Borrelia burgdorferi Genotype Predicts the Capacity for Hematogenous Dissemination during Early Lyme Disease

Gary P. Wormser 2 Dustin Brisson 0 Dionysios Liveris 1 Klra Hanincov 1 Sabina Sandigursky 1 John Nowakowski 2 Robert B. Nadelman 2 Sara Ludin 2 Ira Schwartz 1 0 Department of Biology, University of Pennsylvania , Philadelphia 1 Department of Microbiology and Immunology, New York Medical College , Valhalla , New York 2 Division of Infectious Diseases, Department of Medicine Background. Lyme disease, the most common tickborne disease in the United States, is caused exclusively by Borrelia burgdorferi sensu stricto in North America. The present study evaluated the genotypes of 400 clinical isolates of B. burgdorferi recovered from patients from suburban New York City with early Lyme disease associated with erythema migrans; it is the largest number of borrelial strains from North America ever to be investigated. Methods. Genotyping was performed by restriction fragment-length polymorphism polymerase chain reaction analysis of the 16S-23S ribosomal RNA spacer and reverse line blot analysis of the outer surface protein C gene (ospC). For some isolates, DNA sequence analysis was also performed. Results. The findings showed that the 16S-23S ribosomal spacer and ospC are in strong linkage disequilibrium. Most B. burgdorferi genotypes characterized by either typing method were capable of infecting and disseminating in patients. However, a distinct subset of just 4 of the 16 ospC genotypes identified were responsible for 80% of cases of early disseminated Lyme disease. Conclusions. This study identified the B. burgdorferi genotypes that pose the greatest risk of causing hematogenous dissemination in humans. This information should be considered in the future development of diagnostic assays and vaccine preparations. - Lyme disease is a tickborne zoonosis that occurs globally in the Northern Hemisphere [1, 2]. In North America, Lyme disease appears to be caused exclusively by Borrelia burgdorferi sensu stricto, whereas in Europe other species of Borrelia predominate [3 6]. A variety of typing systems exist to distinguish genetically distinct strains of B. burgdorferi [3]. On the basis of restriction fragment length polymorphism (RFLP) of the 16S23S rRNA intergenic spacer, B. burgdorferi has been classified into 3 distinct genetic subgroups (herein called genotypes), arbitrarily called ribosomal spacer type (RST) 1, RST2, and RST3 [7, 8]. Greater separation into distinct genotypes is achievable on the basis of the sequence heterogeneity of the outer surface protein C gene (ospC) [9, 10]. Genetically distinct genotypes of B. burgdorferi appear to differ ecologically and epidemiologically [11 14], suggesting that genotype classification is relevant to understanding the basic biology of the spirochete. Differential pathogenicity of B. burgdorferi on the basis of genotype has been reported in several studies. For example, in an analysis of 104 borrelial strains recovered from the skin of patients with a diagnosis of erythema migrans in Westchester County, New York, it was found that patients with disseminated infection (i.e., positive blood culture and/or multiple erythema migrans skin lesions) were at least 5 times more likely to have been infected with RST1 strains of B. burgdorferi than with RST3 strains [15]. A subsequent study of patients from Connecticut and Rhode Island with erythema migrans similarly found that infections believed to have disseminated were 5 times more common among RST1infected patients compared with those infected with RST3 strains of B. burgdorferi [16]. In a study from New York State, dissemination of B. burgdorferi to blood or cerebrospinal fluid (CSF) was exclusively associated with ospC genotypes A, B, I, or K [9]. Subsequent studies from other geographic areas, however, have suggested that hematogenous dissemination is not restricted to just these 4 ospC genotypes [16 18]. The present study was undertaken to evaluate the relationship between RST and ospC genotypes and to examine the association between specific genotypes and objective evidence of dissemination of the spirochete in patients, using the largest sample of clinical isolates of B. burgdorferi studied to date. In addition, the frequency distribution of RST and ospC genotypes in the skin or blood of patients with culture-confirmed erythema migrans was compared with that present in local tick populations. Subjects, clinical specimens, and cultures. All human subjects were adults with erythema migrans enrolled in prospective studies at the Lyme Disease Practice of the Westchester Medical Center between 1991 and 2005. This practice serves patients in suburban New York City who live or work in the lower Hudson Valley of New York State. Specimens from skin, whole blood, serum, or plasma were collected and cultured as described elsewhere [15, 19]. Typing of B. burgdorferi strains. B. burgdorferi DNA was isolated from low-passage (15) cultures using a nucleic acid extraction kit (IsoQuick; Orca Research). A 941-bp fragment of the 16S23S intergenic spacer was amplified by polymerase chain reaction (PCR) using primers PA and P95, as described elsewhere [7]. PCR-based RFLP analyses of the 16S23S intergenic spacer were performed using the restriction enzyme Tru1I (Fermentas) [8, 20]. A 522-bp region of ospC was amplified by PCR using external primers OC6() and OC623() and internal primers OC6(Fluo) and OC602() [11, 21]. Amplicons were then probed with ospC typespecific probes by reverse line blot [11, 21]. The ospC amplicons that did not hybridize with any ospC typespecific probes were reamplified and sequenced in both directions (Genewiz) using either primer set ospC-N/ ospC-C [5] or OC6()/OC623(). Isolates that produced ambiguous sequence results were cloned by limiting dilution, and sequence analyses were performed on 2 clones from each isolate. Some of the isolates in the present study have been reported in the context of other investigations of the pathogenicity of particular genotypes of B. burgdorferi [9, 15, 2224]. The ospC typing of B. burgdorferi in extracts from infected ticks was done by reverse line blotting, essentially as described above and elsewhere [11, 21]. Statistical analyses. Differences in the frequency distributions of genotypes were assessed using a 2 analysis [25]. P values were estimated using a Monte Carlo simulation with 100,000 replicates when the marginal values summed to 5, making the 2 distribution inappropriate [26]. The Bonferroni correction for multiple tests (P .05/number of tests) was applied where appropriate in determining statistical significance [25]. Analyses were performed in R [27]. Genotyping of B. burgdorferi isolates from patients with early Lyme disease. B. burgdorferi isolates were obtained from either the skin or blood of 374 adult patients with erythema migrans evaluated at the Lyme Disease Practice of the Westchester Medical Center. RST genotyping was performed on 462 isolates (319 from skin and 143 from blood), and ospC genotyping was performed (...truncated)