Advertisement

Infection

, Volume 23, Issue 3, pp 173–179 | Cite as

Evaluation of the detection ofBorrelia burgdorferi DNA in urine samples by polymerase chain reaction

  • M. Maiwald
  • C. Stockinger
  • H. -G. Sonntag
  • D. Hassler
  • M. von Knebel Doeberitz
Originalia

Summary

It is difficult in some cases to identify an infection caused byBorrelia burgdorferi and to monitor the effect of therapy. Seropositivity will persist even after successful treatment and therefore may suggest ongoing infection. For direct detection ofB. burgdorferi DNA in human urine samples, the polymerase chain reaction (PCR) was evaluated. A published primer system was selected, which amplifies a 259 bp fragment from the gene encoding the 23S rRNA. The lower detection limit of the primer system was 10 fg of extractedB. burgdorferi DNA. Several methods for the pretreatment of urine sample were tested. Of these, the Geneclean® kit (Bio 101, USA) showed the best results. A total of 114 urine samples from 74 patients belonging to three clinical groups was investigated: (i) 51 samples from 26 patients with active Lyme disease, (ii) 36 samples from 27 patients with previous infection but no symptoms at the time the urine was collected, and (iii) 27 samples from 21 seronegative control patients without Lyme disease.B. burgdorferi DNA was detected in 25 urine samples of 17 patients with active disease, whereas 26 samples from this group of patients were negative. Only one asymptomatic case with previous infection showed a positive result, and the urine samples of the patients without Lyme disease were uniformly negative. Two of four patients from whom samples before and directly after onset of therapy were available converted from negative to positive PCR results after initiation of therapy, accompanied by the symptoms of a Jarisch-Herxheimer reaction. It can be concluded from these results that a positive PCR from urine is with high probability an indicator of active Lyme disease. On the other hand, as only 17 of the 26 patients with active infection were positive, a negative PCR result does not exclude active infection.

Keywords

Polymerase Chain Reaction Urine Sample Lyme Disease Active Infection Prime System 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Evaluation des Nachweises vonBorrelia burgdorferi-DNA in Urinproben mittels Polymerase-Kettenreaktion

Zusammenfassung

In manchen Fällen ist es schwierig, Infektionen mitBorrelia burgdorferi zu diagnostizieren und den Effekt einer Therapie abzuschätzen. Seropositivität bleibt selbst nach erfolgreicher Therapie bestehen und kann deshalb ein weiterbestehendes Infektionsgeschehen vortäuschen. Zum Direktnachweis vonB. burgdorferi-DNA in menschlichen Urinproben wurde das Verfahren der Polymerase-Kettenreaktion (PCR) evaluiert. Zur Anwendung kam ein Primersystem, dessen Zielsequenz auf dem Gen für die 23S rRNA liegt (Schwartz et al., J. Clin. Microbiol. 30: 3082–3088; 1992). Die Nachweisgrenze des Primersystems betrug 10 fg an extrahierter DNA vonB. burgdorferi. Zur Probenvorbereitung von Urinproben wurden mehrere Methoden getestet. Davon erwies sich das Geneclean® Kit (Fa. Bio 101) als am besten geeignet. Damit wurden insgesamt 114 Urinproben von Patienten aus drei klinischen Gruppen untersucht: (i) 51 Proben von 26 Patienten mit aktiver Lyme-Borreliose, (ii) 36 Proben von 27 symptomlosen Patienten mit vorangegangener Infektion, und (iii) 27 Proben von 21 seronegativen Kontrollpatienten ohne Lyme-Borreliose. Mit Hilfe der PCR bei Probenaufbereitung mit Geneclean® gelang ein Nachweis von Borrelien-DNA in 25 Proben von 17 Patienten mit aktiver Erkrankung, während 26 Proben aus dieser Gruppe negativ waren. Ein asymptomatischer Fall zeigte ein positives Resultat, und die Proben der Patienten ohne Lyme-Borreliose waren einheitlich negativ. Zwei von vier Patienten, von denen Urinproben vor und direkt nach Therapie verfügbar waren, zeigten eine Konversion von negativen zu positiven PCR-Resultaten nach Therapiebeginn, was auf einen Erregerzerfall und vermehrte Ausscheidung von Borrelien-DNA schließen läßt. Die Positivität unter Therapie war korreliert mit dem klinischen Auftreten einer Jarisch-Herxheimer-Reaktion. Die vorliegenden Ergebnisse lassen schließen, daß zwar ein negatives PCR-Ergebnis nicht gegen Krankheitsaktivität spricht, da nur 17 der 26 Patienten mit aktiver Erkrankung positive Resultate zeigten, jedoch daß ein positives PCR-Resultat als Hinweis auf eine aktive Infektion gewertet werden kann.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Burgdorfer, W., Barbour, A. G., Hayes, S. F., Benach, J. L., Grunwaldt, E., Davis, J. P. Lyme disease — a tick-borne spirochetosis? Science 216 (1982) 1317–1319.Google Scholar
  2. 2.
    Steere, A. C. Lyme disease. N. Engl. J. Med. 321 (1989) 586–596.Google Scholar
  3. 3.
    Stiernstedt, G., Dattwyler, R., Duray, P. H., Hansen, K., Jirous, J., Johnson, R. C., Karlsson, M., Preac-Mursic, V., Schwan, T. G. Diagnostic tests in Lyme borreliosis. Scand. J. Infect. Dis. (Suppl. 77) (1991) 136–142.Google Scholar
  4. 4.
    Wilske, B., Schierz, G., Preac-Mursic, V., Weber, K., Pfister, H. W., Einhäupl, K. Serological diagnosis of erythema migrans disease and related disorders. Infection 12 (1984) 331–337.Google Scholar
  5. 5.
    Persing, D. H., Telford, S. R., Rys, P. N., Dodge, D. E., White, T. J., Malawista, S. E., Spielman, A. Detection ofBorrelia burgdorferi DNA in museum specimens ofIxodes dammini ticks. Science 249 (1990) 1420–1423.Google Scholar
  6. 6.
    Hofmeister, E. K., Markham, R. B., Childs, J. E., Arthur, R. R. Comparison of polymerase chain reaction and culture for detection ofBorrelia burgdorferi in naturally infectedPeromyscus leucopus and experimentally infected C.B-17 scid/scid mice. J. Clin. Microbiol. 30 (1992) 2625–2631.Google Scholar
  7. 7.
    Jaulhac, B., Nicolini, P., Piemont, Y., Monteil, H. Detection ofBorrelia burgdorferi in cerebrospinal fluid of patients with Lyme borreliosis. N. Engl. J. Med. 324 (1991) 1440.Google Scholar
  8. 8.
    Keller, T. L., Halperin, J. J., Whitman, M. PCR detection ofBorrelia burgdorferi DNA in cerebrospinal fluid of Lyme neuroborreliosis patients. Neurology 42 (1992) 32–42.Google Scholar
  9. 9.
    Dodge, D. E., Nersesian, R., Sun, R. Diagnosis of the Lyme disease spirocheteBorrelia burgdorferi. Clinical Immunology Newsletter 12 (1992) 69–73.Google Scholar
  10. 10.
    Goodman, J. L., Jurkovich, P., Kramber, J. M., Johnson, R. C. Molecular detection of persistentBorrelia burgdorferi in the urine of patients with active Lyme disease. Infect. Immun. 59 (1991) 269–278.Google Scholar
  11. 11.
    Lebech, A.-M., Hansen, K. Detection ofBorrelia burgdorferi DNA in urine samples and cerebrospinal fluid samples from patients with early and late Lyme neuroborreliosis by polymerase chain reaction. J. Clin. Microbiol. 30 (1992) 1646–1653.Google Scholar
  12. 12.
    Melchers, W., Meis, J., Rosa, P., Claas, E., Nohlmans, L., Koopman, R., Horrevorts, A., Galama, J. Amplification ofBorrelia burgdorferi DNA in skin biopsies from patients with Lyme disease. J. Clin. Microbiol. 29 (1991) 2401–2406.Google Scholar
  13. 13.
    Schwartz, I., Wormser, G. P., Schwartz, J. J., Cooper, D., Weissensee, P., Gazumyan, A., Zimmermann, E., Goldberg, N. S., Bittker, S., Campbell, G. L., Pavia, C. S. Diagnosis of early Lyme disease by polymerase chain reaction amplification and culture of skin biopsies from erythema migrans lesions. J. Clin. Microbiol. 30 (1992) 3082–3088.Google Scholar
  14. 14.
    Schwartz, J. J., Gazumyan, A., Schwartz, I. rRNA gene organization in the Lyme disease spirochete,Borrelia burgdorferi. J. Bacteriol. 174 (1992) 3757–3765.Google Scholar
  15. 15.
    Wilske, B., Preac-Mursic, V., Schierz, G., von Busch, K. Immunochemical and immunological analysis of EuropeanBorrelia burgdorferi strains. Zbl. Bakt. Hyg. A 263 (1986) 92–102.Google Scholar
  16. 16.
    Barbour, A. Isolation and cultivation of Lyme disease spirochetes. Yale J. Biol. Med. 57 (1984) 521–525.Google Scholar
  17. 17.
    Wilson, K. Preparation of genomic DNA from bacteria. In:Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. (eds.): Current protocols in molecular biology. Greene Publishing and Wiley Interscience, New York 1987, pp. 2.4.1–2.4.5.Google Scholar
  18. 18.
    Sambrook, J., Fritsch, E. F., Maniatis, T. Molecular cloning: a laboratory manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. 1989.Google Scholar
  19. 19.
    Gerritsen, M. J., Olyhoek, T., Smits, M. A., Bokhout, B. A. Sample preparation method for polymerase chain reaction-based semiquantitative detection ofLeptospira interrogans serovar Hardjo subtype Hardjobovis in bovine urine. J. Clin. Microbiol. 29 (1991) 2805–2808.Google Scholar
  20. 20.
    Maiwald, M., Ditton, H.-J., Sonntag, H.-G., von Knebel Doeberitz, M. Characterization of contaminating DNA inTaq polymerase which occurs during amplification with a primer set forLegionella 5S ribosomal RNA. Mol. Cell. Probes 8 (1994) 11–14.Google Scholar
  21. 21.
    Hassler, D., Zöller, L., Haude, M., Hufnagel, H. D., Sonntag, H.-G. Lyme-Borreliose in einem europäischen Endemiegebiet. Antikörperprävalenz und klinisches Spektrum. Dtsch. Med. Wochenschr. 117 (1992) 767–774.Google Scholar
  22. 22.
    Schutzer, S. E. Diagnosing Lyme disease. Am. Fam. Physician 45 (1992) 2151–2156.Google Scholar
  23. 23.
    Zöller, L., Burkard, S., Schäfer, H. Validity of Western immunoblot band patterns in the serodiagnosis of Lyme borreliosis. J. Clin. Microbiol. 29 (1991) 174–182.Google Scholar
  24. 24.
    Pachner, A. R., Ricalton, N., Delaney, E. Comparison of polymerase chain reaction with culture and serology for diagnosis of murine experimental Lyme borreliosis. J. Clin. Microbiol. 31 (1993) 208–214.Google Scholar
  25. 25.
    Hyde, F. W., Johnson, R. C., White, T. J., Shelburne, C. E. Detection of antigens in urine of mice and humans infected withBorrelia burgdorferi, etiologic agent of Lyme disease. J. Clin. Microbiol. 27 (1989) 58–61.Google Scholar
  26. 26.
    McGuire, B. S., Chandler, F. W., Felz, M. W., Huey, L. O., Field, R. S. Detection ofBorrelia burgdorferi in human blood and urine using the polymerase chain reaction. Pathobiology 60 (1992) 163–167.Google Scholar
  27. 27.
    Huppertz, H.-I., Schmidt, H., Karch, H. Detection ofBorrelia burgdorferi by nested polymerase chain reaction in cerebrospinal fluid and urine of children with neuroborreliosis. Eur. J. Pediatr. 152 (1993) 414–417.Google Scholar
  28. 28.
    Liebling, M. R., Nishio, M. J., Rodriguez, A., Sigal, L. H., Jin, T., Louie, J. S. The polymerase chain reaction for the detection ofBorrelia burgdorferi in human body fluids. Arthritis Rheum. 36 (1993) 665–675.Google Scholar
  29. 29.
    Khan, G., Kangro, H. O., Coates, P. J., Heath, R. B. Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA. J. Clin. Pathol. 44 (1991) 360–365.Google Scholar
  30. 30.
    Schwan, T. G., Burgdorfer, W., Schrumpf, M. E., Karstens, R. H. The urinary bladder, a consistent source ofBorrelia burgdorferi in experimentally infected white-footed mice (Peromyscus leucopus). J. Clin. Microbiol. 26 (1988) 893–895.Google Scholar
  31. 31.
    Maiwald, M., Schill, M., Stockinger, C., Helbig, J. H., Lück, P. C., Witzleb, W., Sonntag, H.-G. Detection ofLegionella DNA in human and guinea pig urine samples by the polymerase chain reaction. Eur. J. Clin. Microbiol. Infect. Dis. 14 (1995) 25–33.Google Scholar
  32. 32.
    Gretch, D. R., Wilson, J. J., Carrithers, R. L., dela Rosa, C., Han, J. H., Corey, L. Detection of hepatitis C virus DNA: Comparison of one-stage polymerase chain reaction (PCR) with nested-set PCR. J. Clin. Microbiol. 31 (1993) 289–291.Google Scholar
  33. 33.
    Karch, H., Huppertz, H.-L., Böhme, M., Schmidt, H., Wiebecke, D., Schwarzkopf, A. Demonstration ofBorrelia burgdorferi DNA in urine samples from healthy humans whose sera containB. burgdorferi specific antibodies. J. Clin. Microbiol. 32 (1994) 2312–2314.Google Scholar
  34. 34.
    Sheppard, H. W., Ascher, M. S., Busch, M. P., Sohmer, P. R., Stanley, M., Luce, M. C., Chimera, J. A., Madei, R., Rodgers, G. C., Lynch, C., Khayam-Bashi, H., Murphy, E. L., Eble, B., Bradford, W. Z., Royce, R. A., Winkelstein, W. A multicenter proficiency trial of gene amplification (PCR) for the detection of HIV-1. J. Acquir. Immune Defic. Syndr. 4 (1991) 277–283.Google Scholar

Copyright information

© MMV Medizin Verlag GmbH 1995

Authors and Affiliations

  • M. Maiwald
    • 1
  • C. Stockinger
    • 1
  • H. -G. Sonntag
    • 1
  • D. Hassler
    • 2
  • M. von Knebel Doeberitz
    • 3
  1. 1.Hygiene Institut der UniversitätHeidelberg
  2. 2.AllgemeinarztpraxisKraichtal
  3. 3.Projektgruppe Angewandte TumorvirologieDeutsches KrebsforschungszentrumHeidelbergGermany

Personalised recommendations