Isolation of Borrelia lusitaniae from the blood of a patient with multiple erythema migrans

Abstract

Tropical Diseases of the University Clinical Centre of Serbia with a clinical presentation of disseminated erythema migrans. The patient and the mother could not recall if there had been a tick bite. A sample of blood was taken, and antibiotic therapy with amoxicillin was started immediately. Human serum sample was checked for the presence of IgM antibody against Borrelia burgdorferi sensu lato by commercial ELISA test and the sample was positive for IgM. Blood was collected into the sterile K2EDTA tube, immediately transported to the Laboratory at the Institute for Medical Research, National Institute of the Republic of Serbia, University of Belgrade, and centrifuged twice at 2200 rpm for 17 min at room temperature. After centrifugation, one part of the serum was served for DNA extraction using ammonium hydroxide, ethanol, and sodium acetate while the sediment was inoculated into a 6 mL tube with Barbour-Stoenner-Kelly-H (BSK-H) medium under aseptic conditions and incubated as 33°C. After 16 days of incubation, viable, motile, and spiral-shaped microorganisms were observed in the initial BSK-H culture under dark-field microscopy, and incubation was prolonged for 29 days. DNA from the culture was extracted using centrifugation, dissolving the sediment in the water, and heating at 95°C for 10 minutes. After extracting DNA from the human serum and the culture, rrf-rrl rDNA intergenic spacer and flagellin gene (flaB) were amplified by conventional PCR, and sequencing of obtained PCR products was performed commercially (Macrogen, Amsterdam, the Netherlands). After analysis of the sequences obtained, Borrelia lusitaniae was confirmed in human serum and culture. This is the first isolate of B. lusitaniae from a human blood sample that confirms that B. lusitaniae can disseminate via the hematogenous route.