Tanzania.
Although Y. pestis is sensitive to β-lactam drugs in vitro and these drugs have been efficacious against plague in some animal models, the response to penicillins has been poor in some clinical cases; thus β-lactams and macrolides are not generally recommended as first-line therapy. Chloramphenicol, alone or in combination, is recommended for some focal complications of plague (e.g., meningitis, endophthalmitis, myocarditis) because of its tissue penetration properties. Fluoroquinolones, effective in vitro and in animal models, are recommended in guidelines for possible bioterrorism-associated pneumonic plague and are increasingly used in therapy, although the only human efficacy data available so far are from a case report. Animal and in vitro studies suggest that fluoroquinolones other than levofloxacin, at doses used in systemic gram-negative sepsis, should be effective as therapy for plague: e.g., ciprofloxacin (400 mg twice daily IV, 500 mg twice daily by mouth), ofloxacin (400 mg twice daily IV or by mouth), or moxifloxacin (400 mg/d IV or by mouth).
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In endemic areas, the control of plague in humans is based on reduction of the likelihood of being bitten by infected fleas or exposed to infected droplets from either humans or animals with plague pneumonia. In the United States, residence and outdoor activity in rural areas of western states where epizootics occur are the main risk factors for infection. To assess potential risks to humans in specific areas, surveillance for Y. pestis infection among animal plague hosts and vectors is carried out regularly as well as in response to observed animal die-offs. Personal protective measures include avoidance of areas where a plague epizootic has been identified and publicized (e.g., by warning signs or closure of campsites). Sick or dead animals should not be handled by the general public. Hunters and zoologists should wear gloves when handling wild-animal carcasses in endemic areas. General measures to avoid rodent fleabite during outdoor activity are appropriate and include the use of insect repellant, insecticide, and protective clothing. General measures to reduce peridomestic and occupational human contact with rodents are advised and include rodent-proofing of buildings and food-waste stores and removal of potential rodent habitats (e.g., woodpiles and junk heaps). Flea control by insecticide treatment of wild rodents is an effective means of minimizing human contact with plague if an epizootic is identified in an area close to human habitation. Any attempt to reduce rodent numbers must be preceded by flea suppression to reduce the migration of infected fleas to human hosts. An oral F1-V subunit vaccine using raccoon poxvirus (RCN) as a vector protects prairie dogs against Y. pestis injections and is being investigated for efficacy in preventing disease in wild animals, thus potentially reducing human exposure.
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Patients in whom pneumonic plague is suspected should be managed in isolation, with droplet precautions observed until pneumonia is excluded or effective antimicrobial therapy has been given for 48 h. Review of the literature published before the advent of antimicrobial agents suggests that the main infective risk is posed by patients in the final stages of disease who are coughing up sputum with plentiful visible blood and/or pus. Cotton and gauze masks were protective in these circumstances. Current surgical masks capable of barrier protection against droplets, including large respiratory particles, are considered protective; a particulate respirator (e.g., N95 or greater) is not required.
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Antimicrobial prophylaxis
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Postexposure antimicrobial prophylaxis lasting 7 days is recommended following household, hospital, or other close contact with persons with untreated pneumonic plague. (Close contact is defined as contact with a patient at <2 m.) In animal aerosol-infection studies, levofloxacin and ciprofloxacin are associated with higher survival rates than doxycycline (Table 68-3).
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Studies with candidate plague vaccines in animal models show that neutralizing antibody provides protection against exposure but that cell-mediated immunity is critical for protection and clearance of Y. pestis from the host. A killed whole-cell vaccine used in humans required multiple doses, caused significant local and systemic reactions, and was not protective against pneumonic plague; this vaccine is not currently available in the United States. A live attenuated vaccine based on strain EV76 is still used in countries of the former Soviet Union but has significant side effects. The vaccines closest to licensure are subunit vaccines comprising recombinant F1 (rF1) and various recombinant V (rV) proteins produced in Escherichia coli, which are combined either as a fusion protein or as a mixture, purified, and adsorbed to aluminum hydroxide for injection. This combination protects mice and various nonhuman primates in laboratory models of bubonic and pneumonic plague and has been evaluated in phase 2 clinical trials. Special ethical considerations with controlled clinical studies involving plague in humans make prelicensure field efficacy studies unlikely. In the United States, the FDA is therefore prepared to assess plague vaccines for human use under the Animal Rule, using efficacy data and other results from animal studies as well as antibodies and other correlates of immunity from human vaccine recipients (www.fda.gov/BiologicsBloodVaccines/ScienceResearch/BiologicsResearchAreas/ucm127288.htm). Live attenuated Y. pseudotuberculosis and Salmonella strains expressing Y. pestis–specific antigens have been shown to be protective in laboratory animal models of bubonic and pneumonic plague and could be delivered by the oral route. A wide variety of other delivery mechanisms for Y. pestis antigens are being explored. Antigens other than F1 and V that could be added to subunit vaccines are being investigated. Advances providing impetus for exploration of these antigens are (1) the recovery of F1-negative Y. pestis strains from natural sources and (2) the observation that F1 antigen is not required for virulence in primate models of pneumonic plague.