1. 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

2. 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)

3. 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.

4. 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.

5. 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

6. 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)

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

8. 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

9. 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

10. 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

11. 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
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.

12. 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

13. 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)

14. 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

15. Weaponization of the Plague

16. 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.

17. 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

18. 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
bacteria are enough to cause pneumonic plague, whereas it takes between 1,000-10,000 spores to cause pulmonary anthrax

19. 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.

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

21. 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.

22. 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.