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Identification of Three Species of Borrelia burgdorferi Sensu Lato (B. burgdorferi Sensu Stricto, B. garinii, and B. afzelii) Among Isolates from Acrodermatitis Chronica Atrophicans Lesions

1998; Elsevier BV; Volume: 110; Issue: 3 Linguagem: Inglês

10.1046/j.1523-1747.1998.00130.x

1523-1747

Roger N. Picken, Franc Strle, Maria M. Picken, Eva Ružić–Sabljić, Vera Maraspin, Stanka Lotrič‐Furlan, J. Cimperman,

Insect and Pesticide Research

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 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. Acrodermatitis chronica atrophicans (ACA) is a late cutaneous manifestation of Lyme borreliosis characterized by erythrocyanotic lesions that typically involve acral sites (Åsbrink and Hovmark, 1987Åsbrink E. Hovmark A. Cutaneous manifestations in Ixodes-borne Borrelia spirochetosis.Int J Derm. 1987; 26: 215-223Crossref PubMed Scopus (40) Google Scholar, Åsbrink and Hovmark, 1988Åsbrink E. Hovmark A. Early and late cutaneous manifestations in Ixodes-borne borreliosis (erythema migrans borreliosis, Lyme borreliosis).Ann NY Acad Sci. 1988; 539: 4-15Crossref PubMed Scopus (178) Google Scholar, Abele and Anders, 1990Abele D.C. Anders K.H. The many faces and phases of borreliosis II.J Am Acad Dermatol. 1990; 23: 401-410Abstract Full Text PDF PubMed Scopus (49) Google Scholar). Untreated disease has a long progressive course that comprises both inflammatory and atrophic stages where the evolution of lesions is slow and insidious. In the atrophic phase, generalized thinning of the dermis and epidermis gives the skin a transparent appearance, often with clearly visible veins (Abele and Anders, 1990Abele D.C. Anders K.H. The many faces and phases of borreliosis II.J Am Acad Dermatol. 1990; 23: 401-410Abstract Full Text PDF PubMed Scopus (49) Google Scholar, Åsbrink, 1993Åsbrink E. Acrodermatitis chronica atrophicans.Clin Dermatol. 1993; 11: 369-375Abstract Full Text PDF PubMed Scopus (23) Google Scholar). Unlike other skin manifestations, such as erythema migrans and borrelial lymphocytoma, ACA does not heal without treatment; the inflammatory stage may last for months or years and the atrophic stage can endure for decades (Åsbrink, 1993Åsbrink E. Acrodermatitis chronica atrophicans.Clin Dermatol. 1993; 11: 369-375Abstract Full Text PDF PubMed Scopus (23) Google Scholar). The borrelial etiology of the disease was firmly established in 1984 by the cultivation of Borrelia burgdorferi from an ACA lesion (Åsbrink et al., 1984Åsbrink E. Hovmark A. Hederstedt B. The spirochetal etiology of acrodermatitis chronica atrophicans Herxheimer.Acta Derm Venereol (Stockh). 1984; 64: 506-512PubMed Google Scholar). Within the general taxon B. burgdorferi sensu lato, three species are known to be causative agents of human Lyme borreliosis: B. burgdorferi sensu stricto, B. garinii, and B. afzelii (Baranton et al., 1992Baranton G. Postic D. Saint Girons I. Boerlin P. Piffaretti J.C. Assous M. Grimont P.A.D. Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov. & group VS461 associated with Lyme borreliosis.Int J Syst Bacteriol. 1992; 42: 378-383Crossref PubMed Scopus (675) Google Scholar). There is disparity in their geographic distribution, however; whereas all three species have been encountered among European patient isolates, only B. burgdorferi sensu stricto has thus far been isolated from patients in North America. These findings have led to speculation that the unequal distribution of species might be associated with perceived differences in disease manifestations. For example, ACA has been widely encountered throughout Europe, but there have been few reports from the United States. Of 45 cases of ACA seen at the Mayo Clinic up to 1945, only six patients were born in the United States, and the remainder were immigrants, mostly from Europe (Montgomery and Sullivan, 1945Montgomery H. Sullivan R. Acrodermatitis chronica atrophicans.Arch Dermatol Syph. 1945; 51: 32-45Crossref Scopus (23) Google Scholar). Since then, only a few additional reports from North America have appeared (Steere et al., 1986Steere A.C. Taylor E. Wilson M.L. Levine J.F. Spielman A. Longitudinal assessment of the clinical and epidemiological features of Lyme disease in a defined population.J Infect Dis. 1986; 154: 295-300Crossref PubMed Scopus (179) Google Scholar, Lavoie et al., 1986Lavoie P.E. Wilson A.J. Tuffanelli D.L. Acrodermatitis chronica atrophicans with antecedent Lyme disease in California.Zentralbl Bakteriol Mikrobiol Hyg [A]. 1986; 263: 262-265PubMed Google Scholar, Tuffanelli, 1987Tuffanelli D. Do some patients with morphea and lichen sclerosus et atrophicus have a Borrelia infection?.Am J Dermatopathol. 1987; 9: 371-373Crossref PubMed Scopus (30) Google Scholar, Kaufman et al., 1989Kaufman L.D. Gruber B.L. Philips M.E. Benach J.L. Late cutaneous Lyme disease: Acrodermatitis chronica atrophicans.Am J Med. 1989; 86: 828-830Abstract Full Text PDF PubMed Scopus (25) Google Scholar, Edwards et al., 1992Edwards L. Hoshaw R.A. Burgdorf W.H.C. Acrodermatitis chronica atrophicans.Arch Dermatol. 1992; 128: 858-860Crossref PubMed Scopus (6) Google Scholar). Such observations have led to a search for associations between individual Borrelia spp. and the clinical manifestations of Lyme borre-liosis. In the case of ACA, indications of just such a correlation were found. Molecular studies of ACA isolates or sera from several European countries over the last 4 y have produced evidence suggesting an exclusive association of B. afzelii with ACA (van Dam et al., 1993van Dam A.P. Kuiper H. Vos K. et al.Different genospecies of Borrelia burgdorferi are associated with distinct clinical manifestations of Lyme borreliosis.Clin Infect Dis. 1993; 17: 708-717Crossref PubMed Scopus (484) Google Scholar, Assous et al., 1993Assous M.V. Postic D. Paul G. Nevot P. Baranton G. Western blot analysis of sera from Lyme borreliosis patients according to the genomic species of the Borrelia strains used as antigens.Eur J Clin Microbiol Infect Dis. 1993; 12: 261-268Crossref PubMed Scopus (149) Google Scholar, Anthonissen et al., 1994Anthonissen F.M. De Kesel M. Hoet P.P. Bigaignon G.H. Evidence for the involvement of different genospecies of Borrelia in the clinical outcome of Lyme disease in Belgium.Res Microbiol. 1994; 145: 327-331Crossref PubMed Scopus (66) Google Scholar, Balmelli and Piffaretti, 1995Balmelli T. Piffaretti J.-C. Association between different clinical manifestations of Lyme disease and different species of Borrelia burgdorferi sensu lato.Res Microbiol. 1995; 146: 329340Crossref Scopus (168) Google Scholar, Wienecke et al., 1995Wienecke R. Zochling N. Neubert U. Schlüpen E.-M. Meurer M. Volkenandt M. Molecular subtyping of Borrelia burgdorferi in erythema migrans and acrodermatitis chronica atrophicans.J Invest Dermatol. 1995; 103: 19-22Crossref Scopus (55) Google Scholar, Ohlenbusch et al., 1996Ohlenbusch A. Matuschka F.-R. Richter D. Christen H.-J. Thomssen R. Spielman A. Eiffert H. Etiology of the acrodermatitis chronica atrophicans lesion in Lyme disease.J Infect Dis. 1996; 174: 421-423Crossref PubMed Scopus (43) Google Scholar). To further investigate this phenomenon, we examined 22 new isolates of B. burgdorferi sensu lato derived from Slovene patients with ACA lesions. This report presents data on the molecular subtyping of these strains. All patients presented to the University Medical Center, Lubljana, between July 1992 and June 1995. Criteria for the inclusion of patients in the study were as follows: (i) they had not received antibiotic treatment specifically for ACA, (ii) they had typical lesions on the extremities, (iii) histopathologic findings supported the diagnosis of ACA, (iv) IgG antibody titers against B. burgdorferi sensu lato were detectable, and (v) B. burgdorferi sensu lato was isolated in culture from a skin biopsy. Skin biopsies were taken from areas where the ACA lesions were most prominent. In the case of one patient two separate ACA lesions were cultured. Methods pertaining to the excision of tissue, the inoculation into growth medium, and the isolation of strains have been described previously (Strle et al., 1995aStrle F. Cheng Y. Cimperman J. et al.Persistence of Borrelia burgdorferi sensu lato in resolved erythema migrans lesions.Clin Infect Dis. 1995 a; 21: 380-389Crossref PubMed Scopus (44) Google Scholar). In some patients lumbar puncture was also performed and cerebrospinal fluid (CSF) cultured for B. burgdorferi sensu lato. Standard microbiologic techniques were employed to preclude the possibility of cross-contamination of cultures. Representative B. burgdorferi sensu lato isolates obtained from ACA lesions of patients are listed in Table I along with reference strains used in comparative studies. All patient strains used in this study were low passage isolates.Table IBorrelia spp. and strains used and results of molecular subtyping studiesSpeciesStrain designationLRFPaLRFP, large restriction fragment pattern. Nomenclature follows the system devised previously (Belfaiza et al, 1993) for bands >70 kb in size. Thus, all LRFP derived from restriction endonuclease Mlu I digestion are designated ML, B. burgdorferi sensu stricto LRFP are designated MLb, B. garinii LRFP are designated MLg, and B. afzelii LRFP are designated MLa. U, undesignated LRFP.Biologic originGeographic locationReference strainsDN127 genomic groupDN127UTick (Ixodes pacificus)U.S.A.DN127 genomic group25015UTick (Ixodes scapularis)U.S.A.B. burgdorferi sensu strictB31 [ATCC 35210]MLb1Tick (Ixodes scapularis)U.S.A.B. burgdorferi sensu strict297 [ATCC 53899]MLb2CSFbCSF, cerebrospinal fluid.U.S.A.B. garinii20047MLg1Tick (Ixodes ricinus)FranceB. gariniiPBiMLg2CSFGermanyB. afzeliiVS461MLa1Tick (Ixodes ricinus)SwitzerlandVS116 genomic groupVS116UTick (Ixodes ricinus)SwitzerlandStrains from patientsB. burgdorferi sensu strictSL-91MLb2Skin (ACAcACA, acrodermatitis chronica atrophicans.)SloveniaB. gariniiSL-92MLg2Skin (ACA)SloveniaB. gariniiSL-95MLg2Skin (ACA)SloveniaB. gariniiSL-109MLg2Skin (ACA)SloveniaB. afzeliiSL-94MLa1Skin (ACA)SloveniaB. afzeliiSL-112MLa1Skin (ACA)Sloveniaa LRFP, large restriction fragment pattern. Nomenclature follows the system devised previously (Belfaiza et al., 1993Belfaiza J. Postic D. Bellenger E. Baranton G. Saint Girons I. Genomic fingerprinting of Borrelia burgdorferi sensu lato by pulsed-field gel electrophoresis.J Clin Micriobiol. 1993; 31: 2873-2877PubMed Google Scholar) for bands >70 kb in size. Thus, all LRFP derived from restriction endonuclease Mlu I digestion are designated ML, B. burgdorferi sensu stricto LRFP are designated MLb, B. garinii LRFP are designated MLg, and B. afzelii LRFP are designated MLa. U, undesignated LRFP.b CSF, cerebrospinal fluid.c ACA, acrodermatitis chronica atrophicans. Open table in a new tab For all patients IgM and IgG antibody titers against B. burgdorferi sensu lato were determined on the day of skin biopsy by immunofluorescence assay (Wilske et al., 1984Wilske B. Schierz B. Preac-Mursic V. Weber K. Pfister H.W. Einhaupl K.M. Serological diagnosis in erythema migrans disease and related disorders.Infection. 1984; 12: 331-337Crossref PubMed Scopus (134) Google Scholar). A local isolate of B. afzelii was used as antigen. Titers of 1:256 or higher were interpreted as positive. Isolates were identified to the species level using two independent methods: (i) 16S ribosomal RNA (rRNA)-specific polymerase chain reaction (PCR), (ii) large restriction fragment pattern (LRFP) analysis involving cleavage of genomic DNA with the restriction enzyme MluI and separation of the fragments using pulsed-field gel electrophoresis. Well-characterized reference strains of B. burgdorferi sensu lato were included for comparison (Table I). The strains B31, 20047, and VS461 are the type strains of the species B. burgdorferi sensu stricto, B. garinii, and B. afzelii, respectively. Isolates representing other genomic groups of B. burgdorferi sensu lato included strains DN127 and 25015 (DN127 group), and VS116 (VS116 group). The LRFP produced by restriction enzyme digestion are characteristic of each species (Belfaiza et al., 1993Belfaiza J. Postic D. Bellenger E. Baranton G. Saint Girons I. Genomic fingerprinting of Borrelia burgdorferi sensu lato by pulsed-field gel electrophoresis.J Clin Micriobiol. 1993; 31: 2873-2877PubMed Google Scholar). Both techniques were performed as described previously (Strle et al., 1995aStrle F. Cheng Y. Cimperman J. et al.Persistence of Borrelia burgdorferi sensu lato in resolved erythema migrans lesions.Clin Infect Dis. 1995 a; 21: 380-389Crossref PubMed Scopus (44) Google Scholar, Strle et al., 1995bStrle F. Cheng Y. Nelson J.A. Picken M.M. Bouseman J.K. Picken R.N. Infection rate of Ixodes ricinus Ticks with Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi Sensu Stricto in Slovenia.Eur J Clin Microbiol Infect Dis. 1995 b; 14: 994-1001Crossref PubMed Scopus (49) Google Scholar, Picken et al., 1995Picken R.N. Cheng Y. Han D. et al.Genotypic and phenotypic characterization of Borrelia burgdorferi isolated from ticks and small animals in Illinois.J Clin Microbiol. 1995; 33: 23042315Google Scholar, Picken et al., 1996Picken R.N. Cheng Y. Strle F. et al.Molecular Characterization of Borrelia burgdorferi sensu lato from Slovenia Reveals Significant Differences Between Tick and Human Isolates.Eur J Clin Microbiol Infect Dis. 1996; 15: 313-323Crossref PubMed Scopus (60) Google Scholar). From July 1992 to June 1995, 104 Slovene patients pre- senting with ACA were biopsied and cultured for B. burgdorferi sensu lato. In one of the patients two separate lesions were biopsied. Twenty- four lesions from 23 patients grew spirochetes, for an overall isolation rate of 22%. In addition, spirochetes were concomitantly isolated from the CSF of one patient. Twenty-two ACA isolates from 21 patients were subsequently studied by molecular subtyping methods. The patients comprised 14 females (67%) and seven males (33%), aged 21— 76 y (median 66 y). The duration of ACA lesions varied from 5 mo to more than 20 y (median 2 y). Eighteen of the 21 patients recalled one or more tick bites in the preceding years and seven patients had a prior recollection of erythema migrans, which occurred on the same extremity as the subsequent ACA lesion in five patients. By immunofluorescence assay, all 21 patients had an IgG antibody response to B. burgdorferi sensu lato, and four also demonstrated an IgM response. PCR analysis of the 22 isolates indicated that 17 isolates were B. afzelii, four were B. garinii, and one was B. burgdorferi sensu stricto (Table II). In one patient, where isolates were obtained from two separate ACA lesions, both strains typed as B. garinii. Figure 1 shows the results of PCR amplification of DNA from six patient ACA isolates: B. burgdorferi sensu stricto strain SL-91, B. garinii strains SL-92, SL-95, and SL-109, and two representatives (SL-94, SL- 112) from the 17 B. afzelii isolates. Only one of the two B. garinii isolates obtained from the same patient is shown (SL-95). Eight well- characterized reference strains, representing the DN127 genomic group, B. burgdorferi sensu stricto, B. garinii, B. afzelii, and the VS116 genomic group, are shown for comparison. Note, however, that the 16S rRNA-specific PCR primers are not able to differentiate B. garinii from the VS116 genomic group (Fig 1).Table IIFrequency of isolation of Borrelia spp. from acrodermatitis chronica atrophicans lesionsSpeciesLRFPNo. of isolates obtainedB. burgdorferi sensu strictoMLb21B. gariniiMLg24aTwo isolates were obtained from separate ACA lesions of one patient.B. afzeliiMLa117a Two isolates were obtained from separate ACA lesions of one patient. Open table in a new tab Figure 2 shows the results of LRFP analysis of the same six representative patient ACA isolates and the eight reference strains used in Fig 1. In all cases, results from the two methods of species identification were concordant. Seventeen of the 22 isolates typed as B. afzelii, possessing the same MLa1 LRFP as VS461, the B. afzelii type strain; this LRFP is characterized by fragments of ≈440, 320, and 90 kb (Picken et al., 1996Picken R.N. Cheng Y. Strle F. et al.Molecular Characterization of Borrelia burgdorferi sensu lato from Slovenia Reveals Significant Differences Between Tick and Human Isolates.Eur J Clin Microbiol Infect Dis. 1996; 15: 313-323Crossref PubMed Scopus (60) Google Scholar). Four isolates typed as B. garinii, possessing the same MLg2 LRFP as the B. garinii reference strain PBi; this LRFP is characterized by fragments of ≈390, 220, 100, and 80 kb (Picken et al., 1996Picken R.N. Cheng Y. Strle F. et al.Molecular Characterization of Borrelia burgdorferi sensu lato from Slovenia Reveals Significant Differences Between Tick and Human Isolates.Eur J Clin Microbiol Infect Dis. 1996; 15: 313-323Crossref PubMed Scopus (60) Google Scholar). Three of the B. garinii patient isolates are shown in Fig 2 (SL-92, SL-95, and SL-109). In one patient, where two isolates were obtained from separate ACA lesions, only one of the two strains is shown in Fig 2 (SL-95), because both isolates produced identical macrorestriction patterns. One isolate (SL-91) typed as B. burgdorferi sensu stricto, possessing the same MLb2 LRFP as B. burgdorferi sensu stricto reference strain 297; this LRFP is characterized by fragments of ≈410, 160, 140, and 110 kb (Picken et al., 1996Picken R.N. Cheng Y. Strle F. et al.Molecular Characterization of Borrelia burgdorferi sensu lato from Slovenia Reveals Significant Differences Between Tick and Human Isolates.Eur J Clin Microbiol Infect Dis. 1996; 15: 313-323Crossref PubMed Scopus (60) Google Scholar). The 21 patients and 22 isolates described in this report represent the largest series of new, consecutive ACA patients and strains studied to date. Previously, the largest number of ACA patients investigated was represented by the 18 lesional skin biopsies studied by Wienecke et al., 1995Wienecke R. Zochling N. Neubert U. Schlüpen E.-M. Meurer M. Volkenandt M. Molecular subtyping of Borrelia burgdorferi in erythema migrans and acrodermatitis chronica atrophicans.J Invest Dermatol. 1995; 103: 19-22Crossref Scopus (55) Google Scholar, from all of which B. afzelii-specific sequences were amplified by PCR. In contrast to their results, we found that four isolates typed as B. garinii and one isolate typed as B. burgdorferi sensu stricto. This is the second report of an association between B. burgdorferi sensu stricto and ACA; previously, the Danish isolate DK7, which was described as being derived from an ACA lesion, was typed as B. burgdorferi sensu stricto by an Outer Surface Protein A serotyping method (Wilske et al., 1993Wilske B. Preac-Mursic V. Göbel U.B. et al.An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis.J Clin Microbiol. 1993; 31: 340-350PubMed Google Scholar). To date, only one, highly conserved LRFP (MLa1) has been described for B. afzelii (Belfaiza et al., 1993Belfaiza J. Postic D. Bellenger E. Baranton G. Saint Girons I. Genomic fingerprinting of Borrelia burgdorferi sensu lato by pulsed-field gel electrophoresis.J Clin Micriobiol. 1993; 31: 2873-2877PubMed Google Scholar, Strle et al., 1995bStrle F. Cheng Y. Nelson J.A. Picken M.M. Bouseman J.K. Picken R.N. Infection rate of Ixodes ricinus Ticks with Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi Sensu Stricto in Slovenia.Eur J Clin Microbiol Infect Dis. 1995 b; 14: 994-1001Crossref PubMed Scopus (49) Google Scholar, Picken et al., 1996Picken R.N. Cheng Y. Strle F. et al.Molecular Characterization of Borrelia burgdorferi sensu lato from Slovenia Reveals Significant Differences Between Tick and Human Isolates.Eur J Clin Microbiol Infect Dis. 1996; 15: 313-323Crossref PubMed Scopus (60) Google Scholar). In this study, 17 of 22 isolates investigated possessed this MLa1 LRFP, demonstrating that they belong to the species B. afzelii. Six LRFP have been described for B. garinii (Belfaiza et al., 1993Belfaiza J. Postic D. Bellenger E. Baranton G. Saint Girons I. Genomic fingerprinting of Borrelia burgdorferi sensu lato by pulsed-field gel electrophoresis.J Clin Micriobiol. 1993; 31: 2873-2877PubMed Google Scholar, Strle et al., 1995bStrle F. Cheng Y. Nelson J.A. Picken M.M. Bouseman J.K. Picken R.N. Infection rate of Ixodes ricinus Ticks with Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi Sensu Stricto in Slovenia.Eur J Clin Microbiol Infect Dis. 1995 b; 14: 994-1001Crossref PubMed Scopus (49) Google Scholar, Picken et al., 1996Picken R.N. Cheng Y. Strle F. et al.Molecular Characterization of Borrelia burgdorferi sensu lato from Slovenia Reveals Significant Differences Between Tick and Human Isolates.Eur J Clin Microbiol Infect Dis. 1996; 15: 313-323Crossref PubMed Scopus (60) Google Scholar), of which four have been encountered among Slovene patient isolates and six among Slovene tick isolates (Strle et al., 1995bStrle F. Cheng Y. Nelson J.A. Picken M.M. Bouseman J.K. Picken R.N. Infection rate of Ixodes ricinus Ticks with Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi Sensu Stricto in Slovenia.Eur J Clin Microbiol Infect Dis. 1995 b; 14: 994-1001Crossref PubMed Scopus (49) Google Scholar, Picken et al., 1996Picken R.N. Cheng Y. Strle F. et al.Molecular Characterization of Borrelia burgdorferi sensu lato from Slovenia Reveals Significant Differences Between Tick and Human Isolates.Eur J Clin Microbiol Infect Dis. 1996; 15: 313-323Crossref PubMed Scopus (60) Google Scholar). The four B. garinii strains isolated from ACA lesions in this study all possessed the MLg2 LRFP. This was also found to be the most prevalent B. garinii LRFP isolated from patients and ticks in Slovenia (Strle et al., 1995bStrle F. Cheng Y. Nelson J.A. Picken M.M. Bouseman J.K. Picken R.N. Infection rate of Ixodes ricinus Ticks with Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi Sensu Stricto in Slovenia.Eur J Clin Microbiol Infect Dis. 1995 b; 14: 994-1001Crossref PubMed Scopus (49) Google Scholar, Picken et al., 1996Picken R.N. Cheng Y. Strle F. et al.Molecular Characterization of Borrelia burgdorferi sensu lato from Slovenia Reveals Significant Differences Between Tick and Human Isolates.Eur J Clin Microbiol Infect Dis. 1996; 15: 313-323Crossref PubMed Scopus (60) Google Scholar) and may not therefore reflect a particular association with ACA. PBi, the MLg2 reference strain used in this study, was isolated from the CSF of a patient residing in Germany (Wilske et al., 1993Wilske B. Preac-Mursic V. Göbel U.B. et al.An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis.J Clin Microbiol. 1993; 31: 340-350PubMed Google Scholar). It would therefore seem that B. garinii of this type can be responsible for at least two of the sequelae of Lyme borreliosis, neuroborreliosis, and ACA. Of the three species that infect humans, B. burgdorferi sensu stricto has been shown to be the most heterogeneous in terms of number of LRFP (Belfaiza et al., 1993Belfaiza J. Postic D. Bellenger E. Baranton G. Saint Girons I. Genomic fingerprinting of Borrelia burgdorferi sensu lato by pulsed-field gel electrophoresis.J Clin Micriobiol. 1993; 31: 2873-2877PubMed Google Scholar, Strle et al., 1995bStrle F. Cheng Y. Nelson J.A. Picken M.M. Bouseman J.K. Picken R.N. Infection rate of Ixodes ricinus Ticks with Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi Sensu Stricto in Slovenia.Eur J Clin Microbiol Infect Dis. 1995 b; 14: 994-1001Crossref PubMed Scopus (49) Google Scholar, Picken et al., 1996Picken R.N. Cheng Y. Strle F. et al.Molecular Characterization of Borrelia burgdorferi sensu lato from Slovenia Reveals Significant Differences Between Tick and Human Isolates.Eur J Clin Microbiol Infect Dis. 1996; 15: 313-323Crossref PubMed Scopus (60) Google Scholar). The single B. burgdorferi sensu stricto isolate that we obtained from ACA possessed the MLb2 LRFP; previously, we have also encountered this LRFP among isolates derived from erythema migrans lesions of Slovene patients (Picken et al., 1996Picken R.N. Cheng Y. Strle F. et al.Molecular Characterization of Borrelia burgdorferi sensu lato from Slovenia Reveals Significant Differences Between Tick and Human Isolates.Eur J Clin Microbiol Infect Dis. 1996; 15: 313-323Crossref PubMed Scopus (60) Google Scholar). Borrelia burgdorferi sensu stricto reference strain 297 (ATCC 53899), which also possesses the MLb2 LRFP, was isolated from the CSF of a North American patient. This suggests not only that strains of this type can be responsible for both neuroborreliosis and ACA, but also that the potential for ACA is present in North America. The paucity of case reports from the United States has resulted in ACA being generally regarded as a "European" manifestation of chronic Lyme borreliosis; however, there have been sporadic reports of ACA from the United States over several years (Steere et al., 1986Steere A.C. Taylor E. Wilson M.L. Levine J.F. Spielman A. Longitudinal assessment of the clinical and epidemiological features of Lyme disease in a defined population.J Infect Dis. 1986; 154: 295-300Crossref PubMed Scopus (179) Google Scholar, Lavoie et al., 1986Lavoie P.E. Wilson A.J. Tuffanelli D.L. Acrodermatitis chronica atrophicans with antecedent Lyme disease in California.Zentralbl Bakteriol Mikrobiol Hyg [A]. 1986; 263: 262-265PubMed Google Scholar, Tuffanelli, 1987Tuffanelli D. Do some patients with morphea and lichen sclerosus et atrophicus have a Borrelia infection?.Am J Dermatopathol. 1987; 9: 371-373Crossref PubMed Scopus (30) Google Scholar, Kaufman et al., 1989Kaufman L.D. Gruber B.L. Philips M.E. Benach J.L. Late cutaneous Lyme disease: Acrodermatitis chronica atrophicans.Am J Med. 1989; 86: 828-830Abstract Full Text PDF PubMed Scopus (25) Google Scholar, Edwards et al., 1992Edwards L. Hoshaw R.A. Burgdorf W.H.C. Acrodermatitis chronica atrophicans.Arch Dermatol. 1992; 128: 858-860Crossref PubMed Scopus (6) Google Scholar). Recently, detailed case reports have been published of patients from New York who had clinically classic lesions and/or elevated serum antibody titers to B. burgdorferi with demonstration of spirochetes by antibody staining (Kaufman et al., 1989Kaufman L.D. Gruber B.L. Philips M.E. Benach J.L. Late cutaneous Lyme disease: Acrodermatitis chronica atrophicans.Am J Med. 1989; 86: 828-830Abstract Full Text PDF PubMed Scopus (25) Google Scholar, Edwards et al., 1992Edwards L. Hoshaw R.A. Burgdorf W.H.C. Acrodermatitis chronica atrophicans.Arch Dermatol. 1992; 128: 858-860Crossref PubMed Scopus (6) Google Scholar). The fact that B. burgdorferi sensu stricto has now been found associated with ACA lesions (Wilske et al., 1993Wilske B. Preac-Mursic V. Göbel U.B. et al.An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis.J Clin Microbiol. 1993; 31: 340-350PubMed Google Scholar; this study) supports the notion that cases of ACA can occur in the United States; however, reasons for its low incidence remain obscure. Alternatively, it has also been shown that patients can be concomit- antly infected with more than one Borrelia spp. (Demaerschalk et al., 1995Demaerschalk I. Messaoud A.B. De Kesel M. et al.Simultaneous presence of different Borrelia burgdorferi genospecies in biological fluids of Lyme disease patients.J Clin Microbiol. 1995; 33: 602-608PubMed Google Scholar). Thus, it is conceivable that the ACA lesions from which the B. garinii and B. burgdorferi sensu stricto isolates derived could actually have been caused by a concomitant B. afzelii infection, and that in vitro culture favored the outgrowth of B. garinii and B. burgdorferi sensu stricto. This possibility cannot be ruled out; however, it can be concluded that these latter species were present in the ACA skin lesion at the time of biopsy. Also, in the case where two isolates were obtained from separate ACA lesions of one patient, both strains had identical molecular properties (B. garinii, MLg2 subclass). In the case of another patient, isolates were obtained from both an ACA lesion and CSF; both strains typed as B. afzelii MLa1 (data not shown). We conclude from these findings that in Slovene patients B. afzelii is not the sole etiologic agent of ACA and that this chronic skin manifestation can also result from infection with B. garinii and B. burgdorferi sensu stricto. The finding of involvement by the latter species suggests that the potential for ACA also exists in North America, and recent case reports from the United States appear to support this. It remains to be determined why a higher incidence of ACA is seen among Lyme borreliosis patients in Europe than in North America. This work was supported in part by grant #AR 41517 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (to R.N.P) and by a grant from the Schweppe Foundation (to M.M.P.). The authors are also grateful to Dr. Olivier Peter for supplying the reference strains VS461 and VS116.