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. 1991 Apr;29(4):712–717. doi: 10.1128/jcm.29.4.712-717.1991

Use of a reamplification protocol improves sensitivity of detection of Mycobacterium tuberculosis in clinical samples by amplification of DNA.

C Pierre 1, D Lecossier 1, Y Boussougant 1, D Bocart 1, V Joly 1, P Yeni 1, A J Hance 1
PMCID: PMC269858  PMID: 1909710

Abstract

We have compared the sensitivity and specificity of quantitative mycobacterial culture against results obtained by using the polymerase chain reaction for the detection of DNA from organisms of the Mycobacterium tuberculosis complex in 82 clinical specimens from patients suspected of having tuberculosis. Two amplification protocols were used, a standard amplification protocol, which amplifies a segment of the gene coding for the 65-kDa antigen, and a protocol in which the initial amplification products are reamplified with a second set of nested oligonucleotide primers. Although the standard amplification protocol gave positive results for 18 of 18 samples which grew greater than 100 CFU/ml and gave positive results in 4 of 35 specimens from patients with tuberculosis which were negative by culture, only 1 of 6 samples which grew less than 100 CFU/ml was positive. This lack of sensitivity could not be explained by the presence of inhibitors of Taq polymerase present in the original samples. In contrast, the reamplification protocol gave positive results for 24 of 24 samples which were positive by culture as well as for 13 of 35 samples from patients with tuberculosis which were negative by culture (overall sensitivity, 63%, P less than 0.02, compared with the standard amplification protocol and routine culture). Two of 23 samples from patients not diagnosed as having tuberculosis gave positive results when the standard amplification protocol was used, but no additional false-positive results were seen with the reamplification protocol (overall specificity, 91%). We conclude that the use of a reamplification protocol improves the sensitivity of detection of mycobacterial DNA in clinical samples without sacrificing specificity. The sensitivity of this approach appears to be superior to that of standard culture techniques.

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Selected References

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  1. Bates J. H. Diagnosis of tuberculosis. Chest. 1979 Dec;76(6 Suppl):757–763. doi: 10.1378/chest.76.6_supplement.757. [DOI] [PubMed] [Google Scholar]
  2. Blair E. B., Brown G. L., Tull A. H. Computer files and analyses of laboratory data from tuberculosis patients. II. Analyses of six years' data on sputum specimens. Am Rev Respir Dis. 1976 Apr;113(4):427–432. doi: 10.1164/arrd.1976.113.4.427. [DOI] [PubMed] [Google Scholar]
  3. Brisson-Noël A., Gicquel B., Lecossier D., Lévy-Frébault V., Nassif X., Hance A. J. Rapid diagnosis of tuberculosis by amplification of mycobacterial DNA in clinical samples. Lancet. 1989 Nov 4;2(8671):1069–1071. doi: 10.1016/s0140-6736(89)91082-9. [DOI] [PubMed] [Google Scholar]
  4. Böddinghaus B., Rogall T., Flohr T., Blöcker H., Böttger E. C. Detection and identification of mycobacteria by amplification of rRNA. J Clin Microbiol. 1990 Aug;28(8):1751–1759. doi: 10.1128/jcm.28.8.1751-1759.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. De Wit D., Steyn L., Shoemaker S., Sogin M. Direct detection of Mycobacterium tuberculosis in clinical specimens by DNA amplification. J Clin Microbiol. 1990 Nov;28(11):2437–2441. doi: 10.1128/jcm.28.11.2437-2441.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eisenach K. D., Cave M. D., Bates J. H., Crawford J. T. Polymerase chain reaction amplification of a repetitive DNA sequence specific for Mycobacterium tuberculosis. J Infect Dis. 1990 May;161(5):977–981. doi: 10.1093/infdis/161.5.977. [DOI] [PubMed] [Google Scholar]
  7. Engelke D. R., Hoener P. A., Collins F. S. Direct sequencing of enzymatically amplified human genomic DNA. Proc Natl Acad Sci U S A. 1988 Jan;85(2):544–548. doi: 10.1073/pnas.85.2.544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hance A. J., Grandchamp B., Lévy-Frébault V., Lecossier D., Rauzier J., Bocart D., Gicquel B. Detection and identification of mycobacteria by amplification of mycobacterial DNA. Mol Microbiol. 1989 Jul;3(7):843–849. doi: 10.1111/j.1365-2958.1989.tb00233.x. [DOI] [PubMed] [Google Scholar]
  9. Haqqi T. M., Sarkar G., David C. S., Sommer S. S. Specific amplification with PCR of a refractory segment of genomic DNA. Nucleic Acids Res. 1988 Dec 23;16(24):11844–11844. doi: 10.1093/nar/16.24.11844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hermans P. W., van Soolingen D., Dale J. W., Schuitema A. R., McAdam R. A., Catty D., van Embden J. D. Insertion element IS986 from Mycobacterium tuberculosis: a useful tool for diagnosis and epidemiology of tuberculosis. J Clin Microbiol. 1990 Sep;28(9):2051–2058. doi: 10.1128/jcm.28.9.2051-2058.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kogan S. C., Doherty M., Gitschier J. An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences. Application to hemophilia A. N Engl J Med. 1987 Oct 15;317(16):985–990. doi: 10.1056/NEJM198710153171603. [DOI] [PubMed] [Google Scholar]
  12. Pao C. C., Yen T. S., You J. B., Maa J. S., Fiss E. H., Chang C. H. Detection and identification of Mycobacterium tuberculosis by DNA amplification. J Clin Microbiol. 1990 Sep;28(9):1877–1880. doi: 10.1128/jcm.28.9.1877-1880.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Patel R. J., Fries J. W., Piessens W. F., Wirth D. F. Sequence analysis and amplification by polymerase chain reaction of a cloned DNA fragment for identification of Mycobacterium tuberculosis. J Clin Microbiol. 1990 Mar;28(3):513–518. doi: 10.1128/jcm.28.3.513-518.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Plikaytis B. B., Gelber R. H., Shinnick T. M. Rapid and sensitive detection of Mycobacterium leprae using a nested-primer gene amplification assay. J Clin Microbiol. 1990 Sep;28(9):1913–1917. doi: 10.1128/jcm.28.9.1913-1917.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  16. Shankar P., Manjunath N., Lakshmi R., Aditi B., Seth P., Shriniwas Identification of Mycobacterium tuberculosis by polymerase chain reaction. Lancet. 1990 Feb 17;335(8686):423–423. doi: 10.1016/0140-6736(90)90268-a. [DOI] [PubMed] [Google Scholar]
  17. Sjöbring U., Mecklenburg M., Andersen A. B., Miörner H. Polymerase chain reaction for detection of Mycobacterium tuberculosis. J Clin Microbiol. 1990 Oct;28(10):2200–2204. doi: 10.1128/jcm.28.10.2200-2204.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Smithwick R. W., David H. L. Acridine orange as a fluorescent counterstain with the auramine acid-fast stain. Tubercle. 1971 Sep;52(3):226–231. doi: 10.1016/0041-3879(71)90045-6. [DOI] [PubMed] [Google Scholar]
  19. TACQUET A., TISON F. [New technic of isolation of mycobacteria by sodium laurylsulfate]. Ann Inst Pasteur (Paris) 1961 May;100:676–680. [PubMed] [Google Scholar]
  20. Thierry D., Brisson-Noël A., Vincent-Lévy-Frébault V., Nguyen S., Guesdon J. L., Gicquel B. Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its application in diagnosis. J Clin Microbiol. 1990 Dec;28(12):2668–2673. doi: 10.1128/jcm.28.12.2668-2673.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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