Roger N. Picken

Comparison of Culture-Confirmed Erythema Migrans Caused by Borrelia burgdorferi sensu stricto in New York State and by Borrelia afzelii in Slovenia

The clinical presentations of Lyme borreliosis in the United States and Europe seem to differ (1-7). For example, European patients with Lyme borreliosis may develop certain skin manifestations (acrodermatitis chronica atrophicans and borrelial lymphocytoma) that are extremely rare or nonexistent in the United States (6, 7). One explanation of such differences may be the recently appreciated variation in strains of Borrelia species between the two continents (7-9). All isolates from U.S. patients have thus far been members of the genomic group Borrelia burgdorferi sensu stricto (henceforth referred to as B. burgdorferi), whereas European isolates have included two additional genospecies, B. garinii and B. afzelii. Until now, however, no reports of clinical disparities have been based on the systematic study of large numbers of patients with culture-confirmed infection. To investigate possible differences in the manifestations of Lyme borreliosis in Europe and the United States, we compared epidemiologic, demographic, clinical, and laboratory findings in patients from New York State and patients from Slovenia who had microbiologically confirmed erythema migrans caused by B. burgdorferi and B. afzelii, respectively. Methods Patients To isolate Borrelia species from clinical specimens, we recruited patients with a clinical diagnosis of erythema migrans in two separate prospective studies. The Centers for Disease Control and Prevention surveillance definition of erythema migrans (10) was satisfied in almost all cases, although three Slovenian patients with erythema migrans lesions less than 5 cm in diameter were enrolled. Patients from the United States were seen at the Lyme Disease Diagnostic Center, Westchester Medical Center, Valhalla, New York, from June 1991 through August 1995. Slovenian patients were evaluated at the Lyme Borreliosis Outpatient Clinic, Ljubljana, Slovenia, in 1993. All patients were older than 15 years of age and gave informed consent. Patients were included in the present study if Borrelia organisms were recovered from their skin specimens. Several U.S. patients had repeated culture-confirmed episodes of erythema migrans in separate years; for each of these patients, only the first episode was included. Laboratory Methods All isolates were obtained from biopsy or needle aspiration of erythema migrans lesions (11, 12). Cultures were processed as previously reported in modified Barbour-Stoenner-Kelly medium (11) for U.S. patients and modified Kelly-Pettenkofer medium (12) for Slovenian patients. At the U.S. study site, we identified spirochetes as B. burgdorferi by using polymerase chain reaction (PCR) with specific primers directed at the ribosomal RNA genes of Borrelia species [13]. We identified the species of European isolates by using two independent methods: 1) species-specific PCR with different oligonucleotide primer sequences [14] and 2) pulsed-field gel electrophoretic separation of restriction enzyme MluI digestion fragments (15) (the pattern of large restriction fragments produced is diagnostic of the species). Because most Slovenian isolates by far were B. afzelii (12), cases of erythema migrans due to other species (including four cases due to B. burgdorferi and six due to B. garinii) were excluded. Complete blood counts were done, liver function tests were performed, and the erythrocyte sedimentation rate was measured at the time when cultures were obtained. Baseline serum specimens were tested for antibodies to B. burgdorferi. Convalescent-phase specimens were obtained after 1 week to 1 month for U.S. patients and after 2 months for Slovenian patients. In the United States, serum specimens were tested for antibodies to B. burgdorferi with a polyvalent enzyme-linked immunosorbent assay (Whittaker Bioproducts, Inc., Walkersville, Maryland) (11, 16). In Slovenia, the presence of serum IgM and IgG antibodies to B. afzelii was determined separately by immunofluorescent assay without preabsorption (17); a local skin isolate of B. afzelii was used as antigen. Titers of 1:256 or more were considered to indicate positivity. Statistical Analysis Differences in quantitative data were analyzed by using the median test, and differences in qualitative data were analyzed by using the chi-square test (with the Yates correction) or the Fisher exact test. We used Epi-Info 6 for all analyses (Statcalc, version 6.04a, Centers for Disease Control and Prevention, Atlanta, Georgia). All P values were two-tailed. Results Borrelia afzelii was recovered from the skin specimens of 85 Slovenian patients; none of these patients had been reported previously. Borrelia burgdorferi was cultured from 119 U.S. patients; 77 of these patients had been reported previously (11). Of the U.S. isolates, 30 were randomly selected for species identification by PCR, and all 30 were identified as B. burgdorferi. Thus, it is likely (although not certain) that the remainder of the isolates were also B. burgdorferi. Demographic patient data and the characteristics of erythema migrans are summarized in Table 1. All Slovenian patients and 113 of 119 U.S. patients (95%) were white. Slovenian patients were more likely than U.S. patients to recall a tick bite at the erythema migrans site (63.5% compared with 25.2%; P<0.001). Although the size of the erythema migrans lesions was similar in the two groups of patients, the median duration of erythema migrans at presentation was longer (14 days [range, 1 to 206 days] compared with 4 days [range, 1 to 39 days]; P<0.001) and central clearing was more common (68.2% compared with 35.3%; P<0.001) in Slovenian patients than in U.S. patients. Localized pain or burning at the erythema migrans site was more common in U.S. patients (34.3% compared with 18.8%; P=0.02). The occurrence of multiple erythema migrans lesions was greater in the U.S. patients than in the Slovenian patients, but the difference was not significant (13.4% compared with 7.1%; P>0.2). Table 1. Characteristics of Patients with Culture-Confirmed Erythema Migrans Patients in the United States were more likely than those in Slovenia to have systemic symptoms (68.9% compared with 50.6%; P=0.01), including fatigue (54.6% compared with 32.9%; P=0.003), myalgia (44.5% compared with 21.1%; P=0.001), fever or chills (37.8% compared with 8.2%; P<0.001), and stiff neck (38.7% compared with 8.2%; P<0.001) (Table 2). Objective findings on physical examination were also more common in U.S. patients (57.1% compared with 14.1%; P<0.001) and most frequently manifested as regional lymphadenopathy (seen in 28.6% of U.S. patients compared with 8.2% of Slovenian patients; P<0.001) or fever (body temperature 37.8 C) (seen in 15.1% of U.S. patients compared with 1.2% of Slovenian patients; P<0.001). Slovenian patients were more likely to have hepatomegaly (5.9% compared with 0; P=0.01), but U.S. patients more often had at least one abnormal result on a liver function assay (32.2% compared with 18.3%; P=0.04), were more likely to have erythrocyte sedimentation rates two or more times the upper limit of normal (25% compared with 3.7%; P<0.001), were more likely to seroconvert (84.2% compared with 10.9%; P<0.001), and were more often seropositive at presentation (35.3% compared with 22.4%; P=0.07). Table 2. Selected Clinical and Laboratory Findings in Patients with Culture-Confirmed Erythema Migrans Discussion Our findings establish what has long been believed: Some of the clinical and laboratory features of Lyme disease differ in the United States and Europe. The differences seem to result, in part, from a tendency for B. afzelii to be less virulent than B. burgdorferi. Slovenian patients with B. afzelii were significantly less likely than U.S. patients with B. burgdorferi to be systemically ill (Table 2) and to have fever (P<0.001) or regional lymphadenopathy (P<0.001) on physical examination. Although the duration of erythema migrans at presentation was longer in Slovenian patients, lesion size in Slovenian patients was similar to that in U.S. patients; this implies that B. afzelii spreads more slowly in the skin (11). Because central clearing occurs, in part, as a function of time (2), its higher frequency in Slovenian patients (68.2% compared with 35.3%; P<0.001) is probably related to the longer duration of erythema migrans at presentation. Nonetheless, in contrast to what might have been anticipated from earlier reports (1-6, 18), dissemination to multiple cutaneous sites did not occur significantly more often in U.S. patients than in Slovenian patients in our study (P=0.2). Our observations in patients in Westchester County, New York, agree with those of other studies of North American patients with erythema migrans (1, 4, 5), in which reported rates of systemic illness are often greater than 75%. In contrast, reported rates of systemic illness in Europe are often less than 35% (2, 3, 8). In our study, Slovenian patients were much less likely than U.S. patients to be seropositive (26 of 85 [30.6%] compared with 106 of 119 [89.1%]; P<0.001), principally because seroconversion was less common (7 of 64 patients [10.9%] compared with 64 of 76 patients [84.2%]; P<0.001). The seroconversion rate in our U.S. patients, however, was similar to that seen in other recent studies of U.S. patients with erythema migrans (5). Moreover, the contrasting rates of seropositivity at presentation were particularly evident when patients with longstanding erythema migrans (duration 14 days) were compared; 94% of such patients in the United States (15 of 16) but only 28% of such patients in Slovenia (13 of 47) were seropositive (P<0.001). With rare exceptions (19), European studies have reported low rates of seropositivity (20% to 50%) in patients with erythema migrans (3, 18), despite a long duration of illness (often longer than several weeks) (3). However, because convalescent-phase serologic samples from Slovenian patients were obtained at 2 months (rather th

Clinical Findings for Patients with Lyme Borreliosis Caused by Borrelia burgdorferi Sensu Lato with Genotypic and Phenotypic Similarities to Strain 25015

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.

Molecular characterization of Borrelia burgdorferi sensu lato from Slovenia revealing significant differences between tick and human isolates.

One hundred twenty-nine Slovenian isolates ofBorrelia burgdorferi sensu lato derived from patients (69 strains) orIxodes ricinus ticks (60 strains) were characterized. All of the strains were first- or second-passage isolates obtained in 1992 and 1993 from the same endemic region. The techniques used for the molecular analysis of strains included species-specific polymerase chain reaction (PCR) typing, and pulsed-field gel electrophoretic separation of undigested andMlul-digested genomic DNA. Isolates were identified to the species level by large restriction fragment pattern (LRFP) analysis and the results compared with the species-specific PCR result. Fifty-two patient isolates (75%) were typed asBorrelia afzelii (LRFP MLa1), 6 (9%) asBorrelia garinii (LRFPs MLg1–4), and 11 (16%) asBorrelia burgdorferi sensu stricto. The latter included 9 isolates (13%) with a new LRFP that is not typical ofBorrelia burgdorferi sensu stricto and for which the designation MLx is suggested. In contrast, only 32 of 60 (53%) tick isolates were typed asBorrelia afzelii, while 20 strains (33%) were typed asBorrelia garinii and 8 strains (13%) asBorrelia burgdorferi sensu stricto. Three new LRFPs were found among theBorrelia garinii (MLg5 and 6) andBorrelia burgdorferi sensu stricto (MLb15) tick isolates. Large restriction fragment pattern analysis identified new groups ofBorrelia burgdorferi sensu lato and revealed an apparent difference in the isolation frequency of different species from patients and ticks in the same endemic region.

Single-tube nested polymerase chain reaction assay based on flagellin gene sequences for detection ofBorrelia burgdorferi sensu lato

An inherent drawback of nested PCR systems to increase sensitivity of PCR-based assays is that tubes must be opened after the first round of amplification in order to transfer template molecules to the second amplification reaction; this procedure introduces the risk of carry-over contamination of negative specimens. To obviate this disadvantage, a nested PCR assay for detection ofBorrelia burgdorferi in which both amplifications are performed in a single tube that remains closed throughout the entire process was devised. The assay is based on flagellin gene sequences with previously determined species-wide and species-specific properties. The nested PCR system proved to be 1000 times more sensitive than the conventional assay. Using the nested PCR system, ten spirochaetes could be routinely detected by agarose gel electrophoresis alone, whereas the conventional PCR system could detect only 104 spirochaetes under these conditions. After Southern transfer of amplification products and hybridization with32P- or chemiluminescent-labeled probes, the nested PCR system could easily detect a single spirochaete by both means, whereas the sensitivity of the conventional PCR assay varied from 101 (32P) to 103 (chemiluminescence) spirochaetes. This single-tube nested PCR system should be a useful addition to the current range of diagnostic assays for Lyme borreliosis.

Infection rate ofIxodes ricinus ticks withBorrelia afzelii, Borrelia garinii, andBorrelia burgdorferi sensu stricto in Slovenia

In spring 1993,Ixodes ricinus ticks were collected from six regions of Slovenia to determine their overall rate of infection withBorrelia burgdorferi sensu lato and to assess the frequency of individual species in these tick populations. Ticks were dissected and midgut tissue inoculated into modified Barbour-Stoenner-Kelly (BSK II) medium.Borrelia isolates were differentiated into separate species using species-specific polymerase chain reaction (PCR) primers and by large restriction fragment pattern (LRFP) analysis. Infected ticks were found in all six regions surveyed. Spirochaetes were isolated from 69 of 363 ticks (19 %): the isolation rate from adult female ticks was 35 % (23/66 ticks cultured), from adult male ticks 22 % (20/91), and from nymphal ticks 13 % (26/206). Determination of the species of 60 isolates revealed that 32 wereBorrelia afzelii (53 %), 20 wereBorrelia garinii (33 %), and 8 wereBorrelia burgdorferi sensu stricto (13 %). In the Ljubljana regionBorrelia afzelii andBorrelia garinii predominated (43 % and 40 %, respectively), whereasBorrelia burgdorferi sensu stricto constituted only 17 % of isolates. In three other regions of the countryBorrelia afzeliiwas isolated exclusively, although the number of isolates investigated was small. This study demonstrates the presence of all three European species ofBorrelia burgdorferi sensu lato within the Slovenian tick population and also within a geographic area of less than 100 m2.

Concomitant infection with tick-borne encephalitis virus and Borrelia burgdorferi sensu lato in patients with acute meningitis or meningoencephalitis

SummaryFrom September 1992 to August 1993, 338 patients over the age of 15 years presented to the Department of Infectious Diseases, University Medical Centre Ljubljana, with acute lymphocytic meningitis. In 89 of these patients (26.3%) serum IgM and IgG antibodies against tick-borne encephalitis (TBE) virus were detected, and in 59 patients (17.5%) a borrelial etiology of disease was demonstrated by one or more of the following: presence of intrathecal antibody production, seroconversion to borrelial antigens, presence of erythema migrans, and/or isolation ofBorrelia burgdorferi sensu lato from skin or cerebrospinal fluid. Of the 148 patients who fulfilled criteria for TBE or borrelial infection, concomitant infection with TBE virus andB. burgdorferi sensu lato was demonstrated in 12 patients (3.6% of all patients presenting with acute lymphocytic meningitis). In the majority of patients with concomitant infection the clinical features at presentation were characteristic of, or consistent with, TBE. In addition, during follow-up studies, eight of the 12 patients subsequently developed signs and symptoms compatible with minor and/or major manifestations of Lyme borreliosis. Six patients were diagnosed with neuroborreliosis based on signs or symptoms and/or laboratory tests. These findings show that in patients with acute lymphocytic meningitis or meningoencephalitis, originating in TBE and Lyme borreliosis endemic regions, the possibility of concomitant infection should be considered.

Comparison of Peripheral and Central Biopsy Sites for the Isolation of Borrelia burgdorferi Sensu Lato from Erythema Migrans Skin Lesions

To compare peripheral and central biopsy sites for the isolation of Borrelia burgdorferi sensu lato from typical erythema migrans lesions, two biopsy specimens (one from the margin and the other from the center of the lesion) were taken from each of 53 adult patients. Thirty-four (32.1%) of the 106 biopsy specimens and 23 (43.4%) of the 53 patients were culture-positive. Spirochetes were isolated in 19 (35.9%) of the 53 central and 15 (28.3%) of the 53 peripheral biopsies (nonsignificant difference). In 8 cases only the central specimen, in 4 cases only the peripheral specimen, and in 11 cases both specimens were positive. In a comparison of 36 annular and 17 homogeneous lesions, borreliae were isolated from 15 (41.7%) of the former and 8 (47.1%) of the latter. In the annular-lesion biopsies, 11 central and 12 peripheral specimens were culture-positive. In the homogeneous-lesion biopsies, eight central and three peripheral specimens were culture-positive (nonsignificant difference); there were no culture-positive results solely for the margin of homogeneous lesions. This study demonstrates that B. burgdorferi sensu lato may be isolated from the central aspect of erythema migrans lesions as efficiently as from the peripheral aspect.