1921
Volume 91, Issue 4
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645

Abstract

Abstract.

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.

Loading

Article metrics loading...

The graphs shown below represent data from March 2017
/content/journals/10.4269/ajtmh.13-0603
2014-10-01
2020-10-31
Loading full text...

Full text loading...

/deliver/fulltext/14761645/91/4/709.html?itemId=/content/journals/10.4269/ajtmh.13-0603&mimeType=html&fmt=ahah

References

  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]
http://instance.metastore.ingenta.com/content/journals/10.4269/ajtmh.13-0603
Loading
/content/journals/10.4269/ajtmh.13-0603
Loading

Data & Media loading...

  • Received : 18 Oct 2013
  • Accepted : 13 Jun 2014
  • Published online : 01 Oct 2014
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error