Chotani, 2003 Laboratory Criteria for Identification of B. anthracis n From clinical samples, such as blood, cerebrospinal fluid (CSF), skin lesion (eschar),

Chotani, 2003Chotani, 2003

Laboratory Criteria for Laboratory Criteria for Identification of Identification of B. anthracisB. anthracisLaboratory Criteria for Laboratory Criteria for Identification of Identification of B. anthracisB. anthracis From clinical samples, such as blood, From clinical samples, such as blood,

cerebrospinal fluid (CSF), skin lesion cerebrospinal fluid (CSF), skin lesion (eschar), or oropharyngeal ulcer(eschar), or oropharyngeal ulcer– Encapsulated gram-positive rods on Encapsulated gram-positive rods on

Gram stain Gram stain From growth on sheep blood agar: From growth on sheep blood agar:

– Large gram-positive rodsLarge gram-positive rods– NonmotileNonmotile– NonhemolyticNonhemolytic


Laboratory Criteria for Laboratory Criteria for Identification of Identification of B. anthracisB. anthracisLaboratory Criteria for Laboratory Criteria for Identification of Identification of B. anthracisB. anthracis

Rapid screening assay (PCR- and antigen-Rapid screening assay (PCR- and antigen-detection based) for use on cultures and detection based) for use on cultures and directly on clinical specimensdirectly on clinical specimens

Confirmatory criteria for identification of Confirmatory criteria for identification of B. B. anthracis anthracis – Capsule productionCapsule production– Lysis by gamma-phageLysis by gamma-phage– Direct fluorescent antibody assay (DFA)Direct fluorescent antibody assay (DFA)


Gram Stain Morphologyof B. anthracisGram Stain Morphologyof B. anthracis

Broad, gram-positive rod: 1–1.5 x Broad, gram-positive rod: 1–1.5 x 3–5 μ3–5 μ

Oval, central to subterminal spores: Oval, central to subterminal spores: 1 x 1.5 μ with no significant 1 x 1.5 μ with no significant swelling of cellswelling of cell

Spores usually NOT present in Spores usually NOT present in clinical specimens unless exposed clinical specimens unless exposed to atmospheric Oto atmospheric O22


B. anthracis

Gram-positive, spore-forming, non-motile bacillus


Gram Stain of Blood in Culture MediaGram Stain of Blood in Culture Media

Gram-positive bacilli in long chains (original Gram-positive bacilli in long chains (original magnification 20). Enlargement shows typical "jointed magnification 20). Enlargement shows typical "jointed

bamboo-rod" appearance of bamboo-rod" appearance of Bacillus anthracisBacillus anthracis (original (original magnification 100). Reprinted from Borio et al.magnification 100). Reprinted from Borio et al.3636


Isolate on SBA

Colony morphologyHemolysis

MotilitySpores

Gram stain Malachite green

B. anthracis:Presumptive Identification

Clinical specimen (blood, CSF, etc.)

Gram stainCapsule production


B. anthracis:Confirmatory Identification

Isolate

Phagelysis

Capsule

Bicarbonate media

Horse blood

(M’Fadyean Stain)

(M’Fadyean stain India ink stain)

DFA

Capsule antigenCell wall


Immune Protection Immune Protection Against AnthraxAgainst AnthraxImmune Protection Immune Protection Against AnthraxAgainst Anthrax

Live cellular vaccinesLive cellular vaccines– "Sterne" type live spore (toxigenic, non-capsulating)"Sterne" type live spore (toxigenic, non-capsulating)– Former USSR STI live spore (toxigenic, non-Former USSR STI live spore (toxigenic, non-

capsulating)capsulating)– "Pasteur" type (mixed culture, reduced virulence)"Pasteur" type (mixed culture, reduced virulence)

Sterile, acellular vaccinesSterile, acellular vaccines– US "anthrax vaccine adsorbed" (AVA)—not licensed US "anthrax vaccine adsorbed" (AVA)—not licensed

for use in civilian populationsfor use in civilian populations– UK "anthrax vaccine precipitated" (AVP)UK "anthrax vaccine precipitated" (AVP)

Recombinant PA research vaccinesRecombinant PA research vaccines– AIAI3+3+; Freund’s; Saponin, Monophosphoryl lipid A; ; Freund’s; Saponin, Monophosphoryl lipid A;

RibiRibi


Anthrax in the US - 2001Anthrax in the US - 2001Anthrax in the US - 2001Anthrax in the US - 2001

In October 2001, the first inhalational anthrax In October 2001, the first inhalational anthrax case in the United States since 1976 was case in the United States since 1976 was identified in a media company worker in identified in a media company worker in Florida. Florida.

A national investigation was initiated to identify A national investigation was initiated to identify additional cases and determine possible additional cases and determine possible exposures to exposures to Bacillus anthracisBacillus anthracis. .

Surveillance was enhanced through health-care Surveillance was enhanced through health-care facilities, laboratories, and other means to facilities, laboratories, and other means to identify cases, which were defined as clinically identify cases, which were defined as clinically compatible illness with laboratory-confirmed compatible illness with laboratory-confirmed B. B. anthracisanthracis infection. infection.

From October 4 to November 20, 2001, 22 cases From October 4 to November 20, 2001, 22 cases of anthrax (11 inhalational, 11 cutaneous) were of anthrax (11 inhalational, 11 cutaneous) were identified; 5 of the inhalational cases were fatal. identified; 5 of the inhalational cases were fatal.

Epidemic curve for 22 cases of bioterrorism-Epidemic curve for 22 cases of bioterrorism-

related anthrax, United States, 2001.related anthrax, United States, 2001. Epidemic curve for 22 cases of bioterrorism-Epidemic curve for 22 cases of bioterrorism-

related anthrax, United States, 2001.related anthrax, United States, 2001.


Anthrax MeningitisAnthrax Meningitis

• Hemorrhagic meningitis is a potential Hemorrhagic meningitis is a potential complication of anthrax, and may complication of anthrax, and may accompany cutaneous, accompany cutaneous, gastrointestinal, or inhalational anthrax gastrointestinal, or inhalational anthrax (seen in 50% of inhalational cases)(seen in 50% of inhalational cases)

• Death almost universal within 1-6 days Death almost universal within 1-6 days after onset of illnessafter onset of illness

• Rare survivors have been treated using Rare survivors have been treated using appropriate antibiotics in combination appropriate antibiotics in combination with antitoxin, prednisone or bothwith antitoxin, prednisone or both


Recommended Postexposure Prophylaxis Recommended Postexposure Prophylaxis to Prevent Inhalational Anthraxto Prevent Inhalational AnthraxRecommended Postexposure Prophylaxis Recommended Postexposure Prophylaxis to Prevent Inhalational Anthraxto Prevent Inhalational Anthrax

Initial TherapyInitial Therapy DurationDurationAdults Adults Ciprofloxacin Ciprofloxacin 60 days60 days(including pregnant(including pregnant 500 mg PO BID 500 mg PO BID women andwomen and ORORimmunocompromised)immunocompromised) Doxycycline Doxycycline

100 mg PO BID100 mg PO BIDChildrenChildren Ciprofloxacin*Ciprofloxacin* 60 days 60 days 10–15 mg/kg PO Q 12 hrs10–15 mg/kg PO Q 12 hrs Change to Change to

OROR amoxicillin amoxicillinDoxycycline:Doxycycline: if susceptibleif susceptible

>8 yrs and >45 kg: 100 mg PO BID>8 yrs and >45 kg: 100 mg PO BID >8 yrs and >8 yrs and <<45 kg: 2.2 mg/kg PO BID45 kg: 2.2 mg/kg PO BID

<<8 yrs: 2.2 mg/kg PO BID8 yrs: 2.2 mg/kg PO BID


CategoryCategory Initial Therapy (Oral)Initial Therapy (Oral) DurationDuration

Adults Adults Ciprofloxacin Ciprofloxacin 60 days60 days

(Including pregnant women(Including pregnant women 500 mg BID 500 mg BID and immunocompromised)and immunocompromised) OROR

DoxycyclineDoxycycline 100 mg BID100 mg BID

ChildrenChildren Ciprofloxacin**Ciprofloxacin** 60 days 60 days

(including immuno-(including immuno- 10–15 mg/kg Q 12 hrs10–15 mg/kg Q 12 hrs compromised)compromised) OROR

Doxycycline:Doxycycline:>8 yrs and >45 kg: 100 mg BID>8 yrs and >45 kg: 100 mg BID

>8 yrs and >8 yrs and <<45 kg: 2.2 mg/kg BID45 kg: 2.2 mg/kg BID<<8 yrs: 2.2 mg/kg BID8 yrs: 2.2 mg/kg BID

Cutaneous Anthrax Treatment Protocol* Cutaneous Anthrax Treatment Protocol* for Cases Associated with Bioterrorist Events for Cases Associated with Bioterrorist EventsCutaneous Anthrax Treatment Protocol* Cutaneous Anthrax Treatment Protocol* for Cases Associated with Bioterrorist Events for Cases Associated with Bioterrorist Events


CategoryCategory Initial therapy (intravenous) Initial therapy (intravenous) DurationDuration

Adults Adults Ciprofloxacin Ciprofloxacin Switch to oralSwitch to oral(Including pregnant(Including pregnant 400 mg Q 12 hrs 400 mg Q 12 hrs therapy whentherapy when womenwomen** ** andand OROR clinicallyclinicallyimmunocompromised) immunocompromised) Doxycycline Doxycycline appropriate:appropriate:

100 mg Q 12 hrs100 mg Q 12 hrs Ciprofloxacin 500 mg BID Ciprofloxacin 500 mg BIDANDAND OROR

One or two additional One or two additional Doxycycline 100 mg BIDDoxycycline 100 mg BIDantimicrobialsantimicrobials

Continue for 60 daysContinue for 60 days(IV and PO combined)(IV and PO combined)

Inhalational Anthrax Treatment Inhalational Anthrax Treatment Protocol* for Cases Associated with Protocol* for Cases Associated with Bioterrorist EventsBioterrorist Events

Inhalational Anthrax Treatment Inhalational Anthrax Treatment Protocol* for Cases Associated with Protocol* for Cases Associated with Bioterrorist EventsBioterrorist Events


SmallpoxSmallpox


SmallpoxSmallpox

Worst-case scenario biological agentWorst-case scenario biological agent Highly contagious once rash present (not before)Highly contagious once rash present (not before) World’s population is largely susceptibleWorld’s population is largely susceptible Up to 30% case fatality rate in non-immuneUp to 30% case fatality rate in non-immune Secondary attack rate of 25-40% (10-20 secondary cases Secondary attack rate of 25-40% (10-20 secondary cases

can be expected per index case)can be expected per index case) No specific treatment availableNo specific treatment available

Globally very few physicians currently practicing Globally very few physicians currently practicing have seen actual caseshave seen actual cases

Virus has been weaponized by Soviets, uncertain Virus has been weaponized by Soviets, uncertain who exactly owns viable stockswho exactly owns viable stocks


SmallpoxSmallpox Caused by Variola virus (Orthopox virus)Caused by Variola virus (Orthopox virus)

Immunization of U.S. civilian population suspended in Immunization of U.S. civilian population suspended in 1980, U.S. military recruits in 19891980, U.S. military recruits in 1989

Virus is stable in environmentVirus is stable in environment

Spread primarily by respiratory droplets, also by contact, Spread primarily by respiratory droplets, also by contact, fomitesfomites

Two distinct types of smallpox:Two distinct types of smallpox: Variola Minor (Alastrim): diminutive lesions and mild Variola Minor (Alastrim): diminutive lesions and mild

systemic toxicitysystemic toxicity Variola Major: Ordinary (subtypes discrete, semi-confluent, Variola Major: Ordinary (subtypes discrete, semi-confluent,

confluent), Modified, Flat, Hemorrhagicconfluent), Modified, Flat, Hemorrhagic


SmallpoxSmallpox

Clinical PresentationClinical Presentation 7-17 day incubation period7-17 day incubation period Prodromal phase: 2-4 days of malaise, fever, Prodromal phase: 2-4 days of malaise, fever,

rigors, headache, backache, deliriumrigors, headache, backache, delirium Rash then develops on face, hands, forearms Rash then develops on face, hands, forearms

and legs, including palms and soles and legs, including palms and soles (centrifugal distribution is important (centrifugal distribution is important distinguishing feature).distinguishing feature).

Initial rash is maculopapular. In 1-2 days, Initial rash is maculopapular. In 1-2 days, lesions become vesicular, then evolve into lesions become vesicular, then evolve into round, tense pustules deeply imbedded in the round, tense pustules deeply imbedded in the dermis. Crusts form on 8dermis. Crusts form on 8thth to 9th day of rash to 9th day of rash

Crusts separate to form depressed, Crusts separate to form depressed, hypopigmented scarshypopigmented scars


SmallpoxSmallpox




Smallpox Outbreak, Meschede Hospital, Smallpox Outbreak, Meschede Hospital, Germany, 1970Germany, 1970

Wehrle PF, Bull WHO 1970;43:669


Meschede Hospital OutbreakMeschede Hospital Outbreak


Diagnosis of SmallpoxDiagnosis of Smallpox

Recognition of clinical features in early index cases is key

Identification of a single case of smallpox constitutes an international medical emergency, and should be considered evidence of a bioterrorist attack

Confirmation of diagnosis is made by demonstration of characteristic virions on electron microscopy of vesicular scrapings


Management of SmallpoxManagement of Smallpox

Immediate quarantine of affected and exposed Immediate quarantine of affected and exposed individuals for 17 daysindividuals for 17 days

Only supportive care is available. Cidofovir has Only supportive care is available. Cidofovir has demonstrated demonstrated in vitroin vitro activity activity

Immediate vaccination of all exposed persons with Immediate vaccination of all exposed persons with Vaccinia virus vaccine by inoculation with a bifurcated Vaccinia virus vaccine by inoculation with a bifurcated needle (scarification)needle (scarification)

Administration of Vaccinia Immune Globulin (VIG), 0.6 Administration of Vaccinia Immune Globulin (VIG), 0.6 ml/kg intramuscularly, concomitant with vaccinationml/kg intramuscularly, concomitant with vaccination

Active and passive immunization is effective at Active and passive immunization is effective at preventing disease and death if given within 7 days of preventing disease and death if given within 7 days of exposureexposure

For the past half-century the main concern was For the past half-century the main concern was a nuclear Armageddon caused by atomic a nuclear Armageddon caused by atomic weapons. However, with the discovery of weapons. However, with the discovery of

various offensive biological warfare programs various offensive biological warfare programs around the globe, the concern regarding around the globe, the concern regarding

intentional use of viruses and microorganisms intentional use of viruses and microorganisms as weapons of mass destruction increased as weapons of mass destruction increased

during the past decade. The threat of biological during the past decade. The threat of biological warfare is real. It is now widely acknowledged warfare is real. It is now widely acknowledged

that the biological weapons, in terms of that the biological weapons, in terms of destructiveness and in the generation of panic destructiveness and in the generation of panic and civil disorder could produce an effect that and civil disorder could produce an effect that

is equivalent to that of a nuclear weapon. is equivalent to that of a nuclear weapon.


Medical professionals globally Medical professionals globally should make sure that the world should make sure that the world

does not suffer a catastrophe as a does not suffer a catastrophe as a result of the use of biowarfare result of the use of biowarfare

pathogenspathogens



War on DiseasesWar on DiseasesWar on DiseasesWar on Diseases The recent emphasis on bioterrorism: The recent emphasis on bioterrorism:

important truth about infectious diseasesimportant truth about infectious diseases– Even without the element of intentional Even without the element of intentional

terror, diseases are a huge source of terror, diseases are a huge source of human suffering—and a tremendously human suffering—and a tremendously destabilizing forcedestabilizing force

– Nearly half of the world’s premature deaths Nearly half of the world’s premature deaths (deaths under the age of 45) are caused by (deaths under the age of 45) are caused by infectious diseasesinfectious diseases

– Some 30 million infants in developing Some 30 million infants in developing countries remain unprotected by the countries remain unprotected by the lifesaving childhood vaccines that in the lifesaving childhood vaccines that in the rest of the world are administered rest of the world are administered routinely; a million die each year from routinely; a million die each year from measles alonemeasles alone

Pathogens of BT do threaten Pathogens of BT do threaten humanity but let us NOT loose sight humanity but let us NOT loose sight of the naturally occurring microbes of the naturally occurring microbes that threaten billions of people in a that threaten billions of people in a

world weakened by world weakened by poverty, war, lack poverty, war, lack of clean water, and inattentionof clean water, and inattention

specially during wars & complex specially during wars & complex humanitarian emergencieshumanitarian emergencies



Thank YouThank You

Documents

Chotani, 2003 Laboratory Criteria for Identification of B. anthracis n From clinical samples, such as blood, cerebrospinal fluid (CSF), skin lesion (eschar),