Volume 91, Issue 4
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645



Real-time polymerase chain reaction (qPCR) was optimized for detecting in sputum. Sputum was collected from patients ( = 112) with suspected pulmonary tuberculosis, tested by smear microscopy, decontaminated, and split into equal aliquots that were cultured in Löwenstein-Jensen medium and tested by qPCR for the small mobile genetic element IS. The human sequence was used as an internal control. 3 of 112 (3%) qPCR failed. For the remaining 109 samples, qPCR diagnosed tuberculosis in 79 of 84 patients with culture-proven tuberculosis, and sensitivity was greater than microscopy (94% versus 76%, respectively, < 0.05). The qPCR sensitivity was similar ( = 0.9) for smear-positive (94%, 60 of 64) and smear-negative (95%, 19 of 20) samples. The qPCR was negative for 24 of 25 of the sputa with negative microscopy and culture (diagnostic specificity 96%). The qPCR had 99.5% sensitivity and specificity for 211 quality control samples including 84 non-tuberculosis mycobacteria. The qPCR cost ∼5US$ per sample and provided same-day results compared with 2–6 weeks for culture.


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  1. Master RN, , section editor, 1992. Microbiology. Clinical Microbiology Procedures Handbook. Volume I. Washington, DC: ASM. [Google Scholar]
  2. Steingart KR, Ng V, Henry M, Hopewell PC, Ramsay A, Cunningham J, Urbanczik R, Perkins MD, Aziz MA, Pai M, , 2006. Sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review. Lancet Infect Dis 6: 664674, Review.[Crossref] [Google Scholar]
  3. Katila ML, Katila P, Erkinjuntti-Pekkanen R, , 2000. Accelerated detection and identification of Mycobacteria with MGIT 960 and COBAS AMPLICOR Systems. J Clin Microbiol 38: 960964. [Google Scholar]
  4. Morán Mogue MC, Hernández DA, Pena Montes de Oca PM, Gallegos Arreola MP, Flores Martínez SE, Montoya Fuentes H, Figuera LE, Villa Manzanares L, Sánchez Corona J, , 2000. Detección de Mycobacterium tuberculosis mediante la reacción en cadena de la polimerasa en una población seleccionada del noroccidente de México. Rev Panam Salud Publica 7: 389394.[Crossref] [Google Scholar]
  5. Parimango D, Chávez M, Luján M, Otiniano M, Robles H, Muñoz E, , 2007. Comparación de los medios Ogawa y Löwenstein-Jensen en el aislamiento de Mycobacterium tuberculosis de pacientes con tuberculosis pulmonar. Hospital Regional Docente de Trujillo, Perú. Rev. Med. Vallejiana 4: 2431. [Google Scholar]
  6. Ling DI, Flores LL, Riley LW, Pai M, , 2008. Commercial nucleic-acid amplification tests for diagnosis of pulmonary tuberculosis in respiratory specimens: meta-analysis and meta-regression. PLoS One 3: e1536.[Crossref] [Google Scholar]
  7. Flores LL, Pai M, Colford JM, Jr Riley LW, , 2005. In-house nucleic acid amplification tests for the detection of Mycobacterium tuberculosis in sputum specimens: meta-analysis and meta-regression. BMC Microbiol 5: 5563.[Crossref] [Google Scholar]
  8. Moure R, Muñoz L, Torres M, Santin M, Martín R, Alcaide F, , 2011. Rapid detection of Mycobacterium tuberculosis complex and rifampin resistance in smear-negative clinical samples by use of an integrated real-time PCR method. J Clin Microbiol 49: 11371139.[Crossref] [Google Scholar]
  9. Yuan CC, Miley W, Waters D, , 2001. A quantification of human cells using an ERV-3 real time PCR assay. J Virol Methods 91: 109117.[Crossref] [Google Scholar]
  10. Van Embden JDA, Cave MD, Crawford JT, Dale JW, Eisenach KD, Gicquel B, Hermans P, Martin C, McAdam R, Shinnick TM, Small PM, , 1993. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol 31: 406409. [Google Scholar]
  11. Käser M, Ruf MT, Huaser J, Marsollier L, Pluschke G, , 2009. Optimized method for preparation of DNA from pathogenic and environmental Mycobacteria . Appl Environ Microbiol 75: 414418.[Crossref] [Google Scholar]
  12. Palomino JC, , 2006. Newer diagnostics for tuberculosis and multidrug resistant tuberculosis. Curr Opin Pulm Med 12: 172178.[Crossref] [Google Scholar]
  13. Somoskovi A, Hotaling JE, Fitzgerald M, O'Donnell D, Parsons LM, Salfinger M, , 2001. Lessons from a proficiency testing event for acid-fast microscopy. Chest 120: 250257.[Crossref] [Google Scholar]
  14. Van Deun A, Hamid Salim A, Aung KJ, Hossain MA, Chambugonj N, Hye MA, Kawria A, Declercq E, , 2005. Performance of variations of caebolfuchsin staining of sputum for AFB under field conditions. Int J Tuberc Lung Dis 9: 11271133. [Google Scholar]
  15. Ricaldi JN, Guerra H, , 2008. A simple and improved method for diagnosis of tuberculosis using hypertonic saline and sodium hydroxide (HS–SH) to concentrate and decontaminate sputum. Trop Doct 38: 9799.[Crossref] [Google Scholar]
  16. Altamirano M, Kelly MT, Wong A, Bessuille ET, Black WA, Smith JA, , 1992. Characterization of a DNA probe for detection of Mycobacterium tuberculosis complex in clinical samples by polymerase chain reaction. J Clin Microbiol 30: 21732176. [Google Scholar]
  17. Beige J, Lokies J, Schaberg T, Finckh U, Fischer M, Mauch H, Lode H, Köhler B, Rolfs A, , 1995. Clinical evaluation of a Mycobacterium tuberculosis PCR assay. J Clin Microbiol 33: 9095. [Google Scholar]
  18. Clarridge JE, 3rd Shawar RM, Shinnick TM, Plikaytis BB, , 1993. Large-scale use of polymerase chain reaction for detection of Mycobacterium tuberculosis in a routine mycobacteriology laboratory. J Clin Microbiol 31: 20492056. [Google Scholar]
  19. Cousins DV, Wilton SD, Francis BR, Gow BL, , 1992. Use of polymerase chain reaction for rapid diagnosis of tuberculosis. J Clin Microbiol 30: 255258. [Google Scholar]
  20. Eisenach KD, Cave MD, Bates JH, Crawford JT, , 1990. Polymerase chain reaction amplification of a repetitive DNA sequence specific for Mycobacterium tuberculosis . J Infect Dis 161: 977981.[Crossref] [Google Scholar]
  21. Folgueira L, Delgado R, Palenque E, Noriega AR, , 1993. Detection of Mycobacterium tuberculosis DNA in clinical samples by using a simple lysis method and polymerase chain reaction. J Clin Microbiol 31: 10191021. [Google Scholar]
  22. Kocagöz T, Yilmaz E, Ozkara S, Kocagöz S, Hayran M, Sachedeva M, Chambers HF, , 1993. Detection of Mycobacterium tuberculosis in sputum samples by polymerase chain reaction using a simplified procedure. J Clin Microbiol 31: 14351438. [Google Scholar]
  23. Kox LF, Rhienthong D, Miranda AM, Udomsantisuk N, Ellis K, van Leeuwen J, van Heusden S, Kuijper S, Kolk AH, , 1994. A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol 32: 672678. [Google Scholar]
  24. Moore DF, Curry JL, , 1995. Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by Amplicor PC. J Clin Microbiol 33: 26862691. [Google Scholar]
  25. Beqaj SH, Flesher R, Walker GR, Smith SA, , 2007. Use of the real-time PCR assay in conjunction with MagNA Pure for the detection of mycobacterial DNA from fixed specimens. Diagn Mol Pathol 16: 169173.[Crossref] [Google Scholar]
  26. Piersimoni C, Scarparo C, , 2003. Relevance of commercial amplification methods for direct detection of Mycobacterium tuberculosis complex in clinical samples. J Clin Microbiol 41: 53555365.[Crossref] [Google Scholar]
  27. Lebrun L, Weill FX, Lafendi L, Houriez F, Casanova F, Gutierrez MC, Ingrand D, Lagrange P, Vincent V, Herrmann JL, , 2005. Use of the INNO-LiPA-MYCOBACTERIA assay (version 2) for identification of Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex isolates. J Clin Microbiol 43: 25672574.[Crossref] [Google Scholar]
  28. Richter E, Weizenegger M, Fahr AM, Rüsch-Gerdes S, , 2004. Usefulness of the GenoType MTBC assay for differentiating species of the Mycobacterium tuberculosis complex in cultures obtained from clinical specimens. J Clin Microbiol 42: 43034306.[Crossref] [Google Scholar]
  29. Mazzarelli G, Rindi L, Piccoli P, Scarparo C, Garzelli C, Tortoli E, , 2003. Evaluation of the BDProbeTec ET system for direct detection of Mycobacterium tuberculosis in pulmonary and extrapulmonary samples: a multicenter study. J Clin Microbiol 41: 17791782.[Crossref] [Google Scholar]
  30. Chen X, Yang Q, Kong H, Chen Y, , 2012. Real-time PCR and amplified MTD for rapid detection of Mycobacterium tuberculosis in pulmonary specimens. Int J Tuberc Lung Dis 16: 235239.[Crossref] [Google Scholar]
  31. Eigner U, Veldenzer A, Holfelder M, , 2013. Evaluation of the FluoroType MTB assay for the rapid and reliable detection of Mycobacterium tuberculosis in respiratory tract specimens. Clin Lab 59: 11791181.[Crossref] [Google Scholar]
  32. McHugh TD, Newport LE, Gillespie SH, , 1997. IS6110 homologs are present in multiple copies in mycobacteria other than tuberculosis-causing mycobacteria. J Clin Microbiol 35: 17691771. [Google Scholar]
  33. Thierry D, Cave MD, Eisenach KD, Crawford JT, Bates JH, Gicquel B, Guesdon JL, , 1990. IS6110, an IS-like element of Mycobacterium tuberculosis complex. Nucleic Acids Res 18: 188.[Crossref] [Google Scholar]
  34. Kurepina NE, Sreevatsan S, Plikaytis BB, Bifani PJ, Connell ND, Donnelly RJ, van Sooligen D, Musser JM, Kreiswirth BN, , 1998. Characterization of the phylogenetic distribution and chromosomal insertion sites of five IS6110 elements in Mycobacterium tuberculosis: non-random integration in the dnaA-dnaN region. Tuber Lung Dis 79: 3142.[Crossref] [Google Scholar]
  35. Helb D, Jones M, Story E, Boehme C, Wallace E, Ho K, Kop J, Owens MR, Rodgers R, Banada P, Safi H, Blakemore R, Lan NT, Jones-López EC, Levi M, Burday M, Ayakaka I, Mugerwa RD, McMillan B, Winn-Deen E, Christel L, Dailey P, Perkins MD, Persing DH, Alland D, , 2010. Rapid detection of Mycobacterium tuberculosis and rifampin resistance by use of on-demand, near-patient technology. J Clin Microbiol 48: 229237.[Crossref] [Google Scholar]
  36. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT, , 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55: 611622.[Crossref] [Google Scholar]
  37. Burkardt HJ, , 2000. Standardization and quality control of PCR analyses. Clin Chem Lab Med 38: 8791.[Crossref] [Google Scholar]
  38. De Parseval N, Hiedmann T, , 1998. Physiological knockout of the envelope gene of the single-copy ERV-3 human endogenous retrovirus in a fraction of the caucasian population. J Virol 72: 34423445. [Google Scholar]
  39. Cohen M, Powers M, O'Connell C, Kato N, , 1985. The nucleotide sequence of the env gene from the human provirus ERV3 and isolation and characterization of an ERV3-specific cDNA. Virology 147: 449458.[Crossref] [Google Scholar]
  40. Yang YC, Lu PL, Huang SC, Jenh YS, Jou R, Chang TC, , 2011. Evaluation of the Cobas TaqMan MTB test for direct detection of Mycobacterium tuberculosis complex in respiratory specimens. J Clin Microbiol 49: 797801.[Crossref] [Google Scholar]

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  • Received : 18 Oct 2013
  • Accepted : 13 Jun 2014
  • Published online : 01 Oct 2014

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