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. 1997 Nov;35(11):2802–2806. doi: 10.1128/jcm.35.11.2802-2806.1997

Detection of rifampin resistance by single-strand conformation polymorphism analysis of cerebrospinal fluid of patients with tuberculosis of the central nervous system.

P Scarpellini 1, S Braglia 1, A M Brambilla 1, M Dalessandro 1, P Cichero 1, A Gori 1, A Lazzarin 1
PMCID: PMC230065  PMID: 9350737

Abstract

Mutations in a 69-bp region of the rpoB gene of Mycobacterium tuberculosis are associated with rifampin resistance (Rif[r]). These have been detected with mycobacterial DNA extracted from bacterial suspensions or respiratory specimens that were acid-fast smear positive. We experimented with a strategy for the rapid detection of Rif(r) in cerebrospinal fluid (CSF) samples. The strategy involves the amplification of the 69-bp region of rpoB by means of PCR and the identification of nucleotide mutations by single-strand conformation polymorphism (SSCP) analysis of the amplification products. Sixty-five CSF specimens collected from 29 patients (19 patients were coinfected with human immunodeficiency virus) with culture or autopsy-confirmed (22 patients) or highly probable (7 patients) tuberculosis of the central nervous system (CNS-TB) were processed. Amplified products suitable for evaluation by SSCP analysis were obtained from 37 CSF specimens from 25 subjects (86.2%). PCR-SSCP of CSF correctly identified the rifampin susceptibility phenotype of isolates from all 17 patients for whom the results of susceptibility tests carried out with strains cultured from CSF or respiratory samples were available. Moreover, this assay revealed the rifampin susceptibility genotype of isolates from the eight patients (three patients with culture-confirmed CNS-TB and five patients in whom CNS-TB was highly probable) for whom no susceptibility test results were available; the PCR-SSCP data obtained for these patients were concordant with the outcome after a standard antituberculosis treatment. The evolution of a mutation in the rpoB gene was documented in a patient during the course of treatment. PCR-SSCP analysis of CSF seems to be an efficacious method of predicting Rif(r) and would reduce the time required for susceptibility testing from approximately 4 to 8 weeks to a few days.

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Selected References

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  1. Birch D. E. Simplified hot start PCR. Nature. 1996 May 30;381(6581):445–446. doi: 10.1038/381445a0. [DOI] [PubMed] [Google Scholar]
  2. Bodmer T., Zürcher G., Imboden P., Telenti A. Mutation position and type of substitution in the beta-subunit of the RNA polymerase influence in-vitro activity of rifamycins in rifampicin-resistant Mycobacterium tuberculosis. J Antimicrob Chemother. 1995 Feb;35(2):345–348. doi: 10.1093/jac/35.2.345. [DOI] [PubMed] [Google Scholar]
  3. Boom R., Sol C. J., Salimans M. M., Jansen C. L., Wertheim-van Dillen P. M., van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990 Mar;28(3):495–503. doi: 10.1128/jcm.28.3.495-503.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caugant D. A., Sandven P., Eng J., Jeque J. T., Tønjum T. Detection of rifampin resistance among isolates of Mycobacterium tuberculosis from Mozambique. Microb Drug Resist. 1995 Winter;1(4):321–326. doi: 10.1089/mdr.1995.1.321. [DOI] [PubMed] [Google Scholar]
  5. Dolin P. J., Raviglione M. C., Kochi A. Global tuberculosis incidence and mortality during 1990-2000. Bull World Health Organ. 1994;72(2):213–220. [PMC free article] [PubMed] [Google Scholar]
  6. Felmlee T. A., Liu Q., Whelen A. C., Williams D., Sommer S. S., Persing D. H. Genotypic detection of Mycobacterium tuberculosis rifampin resistance: comparison of single-strand conformation polymorphism and dideoxy fingerprinting. J Clin Microbiol. 1995 Jun;33(6):1617–1623. doi: 10.1128/jcm.33.6.1617-1623.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Honore N., Cole S. T. Molecular basis of rifampin resistance in Mycobacterium leprae. Antimicrob Agents Chemother. 1993 Mar;37(3):414–418. doi: 10.1128/aac.37.3.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hunt J. M., Roberts G. D., Stockman L., Felmlee T. A., Persing D. H. Detection of a genetic locus encoding resistance to rifampin in mycobacterial cultures and in clinical specimens. Diagn Microbiol Infect Dis. 1994 Apr;18(4):219–227. doi: 10.1016/0732-8893(94)90024-8. [DOI] [PubMed] [Google Scholar]
  9. Jin D. J., Gross C. A. Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance. J Mol Biol. 1988 Jul 5;202(1):45–58. doi: 10.1016/0022-2836(88)90517-7. [DOI] [PubMed] [Google Scholar]
  10. Kapur V., Li L. L., Iordanescu S., Hamrick M. R., Wanger A., Kreiswirth B. N., Musser J. M. Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase beta subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas. J Clin Microbiol. 1994 Apr;32(4):1095–1098. doi: 10.1128/jcm.32.4.1095-1098.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kennedy D. H., Fallon R. J. Tuberculous meningitis. JAMA. 1979 Jan 19;241(3):264–268. [PubMed] [Google Scholar]
  12. Kochi A. Global challenge of tuberculosis. Lancet. 1994 Aug 27;344(8922):608–609. [PubMed] [Google Scholar]
  13. Kolk A. H., Schuitema A. R., Kuijper S., van Leeuwen J., Hermans P. W., van Embden J. D., Hartskeerl R. A. Detection of Mycobacterium tuberculosis in clinical samples by using polymerase chain reaction and a nonradioactive detection system. J Clin Microbiol. 1992 Oct;30(10):2567–2575. doi: 10.1128/jcm.30.10.2567-2575.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kwok S., Higuchi R. Avoiding false positives with PCR. Nature. 1989 May 18;339(6221):237–238. doi: 10.1038/339237a0. [DOI] [PubMed] [Google Scholar]
  15. Leonard J. M., Des Prez R. M. Tuberculous meningitis. Infect Dis Clin North Am. 1990 Dec;4(4):769–787. [PubMed] [Google Scholar]
  16. Scarpellini P., Racca S., Cinque P., Delfanti F., Gianotti N., Terreni M. R., Vago L., Lazzarin A. Nested polymerase chain reaction for diagnosis and monitoring treatment response in AIDS patients with tuberculous meningitis. AIDS. 1995 Aug;9(8):895–900. doi: 10.1097/00002030-199508000-00010. [DOI] [PubMed] [Google Scholar]
  17. Telenti A., Imboden P., Marchesi F., Lowrie D., Cole S., Colston M. J., Matter L., Schopfer K., Bodmer T. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet. 1993 Mar 13;341(8846):647–650. doi: 10.1016/0140-6736(93)90417-f. [DOI] [PubMed] [Google Scholar]
  18. Telenti A., Imboden P., Marchesi F., Schmidheini T., Bodmer T. Direct, automated detection of rifampin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis. Antimicrob Agents Chemother. 1993 Oct;37(10):2054–2058. doi: 10.1128/aac.37.10.2054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Whelen A. C., Felmlee T. A., Hunt J. M., Williams D. L., Roberts G. D., Stockman L., Persing D. H. Direct genotypic detection of Mycobacterium tuberculosis rifampin resistance in clinical specimens by using single-tube heminested PCR. J Clin Microbiol. 1995 Mar;33(3):556–561. doi: 10.1128/jcm.33.3.556-561.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Williams D. L., Waguespack C., Eisenach K., Crawford J. T., Portaels F., Salfinger M., Nolan C. M., Abe C., Sticht-Groh V., Gillis T. P. Characterization of rifampin-resistance in pathogenic mycobacteria. Antimicrob Agents Chemother. 1994 Oct;38(10):2380–2386. doi: 10.1128/aac.38.10.2380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. van Embden J. D., Cave M. D., Crawford J. T., Dale J. W., Eisenach K. D., Gicquel B., Hermans P., Martin C., McAdam R., Shinnick T. M. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol. 1993 Feb;31(2):406–409. doi: 10.1128/jcm.31.2.406-409.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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