Anthrax: Bacteriology, Clinical Presentations and Management

ANTHRAX: BACTERIOLOGY, CLINICAL PRESENTATIONS AND MANAGEMENT

Historical Background
Anthrax - History of Current Threat
Microbiology
Epidemiology
Clinical Manifestations
Diagnosis
Control and Prevention
Immunization
Treatment
Emerging/Investigational Therapies
Hospital Infection Control
Future Research
References
View Dr. Nancy Khardori's Presentation on Anthrax (Adobe Acrobat Reader required)

Historical Background

The earliest known description of anthrax was made in 1491 BC in writings from Egypt and Mesopotamia and in the Old Testament's description of the Fifth Plague of Egypt (1). There are descriptions of anthrax involving animals and humans in the early literature of Hindus, Greeks and Romans. The first pandemic in Europe known as "Black Bane" was recorded in 1613 and caused more than 60,000 deaths. The first epidemic in the United States occurred in the early 18th century. Outbreaks of occupational cutaneous and respiratory anthrax were reported from Industrial European countries in the mid-1800's. Cutaneous infection was caused by handling wool, hair and hides. Respiratory disease was caused by processes that created aerosol while handling wool, hair and hides.

Deleford described the microscopic appearance of anthrax bacteria in 1838.
Devain demonstrated the infectivity of anthrax in 1868.
Anthrax became the first human disease attributed to a specific etiological when Koch showed it to fulfil his "postulates" in 1877.
Pasteur first tested the attenuated spore vaccines in sheep in 1881.
Decreased use of imported potentially contaminated animal products and improved industrial and animal husbandry practices led to a steady decrease in annual numbers of cases in the developed countries in the early 1900's.
Sterne reported the development of an animal vaccine from the spore suspension of an avirulent, noncapsulated live strain of Bacillus anthralis in 1939.
Cell free anthrax vaccine for humans - a sterile filtrate of cultures from an avirulent noncapsulated strain that elaborates protective antigen was licensed in the United States in 1970.
Both live attenuated and killed vaccines have been developed. In the former Soviet Union, the human live anthrax vaccine has undergone many field trials (2).
The largest recorded outbreak of anthrax in humans and likely the largest among animals occurred in Zimbabwe in 1978-1980 during the time of its Civil War (3). 10,000 human cases and 151 deaths were documented.

Top

Anthrax - History of Current Threat

Research on anthrax as a biological weapon started > 80 years ago (4).
Today 17 nations are believed to have offensive biological weapons programs.
Iraq has acknowledged producing and weaponizing anthrax between 1955 and 1991.
Aerosols of anthrax bacteria and botulism toxin dispersed in Tokyo on 8 occasions failed to produce illness.
WHO's expert committee (1970) estimated that an aircraft release of 50 kg of anthrax over a 5 million population would kill 250,000 - 100,000 of whom would die without treatment.
Accidental aerosolized release of anthrax spores in the Soviet Union in 1979 resulted in 79 cases and 68 deaths.
Outdoor aerosol release could be a threat to people indoors (5).
US Congressional Office of Technology Assessment (1993) estimated that between 130,000 and 3 million deaths could follow the aerosolized release of 100 kg of anthrax spore upwind of the Washington, D.C. area. The lethality would match or exceed that of a hydrogen bomb.
The CDC economic model estimated - $26 billion per 100,000 persons exposed.

Top

Microbiology

Bacillus anthracis - anthrakis is the Greek word for coal because anthrax causes black, coal-like skin lesions. All Bacillus species are aerobic, gram-positive, spore forming, non motile, bacteria. The spore size of B. anthracis is about 1 :g. Factors that favor sporulation include: alkaline soils (pH grater than 6.0); high nitrogen levels in the soil caused by decaying vegetation; alternating periods or rain and drought and; temperatures in excess of 15.5OC. Spores grow readily at 37OC on ordinary laboratory media with "curled hair" colony morphology and "Jointed Bamboo rod" cellular appearances on staining.

Spores germinate in an environment rich in amino acids, nucleosides and glucose into rapidly multiplying vegetative bacteria. The vegetative cell is nonflagellated and large (1 - 8 :m in length and 1 - 1.5 :m in breadth.) Full virulence requires the presence of a capsule and a three component toxin - protective antigen, lethal factor and edema factor. The toxin has two enzymatic components. The first or edema factor (EF) is an adenylate cyclase that leads to increase in cyclic AMP resulting in edema at the site of infection. The second or lethal factor (LF) is a protease that appears to alter the production of cytokines by macrophages and to induce macrophage lysis and lethal effects of anthrax in animals.(6,7). A third non-enzymatic component, the protective antigen (PA) helps in the delivery of the two enzymatic components into the cells (8 ). Adding a mutant PA can prevent release of EF/LF inside the cells (9). The gene coding for major virulence factors of B. anthracis reside on plasmids (10,11). The sequences of the virulence plasmids, p x 01 and p x 02 in B. anthracis have already been completed ( 12,13). The work on the anthrax genome itself is still underway. Vegetative bacteria survive poorly outside the animal or human host and form spores after local nutrients are exhausted, e.g., infected body fluids exposed to ambient air. The hardy spores can survive for decades in the environment.

Top

Epidemiology

Anthrax is a disease of herbivores acquired by ingesting spores from the soil However, few if any warm blooded species are entirely immune to it. Prior to animal vaccine and antibiotics, the disease was one of the foremost causes of uncontrolled mortality in cattle, sheep, goats, horses and pigs worldwide (14). Animal vaccination programs have drastically reduced animal mortality. Humans are incidental hosts. Anthrax spores continue to be documented in soil samples from throughout the world.

Sources of Animal Anthrax

Grazing in "incubator areas" (soil contaminated with B. anthracis spores and organisms.)
Excreta and saliva from dying or dead animals.
Imported bone meals and vegetable protein (e.g. groundnut).
Wool, hair wastes.
Cleanings used in fertilizers
Tannery effluents
Commercial animal fee (rare in US - last outbreak among swine in 1952).
Blood-sucking flies.
Carrion eaters.

Natural disease in humans is acquired by contact with anthrax-infected animals or contaminated animal products. Anthrax remains a problem in developing countries. Human cases occur in industrial or agricultural environments. The incubation period is 2- 5 days. Older observations showed that unimmunized workers in wool mills could inhale several hundred spores daily without developing disease. The LD50 for aerosolized anthrax spores is around 8000 colony forming units in experiments done by the US Army in cynomolgous monkeys (15). Fatality rate under these experimental conditions is 20 - 80 %. However, the occurrence of sporadic cases in people with a low dose contact may be explained by differences in the virulence of strains and the role of host factors.

Top

Clinical Manifestations

Cutaneous anthrax -

The most common naturally occurring form - 95% of anthrax cases in developed countries, 224 cases in the US between 1944 and 1994.
Exposed areas on the arms and hands followed by face and neck
Pruritic papule ulcer surrounded by vesicles black necrotic central eschar with edema.
After 1 - 2 weeks, eschar dries, loosens, separates, leaving a permanent scar.
Regional lymphangitis and lymphadenitis and systemic symptoms. Mortality rate for untreated disease - 20%.
Antibiotics decrease edema and systemic symptoms.
Differential diagnosis - Plague and Tularemia.

Respiratory anthrax -

Inhalational anthrax follows deposition of spore-bearing particles of 1 - 5 : into alveolar spaces. The size of a B. anthracis spore is 1 :m.
Macrophages ingest spores resulting in their lysis and destruction.
Surviving spores are transported to mediatinal lymph nodes.
Germination may occur up to 60 days.
Following germination, disease follows rapidly.
Toxins released by replicating bacteria cause hemorrhage, edema and necrosis.
Typical bronchopneumonia does not occur. Chest x-ray findings and absence of hemoptysis differentiates inhalational anthrax from pneumonic plague.
LD50 is 2500 to 55,000 inhaled spores.
Hemorrhagic thoracic lymphadenitis, hemorrhagic mediastinitis (all patients) and hemorrhagic meningitis (50% of patients) are the pathological hallmarks of disease.
Clinical presentation shows a biphasic pattern - non specific symptoms followed by fever, dyspnea, diaphoresis and shock.
Morality rate is 80 - 90%, when untreated.
Aggressive, early antimicrobial therapy and improved supportive care improves prognosis.

Gastrointestinal anthrax-

Oropharynegeal anthrax
Oral or esophageal ulcer - regional lymphadenopathy, edema and sepsis
Abdominal anthrax Predominantly terminal ileum or cecum. Nausea, vomiting, malaise progressing to bloody diarrhea, acute abdomen and sepsis. Mortality rate is high.

Top

Diagnosis

Cutaneous - Vesicular fluid from skin lesions - gram stain and culture

Inhalational - Chest X-ray - widened mediastinum

Blood - Gram stain and culture

Biopsy - histopathology and culture

Gastrointestinal - Biopsy - histopathology and culture Rapid diagnostic tests - EIA, PCR - for confirmation

New rapid molecular diagnostic tests are being extensively studied. More than 1200 strains of B. anthracis have been identified around the world over the years. Dr. Paul Keim's genetics laboratory at Northern Arizona University in Flagstaff, has used amplified fragment length polymorphism (AFLP) to examine all of them (16). His laboratory has also adapted some precise assays like VNTR (Variable Number Tandem Repeat) and MLVA to study 400 of the 1200 known strains of B. anthracis. It takes 12 hours for this laboratory to analyze an anthrax sample. Detection of B. anthracis DNA by light cycler Polymerase Chain Reaction after autoclaving (17) and by Rapid Cycle Real Time Polymerase Chain Reaction - The Mayo Roche Rapid Anthrax Test (18) were reported recently.

Top

Control and Prevention

Formaldehyde disinfection
Industrial hygiene - dust collecting equipment and effective environmental clean up procedures.
Environmental decontamination - paraformaldehyde vapor.
Spores can persist and remain viable for 36 years.
Surface contamination - 5% hypochlorite or 5% phenol.
Forbidding the sale of meat from sick animals.
Cooking all meats thoroughly.
Control of anthrax in animals - vaccination and reporting of disease.

Top

Immunization

Human attenuated live anthrax vaccine used in former Soviet Union.
Human killed anthrax vaccine.
Sterile filtrate of cultures from an avirulent noncapsulated strain that elaborates protective antigen - human vaccine in use in the US.
The vaccine was field tested in employees of four textile mills in the US - "Anthrax Vaccine Adsorbed" (AVA).
Effectiveness - 92.5%.
Given SQ 0.5 ml at 0,2,4 weeks and 6, 12, 19 months followed by annual boosters.
Used for people exposed to contaminated materials or environments.
US Armed Forces - 1998 Vaccinate every member - 1.4 million active duty troops and 1 million reservists.
The vaccine is produced by Bioport Corp., Lansing, Michigan. Current information available at the Bureau of Disease Control and Laboratory Services, Michigan Department of Public Health, PO Box 30035, 3500 N. Logan Street, Lansing, Michigan.
Future vaccines - Recombinant anthrax toxin, PA toxoid vaccines, Pa - producing live vaccines.

Friedlander and others at the USAMRIID in Fort Detrick, Maryland, have shown that recombinant PA, produced by non- spore- forming B. anthracis protects rhesus monkeys against inhalational anthrax (20). AVANT Immunotherapeutics, Needham, Massachusetts, is developing an oral one dose anthrax vaccine. This vaccine is made from attenuated V. cholerae that produces PA and acts rapidly.

CHEMOPROPHYLAXIS/POST-EXPOSURE PROPHYLAXIS*

Drug	Adults	Children
Oral Fluoroquinolones Ciprofloxacin (Cipro)1	500 mg bid	10 - 15 mg/kg bid2
Oral tetracyclines3 Doxycycline (Vibramycin, others)4	100 mg bid	2.2 ,g/kg bid2
Oral Penicillins 3,5 Amoxicillin (Amoxil, others)6	500 mg tid	80 mg/kg/day divided into 3 doses

1 Other fluoroquinolones such as ofloxacin (Floxin) 400 mg bid or Levofloxacin (Levaquin) 500 mg once daily may also be effective. Ciprofloxacin approved by FDA in 2000.
2 Should be changed to Amoxicillin as soon as susceptibility to penicillin has been confirmed.
3 Susceptible strains
4 Tetracycline 500 mg qid should also be effective
5 Penicillin resistance could emerge during treatment, but should not be a problem in prophylaxis
6 Penicillin VK1 7.5 mg/kg in adults, or 12.5 mg/kg qid, should also be effective for prophylaxis
* Medical Letter, October 29, 2001 (21)

Post exposure vaccination following a biological attack with anthrax is recommended to protect against residual retained spores after chemoprophylaxis. This approach may also reduce the duration of antibiotic prophylaxis to 30 - 45 days.

Mycoplasma contamination of AVA had been suggested as a possible cause of Persian Gulf Illness. Recent studies by nonmilitary laboratories did not show any mycoplasma or mycoplasma DNA and did not support its survival in the vaccine (19).

Top

Treatment

No clinical studies of the treatment of inhalational anthrax in humans.
Most anthrax strains are sensitive to penicillin - preferred antibiotic for treatment in the past ($-lactamase production).
Penicillin and doxycycline approved by the FDA
Engineered vaccine strain resistant to penicillin and tetracycline.
All fluoroquinolones active in vitro. Ciprofloxacin excellent efficacy in animal models.
Combination antibiotic therapy may have a role.
Other antibiotic choices include streptomycin, erythromycin, chloramphenicol, vancomycin, clindamycin, and first generation cephalosporins.
Treat for 60 days because of risk of delayed germination of spores.
Treatment of cutaneous anthrax does not alter the evolution of eschar but prevents systemic disease.
Systemic steroids for cervical edema and meningitis

Top

Emerging/Investigational Therapies

The CDC and other federal agencies are discussing the use of "antitoxin" therapy as an adjunct to antimicrobial therapy (22). Currently a limited supply of plasma collected from vaccinated military personnel is available. There are plans to collect a second larger batch from vaccinated volunteers for use in treatment and for animal studies. Maynard et al used in vitro DNA manipulation and an E. coli expression system to create an antibody library. Selected antibodies from this library were shown to bind to PA of B. anthracis with high affinity, prevented anthrax toxin from binding to macrophages and protected rats from a lethal challenge (23). Iverson and Georgiou at the University of Texas, Austin, have reported the production of a monoclonal antibody against anthrax toxin (unpublished data). This antibody reportedly has responded 40-fold better affinity for the toxin and protected rats injected with the toxin (22). The other approach would be to design a polyvalent inhibitor of anthrax toxin. Mourez et. al. isolated a peptide from a phage display library that binds weakly to the heptameric cell-binding subunit of anthrax toxin and prevents the interaction between cell-binding and enzymatic moieties. (24) A molecule consisting of multiple copies of this non natural peptide prevented assembly of the toxin and blocked its action in an animal model. A number of investigators have identified the cellular receptor for protective antigen (25) and the crystal structure of the lethal factor (26,27). These advances have the potential of helping design new drugs and or antitoxin therapies.

Top

Hospital Infection Control

Standard barrier precautions for all forms.
Contact isolation for draining lesions.
Notify Microbiology Laboratory - BSL 2 condition.
Hypochlorite for environmental cleaning.
Proper burial or cremation of humans and animals.
Autopsy related instruments and materials autoclaved or incinerated.

Top

Future Research

Improved rapid diagnostic techniques
Improved prophylactic and therapeutic regimens
Improved second generation vaccine
Impact of B. cereus genes on vaccine induced immunity.
Improved capability to distinguish between highly similar types of B. anthracis.

The entire DNA sequence of the "Florida" strain taken from the first 2001 victims has now been read and some rare distinguishing features have been identified (28).

Top

References

Koch R. The etiology of anthrax, based on the life history of Bacillus anthracis. In: Brock TD (ed). Milestones in Microbiology. Washington, D.C., American Society for Microbiology, 1961, pp. 89-90.
Turnbull PC. Anthrax vaccines: past, present and future. Vaccine. 1991; 9:533-539
Kobuch WE, Davis J, Fleischer K, et al. A clinical and epidemiological study of 621 patients with anthrax in western Zimbabwe. Salisbury Medical Bulletin. Proceedings of the International Workshop on anthrax. 1990; 68 (suppl):34-38.
Inglesby TV, Henderso DA, Bartlett JG et. al. Anthrax as a Biological Weapon. Medical and Public Health Management. JAMA 1999; 281:1735-1745.
Cristy GA, Chester CV. Emergency protection against aerosols. Oak Ridge, Tenn:Oak Ridge National Laboratory; 1981.
Hana PC, Acosta D, Collier RJ. Proc Natl. Acad. Sci. USA 1993; 90:10198.
Erwin JL, et al. Infect Immun. 2001; 292:675.
Sellman et al. Science. 2001; 292:695.
Enserink M. This time it was real: Knowledge of anthrax put to the test. Science. 2001; 294:490-491.
Mikesell P, Ivins BE, Ristropn JD, et al. Infect Immun. 1983; 39:371.
Green BD, Battisti C, Koehler TM, et al. Infect Immun. 1985; 49:291.
Okinaka RT, et al. Journal of Bacteriology. 1999; 181:6509.
Okinaka RT, et al. J. Appl. Microbiol. 1999; 87:261.
World Health Organization. Guidelines for the Surveillance and Control of Anthrax in Humans and Animals, Geneva Switzerland. 1998 WHO/EMC/ZDI/98.6.
Peters CJ, Hartley DM. Anthrax Inhalation and lethal human infection. The Lancet. 2002; 359:710.
Dalton R. Genetic sleuths rush to identify anthrax strains in mail attacks. Nature. 2001; 413:657-658.
Espy MJ, Uhl JR, Sloan LM et. Al. Detection of vaccinia virus, herpes simplex virus, varicella-zoster virus, herpes and Bacillus anthracis DNA by light cycler Polymerase Chain Reaction after autoclaving: Implications for biosafety of bioterrorism agents. Mayo Clin Proc. 2002; 77:624-628.
Uhl JR, Constance BA, Lynne MS, et. Al. Application of rapid-cycle real-time polymerase chain reaction for the detection of microbial pathogens: The Mayo-Roche rapid anthrax test. Mayo Clin Proc. 2002; 77:673-680.
Hart MK, Del Giudice RA, Korch GW. Absence of mycoplasm contamination in the anthrax vaccine. Emerging Infectious Diseases. 2002; 8:94-96.
Enserink M, Marshall E. New anthrax vaccine gets a green light. Science. 2002; 296:639-640.
The Medical Letter. Post-exposure anthrax prophylaxis. 2001; 43:91-92.
Enserink M. 'Borrowed Immunity' may save future victims. Science. 2002; 295:777.
Maynarud JA et.al.Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity. Nature Biotechnol. 2002; 20:597-601
Moure ZM, Kane RS, Mogridge J. Designing a polyvalent inhibitor of anthrax toxin. Nature Biotechnol. 2001; 19:9580961.
Bradley KA, Mogridge J, Maurez M et. el. Identification of the cellular receptor for anthrax toxin. Nature. 2001; 414:225-229.
Pannifer AD, Wong TY, Schwarzenbacher R, et.al. Crystal structure of the anthrax lethal factor. Nature. 2001; 414:229-232.
Friedlander A. Tackling anthrax. Nature. 2001; 414:160-161.
Read TD, et al. Comparative genome sequencing for discovery of novel polymorphisms in Bacillus anthracis. Sciencexpres, doi; 10:1126/Science. 1071737 (2002).

Top