Identification of Cowpox Infection in a 13-year-old Danish Boy

developed into a pustule. He initially had systemic symptoms with high-grade fever up to 39.5oC and flu-like symptoms. At this stage he sought medical attention by an otologist who initially prescribed dicloxacillin and, when this had no effect, erythromycin, as bacterial infection was suspected. The fever abated in 4–5 days, but the neck lesion continued to enlarge and ulcerated during the second week. At the time of referral the patient presented with an indurated, indolent ulcer, 3 cm in diameter, with a hard, black eschar surrounded by erythema and local lymphadenopathy. He was treated with oral antibiotics until the viral aetiology was established and local antiseptic treatment was continued to prevent secondary infection. The lymphadenopathy gradually subsided and the ulcer healed in 6–8 weeks with some scarring. Laboratory investigations revealed a slightly raised erythrocyte sedimentation rate, while leukocyte counts, C-reactive protein and liver function tests were normal. Cutaneous anthrax and impetigo were excluded by negative swab culture from the ulcer. Swabs from the lesion were negative, as were serological tests for Bartonella henselae and Francisella tularensensis. Biopsy samples obtained for Mycobacterium tuberculosis culture were also negative. Two 4-mm punch biopsies were taken from the rim of the ulcer. Light microscopy of haematoxylin and eosin (H&E)-stained sections showed non-specific inflammatory changes in the epidermis and dermis. No cytoplasmic inclusions were seen. The swab sample was analysed initially using MagNA Pure extraction method (Total NA Isolation Kit, no. 3 038 505, Roche, Mannheim, Germany). To improve sensitivity, an additional extraction was performed subsequently, using increased sample volumes (1000 µl) combined with decreased elution volumes (50 µl). Ulcer biopsy material was split and prepared for electron microscopy and cultivation of mycobacteria, for which the latter was treated with BLL™ MycoPrep™ (BD Diagnostics, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. This material was extracted using the modified protocol.