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Oculocutaneous anthrax: detection and treatment.

Anthrax, a zoonotic disease that primarily affects herbivores, has received recent attention as a potential agent of bioterrorism. We report a patient who presented with a 4-day history of pain, watering and difficulty in opening the left upper and lower eyelids, and fever. Clinical examination revealed brawny nonpitting edema with serosanguinous discharge. The history of the death of his sheep 1 week prior to the illness provided the clue to the diagnosis. Although standard cultures of the blood and the serous fluid from the lesion were negative, probably as a result of prior treatment, the diagnosis of cutaneous anthrax was made by a polymerase chain reaction (PCR) test of the serous fluid. Serial photographs demonstrating resolution of the lesion with appropriate antibiotic therapy are presented.


Introduction
Anthrax, a zoonotic disease caused by Bacillus anthracis (B. anthracis), primarily affects herbivores including sheep, cattle, horses, and other domestic animals. 1 Humans may be affected as a result of exposure to an infected animal or animal products. This organism has received recent attention as a potential agent of bioterrorism. 1 Three forms are described: cutaneous, inhalational, and gastrointestinal. 1 We describe a patient with oculocutaneous anthrax.

Case description
A 39-year-old male presented to our outpatient clinic with a 4-day history of fever and swelling of the left upper and lower eyelids associated with pain, watering, and difficulty in opening the eyelids. He reported that his sheep had died suddenly 1 week prior to his illness. There was no past history of any medical illness. The patient reported that he was prescribed tablets and injections by a general practitioner, but was unable to provide details of this treatment to us.
On examination, there was brawny nonpitting edema of the upper and lower eyelids of the left eye and left side of the face (Figure 1). There was an ulceration and black discoloration along the left lower lid margin with serosanguinous discharge. His visual acuity was 20/20 in both the eyes. The anterior and posterior segments of both the eyes were within normal limits. A diagnosis of oculocutaneous anthrax was considered based on the history and clinical findings. Serous fluid from the eyelid and blood were collected and sent for conventional cultural methods and polymerase chain reaction (PCR) test. The Gram stain of the serous fluid revealed no leucocytes or

PCR method for the detection of B. anthracis
The patient's DNA was extracted using the QIA amp DNA blood mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instruction. A 596-bp fragment of the protective antigen (PA) gene, PA (F)-TCC TAA CAC TAA CGA AGT CG and PA (R)-GAG GTA GAA GGA TAT ACG GT and a 846-bp fragment of the capsule (CAP) gene, CAP (F)-CTG AGC CAT TAA TCG ATA TG and CAP (R) 2,3 of B. anthracis were used as the target for anthrax PCR. The DNA was amplified in a total volume of 50 µL with the above mentioned primers, 200 µm (each) deoxynucleotide triphosphates, 1X PCR buffer, 1.5 mM MgCL 2 and 2.5 units of Taq polymerase (Amplitaq Gold-Applied Biosystems, California, USA) and followed by the addition of 5 µL of the template DNA. Thermal cycling was performed at 94°C for 5 minutes (initial denaturation) followed by 35 cycles at 94°C for 1 minute, 52°C for 1 minute (annealing), 72°C for 2 minutes (extension) and the final extension at 72°C for 5 minutes; cooled to 4°C. 4 Thermal cycling was done in the Gene AMP PCR system 9200 (Applied Biosystems, California, USA).
A 15 µL portion of the PCR product and 5 µL of 10% tracking dye (0.02% xylene cyanol, 0.02% bromophenol blue and 50% glycerol) was added to the PCR tube. Fifteen microliters of this mix were loaded into the wells in the agarose (Amersham Pharmacia, New Jersey, USA). The PCR products were analyzed by submarine gel electrophoresis through a 1% agarose gel in 1X TAE buffer. The gel was run at 120 volts for 60 minutes, stained with 0.5 µg/mL of ethidium bromide (Amersham Pharmacia, New Jersey, USA), visualized and recorded under UV 4 in the Gel Doc 2000 System (Bio-Rad, California, USA). The Gene Ruler TM 100-bp (3000-100bp) DNA Ladder Plus (MBI Fermentas, USA) was used as the molecular standard. The PCR from the fluid and the blood were positive for the gene encoding the protective antigen (PA gene) and the capsule (CAP gene) of B. anthracis (Figure 2).

Clinical course of the patient
The patient was started on injection crystalline penicillin, 2 million international units (1250 mg) every 2 hours intravenously. No topical therapy was prescribed, however, the patient was given anti-inflammatory agents (ibuprofen). The patient responded well to antibiotic therapy with reduction of fever and lid edema. A black eschar developed within a few days of treatment ( Figure 3). On completion of a 10-day course of intravenous antibiotics, he was discharged on oral penicillin G 500 mg every 6 hours. The total duration of antibiotic therapy including intravenous antibiotics was 6 weeks. Although the eschar reduced in size over time, it was firmly adhered to the underlying tissue. When the eschar was removed after 1 month of the onset of illness, an ulcerated undersurface was observed (Figure 4). The ulceration healed over the next 2 weeks with scarring and ectropion of the left lower lid ( Figure 5). The low grade lid edema and mechanical ptosis of the left upper eyelid resolved over the next few weeks.

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Oculocutaneous anthrax Comments B. anthracis is commonly found in agricultural environments. It is a Gram-positive, aerobic, endospore-forming bacilli that, in the vegetative form, has a poor survival rate outside an animal or human host. 5 It is capable of producing fatal infection in livestock and in humans. 1 Anthrax is considered to be one of most dangerous biological weapons. 6 Humans are infected when spores are introduced into the body by contact with infected animals or animal products. 1 Cutaneous anthrax has been reported to occur as preseptal cellulitis in the eyelids. 7,8 Cutaneous anthrax, if untreated, leads to lifethreatening septicemia. 1 This report highlights that a high index of clinical suspicion and prompt institution of appropriate therapy are essential for a successful outcome. Since lesions can become sterile within 24 hours of antimicrobial therapy, confirmation of diagnosis can be made using PCR, [2][3][4]9 as was done in our patient, when the conventional cultural methods fail to identify the etiology or when patients have already received antimicrobial therapy.

Disclosures
The authors report no conflicts of interest in this work.

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