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Cutaneous anthrax is a rare zoonotic disease in the United States. The clinical diagnosis traditionally has been established by conventional microbiological methods, such as culture and gram staining. However, these methods often yield negative results when patients have received antibiotics. During the bioterrorism event of 2001, we applied two novel immunohistochemical assays that can detect Bacillus anthracis antigens in skin biopsy samples even after prolonged antibiotic treatment. These assays provided a highly sensitive and specific method for the diagnosis of cutaneous anthrax, and were critical in the early and rapid diagnosis of 8 of 11 cases of cutaneous anthrax during the outbreak investigation. Skin biopsies were obtained from 10 of these 11 cases, and histopathological findings included various degrees of ulceration, hemorrhage, edema, coagulative necrosis, perivascular inflammation, and vasculitis. Serology was also an important investigation tool, but the results required several weeks because of the need to test paired serum specimens. Other tests, including culture, special stains, and polymerase chain reaction assay, were less valuable in the diagnosis and epidemiological investigation of these cutaneous anthrax cases. This report underscores the critical role of pathology in investigating potential bioterrorism events and in guiding epidemiological studies, Anthrax captured worldwide attention and aroused serious public health concerns following the intentional release of the etiological agent Bacillus anthracis in the United States postal system during Fall 2001, 1,2 resulting in 11 cases of inhalational anthrax and 11 cases of cutaneous anthrax. [3][4][5][6][7] In its conventional form, cutaneous anthrax accounts for 95% of all naturally occurring B. anthracis infections in the United States. 8 -11 Patients often have a history of occupational contact with animals or animal products contaminated with B. anthracis spores. 12-14 These pathogenic spores are introduced through a cutaneous cut or abrasion, with the most common areas of exposure are the head, neck, and extremities, although any area can be involved. Bacteremia and toxemia following cutaneous infection can occur with a fatality rate of 20% to 25% among untreated cases. [15][16][17] Cutaneous anthrax is characterized by the formation of a black eschar surrounded by prominent edema and vesicles, which may resemble many other skin lesions, such as the brown recluse spider bite, 18 -21 ulceroglandular tularemia, 22,23 plague, 24,25 ecthyma gangrenosum, 26,27 various spotted fever group rickettsial infections, 28 -31 and scrub typhus. 32,33 The clinical diagnosis of cutaneous anthrax is traditionally established by microbiological methods (eg, demonstrating gram-positive, capsulated bacilli on the smear of the lesion or isolating B. anthracis in culture). 34,35 However, gram stain and culture for B. anthracis can be unrevealing for patients who receive antibiotic therapy before specimens are obtained. 36,37 Historically, skin biopsies ...
Cutaneous anthrax is a rare zoonotic disease in the United States. The clinical diagnosis traditionally has been established by conventional microbiological methods, such as culture and gram staining. However, these methods often yield negative results when patients have received antibiotics. During the bioterrorism event of 2001, we applied two novel immunohistochemical assays that can detect Bacillus anthracis antigens in skin biopsy samples even after prolonged antibiotic treatment. These assays provided a highly sensitive and specific method for the diagnosis of cutaneous anthrax, and were critical in the early and rapid diagnosis of 8 of 11 cases of cutaneous anthrax during the outbreak investigation. Skin biopsies were obtained from 10 of these 11 cases, and histopathological findings included various degrees of ulceration, hemorrhage, edema, coagulative necrosis, perivascular inflammation, and vasculitis. Serology was also an important investigation tool, but the results required several weeks because of the need to test paired serum specimens. Other tests, including culture, special stains, and polymerase chain reaction assay, were less valuable in the diagnosis and epidemiological investigation of these cutaneous anthrax cases. This report underscores the critical role of pathology in investigating potential bioterrorism events and in guiding epidemiological studies, Anthrax captured worldwide attention and aroused serious public health concerns following the intentional release of the etiological agent Bacillus anthracis in the United States postal system during Fall 2001, 1,2 resulting in 11 cases of inhalational anthrax and 11 cases of cutaneous anthrax. [3][4][5][6][7] In its conventional form, cutaneous anthrax accounts for 95% of all naturally occurring B. anthracis infections in the United States. 8 -11 Patients often have a history of occupational contact with animals or animal products contaminated with B. anthracis spores. 12-14 These pathogenic spores are introduced through a cutaneous cut or abrasion, with the most common areas of exposure are the head, neck, and extremities, although any area can be involved. Bacteremia and toxemia following cutaneous infection can occur with a fatality rate of 20% to 25% among untreated cases. [15][16][17] Cutaneous anthrax is characterized by the formation of a black eschar surrounded by prominent edema and vesicles, which may resemble many other skin lesions, such as the brown recluse spider bite, 18 -21 ulceroglandular tularemia, 22,23 plague, 24,25 ecthyma gangrenosum, 26,27 various spotted fever group rickettsial infections, 28 -31 and scrub typhus. 32,33 The clinical diagnosis of cutaneous anthrax is traditionally established by microbiological methods (eg, demonstrating gram-positive, capsulated bacilli on the smear of the lesion or isolating B. anthracis in culture). 34,35 However, gram stain and culture for B. anthracis can be unrevealing for patients who receive antibiotic therapy before specimens are obtained. 36,37 Historically, skin biopsies ...
The capsule of Bacillus anthracis, composed of poly-␥-D-glutamic acid (␥DPGA), is an essential virulence factor of B. anthracis. The capsule inhibits innate host defense through its antiphagocytic action. ␥DPGA is a poor immunogen, but when covalently bound to a carrier protein, it elicits serum antibodies. To identify the optimal construct for clinical use, synthetic ␥DPGAs of different lengths were bound to carrier proteins at different densities. The advantages of the synthetic over the natural polypeptide are the homogeneous chain length and end groups, allowing conjugates to be accurately characterized and standardized and their chemical compositions to be related to their immunogenicities. In the present study, we evaluated, in addition to methods reported by us, hydrazone, oxime, and thioether linkages between ␥DPGA and several proteins, including bovine serum albumin, recombinant Pseudomonas aeruginosa exotoxin A, recombinant B. anthracis protective antigen (rPA), and tetanus toxoid (TT). The effects of the dosage and formulation on the immunogenicities of the conjugates were evaluated in mice. All conjugates were immunogenic. The optimal ␥DPGA chain length of 10 to 15 amino acids and the density, an average of 15 mol ␥DPGA per mol of protein, were confirmed. The thioether bond was the optimal linkage type, and TT and rPA were the best carriers. The optimal dosage was 1.2 to 2.5 g of ␥DPGA per mouse, and adsorption of the conjugates onto aluminum hydroxide significantly increased the antibody response to the protein with a lesser effect on anti-␥DPGA levels.
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