In 1994, we isolated Borrelia burgdorferi sensu lato from 231 patients with erythema migrans who presented to the University Medical Center in Ljubljana, Slovenia. Samples of erythema migrans-affected skin were placed in media to support the growth of Borrelia species and evaluated in Ljubljana and Chicago. Patients whose cultures were positive included 132 women and 99 men; 136 of these 231 patients recalled a tick bite. Patients noted a rash an average of 24 days after a bite and presented a mean of 34 days after the bite with erythema migrans (mean diameter. 16 cm). Itching (44%) burning (18%), and pain (11%) were the most common local symptoms. Systemic complaints (40%) included headache, fatigue, malaise, and arthralgia. Other than erythema migrans, findings on physical examination were minimal (< 5% had fever, and in < 10% local lymph nodes were affected). Serial serological studies using indirect immunofluorescence assay, ELISA, and Western blot methods were performed, and antibodies to B, burgdorferi sensu lato were detected in < 50% of samples from patients. This is the largest series reported to date of patients with culture-confirmed Lyme borreliosis. It highlights the deficiencies of serological tests in early disease, demonstrates the sensitivity of direct detection methods for evaluation of patients with erythema migrans, and suggests that patients with early Lyme borreliosis in Slovenia may suffer a milder illness than those in the United States.
In Europe, at least three species of Borrelia are known to be causative agents of Lyme borreliosis: B. burgdorferi sensu stricto, B. garinii, and B. afzelii. Observable differences in the molecular characteristics of the three species have led to speculation that they may also differ in their pathogenic potential and/or tissue tropisms. Several studies have found an association between the chronic skin manifestation of Lyme borreliosis, acrodermatitis chronica atrophicans, and infection by B. afzelii. We sought to find further evidence for such a correlation by studying the genetic profiles of 22 strains of B. burgdorferi sensu lato derived from 21 patients who presented to the University Medical Center, Ljubljana, Slovenia between 1992 and 1995. Strains were isolated in culture from skin biopsies of acrodermatitis chronica atrophicans lesions; in the case of one patient two separate acrodermatitis chronica atrophicans lesions were cultured. All 21 patients had clinically typical lesions with "classic" histopathology and high IgG antibody titers to B. burgdorferi sensu lato. Strains were characterized and typed by 16S ribosomal RNA-specific polymerase chain reaction and determination of their large restriction fragment patterns using pulsed-field gel electrophoresis of MluI-digested genomic DNA. Of the 22 isolates studied, 17 possessed the highly conserved MLa1 pattern characteristic of B. afzelii. The remaining five isolates possessed large restriction fragment patterns that were typical of B. garinii (MLg2, four isolates from three patients) and B. burgdorferi sensu stricto (MLb2, one isolate). The results of 16S ribosomal RNA-specific polymerase chain reaction were concordant with these species designations. These data show that B. afzelii is the predominant, but not the exclusive, etiologic agent of acrodermatitis chronica atrophicans.
In the course of performing culture isolation of Borrelia burgdorferi sensu lato for the diagnosis of Lyme borreliosis in Slovenia, we encountered nine patients who were infected with atypical strains. Molecular analyses of these strains suggested that they were more closely related to the North American tick isolate, strain 25015 (which belongs to the DN127 genomic group of B. burgdorferi sensu lato), than they were to the three species (B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii) hitherto found to be associated with European Lyme borreliosis. Review of the case histories of these patients revealed some atypical clinical features and variability in clinical presentation. In this study, we present the clinical findings for these patients and discuss their significance for the diagnosis of Lyme borreliosis. The DN127 genomic group shares with B. burgdorferi sensu stricto the distinction of being present in both the Old and New Worlds.
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