Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Rapid Detection and Monitoring Therapeutic Efficacy of Mycobacterium tuberculosis Complex Using a Novel Real-Time Assay

  • Jiang, Li Juan (State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University) ;
  • Wu, Wen Juan (Shanghai Public Health Clinical Center) ;
  • Wu, Hai (State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University) ;
  • Ryang, Son Sik (State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University) ;
  • Zhou, Jian (Shanghai Public Health Clinical Center) ;
  • Wu, Wei (State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University) ;
  • Li, Tao (Shanghai Public Health Clinical Center) ;
  • Guo, Jian (Shanghai Public Health Clinical Center) ;
  • Wang, Hong Hai (State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University) ;
  • Lu, Shui Hua (Shanghai Public Health Clinical Center) ;
  • Li, Yao (State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University)
  • Received : 2012.02.17
  • Accepted : 2012.04.21
  • Published : 2012.09.28
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Abstract

We combined real-time RT-PCR and real-time PCR (R/P) assays using a hydrolysis probe to detect Mycobacterium tuberculosis complex (MTBC)-specific 16S rRNA and its rRNA gene (rDNA). The assay was applied to 28 non-respiratory and 207 respiratory specimens from 218 patients. Total nucleic acids (including RNA and DNA) were extracted from samples, and results were considered positive if the repeat RT-PCR threshold cycle was ${\leq}35$ and the ratio of real-time RT-PCR and real-time PCR load was ${\geq}1.51$. The results were compared with those from existing methods, including smear, culture, and real-time PCR. Following resolution of the discrepant results between R/P assay and culture, the overall sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) of all samples (including non-respiratory and respiratory specimens) were 98.2%, 97.2%, 91.7%, and 99.4%, respectively, for R/P assay, and 83.9%, 89.9%, 72.3%, and 94.7%, respectively, for real-time PCR. Furthermore, the R/P assay of four patient samples showed a higher ratio before treatment than after several days of treatment. We conclude that the R/P assay is a rapid and accurate method for direct detection of MTBC, which can distinguish viable and nonviable MTBC, and thus may guide patient therapy and public health decisions.

Keywords

References

  1. Al Zahrani, K., H. Al Jahdali, L. Poirier, P. Rene, M. Gennaro, and D. Menzies. 2000. Accuracy and utility of commercially available amplification and serologic tests for the diagnosis of minimal pulmonary tuberculosis. Am. J. Respir. Crit. Care Med. 162: 1323-1329.
  2. Araj, G., R. Talhouk, L. Itani, W. Jaber, and G. Jamaleddine. 2000. Comparative performance of PCR-based assay versus microscopy and culture for the direct detection of Mycobacterium tuberculosis in clinical respiratory specimens in Lebanon. Int. J. Tuberc. Lung Dis. 4: 877-881.
  3. Broccolo, F., P. Scarpellini, G. Locatelli, A. Zingale, A. M. Brambilla, P. Cichero, et al. 2003. Rapid diagnosis of mycobacterial infections and quantitation of Mycobacterium tuberculosis load by two real-time calibrated PCR assays. J. Clin. Microbiol. 41: 4565-4572. https://doi.org/10.1128/JCM.41.10.4565-4572.2003
  4. Bustin, S. A., V. Benes, J. A. Garson, J. Hellemans, J. Huggett, M. Kubista, et al. 2009. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55: 611-622. https://doi.org/10.1373/clinchem.2008.112797
  5. Cloud, J., H. Neal, R. Rosenberry, C. Turenne, M. Jama, D. Hillyard, et al. 2002. Identification of Mycobacterium spp. by using a commercial 16S ribosomal DNA sequencing kit and additional sequencing libraries. J. Clin. Microbiol. 40: 400-406. https://doi.org/10.1128/JCM.40.2.400-406.2002
  6. DesJardin, L. E., M. D. Perkins, K. Wolski, S. Haun, L. Teixeira, Y. Chen, et al. 1999. Measurement of sputum Mycobacterium tuberculosis messenger RNA as a surrogate for response to chemotherapy. Am. J. Respir. Crit. Care Med. 160: 203-210. https://doi.org/10.1164/ajrccm.160.1.9811006
  7. Espy, M., J. Uhl, L. Sloan, S. Buckwalter, M. Jones, E. Vetter, et al. 2006. Real-time PCR in clinical microbiology: Applications for routine laboratory testing. Clin. Microbiol. Rev. 19: 165-256. https://doi.org/10.1128/CMR.19.1.165-256.2006
  8. Foongladda, S., S. Pholwat, B. Eampokalap, P. Kiratisin, and R. Sutthent. 2009. Multi-probe real-time PCR identification of common Mycobacterium species in blood culture broth. J. Mol. Diagn. 11: 42-48. https://doi.org/10.2353/jmoldx.2009.080081
  9. Hahn, D., R. I. Amann, W. Ludwig, A. D. L. Akkermans, and K. H. Schleifer. 1992. Detection of micro-organisms in soil after in situ hybridization with rRNA-targeted, fluorescently labelled oligonucleotides. J. Gen. Microbiol. 138: 879-887. https://doi.org/10.1099/00221287-138-5-879
  10. Harries, A. D. and C. Dye. 2006. Tuberculosis. Ann. Trop. Med. Parasitol. 100: 415-431. https://doi.org/10.1179/136485906X91477
  11. Hellyer, T., L. DesJardin, G. Hehman, M. Cave, and K. Eisenach. 1999. Quantitative analysis of mRNA as a marker for viability of Mycobacterium tuberculosis. J. Clin. Microbiol. 37: 290-295.
  12. Hellyer, T., L. DesJardin, L. Teixeira, M. Perkins, M. Cave, and K. Eisenach. 1999. Detection of viable Mycobacterium tuberculosis by reverse transcriptase-strand displacement amplification of mRNA. J. Clin. Microbiol. 37: 518-523.
  13. Hofmann-Thiel, S., L. Turaev, and H. Hoffmann. 2010. Evaluation of the hyplex (R) TBC PCR test for detection of Mycobacterium tuberculosis complex in clinical samples. BMC Microbiol. 10: 95. https://doi.org/10.1186/1471-2180-10-95
  14. Kraus, G., T. Cleary, N. Miller, R. Seivright, A. K. Young, G. Spruill, et al. 2001. Rapid and specific detection of the Mycobacterium tuberculosis complex using fluorogenic probes and real-time PCR. Mol. Cell. Probes 15: 375-383. https://doi.org/10.1006/mcpr.2001.0385
  15. Kubista, M., J. M. Andrade, M. Bengtsson, A. Forootan, J. Jonak, K. Lind, et al. 2006. The real-time polymerase chain reaction. Mol. Aspects Med. 27: 95-125. https://doi.org/10.1016/j.mam.2005.12.007
  16. Li, L., C. Mahan, M. Palaci, L. Horter, L. Loeffelholz, J. Johnson, et al. 2010. Sputum Mycobacterium tuberculosis mRNA as a marker of bacteriologic clearance in response to antituberculosis therapy. J. Clin. Microbiol. 48: 46-51. https://doi.org/10.1128/JCM.01526-09
  17. Mackay, I. M. 2004. Real-time PCR in the microbiology laboratory. Clin. Microbiol. Infect. 10: 190-212. https://doi.org/10.1111/j.1198-743X.2004.00722.x
  18. Mdivani, N., H. Li, M. Akhalaia, M. Gegia, L. Goginashvili, D. S. Kernodle, et al. 2009. Monitoring therapeutic efficacy by real-time detection of Mycobacterium tuberculosis mRNA in sputum. Clin. Chem. 55: 1694-1700. https://doi.org/10.1373/clinchem.2009.124396
  19. Philipp, S., H. P. Huemer, E. U. Irschick, and C. Gassner. 2010. Obstacles of multiplex real-time PCR for bacterial 16S rDNA: Primer specifity and DNA decontamination of Taq polymerase. Transfus. Med. Hemother. 37: 21-28. https://doi.org/10.1159/000265571
  20. Piersimoni, C. and C. Scarparo. 2003. Relevance of commercial amplification methods for direct detection of Mycobacterium tuberculosis complex in clinical samples. J. Clin. Microbiol. 41: 5355-5365. https://doi.org/10.1128/JCM.41.12.5355-5365.2003
  21. Pounder, J. I., W. K. Aldous, and G. L. Woods. 2006. Comparison of real-time polymerase chain reaction using the Smart Cycler and the Gen-Probe amplified Mycobacterium tuberculosis direct test for detection of M. tuberculosis complex in clinical specimens. Diagn. Micrbiol. Infect. Dis. 54: 217-222. https://doi.org/10.1016/j.diagmicrobio.2005.05.018
  22. Rogers, G. B., F. A. Stressmann, G. Koller, T. Daniels, M. P. Carroll, and K. D. Bruce. 2008. Assessing the diagnostic importance of nonviable bacterial cells in respiratory infections. Diagn. Microbiol. Infec. Dis. 62: 133-141. https://doi.org/10.1016/j.diagmicrobio.2008.06.011
  23. Sontakke, S., M. B. Cadenas, R. G. Maggi, P. P. V. P. Diniz, and E. B. Breitschwerdt. 2009. Use of broad range 16S rDNA PCR in clinical microbiology. J. Microbiol. Methods 76: 217-225. https://doi.org/10.1016/j.mimet.2008.11.002
  24. Valasek, M. A. and J. J. Repa. 2005. The power of real-time PCR. Adv. Physiol. Educ. 29: 151-159. https://doi.org/10.1152/advan.00019.2005
  25. Van der Vliet, G. M., P. Schepers, R. Schukkink, B. Van Gemen, and P. R. Klatser. 1994. Assessment of mycobacterial viability by RNA amplification. Antimicrob. Agents Chemother. 38: 1959-1965. https://doi.org/10.1128/AAC.38.9.1959
  26. Young, G., S. Turner, J. K. Davies, G. Sundqvist, and D. Figdor. 2007. Bacterial DNA persists for extended periods after cell death. J. Endod. 33: 1417-1420. https://doi.org/10.1016/j.joen.2007.09.002

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