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. 1995 Mar;33(3):668–674. doi: 10.1128/jcm.33.3.668-674.1995

Use of a multiplex PCR to detect and identify Mycobacterium avium and M. intracellulare in blood culture fluids of AIDS patients.

J K Kulski 1, C Khinsoe 1, T Pryce 1, K Christiansen 1
PMCID: PMC228011  PMID: 7751375

Abstract

The presence of mycobacteria in blood culture fluids (BACTEC) of AIDS patients with positive growth indices (GIs, > 20 U) was investigated by using a multiplex PCR to detect and identify members of the genus Mycobacterium, M. avium, M. intracellulare, and M. tuberculosis. Three different methods of extracting mycobacterial DNA from blood culture fluid were compared for use with the multiplex PCR. Mycobacterial cells were pelleted from a small aliquot of blood culture fluid by centrifugation, and the DNA was extracted from cells by heat lysis or a sodium iodide-isopropanol or a phenol-chloroform method. DNAs of different sizes were amplified from a region of the MPB70 gene of M. tuberculosis (372 bp) and from a region of the 16S rRNA gene of members of the genus Mycobacterium (1,030 bp), M. intracellulare (850 bp), or M. avium (180 bp) as a multiplex PCR in a single tube. The amplified DNA products were detected by agarose gel electrophoresis and ethidium bromide staining in all 41 (100%) positive cultures after sodium iodide-isopropanol extraction, in 18 (44%) after heat lysis, and in 5 (12%) after phenol-chloroform extraction. Of the 41 positive cultures, 38 were identified as M. avium and 2 were identified as M. intracellulare by both routine methods and multiplex PCR. The remaining mycobacterium was identified as M. intracellulare by routine methods and as M. avium by the multiplex PCR. Another six blood cultures that were negative for the presence of acid-fast bacilli after Ziehl-Neelson staining were also negative by PCR.(ABSTRACT TRUNCATED AT 250 WORDS)

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Selected References

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  1. Body B. A., Warren N. G., Spicer A., Henderson D., Chery M. Use of Gen-Probe and Bactec for rapid isolation and identification of mycobacteria. Correlation of probe results with growth index. Am J Clin Pathol. 1990 Mar;93(3):415–420. doi: 10.1093/ajcp/93.3.415. [DOI] [PubMed] [Google Scholar]
  2. Brisson-Noel A., Aznar C., Chureau C., Nguyen S., Pierre C., Bartoli M., Bonete R., Pialoux G., Gicquel B., Garrigue G. Diagnosis of tuberculosis by DNA amplification in clinical practice evaluation. Lancet. 1991 Aug 10;338(8763):364–366. doi: 10.1016/0140-6736(91)90492-8. [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. Cormican M. G., Barry T., Gannon F., Flynn J. Use of polymerase chain reaction for early identification of Mycobacterium tuberculosis in positive cultures. J Clin Pathol. 1992 Jul;45(7):601–604. doi: 10.1136/jcp.45.7.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Drake T. A., Hindler J. A., Berlin O. G., Bruckner D. A. Rapid identification of Mycobacterium avium complex in culture using DNA probes. J Clin Microbiol. 1987 Aug;25(8):1442–1445. doi: 10.1128/jcm.25.8.1442-1445.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ellner J. J., Goldberger M. J., Parenti D. M. Mycobacterium avium infection and AIDS: a therapeutic dilemma in rapid evolution. J Infect Dis. 1991 Jun;163(6):1326–1335. doi: 10.1093/infdis/163.6.1326. [DOI] [PubMed] [Google Scholar]
  8. Ellner P. D., Kiehn T. E., Cammarata R., Hosmer M. Rapid detection and identification of pathogenic mycobacteria by combining radiometric and nucleic acid probe methods. J Clin Microbiol. 1988 Jul;26(7):1349–1352. doi: 10.1128/jcm.26.7.1349-1352.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fries J. W., Patel R. J., Piessens W. F., Wirth D. F. Detection of untreated mycobacteria by using polymerase chain reaction and specific DNA probes. J Clin Microbiol. 1991 Aug;29(8):1744–1747. doi: 10.1128/jcm.29.8.1744-1747.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fries J. W., Patel R. J., Piessens W. F., Wirth D. F. Genus- and species-specific DNA probes to identify mycobacteria using the polymerase chain reaction. Mol Cell Probes. 1990 Apr;4(2):87–105. doi: 10.1016/0890-8508(90)90011-n. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Horsburgh C. R., Jr Mycobacterium avium complex infection in the acquired immunodeficiency syndrome. N Engl J Med. 1991 May 9;324(19):1332–1338. doi: 10.1056/NEJM199105093241906. [DOI] [PubMed] [Google Scholar]
  13. Hoy J., Mijch A., Sandland M., Grayson L., Lucas R., Dwyer B. Quadruple-drug therapy for Mycobacterium avium-intracellulare bacteremia in AIDS patients. J Infect Dis. 1990 Apr;161(4):801–805. doi: 10.1093/infdis/161.4.801. [DOI] [PubMed] [Google Scholar]
  14. Iralu J. V., Sritharan V. K., Pieciak W. S., Wirth D. F., Maguire J. H., Barker R. H., Jr Diagnosis of Mycobacterium avium bacteremia by polymerase chain reaction. J Clin Microbiol. 1993 Jul;31(7):1811–1814. doi: 10.1128/jcm.31.7.1811-1814.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ishizawa M., Kobayashi Y., Miyamura T., Matsuura S. Simple procedure of DNA isolation from human serum. Nucleic Acids Res. 1991 Oct 25;19(20):5792–5792. doi: 10.1093/nar/19.20.5792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kulski J. K. Quantitation of human cytomegalovirus DNA in leukocytes by end-point titration and duplex polymerase chain reaction. J Virol Methods. 1994 Sep;49(2):195–208. doi: 10.1016/0166-0934(94)90044-2. [DOI] [PubMed] [Google Scholar]
  17. Lubahn D. B., Brown T. R., Simental J. A., Higgs H. N., Migeon C. J., Wilson E. M., French F. S. Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9534–9538. doi: 10.1073/pnas.86.23.9534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Masur H. Recommendations on prophylaxis and therapy for disseminated Mycobacterium avium complex disease in patients infected with the human immunodeficiency virus. Public Health Service Task Force on Prophylaxis and Therapy for Mycobacterium avium Complex. N Engl J Med. 1993 Sep 16;329(12):898–904. doi: 10.1056/NEJM199309163291228. [DOI] [PubMed] [Google Scholar]
  19. Nightingale S. D., Byrd L. T., Southern P. M., Jockusch J. D., Cal S. X., Wynne B. A. Incidence of Mycobacterium avium-intracellulare complex bacteremia in human immunodeficiency virus-positive patients. J Infect Dis. 1992 Jun;165(6):1082–1085. doi: 10.1093/infdis/165.6.1082. [DOI] [PubMed] [Google Scholar]
  20. Nolte F. S., Metchock B., McGowan J. E., Jr, Edwards A., Okwumabua O., Thurmond C., Mitchell P. S., Plikaytis B., Shinnick T. Direct detection of Mycobacterium tuberculosis in sputum by polymerase chain reaction and DNA hybridization. J Clin Microbiol. 1993 Jul;31(7):1777–1782. doi: 10.1128/jcm.31.7.1777-1782.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Panaccio M., Lew A. PCR based diagnosis in the presence of 8% (v/v) blood. Nucleic Acids Res. 1991 Mar 11;19(5):1151–1151. doi: 10.1093/nar/19.5.1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Radford A. J., Duffield B. J., Plackett P. Cloning of a species-specific antigen of Mycobacterium bovis. Infect Immun. 1988 Apr;56(4):921–925. doi: 10.1128/iai.56.4.921-925.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ratnam S., Stead F. A., Howes M. Simplified acetylcysteine-alkali digestion-decontamination procedure for isolation of mycobacteria from clinical specimens. J Clin Microbiol. 1987 Aug;25(8):1428–1432. doi: 10.1128/jcm.25.8.1428-1432.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rogall T., Flohr T., Böttger E. C. Differentiation of Mycobacterium species by direct sequencing of amplified DNA. J Gen Microbiol. 1990 Sep;136(9):1915–1920. doi: 10.1099/00221287-136-9-1915. [DOI] [PubMed] [Google Scholar]
  25. Salfinger M., Stool E. W., Piot D., Heifets L. Comparison of three methods for recovery of Mycobacterium avium complex from blood specimens. J Clin Microbiol. 1988 Jun;26(6):1225–1226. doi: 10.1128/jcm.26.6.1225-1226.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stahl D. A., Urbance J. W. The division between fast- and slow-growing species corresponds to natural relationships among the mycobacteria. J Bacteriol. 1990 Jan;172(1):116–124. doi: 10.1128/jb.172.1.116-124.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tsukamura M., Yamori S. Differentiation between Mycobacterium avium and M. intracellulare by phenetic characteristics. Tubercle. 1991 Dec;72(4):308–309. doi: 10.1016/0041-3879(91)90065-z. [DOI] [PubMed] [Google Scholar]
  28. Vaneechoutte M., De Beenhouwer H., Claeys G., Verschraegen G., De Rouck A., Paepe N., Elaichouni A., Portaels F. Identification of Mycobacterium species by using amplified ribosomal DNA restriction analysis. J Clin Microbiol. 1993 Aug;31(8):2061–2065. doi: 10.1128/jcm.31.8.2061-2065.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wards B. J., Collins D. M., de Lisle G. W. Restriction endonuclease analysis of members of the Mycobacterium avium-M. intracellulare-M. scrofulaceum serocomplex. J Clin Microbiol. 1987 Dec;25(12):2309–2313. doi: 10.1128/jcm.25.12.2309-2313.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wilton S., Cousins D. Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube. PCR Methods Appl. 1992 May;1(4):269–273. doi: 10.1101/gr.1.4.269. [DOI] [PubMed] [Google Scholar]
  31. Wright P. A., Wynford-Thomas D. The polymerase chain reaction: miracle or mirage? A critical review of its uses and limitations in diagnosis and research. J Pathol. 1990 Oct;162(2):99–117. doi: 10.1002/path.1711620203. [DOI] [PubMed] [Google Scholar]
  32. Yeager H., Jr, Lacy J., Smith L. R., LeMaistre C. A. Quantitative studies of mycobacterial populations in sputum and saliva. Am Rev Respir Dis. 1967 Jun;95(6):998–1004. doi: 10.1164/arrd.1967.95.6.998. [DOI] [PubMed] [Google Scholar]

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