Acrodermatitis chronica atrophicans and serologic confirmation of infection due to Borrelia afzelii and/or Borrelia garinii by immunoblot
1998; Elsevier BV; Volume: 4; Issue: 3 Linguagem: Inglês
10.1111/j.1469-0691.1998.tb00381.x
ISSN1469-0691
AutoresViviane A. Dunand, Anne-Gabrielle Bretz, André Suard, Gérard Praz, Eric Dayer, Olivier Péter,
Tópico(s)Animal Ecology and Behavior Studies
ResumoAcrodermatitis chronica atrophicans (ACA) is a late chronic cutaneous manifestation of Lyme borreliosis [1.Stecre AC Lyme disease.N Engl J Med. 1989; 321: 586-596Crossref PubMed Scopus (1304) Google Scholar], recognized almost exclusively in Europe [2.Canica MM Nato F Du Merle L Mazie JC Baranton G Postic D Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis.Scand J Infect Dis. 1993; 25: 441-448Crossref PubMed Scopus (335) Google Scholar]. Since 1992, European isolates of Borrelia burgdorferi sensu lato, the agent causing Lyme disease have been subdivided into three major genospecies: B. burgdorferi sensu stricto. B. garinii and B. afzelii [2.Canica MM Nato F Du Merle L Mazie JC Baranton G Postic D Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis.Scand J Infect Dis. 1993; 25: 441-448Crossref PubMed Scopus (335) Google Scholar, 3.Baranton G Postic D Saint-Girons I et al.Delincation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov. and group VS 461 associated with Lyme borreliosis.Int J Syst Bacteriol. 1992; 42: 378-383Crossref PubMed Scopus (675) Google Scholar]. Whereas B. burgdorferi sensu stricto seems to be the major if not the only genospecies of clinical importance in North America, European isolates belong to three genospecies, mostly B. garinii or B. afzelii [4.Dressler F Ackermann R Steere AC Antibody responses to the three genomic groups of Borrelia burgdorferi in European Lyme borreliosis.J Infect Dis. 1994; 169: 313-318Crossref PubMed Scopus (88) Google Scholar]. Recent findings suggest that these three genospecies are associated with different late clinical manifestations, respectively to Lyme arthritis, neuroborreliosis and ACA [2.Canica MM Nato F Du Merle L Mazie JC Baranton G Postic D Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis.Scand J Infect Dis. 1993; 25: 441-448Crossref PubMed Scopus (335) Google Scholar,5.Anthonissen FM De Kesel M Hoet PP Bigaignon GH 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, 6.Assous MV Postic D Paul G Névot 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, 7.Péter O Bretz AG Postic D Dayer E Association of distinct species of Borrelia burgdorferi sensu lato with neuroborreliosis in Switzerland.Clin Microbiol Infect. 1997; 3: 423-431Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar]. Because of its sometimes debilitating characteristics and its poor prognosis, ACA should be diagnosed and treated as soon as possible. Although B. burgdorferi sensu lato has been successfully cultured from various clinical lesions, culture remains a difficult procedure that is not practicable for routine diagnosis. Clinical diagnosis is currently supported by measuring the antibody response to the spirochete: indirect immunofluorescence (IF) or enzyme-linked immunosorbent assays (ELISA) are routinely used. In these screening tests, whole cells or whole cell sonicated extract are used as antigens; this may give rise to false-positive results due to cross-reaction with irrelevant antigens in the preparation. Apart from the problem of specificity, which can be partly resolved with ELISAs which use purified antigens such as the flagellum or the OspC or recombinant antigens such as p39 or an internal fragment of flagellin, these tests entail a loss of sensitivity in the specific antibody response to a single Borrelia species. The aim of the study was to develop a type-specific immunoblot assay using whole cell lysates of each of the three genospecies to analyze the serologic response of patients with ACA. A total of 25 patients were retrospectively enrolled on the basis of dermatologic symptoms compatible with ACA and a positive screening test by ELISA (VIDAS Lyme IgG + IgM BioMérieux, France). Results were confirmed by immunoblot using the detection of specific IgG to the three species of Borrelia. All 25 patient sera were screened for antibody to B. burgdorferi sensu lato using five other commercial ELISA tests. The isolates used as antigens in the immunoblot assays were: VS 215 (B. burgdorferi sensu stricto), VS 102 (B. garinii), and VS 461 (B. afzelii). Isolates used in this study were all low-passage strains (fewer than nine passages in BSK-11 medium) isolated from Ixodes ricinus [8.Péter O Bretz AG Polymorphism of outer surface proteins of Borrelia burgdoferi as a tool for classification.Zentralbl Bakteriol. 1992; 277: 28-33Crossref PubMed Scopus (56) Google Scholar]. The three isolates were typed by phenotypic analysis [8.Péter O Bretz AG Polymorphism of outer surface proteins of Borrelia burgdoferi as a tool for classification.Zentralbl Bakteriol. 1992; 277: 28-33Crossref PubMed Scopus (56) Google Scholar] and genotypic analysis according to the method of Postic et al [9.Postic D Assous M Grimont PAD Baranton G Diversity of Borrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf(5S)?m(23s) intergenic spacer amplicons.Int J Syst Bacteriol. 1994; 44: 743-752Crossref PubMed Scopus (446) Google Scholar]. The SDS-PAGE and immunoblot assays were per-formed as previously described [8.Péter O Bretz AG Polymorphism of outer surface proteins of Borrelia burgdoferi as a tool for classification.Zentralbl Bakteriol. 1992; 277: 28-33Crossref PubMed Scopus (56) Google Scholar]. The criterion for a positive immunoblot with human serum was at least five bands including flagellin and two of the following specific bands: OspC, OspA, p39, 93 kDa (p100). as previously described [7.Péter O Bretz AG Postic D Dayer E Association of distinct species of Borrelia burgdorferi sensu lato with neuroborreliosis in Switzerland.Clin Microbiol Infect. 1997; 3: 423-431Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar]. Immunoblots for the three species were performed with three strips, one for each species (VS 215, B. burgdorferi sensu stricto, VS 102, B. garinii, and VS 461, B. afzelii). They were prepared and incubated with scrum from each patient and were processed through all stages in the same strip tray. For comparison between the three immunoblots. scores were allocated (1)-3 points) according to the presence and intensity of the reaction to seven borrelia proteins. as shown in Figure 1(a,b), 93 kDa (p100), flagellin, p39, OspD, OspD, OspC and 18 kDa. This scoring system has been described and validated for neuroborreliosis [7.Péter O Bretz AG Postic D Dayer E Association of distinct species of Borrelia burgdorferi sensu lato with neuroborreliosis in Switzerland.Clin Microbiol Infect. 1997; 3: 423-431Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar]. A total score superior by two points for one individual species compared with the other species was defined as a specific reaction, based on a statistical analysis previously described [7.Péter O Bretz AG Postic D Dayer E Association of distinct species of Borrelia burgdorferi sensu lato with neuroborreliosis in Switzerland.Clin Microbiol Infect. 1997; 3: 423-431Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar] (for example, B 10, A 18, G 11 was typed as B. afzelii). This criterion was based on the significant differences observed in the statistical analysis of the mean scores. The sera of 23 patients with ACA were positive with six commercial ELISAs, while two sera were unreactive, each with one different ELISA, even though they were strongly reactive with the other five ELISAs (Table 1).TABLE 1.Serologic results of 25 patients with acrodermatitis chronica atrophicansPatient no.ELISAImmunoblot scoresInterpretationBAG1+++121411B. afzelii2+++131812B. afzelii3+++61514B. afzelii-B. garinii4++s+131418B. garinii5+++121814B. afzelii6++121912B. afzelii7+++81310B.afzelii8++92111B. afzelii9-/+++91017B. garinii10+++151912B. afzelii111++101414B. afzelii-B. garinii12+++81613B. afzelii13+++111611B. afzelii14+++131714B. afzelii15+++71810B. afzelii16+++81313B. afzelii-B. garinii17+++111814B. afzelii18++142014B. afzelii19+++111512B. afzelii20+++141715B. afzelii21+++91510B. afzelii22+++131511B. afzelii23-/+++101512B. afzelii24+++131813B. afzelii25+++101412B. afzeliiTotal271403319Immunoblot: B, B. burgdorferi sensu stricto; A, B. afzelii; G, B. garinii. Open table in a new tab Immunoblot: B, B. burgdorferi sensu stricto; A, B. afzelii; G, B. garinii. The overall total scores established for the 25 patients by immunoblot were as follows: 403 points for B. afzelii, 319 points for B. garinii and 271 points for B. burgdorferi sensu stricto. Among the 25 patients reacting specifically with B. burgdorferi sensu lato, 20, clearly demonstrated a stronger reaction to B. afzelii (80%) (Figure 1a), with a mean score of 4 above the other two species. Obvious specific reactions to B. garinii were observed in two patients (8%) (Table 1, nos. 4 and 9; Figure 1b) and reactions to both B. afzelii and B. garinii were found in three patients (12%) (Table 1, nos. 3, 11 and 16). No preferential reactivity to B. burgdorferi sensu stricto was observed. This study clearly demonstrated the association between B. afzelii and ACA. Differentiation of the specific reactivity was quite obvious in the immunoblots, as observed by Assous et al [6.Assous MV Postic D Paul G Névot 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]. The numerical scores allowed us to define the specific reactivities on a semiquantitative basis. In 20 of 25 patients (80%) the score was definitely higher for B. afzelii. In three additional patients we may suspect on the basis of the serologic reactions a dual infection by B. afzelii and B. garinii, as previously described by Demaerschalk et al [10.Demaerschalk I Ben Messaoud A De Kesel M et al.Simultaneous presence of different Borrelia burgdoferi genospecies in biological fluids of Lyme disease patients.J Clin Microbiol. 1995; 33: 602-608PubMed Google Scholar] in patients with neuroborreliosis. Two of the 25 cases (8%) showed an unequivocally stronger reaction to B. garinii. Recently, Rijpkema et al [11.Rijpkema SGT Tazelaar DJ Molkenboer MJCH et al.Detection of Borrelia afzelii, Borrelia burgdorferi senus stricto, Borrelia garinii and group VS 116 by PCR in skin biopsies of patients with erythema migrans and acrodermatitis chronica atrophicans.Clin Microbiol infect. 1997; 3: 109-116Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar] amplified by polymerase chain reaction (PCR) B. garinii genomic material from the skin of patients with ACA and also detected mixed infection by B. afzelii and B. garinii. These results seem to indicate that the association between particular Borrelia genospecies and clinical manifestations is real but not absolute. Unfortunately, no skin biopsy was available for PCR confirmation in the two above mentioned cases. A similar association between B. garinii and neuroborreliosis was observed among a group of 28 patients with neuroborreliosis. In this study [7.Péter O Bretz AG Postic D Dayer E Association of distinct species of Borrelia burgdorferi sensu lato with neuroborreliosis in Switzerland.Clin Microbiol Infect. 1997; 3: 423-431Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar], we used the same immunoblots, and we showed that scores were higher for B. garinii (353 points) than for B. afzelii (291 points) and for B. burgdorferi sensu stricto (283 points). Overall, 64% (18/28) of these patients with neuroborreliosis showed a stronger antibody reaction to B. garinii, 11% (3/28) to B. burgdorferi sensu stricto and 7% (2/28) to B. afzelii These results were significantly different (p<0.02) from those of a control group of 20 patients with erythema migrans. Although the diagnosis of Lyme discase is primarily based on clinical manifestations, laboratory data are frequently necessary to elucidate borderline clinical presentations. The direct detection of the causative agent is impaired by the low number of borreliae present in tissue and especially in body fluids. Even the highly sensitive PCR seems not to be superior to the conventional culture isolation methods [12.Schwartz I Wormser GP Schwartz JJ et al.Diagnosis of early Lyme disease by polymerase chain reaction amplification and culture of skin biopsies from erythema migrans skin lesions.J Clin Microbiol. 1992; 30: 3082-3088PubMed Google Scholar], except for synovial fluid, from which borreliae have rarely been isolated but have been demonstrated by PCR in up to 96% of untreated Lyme arthritis cases [13.Nocton JJ Dressler F Rutledge BJ et al.Detection of Borrelia burgdorferi DNA by polymerase chain reaction in synovial fluid from patients with Lyme arthritis.N Engl J Med. 1994; 330: 229-234Crossref PubMed Scopus (455) Google Scholar]. Serologic techniques are, therefore, the most widely used methods to establish the diagnosis of Lyme borreliosis. Several attempts to increase the sensitivity and specificity of serologic tests have included absorption procedures with Treponema phagedenis, use of purified antigens or recombinant antigens and the association of different recombinant antigens selected from different strains in the same immunoblot [14.Wilske B Fingerle V Preac-Mursic V et al.Immunoblot using recombinant antigens derived from different genospecies of Borrelia burgdorferi sensu lato.Med Microbiol Immunol. 1994; 183: 43-59Crossref PubMed Scopus (59) Google Scholar]. The four antigens studied by Wilske et al were flagellin, an internal flagellin fragment, the 93-kDa protein called p100, the plasmidencoded outer-surface proteins OspA and OspC. The authors concluded that recombinant antigens from different genospecies of B. burgdorferi sensu lato are serologically different, and that specific clinical manifestations are correlated with each of the three genospecies. Therefore, genospecific tests for each clinical indication could provide more sensitivity and specificity than a single common test. The type-specific assay described here is based on seven antigens-93 kDa or p100, flagellin, p39, OspA, OspD, OspC and 18-kDa proteins specifically recognized in each of the three genospecies. In this study, each serum was tested for the presence of specific IgG against all these antigenic proteins of each of the three genospecies. The serologic data in the present study show a clear association of B. afzelii with ACA and confirm the results of isolations typed as B. afzelii from the skin of patients with ACA [2.Canica MM Nato F Du Merle L Mazie JC Baranton G Postic D Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis.Scand J Infect Dis. 1993; 25: 441-448Crossref PubMed Scopus (335) Google Scholar, 15.Balmelli T Piffaretti JC Association between different clinical manifestations of Lyme disease and different species of Borrelia burgdorferi sensu lato.Res Microbiol. 1995; 146: 329-340Crossref PubMed Scopus (168) Google Scholar]. Furthermore, our data strongly suggest that B. garinii is also able to produce chronic dermatologic disorders such as ACA, though at a much lower frequency than B. afzelii. This is, to the best of our knowledge, the first report of serologic determination of B. garinii as the causative agent of ACA. Dual infection with B. afzelii and B. garinii or reinfection with one or the other genospecies may be suspected in patients reacting with both B. afzelii and B. garinii. The immunoblot described here also allows the discrimination of false-positive reactions in screening tests and occasionally the confirmation of a chronic Lyme borrcliosis in spite of a negative ELISA. It therefore seems to be a sensitive and specific confirmatory test in the laboratory diagnosis of chronic Lyme borreliosis. We thank G. Baranton, A. G. Barbour. W. T. Golde, T. Schwan and B. Wilske for providing the monoclonal antibodies, as well as M. C. Mottiez and A. Pierroz for technical assistance. This work was supported by the Fonds National Suisse de la Recherche Scientifique (grant no. 32. 40605.94). Acrodermatitis chronica atrophicans and serologic confirmation of infection due to Borrelia afzelii and/or Borrelia garinii by immunoblotClinical Microbiology and InfectionVol. 4Issue 6PreviewOn page 161, we regret that Figure 1 was published with parts (a) and b transposed. We reproduce the figure correctly below. Full-Text PDF Open Archive
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