Differential Diagnosis Pneumonic Plague Septicemic Plague ■ Inhalation anthrax ■ Tularemia ■ Viral Pneumonia Influenza, Hantavirus, RSV, CMV ■ Other Bacterial Pneumonia ■ Q Fever ■ Meningococcemia ■ Septicemia caused by other Gram-Negative Bacteria

Diagnosis Suspected Plague Both of the following conditions are met: 1. Clinical symptoms of Plague in person who resides in or has recently traveled to a plague-endemic region 2. Smear* taken from affected tissues shows small gram-negative and/or bipolar-staining coccobacilli (Polychromic stains: Wright, Giemsa, or Wayson stain ) * Sample taken from Bubo (bubonic plague), Blood (septicemic plague), or Tracheal/lung aspirate (pneumonic plague) Culture isolates will be forwarded to a reference laboratory for definitive identification, which may involve antigen detection, IgM enzyme immunoassay, or polymerase chain reaction. Antibiotic susceptibility testing of Y. pestis is also undertaken at reference laboratories, which are better equipped for this purpose than most hospital laboratories. Numerous other ways for detection, confirmation and characterization (ELISA, PCR, mouse inoculation, IHC, Dipstick Assay, Bacteriological culture, gram stain)

Diagnosis Presumptive Plague One or both of the following conditions are met: 1. Immunofluorescence stain of smear +ve for the presence of Yersinia pestis F1 antigen. 2. Only a single serum specimen is tested & the anti-F1 antigen titer by agglutination is >1:10.* Smears should be sent to a reference lab for fluorescent antibody microscopy. Test can be done in < 2hrs… but F1 expressed at 37C, so refrigerated samples or samples from cultures that have been incubated at lower temps would test negative *Agglutination testing must be shown to be specific to Y. pestis F1 antigen by hemagglutination inhibition.

Diagnosis Confirmed Plague One of the following conditions is met: 1. Isolated culture lysed by specific bacteriophage. 2. 2 serum specimens demonstrate a 4 fold anti-F1 antigen titer difference by agglutination testing.* 3. Single serum specimen tested by agglutination has a titer of >1:128 and the patient has no known previous plague exposure or vaccination history.* 2. Smaller elevations are considered a presumptive diagnosis. These techniques are slow…Y Pestis grows slowly in culture & antibodies can take a number of days or weeks after disease onset to dev’t… usually good for a retrospective confirmation of plague *Agglutination testing must be shown to be specific to Y. pestis F1 antigen by hemagglutination inhibition.

Treatment Isolation: Antibiotics: ■ For the first 48 hours following treatment, in case pneumonia develops ■ By law, patients with pneumonic plague must be isolated ■ If patients have no pneumonia or draining lesions at 48 hours, they may be taken out of strict isolation. Antibiotics: ■ For a minimum of 10 days (or 3-4 days after clinical recovery) b/c tests take so long, treatment should begin as soon as plague is suspected… isolation 72 hours after starting antibiotic therapy. If treated with antibiotics, buboes typically recede in 10 to 14 days and do not require drainage. Strep… FDA approved but limited use in usa, so small supply

Antibiotic Therapy Adult Child Pregnant ♀ Streptomycin Gentamicin * 1g IM or IV x1/day 15mg/kg IM x2/day --------------------------- Gentamicin 5mg/kg IM or IV x1/day 2.5mg/kg IM or IV x3/day 5mg/kg IM or IV Doxycycline 100 mg IM x2/day or 200mg IV x1/day 2.2mg/kg IV x2/day 100mg IV x2/day or 200mg IV x1/day Ciprofloxacin 400 mg IV x2/day 15mg/kg IV x2/day 400mg IV x2/day Chloramphenicol 25mg/kg IV x4/day 25mg/kg IV x4/day (but not if < 2 years of age) *Preferred Treatment (others are alternatives)

Antibiotics – Mechanism of Action Aminoglycosides (Streptomycin, Gentamycin); Tetracyclines (Doxycycline); Quinolones (Ciprofloxacin); Chloramphenicol; Pt should have f/u

Prognosis  Fatality Bubonic 1 or 2 Septicemic 1 or 2 Pneumonic Untreated 50%-90% 50%-100% 100% most within 48 hrs of onset Treated 5%-20% 30%-50% ? treatment must begin w/i 18hrs of onset People continue to die of plague, not because the bacilli have become resistant but, most often, because physicians do not include plague in their differential Diagnosis (in the United States) or because treatment is absent or delayed (in underdeveloped countries). Pneumonic plague is the most rapidly fatal form of plague, and most victims will die if they do not receive antibiotics within the first 18 hours after symptoms begin. Bubonic: most deaths occurring from sepsis in 3 to 5 days. Primary or secondary septicemic plague (infection active in the bloodstream and the victim has symptoms of shock) has a 40% death rate, even when treated.  1 Pneumonic Plague progresses the most rapidly

Prevention ■ Integrated Vector Management ■ Education on modes of (surveillance of animal reservoirs) ■ Education on modes of transmission ■ Control Rat & Flea Populations ( traps, insecticides) Insecticide application must always precede rodent control Rodent control (by trapping, gassing or poisoning) must be undertaken with caution… overkill make lead to new reservoirs San Jose, CA; 1991

Prevention Personal Sanitation Measures ■ Veterinary workers in endemic areas: gloves, eye protection, surgical masks when treating suspect cats ■ Hunters and Outdoorsmen: avoid rodent nests, use insect repellents/insecticides, wear gloves when handling potentially infected animals ■ In the Lab: - Standard Control: when handling Y. Pestis organisms - Biosafety Level 2: when processing clinical specimens & cultures - Biosafety Level 3: with large amounts of bacteria or with potential for aerosolization

Prevention Existing Vaccines – None Available for Use! Vaccine Strategy Status Remarks EV76 Live Attenuated Mutant Strain Introduced in 1908 Limited availability in former Soviet Union - Not avirulent - Questionable Safety - No protection Pnemonic form USP Formalin-Inactivated Whole Cell Manufactured 1940-1999 No Longer Available - Induces F1 antigen response - Not tested in controlled studies - Severe inflammatory reactions - Multiple Booster Shots - No protection Pneumonic form Used in US military during Vietnam war… only 8 cases of immunized servicemen reported. but severe inflammatory reactions are frequent. Primary IM injection followed by boosters at 3-5 mos then another booster at 5-6 mos then 3 more booster shots at 6 mos intervals followed by 1-2 year intervals until not needed.

Prevention Improved Vaccines – In Development Vaccine Strategy Status Remarks F1/V Fusion Protein Recombinant Subunit Preclinical testing in mice and nonhuman primates Effective in aerosol challenge in mice & monkeys F1/V Combo (separate proteins) Phase 1 clinical trials completed (phase 2 trials due to begin in late 2002/early 2003) - 2:1 F1/V ratio - Protection via IgG - Also F1V-specific IgA response - Adjuvanted with alhydrogel Protection Bubonic & Pneumonic V antigen in Plasmid DNA construct DNA Preclinical testing in mice mice: immunization plus booster shot with recombinant antigens developed protective immunity against lethal dose

Prevention Prophylactic Antibiotics ■ only following high-risk exposure to pneumonic plague Adult Child Pregnant ♀ Doxycycline * 100 mg PO x2/day 2.2mg/kg POx2/day Ciprofloxacin 500 mg PO x2/day 20mg/kg PO x2/day Chloramphenicol 25mg/kg PO x4/day 25mg/kg PO x4/day (not < 2 yrs of age) Prevent it from developing into a disease; Recommendations are for mass casualty setting where number of pts. too great for all to receive IV antibiotics *Preferred Treatment (others are alternatives) ■ treat for 10 days (if fever/cough develops during prophylactic treatment, then follow standard therapy for Y. Pestis)

Caution: Drug Resistant Strains in Patients Y. Pestis Strain Isolated Drug Resistance How New Genes 16/95 Madagascar, 1995 Streptomycin streptomycin phosphotransferase Genes in the acquired self-transferable plasmid pIP1203 17/95 1997 1. Ampicillin 2. Chloramphenicol 3. Kanamycin 4. Minocycline 5. Streptomycin 6. Spectinomycin 7. Sulfonamides 8. Tetracycline 1. production  Lactamase 2. Production chloramphenicol acetyltransferase 3. synthesis of a type I 3'-aminoglycoside phosphotransferase 5. & 6. 3''-9 Aminoglycoside adenylyltransferase Genes in the acquired conjugative plasmid pIP1202 Rare point out adenylylation vs. phosphorylation; 150kb vs 40kb Where:? Contact with gut bacteria in dead human host, contact with microorganisms in flea midgut We need to find: molecular and genetic basis origins and evolution of the resistance potential for the spread of resistant Y. pestis in its natural cycle

Weaponization of the Plague

History of Weaponization Mongols throw plague infected bodies over the walls of the besieged city of Kaffa in 1346 In WWII, Japanese army dropped Plague-infected fleas packed into bombs over Manchuria and infected their water supply resulting in an outbreak. During The Cold War, the U.S. and Soviet Union developed methods of aerosolizing Plague-thereby eliminating the flea vector.

CDC Classification of Plague Plague is in Category A, it is a high-priority organism High-priority agents include organisms that pose a risk to national security because: can be easily disseminated or transmitted from person to person result in high mortality rates and have the potential for major public health impact might cause public panic and social disruption require special action for public health preparedness

Plague is a Suitable Pathogen For Use As a Weapon Because… It is accessible, simple to reproduce, economical and efficient. It can be delivered in aerosol form Pneumonic plague causes serious illness with a high case fatality rate Pneumonic plague is communicable 100-500 bacteria are enough to cause pneumonic plague, whereas it takes between 1,000-10,000 spores to cause pulmonary anthrax

Are We Prepared? A 1970 WHO report estimated that an aerosol release of 50kg of Y.Pestis over a city of 5 million people would produce 150,000 illnesses and up to 36,000 deaths. (This report didn’t take into account the secondary cases that would occur through person-to-person contact. A simulated bioterror attack (TOPOFF) involving aerosolization of the plague was carried out in May 2001, in Colorado. By the end of the third day, 783 people had contracted pneumonic plague, by the next day the number of plague cases had risen to 1,871 and by the third day the number stood at 3,060. At the end of the exercise 950 people had “died” of pneumonic plague.

In The Event of An Attack… Early treatment with antibiotics (gentamicin, streptomycin, tetracycline, fluoroquinoline) Use of surgical masks to prevent further transmission.

The Bad News… Resistance Sequence of Y.pestis could have boomerang effect, enabling terrorists to create antibiotic resistant strands. According to Alastair Hay, the Soviet Union has already developed a form of Yersinia pestis that was resistant to 16 different antibiotics. Right: Picture of Staphylococcus Aureus next to Y. Pestis. A transfer of antibiotic resistant genes from Staph to Y. Pestis could result in a uncontrollably lethal bacterium.

The Good News… Requires a high level of knowledge to distinguish between virulent and non-virulent strain, efficiently produce the virulent strains, and aerosolize it. Plague bacteria is a fragile organism because it is non-spore forming, so it can only remain viable for only about 1hr after aerosolization.