2Diagnosis 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 region2. Smear* taken fromaffected tissues showssmall gram-negativeand/or bipolar-stainingcoccobacilli(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)
3Diagnosis Presumptive Plague One or both of the following conditions are met:1. Immunofluorescence stain of smear+ve for the presence of Yersinia pestisF1 antigen.2. Only a single serum specimen istested & 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 byhemagglutination inhibition.
4Diagnosis 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-F1antigen 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 antigenby hemagglutination inhibition.
5Treatment Isolation: Antibiotics: ■ For the first 48 hours following treatment, in case pneumoniadevelops■ 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
6Antibiotic Therapy Adult Child Pregnant ♀ Streptomycin Gentamicin *1g IM or IVx1/day15mg/kg IMx2/dayGentamicin5mg/kg IM or IV x1/day2.5mg/kg IM or IV x3/day5mg/kg IM or IVDoxycycline100 mg IM x2/day or 200mg IV x1/day2.2mg/kg IV x2/day100mg IV x2/day or 200mg IV x1/dayCiprofloxacin400 mg IV x2/day15mg/kg IV x2/day400mg IV x2/dayChloramphenicol25mg/kg IV x4/day25mg/kg IV x4/day (but not if < 2 years of age)*Preferred Treatment (others are alternatives)
7Antibiotics – Mechanism of Action Aminoglycosides (Streptomycin, Gentamycin); Tetracyclines (Doxycycline); Quinolones (Ciprofloxacin); Chloramphenicol; Pt should have f/u
8Prognosis Fatality Bubonic 1 or 2 Septicemic 1 or 2 Pneumonic Untreated50%-90%50%-100%100%most within48 hrs of onsetTreated5%-20%30%-50%?treatment must begin w/i 18hrs of onsetPeople 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
9Prevention ■ Integrated Vector Management ■ Education on modes of (surveillance of animal reservoirs)■ Education on modes oftransmission■ Control Rat & FleaPopulations( traps, insecticides)Insecticide application must always precede rodent controlRodent control (by trapping, gassing or poisoning) must be undertaken with caution… overkill make lead to new reservoirsSan Jose, CA; 1991
10Prevention Personal Sanitation Measures ■ Veterinary workers in endemic areas:gloves, eye protection, surgical masks when treatingsuspect 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 foraerosolization
11Prevention Existing Vaccines – None Available for Use! Vaccine StrategyStatusRemarksEV76Live AttenuatedMutant StrainIntroduced in 1908Limited availability in former Soviet Union- Not avirulent- Questionable Safety- No protection Pnemonic formUSPFormalin-Inactivated Whole CellManufacturedNo Longer Available- Induces F1 antigen response- Not tested in controlled studies- Severe inflammatory reactions- Multiple Booster Shots- No protection Pneumonic formUsed 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.
12Prevention Improved Vaccines – In Development Vaccine Strategy Status RemarksF1/V Fusion ProteinRecombinant SubunitPreclinical testing in mice and nonhuman primatesEffective in aerosol challenge inmice & monkeysF1/VCombo(separateproteins)Phase 1 clinical trialscompleted (phase 2trials due to begin in late 2002/early 2003)- 2:1 F1/V ratio- Protection via IgG- Also F1V-specific IgA response- Adjuvanted with alhydrogelProtection Bubonic & PneumonicV antigen in Plasmid DNA constructDNAPreclinical testing in micemice: immunization plusbooster shot with recombinantantigens developed protectiveimmunity against lethal dose
13Prevention Prophylactic Antibiotics ■ only following high-risk exposure to pneumonic plagueAdultChildPregnant ♀Doxycycline*100 mg PO x2/day2.2mg/kg POx2/dayCiprofloxacin500 mg PO x2/day20mg/kg PO x2/dayChloramphenicol25mg/kg PO x4/day25mg/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)
14Caution: Drug Resistant Strains in Patients Y. Pestis StrainIsolatedDrugResistanceHowNew Genes16/95Madagascar,1995Streptomycinstreptomycin phosphotransferaseGenes in the acquired self-transferable plasmid pIP120317/9519971. Ampicillin2. Chloramphenicol3. Kanamycin4. Minocycline5. Streptomycin6. Spectinomycin7. Sulfonamides8. Tetracycline1. production Lactamase2. Productionchloramphenicolacetyltransferase3. synthesis of a type I 3'-aminoglycoside phosphotransferase5. & 6. 3''-9AminoglycosideadenylyltransferaseGenes in theacquiredconjugativeplasmidpIP1202Rare point out adenylylation vs. phosphorylation; 150kb vs 40kbWhere:? Contact with gut bacteria in dead human host, contact with microorganisms in flea midgutWe need to find:molecular and genetic basisorigins and evolution of the resistancepotential for the spread of resistant Y. pestis in its natural cycle
16History of Weaponization Mongols throw plague infected bodies over the walls of the besieged city of Kaffa in 1346In 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.
17CDC Classification of Plague Plague is in Category A, it is a high-priority organismHigh-priority agents include organisms that pose a risk to national security because:can be easily disseminated or transmitted from person to personresult in high mortality rates and have the potential for major public health impactmight cause public panic and social disruptionrequire special action for public health preparedness
18Plague 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 formPneumonic plague causes serious illness with a high case fatality ratePneumonic plague is communicablebacteria are enough to cause pneumonic plague, whereas it takes between 1,000-10,000 spores to cause pulmonary anthrax
19Are 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.
20In The Event of An Attack… Early treatment with antibiotics (gentamicin, streptomycin, tetracycline, fluoroquinoline)Use of surgical masks to prevent further transmission.
21The 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.
22The 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.