ANTHRAX: A REVIEW. 1Khalifa Sifaw Ghenghesh, MSc, PhD, DipBact, 2Taher Rezgalla, MBBS, MD, and 3Rajab El-Tobgi, MSc, PhD, DipBact 1Dept. of Medical Microbiology, Faculty of Medicine, Al-Fateh

University, 2National Tuberculosis Center,Tripoli, 3National

Tuberculosis Center, Benghazi-Libya

Correspondence:

Prof. Khalifa Sifaw Ghenghesh, MSc, PhD, DipBact Dept. of Medical Microbiology, Faculty of Medicine,

Al-Fateh University,

P.O.Box 80013,

Tripoli-Libya.

E-mail: ghenghesh_micro@yahoo.com

To cite this article: Ghenghesh KS., Rezgalla T., and Tobgi R. 2002. Anthrax: A Review. Jamahiriya Med J; 2 (1): 17-23.

ABSTRACT: Anthrax is a zoonotic disease that affects mainly large domesticated animals

and caused by the bacterium Bacillus anthracis. Man acquires the disease

accidentally through contact with infected animals or their products, often by

the cutaneous route and only rarely by the respiratory or gastrointestinal

routes. B. anthracis is on the top of the list of agents used in biological

weapons programs in many countries. Recent events that occurred in mid-

September last year in United States involving the intentional distribution of B.

anthracis spores through postal system has resulted in a worldwide interest in

anthrax. This article was intended to give a fair idea on the causative agent

and its epidemiology, determinants of pathogenicity, clinical manifestations,

clinical and laboratory diagnosis, treatment, and prevention and control of the

disease.

INTRODUCTION: Anthrax is a zoonotic disease that affects mainly large domesticated

animals. Man acquires the disease accidentally through contact with infected

animals or their products, often by the cutaneous route and only rarely by the

respiratory or gastrointestinal routes (1). Bacillus anthracis, a gram-positive,

rod-shaped bacterium, is the causative agent of anthrax. Although, the disease

was known to man since ancient Egyptians and may have been responsible

for two of the plagues that afflicted Egypt around 3500 years ago (2), the

causative organism was not known until Robert Koch in 1877 was able to grow

it in vitro. Also, by inoculating pure cultures of the organism in healthy animals

and inducing the disease in them, Koch established the famous postulates that

must be fulfilled before an agent can be identified as the cause of a specific

infection (3). Furthermore, in 1881 Pasteur employed the first successful

bacterial vaccine in animals by using a live heat attenuated culture of B.

anthracis (4).

B. anthracis has been used for more than 80 years as a biological

weapon. During the 1st World War the Germans used B. anthracis to infect

livestock and contaminate animal feed to be exported to Allied forces (5). From

1932 to the end of the 2nd World War the Japanese experimented with B.

anthracis and other bacteria agents on prisoners of war with an estimated

death toll of 10,000 prisoners (6, 7). United States, United Kingdom and others

also used B anthracis in their biological weapons programs during World War

II and afterwards. However, It was not until the Gulf War, that the world

attention was seriously drawn to B. anthracis and other biological weapons

with allegations that Iraq possess such weapons and the fear that it might use

them against US lead forces in the Gulf. At present, it is believed that at least

17 countries have offensive biological weapon programs (8). B. anthracis

spores are still at the top of the list of organisms used in these programs and

also the favorite agent used by terrorist organisations and individuals to cause

panic and fear in civilian populations. This is supported by events that occurred

in mid-September last year in the US involving the intentional distribution of B.

anthracis spores through the postal system (9). This resulted in more than 22

individuals developing anthrax and at least 5 of them have died (10, 11, 12, 13,

14). Men and women, old and young including an infant were among the

affected persons. What happened in the US can occur anywhere else

regardless of the political, religious, cultural and geographical differences that

may exist in the world we live in today. This review, therefore, was intended to

give a fair idea on the causative agent and its epidemiology, determinants of

pathogenicity, clinical manifestations, clinical and laboratory diagnosis,

treatment, and prevention and control of the disease.

BACTERIOLOGY: Bacillus anthracis is a gram-positive (Figure 1), non-motile, spore-

forming bacillus, 3-5 μm long and 1-1.2 μm wide. The organisms usually are

found singly or in pairs when examined in smears from the blood or tissues of

an infected animal. Although the organisms grow well on general laboratory

media, blood agar is preferred for demonstration of characteristic colonial

morphology. After incubation on blood agar at 37oC for 24 hours the organisms

produce non-hemolytic typical colonies, 2-3 mm in diameter, with wavy margin

and small projections (a "medusa's head" appearance) (Figure 2). B. anthracis

produces a capsule in the host but not on ordinary cultural media. However, if

sodium bicarbonate is incorporated into such a media and incubated in the

presence of 5% CO2 the organisms form mucoid colonies as a result of

capsule production. The capsule is visible when the organism is stained with

polychrome methylene blue by McFadyean's method (15) (Figure 3).

Although, spores produced by B. anthracis are extremely resistant to

adverse chemical and physical conditions, they can be destroyed by

autoclaving at 121oC for 15 minutes or by other methods of sterilization. In

contaminated pastures the spores may remain viable for many years and

become a source of infection for grazing animals for long periods of time. EPIDEMIOLOGY: In animals:

Anthrax mainly occurs in herbivores (cattle, sheep, camels, horses,

etc.), however practically all animals are susceptible to some degree. The

disease in herbivores tends to be severe, with a high mortality rate (2). Among

birds only ostriches are known to be susceptible to anthrax (16). Omnivores,

such as man, swine, and carnivores such as dogs, possess greater natural

resistance to anthrax infections than herbivores. Terminally ill animals tend to

bleed from orifices resulting in pasture, soil or water sources being

contaminated by B. anthracis that can subsequently sporulate and persist in

the environment. Sporadic outbreaks involving small numbers of animals as a

result of grazing on contaminated forage plants still occur all over the world.

However, large outbreaks have been reported mainly from developing

countries in Africa and Asia (17, 18, 19, 20), where vaccination programs

either do not exist or are sporadic. In 1945 an outbreak of anthrax in Iran

resulted in the death of 1 million sheep (21).

In humans: Three major forms of anthrax occur in humans: inhalation,

gastrointestinal and cutaneous with the later accounting for 95 percent of

cases (22). Occurrence of human anthrax in developed countries has been

reduced dramatically as a result of vaccination of livestock. This can be

demonstrated clearly in the US were the total annual incidence has fallen from

an average of nearly 130 cases in the early part of 20th century to less than 1

case annually over the last 20 years of the same century (23, 24). Sporadic

cases also, still occur in Europe and other developed countries and most of

them associated with exposure to animal products (goat hair, hides, etc.)

imported from Turkey, Sudan, and Pakistan (3). An exception to this is the

outbreak that occurred in Sverdlovsk, in the former USSR in 1979. This

outbreak resulted from an accidental release at a military microbiology facility

with at least 66 deaths (25). In developing countries sporadic and large

outbreaks had been reported mainly from Africa and Asia (17, 18, 19, 26, 27).

These outbreaks usually resulting from either contact with infected animals or

consumption of their meat. One of the largest outbreaks reported in the last

century occurred in Zimbabwe between 1979 and 1985 with more than 10,000

cases, virtually all of them were cutaneous anthrax (28).

In Africa, biting flies have been implicated in the transmission of anthrax

particularly in the younger age groups (29). Also, some investigators believe

that vultures are the main agents for long-distance dispersal, but that blowflies

cause local dissemination with a potential for explosive spread within a region

(30). Human to human transmission of anthrax is unlikely to occur and there is

no evidence of spread to health workers caring for patients with the disease

(29).

VIRULENCE FACTORS:

The ability of B. anthracis to cause disease depends on two important

virulence factors:

i. The anthrax toxin: encoded by plasmid pXO1 (184.5 kilobase pairs [kbp])

and composed of three proteins: edema factor (EF) which is a calmodulin-

dependent adenylate cyclase (31, 32), lethal factor (LF) which is a zinc

metalloprotease (33, 34) that inactivates mitogen-activated protein kinase

kinase in vitro (35, 36), and protective antigen (PA) which acts as the receptor-

binding component mediating entry of either EF or LF into target cells (32).

The three components of anthrax toxin act in binary combinations to produce

two distinct reactions in experimental animals (37). Coinjection of PA and EF

(a combination termed ''edema toxin") intradermally produces edema, while

coinjection of PA and LF (lethal toxin) causes death in susceptible animals.

However, none of the three individual proteins is toxic to animals. The anthrax

toxin is thought to hinder the immune response mounted by the host against

the infection.

ii. The capsule: encoded by plasmid pXO2 (95.3 kbp), a poly-D-glutamic

polymer that interferes with phagocytosis.

The infection starts when the endospores of B. anthracis enter the body

through abrasions, inhalation or ingestion. They are then phagocytosed by

macrophages and carried to the regional lymph nodes. Inside the

macrophages they germinate and become vegetative bacteria (38, 39), that

are then released from macrophages and reach the blood stream, after

multiplying in the lymphatic system, causing massive septicaemia. Both

virulence factors are expressed by the organism in this process and the

resulting toxaemia has systematic effect that lead to the death of the host (40). CLINICAL MANIFESTATIONS: Cutaneous anthrax:

Occurs when spores are deposed on breached areas of the skin

resulting from abrasions or cuts. The areas most frequently affected are arms

(Figure 4), hands, face, and neck. The infection starts within 1-7 days

(maximum 12 days) as painless small papule that develops within a few days

into a vesicle filled with dark bluish black fluid that contains numerous, large

gram-positive bacilli. The vesicle enlarges and satellite vesicles may develop.

The vesicle ruptures and reveals a black eschar (sometimes referred to as a

malignant pustule). When the lesions are on the face or neck, the edema may

become massive and occasionally multiple bullae develop along with marked

toxic effect (41). Within one to two weeks the eschar dries and falls off, most,

often leaving no permanent scar. Regional lymphadenopathy is usually

present in the first few days. Although, most patients experience headache,

malaise, and low-grade fever, some may be asymptomatic. Mortality resulting

from cutaneous anthrax may reach as high as 20% in untreated cases. With

antibiotic treatment, however, death is very rare. In humans, anthrax infection

provides permanent immunity; second attacks are rare and tend to be much

milder (42).

Inhalation anthrax: Occurs when spores-bearing particles are inhaled and deposited into

alveolar spaces (43, 44). Spores are then ingested by macrophages and

transported via lymphatics to mediastinal lymphnodes. Germination of spores

then takes place (up to 60 days) and the disease follows rapidly. Hemorrhage,

edema, and necrosis ensue as a result of toxins released from replicating

bacteria. Inhalation anthrax has been clinically described as a two stage-illness

(45). After an incubation period of one to six days, initially, patients experience

non-specific symptoms that include fever, malaise, myalgia, cough, chest pain

and abdominal pain. Within two to three days, patients enter into the second

stage that starts abruptly with fever, acute dyspnea, diaphoresis, cyanosis, and

shock. Some cases are presenting with stridor due to massive

lymphadenopathy and expansion of the mediastinum (46, 47). Hemorrhagic

meningitis, with associated obtundation and nuchal rigidity, has been reported

in nearly fifty percent of patients. Death occurs within a few to 36 hours.

Prognosis is poor in inhalation anthrax even with intensive antibiotic therapy.

Without antibiotic therapy, mortality is fundamentally 100% (48, 49). Data from

non-human primates exposed to anthrax spores, has shown that for humans

the LD 50 (lethal dose causing 50% mortalities to individuals exposed to it) is

2500 to 55000 inhaled spores (50).

Gastrointestinal anthrax: The intestinal form of anthrax occurs after ingestion of under-cooked

spores-contaminated meat. Symptoms, appearing 2-5 days from ingestion,

include nausea, vomiting, fever, and abdominal pain. Hematemesis, massive

ascites and bloody diarrhoea may follow rapidly. At this stage of infection

symptoms may appear similar to the sepsis syndrome occurring in inhalation

or cutaneous anthrax (51). A feature of intestinal anthrax is the presence of

hemorrhagic mesenteric lymphadenitis (41). Given the difficulty of early

diagnosis, mortality from gastrointestinal anthrax can exceed 50 percent.

Other forms of anthrax: Anthrax meningitis: Despite intensive antibiotic therapy, this form of

anthrax is almost always fatal. It occurs as a result of bacteremia after any of

the other forms of anthrax. The findings of postmortem examination are

compatible with hemorrhagic meningitis, with extensive edema, inflammatory

infiltrates, and numerous gram-positive bacilli in the leptomeninges (52, 53).

Oral-oropharyngeal anthrax: This is an unusual manifestation of

anthrax in humans and with no symptoms or signs suggestive of cutaneous,

inhalation, or gastreointestinal anthrax nor septicemic or meningeal

complications. The syndrome is potentially fatal and characterized by a

mucosal lesion in the oral cavity and/or oropharynx that can progress to

pseudomembranous necrosis, and to cervical adenopathy and edema (54).

DIFFERENTIAL AND LABORATORY DIAGNOSIS: Human anthrax is not a common disease, particularly in developed

countries, and very few medical doctors have ever seen a case, which makes

the diagnosis of sporadic cases of anthrax clinically not an easy task. In

developing countries, in addition to clinical findings, history of exposure to

animals or their products is the basis of diagnosis. In cutaneous anthrax,

other skin lesions caused by pyogenic and other bacteria should be taken into

account. For example, staphylococcal lymphadenitis should be considered the

most likely cause if the lesion is purulent and the regional lymph nodes are

palpable, although superinfected cutaneous anthrax lesion with pyogenic

bacteria may occur (55). In inhalation anthrax, chest radiography showing

widened mediastinum (Figure 5) in a previously healthy patient with flu-like

symptoms, without clinical evidence of pneumonia, of recent origin should

suggest the diagnosis. In gastrointestinal anthrax, symptoms of acute

abdominal pain in patients suspected of eating contaminated meat should be

considered as possible early signs of the disease (40).

Rapid screening methods, such as polymerase chain reaction (PCR)

and enzyme-linked immunosorbent assay (ELISA) for protective antigen,

directly used on clinical and environmental materials have been developed,

however, these methods are available in reference and research laboratories

and mainly in developed countries. On the hospital laboratory level, initial

identification can be made by direct Gram's-stained smear of a skin lesion

(vesicular fluid or eschar), cerebrospinal or pleural fluid, unspun peripheral

blood showing encapsulated, broad, gram-positive bacilli (41, 56). Initial

identification also made by plating the above clinical specimens on blood agar

and observing the characteristic cellular and colonial morphology (see

bacteriology section above). Furthermore, blood cultures systems (blood

bottles) can be used for blood samples obtained from patients suspected with

anthrax and this may increase the chances of isolating the causative organism.

Identification of B. anthracis can be achieved by using API 50 CH test strips

(57). However, if laboratories are not alerted to the possibility of anthrax, B.

anthracis may not be correctly identified (56). Confirmatory tests can be

carried out in a local (if available) or international reference laboratory.

TREATMENT: Because of its rarity early diagnosis of anthrax will be difficult and in

some cases may result in misdiagnosis. Therefore, individual cases of anthrax

will always be a challenge for clinicians and delayed intervention is the norm.

In the past, penicillin, in large doses, was considered the drug of choice in

treating human anthrax. Alternative antibiotics, for patients allergic to penicillin,

include erythromycin and tetracycline. Other antibiotics that can also be used

include the aminoglycosides (gentamicim, tobramycin, amikacin)

chloramphenicol, clindamycin, fluoroquinolones (ciprofloxacin, ofloxacin,

levofloxacin), impinem, rifampin and vancomycin). B. anthracis is resistant to

cefuroxime, third generation cephalosporins (cefotaxime, ceftazidime, etc.),

aztreonam, trimethoprim and sulphamethoxazole. Cutaneous anthrax will

respond excellently to antibiotic treatment with mortality rate reduced to less

than 1%. Untreated inhalation anthrax is almost always fatal, however, even if

antibiotic treatment started early in the course of the disease, high fatality rates

will occur among the affected persons. However, early institution of antibiotics

and aggressive supportive therapy appears to be the keys to successful

management of inhalation anthrax (58). Other procedures that include chest

tube drainage of the recurrent pleural effusions, which are typically

haemorrhagic, can result in a dramatic clinical improvement (58).

Recently the Centers for Disease Control and Prevention (CDC),

published recommendations for the antimicrobial treatment and prophylaxis of

inhalation and cutaneous anthrax (59). These recommendations are

summarized in Table 1 and 2.

CONTROL AND PREVENTION: Control of anthrax in animals is a pre-requisite for its control in humans.

A living spore vaccine derived from a noncapsulated strain of B. anthracis

(Sterne strain) is available for use in livestock. The vaccine is administered to

animals as a single dose with a yearly booster. Although, a human anthrax

vaccine from non-capsulated strain of B. anthracis was available since 1943

and developed by the Sanitary Technical Institute in Russia (USSR previously)

(60), the most widely used and studied in recent years is anthrax vaccine

adsorbed (AVA). AVA consists of a noninfectious sterile filtrate from the culture

of an attenuated strain, adsorbed to the adjuvant, aluminium hydroxide (23).

The vaccine is administered subcutaneously at 0, 2, and 4 weeks and 6, 12,

and 18 months with yearly boosters. AVA has been licensed by the Drug and

Food Agency (FDA) in the US since 1970 and recommended for goat hair and

woolen mill workers, veterinarians, laboratory workers, and live stock handlers

who are at risk due to occupational exposure (61). The vaccine is not indicated

for pregnant women, persons with any active infection or acute illness, or

taking immune-suppressing drugs. Although, mild, moderate, and severe

reactions have been reported in 3-20%, 1-3%, and in <1%, respectively, of

individuals given AVA (62), there is strong evidence that the vaccine is safe

and effective against inhalation anthrax (61).

Education of the public is an important step in the fight against anthrax.

Generally poor knowledge about anthrax in a community and its healthcare

providers may play a major role in the increase of morbidity and mortality rates

during outbreaks and epidemics. Also the use of folk and herbal medicine can

result in the increase of patients presenting with severe symptoms compared

with those seeking antibiotic treatment (27). Public education campaign

involving both veterinary and local health personnel on the actual cause of and

prevention of anthrax could reduce outbreaks of the disease in people (63).

Table 1. Recommendations for antimicrobial treatment and postexposure prophylaxis of inhalation anthrax.a -------------------------------------------------------------------------------------------------------- Type of therapy Adults Children -------------------------------------------------------------------------------------------------------- Treatmentb Cirprofloxacin, 400mg IV every 12hr Ciprofloxacin, 10-15mg/kg IV, every 12hr (for 60 days) or or Doxycycline, 100mg IV every 12hrc Doxycycline:

>8yr and >45kg,100mg every 12hr >8yr and <45kg, 2.2mg/kg every 12hr <8yr, 2.2mg/kg every 12hr

and and one or two additional antibioticsd one or two additional antibiotics

----------------------------------------------------------------------------------------------------------------------------- Prophylaxis Ciprofloxacin, 500mg orally every 12hr Ciprofloxacin, 10-15mg/kg every 12hr (for 60 days) or or Doxycycline, 100mg orally every 12hr. Doxycycline:

>8yr and >45kg, 100mg orally BID >8yr and <45kg, 2.2mg/kg orally BID <8yr, 2.2mg/kg orally BID

-------------------------------------------------------------------------------------------------------- a. For patients with severe edema and for meningitis, steroides may be used as an adjunct therapy. Therapy for pregnant women is the same as for adults. b. Also recommended for gastrointestinal and oropharyngeal anthrex. After initial IV treatmen, oral antibiotic therapy could be used when clinically appropriate. c. Tetracyclines are not recommended during pregnancy and for young children, however, their use may be indicated for life-threatening illness. d. Chloramphenicol, clindamycin, imipenem, rifampin, and vancomycin. Also, penicillin and ampicillin, but should not be used alone. Consultation with infectious disease specialist is recommended. Table 2. Recommendations for the treatment of cutaneous anthraxa -------------------------------------------------------------------------------------------------------- Type of therapy Adults Children -------------------------------------------------------------------------------------------------------- Treatment Ciprofloxacin, 500mg orally BID Ciprofloxacin, 10-15mg/kg orally BID (7-10 days for or or naturally occurring, Doxycycline, 100mg orally BID Doxycycline: and 60 days if associated >8yr and >45kg, 100mg orally BID with inhalation anthrax) >8yr and <45kg, 2.2mg/kg orally BID <8yr, 2.2mg/kg orally BID -------------------------------------------------------------------------------------------------------- a. Therapy of cutaneous anthrax with signs of systemic involvement, extensive edema, or lesion on the head or neck is the same as for inhalation anthrax (Table 1). Also, see footnotes in Table 1.

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