Borrelia spielmanii Erythema Migrans, Hungary

To the Editor: Lyme disease is the most frequent tickborne human infection in the northern hemisphere. At least 5 species of the Borrelia burgdorferi sensu lato complex, B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. bissettii, and B. lusitaniae, have a pathogenic role in human Lyme disease in central Europe (1–3). A sixth pathogenic strain, A14S, has been isolated from 1 Dutch (4) and 2 German patients with erythema migrans (5). This strain was also detected in 4 questing Ixodes ricinus ticks in Germany (6,7) and 1 in the Czech Republic (8). A14S has recently been described as a new species, B. spielmanii (9); its main reservoir host is probably the garden dormouse (Eliomys quercinus), but B. spielmanii could not be detected in mice or voles. Richter et al. (9) could not find ticks harboring B. spielmanii in 3 of 5 examined areas in Germany. They were present almost exclusively in a single area where the prevalence of infection with this genotype was 15 (6%) of 251. We describe the isolation of this novel Lyme disease spirochete from a human patient with erythema migrans in Hungary. Since 1999, we have regularly isolated Borrelia burgdorferi sensu lato from skin biopsy specimens of erythe-ma migrans and acrodermatitis chron-ica atrophicans taken from patients at the Center for Tick-borne Diseases, Budapest, Hungary. To identify the Borrelia species occurring in Hungarian Lyme disease patients, we have started to molecularly analyze cultured isolates that originate from erythema migrans of different patients. DNA was isolated from 8 bacterial pellets by using QIAamp DNA mini kit (Qiagen, Hilden, Germany). Primers BSL-F and BSL-R were used; these amplify an ≈250-bp region of the outer surface protein (osp) A gene from all Lyme disease spirochetes (10). We added 2 µL extracted DNA to a 20-µL reaction mixture composed of 1.0 U HotStart-Taq DNA polymerase, 200 µmol/L of each dNTP, 25 pmol of each primer, and 1.5 mmol/L MgCl 2 (HotStartTaq Master Mix, Qiagen). An initial denaturation step at 94°C for 15 min was followed by 40 cycles of denatu-ration at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s. Final extension was done at 72°C for 5 min. Amplified DNA was subjected to electrophoresis in a 1.5% agarose gel that was prestained with ethidium bromide and viewed under UV light. After purification, the dideoxy chain termination (Applied Biosystems Division, Foster City, …