Improvement in the Laboratory Recognition of Lyme Borreliosis with the Combination of Culture and PCR Methods
Tóm tắt
Background: Lyme disease is a multisystem, multistage infection caused by three genospecies of the Borrelia burgdorferi sensu lato species. The diagnosis of Lyme disease is based on a history of tick-bite, physical examination, and serological tests. In the seronegative patients with Lyme borreliosis symptoms, additional testing should be introduced.
Methods: The study group was composed of 240 hospitalized patients presented with various clinical symptoms suggesting Lyme borreliosis: 221 of the patients with neurological abnormalities and 19 with oligoarticular arthritis. Citrated blood and serum samples were collected from the patients for culture and serological examination, respectively. Moreover, 173 cerebrospinal and 6 synovial fluid samples were tested. New oligonucleotide primers based on B. burgdorferi sensu lato 16SrRNA gene sequences were designed for the detection of the bacteria in blood, cerebrospinal, and synovial fluid specimens with PCR. Levels of specific antibodies were measured in serum, cerebrospinal fluid and synovial fluid samples using ELISA and Western blot. B. burgdorferi spirochetes from blood, cerebrospinal fluid, and synovial fluid samples were cultured in cell line. Extracted and purified B. burgdorferi DNA was identified by PCR with new oligonucleotide primers. Then three genospecies were identified by PCR amplification with other primer sets specific for 16S rDNA and/or by the restriction fragment length polymorphism of 23S(rrl)-5S(rrf).
Results: Bacterial DNA were found in samples from 32 patients, including 28 patients with neuroborreliosis and 4 with Lyme arthritis. B. burgdorferi-sipecific IgM and/or IgG serum antibodies were detected in 14 of these patients. Fourteen strains of Borrelia garini, 4 strains of Borrelia afzelii and 1 strain of B. burgdorferi sensu stricto were identified by PCR. Genospecies were not recognized in 13 specimens.
Conclusions: The procedure can be a rapid and sensitive diagnostic method for the detection of etiological agents in clinical materials derived from patients with the clinical symptoms of Lyme borreliosis. It can be utilized for both basic research as well as routine laboratory diagnosis.
Tài liệu tham khảo
Belfaiza J, Postic D, Bellenger E, et al. Genomic fingerprinting of Borrelia burgdorferi sensu lato by pulsed-field gel electrophoresis. J Clin Microbiol 1993; 31: 2873–7
Centers for Disease Control and Prevention. Case definitions for public health surveillance. MMWR Morb Mortal Wkly Rep 1990; 39(RR-13): 19–21
Stanek G, O’Connel S, Cimmino M, et al. European Union concerted action on risk assesment in Lyme borreliosis: clinical case definitions for Lyme borreliosis. Wien Klin Wochenschr 1996; 108: 741–7
Karlsson M, Hovind-Hougen K, Svenungsson B, et al. Cultivation and characterization of spirochetes from cerebrospinal fluid of patients with Lyme borreliosis. J Clin Microbiol 1990; 28: 473–9
Nadelman RB, Schwartz I, Wormser GP. Detecting Borrelia burgdorferi in blood from patients with Lyme disease. J Infect Dis 1994; 169: 1410–1
Oliver JH JR, Kollars TM JR, Chandler FW JR, et al. First isolation and cultivation of Borrelia burgdorferi sensu lato from Missouri. J Clin Microbiol 1998; 36: 1–5
Schmidli J, Hunziker T, Moesli P, et al. Cultivation of Borrelia burgdorferi from joint fluid three months after treatment of facial palsy due to Lyme borreliosis. J Infect Dis 1988; 158: 905–6
Wallach FR, Forni AL, Hariprashad J, et al. Circulating Borrelia burgdorferi in patients with acute Lyme disease: result of blood cultures and serum DNA analysis. J Infect Dis 1993; 168: 1541–3
Wormser GP, Nowakowski J, Nadelman RB, et al. Improving the yield of blood cultures for patients with early Lyme disease. J Clin Microbiol 1998; 36: 296–8
Nocton JJ, Bloom BJ, Rutledge BJ, et al. Detection of Borrelia burgdorferi DNA by polymerase chain reaction in cerebrospinal fluid in Lyme neuroborreliosis. J Infect Dis 1996; 174: 623–7
Oksi J, Uksila J, Marjamaki M, et al. Antibodies against whole sonicated Borrelia burgdorferi spirochetes, 41-kilodalton flagelin, and P39 protein in patients with PCR- or culture-proven late Lyme borreliosis. J Clin Microbiol 1995; 33: 2260–4
Tylewska-Wierzbanowska S, Chmielewski T. The isolation of Borrelia burgdorferi spirochetes from clinical material in cell line cultures. Zbl Bakt 1997; 286: 363–70
Postic D, Assous MV, Grimont PAD, et al. Diversity of Borrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf(5S)-rrl(23S) intergenic spacer amplicons. Int J Syst Bacteriol 1994; 44: 743–52
Fukunaga M, Koreki Y. A phylogenetic analysis of Borrelia burgdorferi sensu lato isolates associated with Lyme disease in Japan by flagellin gene sequence determination. Int J Syst Bacteriol 1996; 46: 416–21
Bruckbauer HR, Preac-Mursic V, Fuchs R, et al. Cross-reactive proteins of Borrelia burgdorferi. Eur J Clin Microbiol Infect Dis 1992; 11: 224–32
Marconi RT, Garon CF. Phylogenetic analysis of the genus Borrelia: a comparison of North American and European isolates of Borrelia burgdorferi. J Bacteriol 1992; 174: 241–4
Marconi RT, Garon CF. Identification of a third genomic group of Borrelia burgdorferi through signature nucleotide analysis and 16S rRNA sequence determination. J Gen Microbiol 1992; 138: 533–6
Marconi RT, Lubke L, Hauglum W, et al. Species-specific identification of and distinction between Borrelia burgdorferi genomic groups by using 16S rRNA-directed oligonucleotide probes. J Clin Microbiol 1992; 30: 628–32
Liebisch G, Sohns B, Bautsch W. Detection and typing of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks attached to human skin by PCR. J Clin Microbiol 1998; 36: 3355–8
Marconi RT, Garon CF. Development of polymerase chain reaction primer sets for diagnosis of Lyme disease and for species-specific identification of Lyme Disease isolates by 16S rRNA signature nucleotide analysis. J Clin Microbiol 1992; 30: 2830–4
Schwan TG, Burgdorfer W, Garon C. Changes in infectivity and plasmid profile of the Lyme disease spirochete, Borrelia burgdorferi, as a result of in vitro cultivation. Infect Immun 1988; 56: 1831–6
Wilske B, Preac-Mursic V, Gobel UB, et al. An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis. J Clin Microbiol 1993; 31: 340–50
Wallich R, Moter SE, Simon MM, et al. The Borrelia burgdorferi flagellum-associated 41-kilodalton antigen (flagellin): molecular cloning, expression, and amplification of the gene. Infect Immun 1990; 58: 1711–9
Amouriaux P, Assous M, Margarita D, et al. Polimerase chain reaction with the 30-kb circular plasmid of Borrelia burgdorferi B31 as a target for detection of the Lyme borreliosis agents in cerebrospinal fluid. Res Microbiol 1993; 144: 211–9
Schwaiger M, Peter O, Cassinotti P. Routine diagnosis of Borrelia burgdorferi (sensu lato) infections using a real-time PCR assay.Clin Microbiol Infect 2001; 7:461–9
Busch U, Hizo-Teufel C, Boehmer R, et al. Three species of Borrelia burgdorferi sensu lato (Borrelia burgdorferi sensu stricto, B. afzelii, B. garini) identified from cerebrospinal fluid isolates by Pulsed-Field Gel Electrophoresis and PCR. J Clin Microbiol 1996; 34: 1072–8
Wang G, van-Dam AP, Schwartz I, et al. Molecular typing of Borrelia burgdorferi sensu lato: taxonomic, epidemiological, and clinical implications. Clin Microbiol Rev 1999; 12: 633–53
Keller TL, Halperin JJ, Whitman M. PCR detection of Borrelia burgdorferi DNA in cerebrospinal fluid of Lyme neuroborreliosis patients. Neurology 1992; 42: 32–42
Zore A, Ruzic-Sabljic E, Maraspin V, et al. Sensitivity of culture and polymerase chain reaction for the etiologic diagnosis of erythema migrans. Wien Klin Wochenschr 2002; 114: 606–9
Dattwyler RJ, Volkman DJ, Luft BJ, et al. Seronegative Lyme disease. N Engl J Med 1988; 319: 1441–6
Brunner M, Sigal LH. Use of serum immune complexes in a new test that accurately confirms early Lyme disease and active infection with Borrelia burgdorferi. J Clin Microbiol 2001;39:3213–21
Schutzer SE, Coyle PK, Belman AL, et al. Sequestration of antibody to Borrelia burgdorferi in immune complexes in seronegative Lyme disease. Lancet 1990; 335: 312–5