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. 1992 Jul;30(7):1646–1653. doi: 10.1128/jcm.30.7.1646-1653.1992

Detection of Borrelia burgdorferi DNA in urine samples and cerebrospinal fluid samples from patients with early and late Lyme neuroborreliosis by polymerase chain reaction.

A M Lebech 1, K Hansen 1
PMCID: PMC265358  PMID: 1629318

Abstract

A polymerase chain reaction (PCR) was developed for use in the identification of a 248-bp fragment of the Borrelia burgdorferi flagellin gene in urine and cerebrospinal fluid (CSF) from patients with Lyme neuroborreliosis. The specificities of the PCR products were confirmed by DNA-DNA hybridization with an internal probe. The assay had a detection limit of 10 in vitro-cultivated B. burgdorferi. The PCR assay seemed to be species wide as well as species specific, since DNA from all 21 B. burgdorferi isolates from humans tested but not from Borrelia hermsii or Treponema pallidum could be amplified. We tested 10 consecutively diagnosed patients with untreated neuroborreliosis. There was lymphocytic pleocytosis and intrathecal B. burgdorferi-specific antibody synthesis in the CSF of all patients. Urine and CSF samples were investigated by PCR before, during, and up to 8.5 months after therapy. B. burgdorferi DNA was detected in urine samples from nine patients; five patients, including two patients with chronic neuroborreliosis, were PCR positive prior to treatment, whereas urine samples from the remaining four patients obtained 3 to 6 days after the onset of therapy became PCR positive. All urine samples obtained greater than 4 weeks after therapy were negative by PCR. PCR of CSF was less sensitive, and samples from only four patients, including one with chronic neuroborreliosis, were positive. We conclude that urine is a more suitable sample source than CSF for use in B. burgdorferi DNA detection by PCR. Normalization of inflammatory CSF changes and the negative PCR results during follow-up even in patients with chronic neuroborreliosis do not point to a persistent infection. The future role of PCR as a diagnostic tool for Lyme neuroborreliosis is still uncertain.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ackermann R., Gollmer E., Rehse-Küpper B. Progressive Borrelien-Enzephalomyelitis. Chronische Manifestation der Erythema-chronicum-migrans-Krankheit am Nervensystem. Dtsch Med Wochenschr. 1985 Jun 28;110(26):1039–1042. doi: 10.1055/s-2008-1068956. [DOI] [PubMed] [Google Scholar]
  2. Asbrink E., Hovmark A., Hederstedt B. The spirochetal etiology of acrodermatitis chronica atrophicans Herxheimer. Acta Derm Venereol. 1984;64(6):506–512. [PubMed] [Google Scholar]
  3. Aurelius E., Johansson B., Sköldenberg B., Staland A., Forsgren M. Rapid diagnosis of herpes simplex encephalitis by nested polymerase chain reaction assay of cerebrospinal fluid. Lancet. 1991 Jan 26;337(8735):189–192. doi: 10.1016/0140-6736(91)92155-u. [DOI] [PubMed] [Google Scholar]
  4. Barbour A. G. Isolation and cultivation of Lyme disease spirochetes. Yale J Biol Med. 1984 Jul-Aug;57(4):521–525. [PMC free article] [PubMed] [Google Scholar]
  5. Benach J. L., Bosler E. M., Hanrahan J. P., Coleman J. L., Habicht G. S., Bast T. F., Cameron D. J., Ziegler J. L., Barbour A. G., Burgdorfer W. Spirochetes isolated from the blood of two patients with Lyme disease. N Engl J Med. 1983 Mar 31;308(13):740–742. doi: 10.1056/NEJM198303313081302. [DOI] [PubMed] [Google Scholar]
  6. Bergström S., Bundoc V. G., Barbour A. G. Molecular analysis of linear plasmid-encoded major surface proteins, OspA and OspB, of the Lyme disease spirochaete Borrelia burgdorferi. Mol Microbiol. 1989 Apr;3(4):479–486. doi: 10.1111/j.1365-2958.1989.tb00194.x. [DOI] [PubMed] [Google Scholar]
  7. Burgdorfer W., Barbour A. G., Hayes S. F., Benach J. L., Grunwaldt E., Davis J. P. Lyme disease-a tick-borne spirochetosis? Science. 1982 Jun 18;216(4552):1317–1319. doi: 10.1126/science.7043737. [DOI] [PubMed] [Google Scholar]
  8. Burstain J. M., Grimprel E., Lukehart S. A., Norgard M. V., Radolf J. D. Sensitive detection of Treponema pallidum by using the polymerase chain reaction. J Clin Microbiol. 1991 Jan;29(1):62–69. doi: 10.1128/jcm.29.1.62-69.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Champion C. I., Miller J. N., Lovett M. A., Blanco D. R. Cloning, sequencing, and expression of two class B endoflagellar genes of Treponema pallidum subsp. pallidum encoding the 34.5- and 31.0-kilodalton proteins. Infect Immun. 1990 Jun;58(6):1697–1704. doi: 10.1128/iai.58.6.1697-1704.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Collins C., Peltz G. Immunoreactive epitopes on an expressed recombinant flagellar protein of Borrelia burgdorferi. Infect Immun. 1991 Feb;59(2):514–520. doi: 10.1128/iai.59.2.514-520.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Debue M., Gautier P., Hackel C., Van Elsen A., Herzog A., Bigaignon G., Bollen A. Detection of Borrelia burgdorferi in biological samples using the polymerase chain reaction assay. Res Microbiol. 1991 Jun;142(5):565–572. doi: 10.1016/0923-2508(91)90189-h. [DOI] [PubMed] [Google Scholar]
  12. Dorward D. W., Schwan T. G., Garon C. F. Immune capture and detection of Borrelia burgdorferi antigens in urine, blood, or tissues from infected ticks, mice, dogs, and humans. J Clin Microbiol. 1991 Jun;29(6):1162–1170. doi: 10.1128/jcm.29.6.1162-1170.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gassmann G. S., Kramer M., Göbel U. B., Wallich R. Nucleotide sequence of a gene encoding the Borrelia burgdorferi flagellin. Nucleic Acids Res. 1989 May 11;17(9):3590–3590. doi: 10.1093/nar/17.9.3590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goodman J. L., Jurkovich P., Kramber J. M., Johnson R. C. Molecular detection of persistent Borrelia burgdorferi in the urine of patients with active Lyme disease. Infect Immun. 1991 Jan;59(1):269–278. doi: 10.1128/iai.59.1.269-278.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grimprel E., Sanchez P. J., Wendel G. D., Burstain J. M., McCracken G. H., Jr, Radolf J. D., Norgard M. V. Use of polymerase chain reaction and rabbit infectivity testing to detect Treponema pallidum in amniotic fluid, fetal and neonatal sera, and cerebrospinal fluid. J Clin Microbiol. 1991 Aug;29(8):1711–1718. doi: 10.1128/jcm.29.8.1711-1718.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Guy E. C., Stanek G. Detection of Borrelia burgdorferi in patients with Lyme disease by the polymerase chain reaction. J Clin Pathol. 1991 Jul;44(7):610–611. doi: 10.1136/jcp.44.7.610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hansen K., Bangsborg J. M., Fjordvang H., Pedersen N. S., Hindersson P. Immunochemical characterization of and isolation of the gene for a Borrelia burgdorferi immunodominant 60-kilodalton antigen common to a wide range of bacteria. Infect Immun. 1988 Aug;56(8):2047–2053. doi: 10.1128/iai.56.8.2047-2053.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hansen K., Cruz M., Link H. Oligoclonal Borrelia burgdorferi-specific IgG antibodies in cerebrospinal fluid in Lyme neuroborreliosis. J Infect Dis. 1990 Jun;161(6):1194–1202. doi: 10.1093/infdis/161.6.1194. [DOI] [PubMed] [Google Scholar]
  19. Hansen K., Hindersson P., Pedersen N. S. Measurement of antibodies to the Borrelia burgdorferi flagellum improves serodiagnosis in Lyme disease. J Clin Microbiol. 1988 Feb;26(2):338–346. doi: 10.1128/jcm.26.2.338-346.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hansen K., Lebech A. M. Lyme neuroborreliosis: a new sensitive diagnostic assay for intrathecal synthesis of Borrelia burgdorferi--specific immunoglobulin G, A, and M. Ann Neurol. 1991 Aug;30(2):197–205. doi: 10.1002/ana.410300212. [DOI] [PubMed] [Google Scholar]
  21. Hansen K., Lebech A. M. The clinical and epidemiological profile of Lyme neuroborreliosis in Denmark 1985-1990. A prospective study of 187 patients with Borrelia burgdorferi specific intrathecal antibody production. Brain. 1992 Apr;115(Pt 2):399–423. doi: 10.1093/brain/115.2.399. [DOI] [PubMed] [Google Scholar]
  22. Hansen K., Pii K., Lebech A. M. Improved immunoglobulin M serodiagnosis in Lyme borreliosis by using a mu-capture enzyme-linked immunosorbent assay with biotinylated Borrelia burgdorferi flagella. J Clin Microbiol. 1991 Jan;29(1):166–173. doi: 10.1128/jcm.29.1.166-173.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hay P. E., Clarke J. R., Strugnell R. A., Taylor-Robinson D., Goldmeier D. Use of the polymerase chain reaction to detect DNA sequences specific to pathogenic treponemes in cerebrospinal fluid. FEMS Microbiol Lett. 1990 Mar 15;56(3):233–238. doi: 10.1111/j.1574-6968.1990.tb13943.x. [DOI] [PubMed] [Google Scholar]
  24. Hindersson P., Knudsen J. D., Axelsen N. H. Cloning and expression of treponema pallidum common antigen (Tp-4) in Escherichia coli K12. J Gen Microbiol. 1987 Mar;133(3):587–596. doi: 10.1099/00221287-133-3-587. [DOI] [PubMed] [Google Scholar]
  25. Hyde F. W., Johnson R. C., White T. J., Shelburne C. E. Detection of antigens in urine of mice and humans infected with Borrelia burgdorferi, etiologic agent of Lyme disease. J Clin Microbiol. 1989 Jan;27(1):58–61. doi: 10.1128/jcm.27.1.58-61.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kariya Y., Kato K., Hayashizaki Y., Himeno S., Tarui S., Matsubara K. Expression of human gastrin gene in normal and gastrinoma tissues. Gene. 1986;50(1-3):345–352. doi: 10.1016/0378-1119(86)90338-0. [DOI] [PubMed] [Google Scholar]
  27. Karlsson M., Hovind-Hougen K., Svenungsson B., Stiernstedt G. Cultivation and characterization of spirochetes from cerebrospinal fluid of patients with Lyme borreliosis. J Clin Microbiol. 1990 Mar;28(3):473–479. doi: 10.1128/jcm.28.3.473-479.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kristoferitsch W. Neurological manifestations of Lyme borreliosis: clinical definition and differential diagnosis. Scand J Infect Dis Suppl. 1991;77:64–73. [PubMed] [Google Scholar]
  29. Krüger H., Reuss K., Pulz M., Rohrbach E., Pflughaupt K. W., Martin R., Mertens H. G. Meningoradiculitis and encephalomyelitis due to Borrelia burgdorferi: a follow-up study of 72 patients over 27 years. J Neurol. 1989 Sep;236(6):322–328. doi: 10.1007/BF00314373. [DOI] [PubMed] [Google Scholar]
  30. Krüger W. H., Pulz M. Detection of Borrelia burgdorferi in cerebrospinal fluid by the polymerase chain reaction. J Med Microbiol. 1991 Aug;35(2):98–102. doi: 10.1099/00222615-35-2-98. [DOI] [PubMed] [Google Scholar]
  31. Lebech A. M., Hindersson P., Vuust J., Hansen K. Comparison of in vitro culture and polymerase chain reaction for detection of Borrelia burgdorferi in tissue from experimentally infected animals. J Clin Microbiol. 1991 Apr;29(4):731–737. doi: 10.1128/jcm.29.4.731-737.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Malloy D. C., Nauman R. K., Paxton H. Detection of Borrelia burgdorferi using the polymerase chain reaction. J Clin Microbiol. 1990 Jun;28(6):1089–1093. doi: 10.1128/jcm.28.6.1089-1093.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Martin R., Ortlauf J., Sticht-Groh V., Bogdahn U., Goldmann S. F., Mertens H. G. Borrelia burgdorferi--specific and autoreactive T-cell lines from cerebrospinal fluid in Lyme radiculomyelitis. Ann Neurol. 1988 Oct;24(4):509–516. doi: 10.1002/ana.410240406. [DOI] [PubMed] [Google Scholar]
  34. Melchers W., Meis J., Rosa P., Claas E., Nohlmans L., Koopman R., Horrevorts A., Galama J. Amplification of Borrelia burgdorferi DNA in skin biopsies from patients with Lyme disease. J Clin Microbiol. 1991 Nov;29(11):2401–2406. doi: 10.1128/jcm.29.11.2401-2406.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nielsen S. L., Young K. K., Barbour A. G. Detection of Borrelia burgdorferi DNA by the polymerase chain reaction. Mol Cell Probes. 1990 Feb;4(1):73–79. doi: 10.1016/0890-8508(90)90041-w. [DOI] [PubMed] [Google Scholar]
  36. Noordhoek G. T., Wolters E. C., de Jonge M. E., van Embden J. D. Detection by polymerase chain reaction of Treponema pallidum DNA in cerebrospinal fluid from neurosyphilis patients before and after antibiotic treatment. J Clin Microbiol. 1991 Sep;29(9):1976–1984. doi: 10.1128/jcm.29.9.1976-1984.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pallesen L., Hindersson P. Cloning and sequencing of a Treponema pallidum gene encoding a 31.3-kilodalton endoflagellar subunit (FlaB2). Infect Immun. 1989 Jul;57(7):2166–2172. doi: 10.1128/iai.57.7.2166-2172.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pedersen N. S., Axelsen N. H., Petersen C. S. Antigenic analysis of Treponema pallidum: cross-reactions between individual antigens of T. pallidum and T. Reiter. Scand J Immunol. 1981;13(2):143–150. doi: 10.1111/j.1365-3083.1981.tb00120.x. [DOI] [PubMed] [Google Scholar]
  39. Persing D. H., Telford S. R., 3rd, Spielman A., Barthold S. W. Detection of Borrelia burgdorferi infection in Ixodes dammini ticks with the polymerase chain reaction. J Clin Microbiol. 1990 Mar;28(3):566–572. doi: 10.1128/jcm.28.3.566-572.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pfister H. W., Einhäupl K., Preac-Mursic V., Wilske B., Schierz G. The spirochetal etiology of lymphocytic meningoradiculitis of Bannwarth (Bannwarth's syndrome). J Neurol. 1984;231(3):141–144. doi: 10.1007/BF00313682. [DOI] [PubMed] [Google Scholar]
  41. Picken R. N. Polymerase chain reaction primers and probes derived from flagellin gene sequences for specific detection of the agents of Lyme disease and North American relapsing fever. J Clin Microbiol. 1992 Jan;30(1):99–114. doi: 10.1128/jcm.30.1.99-114.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rosa P. A., Schwan T. G. A specific and sensitive assay for the Lyme disease spirochete Borrelia burgdorferi using the polymerase chain reaction. J Infect Dis. 1989 Dec;160(6):1018–1029. doi: 10.1093/infdis/160.6.1018. [DOI] [PubMed] [Google Scholar]
  43. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  44. Schluesener H. J., Martin R., Sticht-Groh V. Autoimmunity in Lyme disease: molecular cloning of antigens recognized by antibodies in the cerebrospinal fluid. Autoimmunity. 1989;2(4):323–330. doi: 10.3109/08916938908997158. [DOI] [PubMed] [Google Scholar]
  45. Steere A. C., Grodzicki R. L., Kornblatt A. N., Craft J. E., Barbour A. G., Burgdorfer W., Schmid G. P., Johnson E., Malawista S. E. The spirochetal etiology of Lyme disease. N Engl J Med. 1983 Mar 31;308(13):733–740. doi: 10.1056/NEJM198303313081301. [DOI] [PubMed] [Google Scholar]
  46. Stiernstedt G. T., Granström M., Hederstedt B., Sköldenberg B. Diagnosis of spirochetal meningitis by enzyme-linked immunosorbent assay and indirect immunofluorescence assay in serum and cerebrospinal fluid. J Clin Microbiol. 1985 May;21(5):819–825. doi: 10.1128/jcm.21.5.819-825.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wallich R., Moter S. E., Simon M. M., Ebnet K., Heiberger A., Kramer M. D. The Borrelia burgdorferi flagellum-associated 41-kilodalton antigen (flagellin): molecular cloning, expression, and amplification of the gene. Infect Immun. 1990 Jun;58(6):1711–1719. doi: 10.1128/iai.58.6.1711-1719.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wilske B., Anderson J. F., Baranton G., Barbour A. G., Hovind-Hougen K., Johnson R. C., Preac-Mursic V. Taxonomy of Borrelia spp. Scand J Infect Dis Suppl. 1991;77:108–129. [PubMed] [Google Scholar]

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