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Development of Real-Time Multiplex NASBA® for the - Journal of ...

Development of Real-Time Multiplex NASBA® for the - Journal of ...

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350 355 360 365 370 in multiplex as well as in mono NASBA reactions, while 4 PCR negative specimens were NASBA positive 2 of which also in the multiplex format. The differences between the PCR and the NASBA results are significant (p=0.01 and p= 0.05), respectively, but there is no difference between the two NASBA reactions (p=0.2). For 7 specimens the negative NASBA result is due to RNA degradation as shown by a negative U1A mRNA NASBA. In five cases (nos. 22, 23, 24, 25 and 26. Table 2), the PCR result was confirmed either by a positive IgM result or by a positive NASBA result on a second specimen from the same patient or by culture. For the remaining patients (nrs. 27, 28, 29, 30, 31, 32 and 33. Table 2) the discordant amplification results for M. pneumoniae could not be resolved by serology or culture. Re-extraction and re-amplification confirmed all PCR and NASBA positive results, classifying the results of the latter patients as true M. pneumoniae positive. The archived nature of the specimens used in this study may be responsible for the negative results in some of the repeat PCRs and for some contradictions between PCR and real-time NASBA results. NASBA is inherently more sensitive to storage conditions than PCR, since RNA is more easily degraded than DNA. A prospective study whereby specimens are put in lysis buffer immediately after production might avoid such contradictions. ACCEPTED We conclude that the proposed real-time multiplex NASBA assay, although marginally less sensitive than the real-time mono NASBA assay, except for Legionella spp where there is a significant difference between real-time mono and multiplex NASBA, is a promising tool for the detection of M. pneumoniae, and C. pneumoniae and Legionella spp. in respiratory specimens, regarding handling, speed and number of samples that can be analyzed in a single run. A large number of clinical specimens from patients with community-acquired pneumonia should be analyzed for further evaluation of the assay. 1.4 Acknowledgements This study was supported by European Commission (Framework V) grant no QLK2-CT- 2000-00294. 14

375 380 385 390 395 400 405 1.5 References 1. Abele-Horn, M., U. Busch, H. Nitschko, E. Jacobs, R. Bax, F. Pfaff, B. Schaffer, and J. Heesemann. 1998. Molecular approaches to diagnosis of pulmonary diseases due to Mycoplasma pneumoniae. J. Clin. Microbiol. 36:548-551. 2. Bernander, S., H-S. Hanson, B. Johansson, and L.-V. von Stedingk. 1997. A nested polymerase chain reaction for detection of Legionella pneumophila in clinical specimens. Clin. Microbiol. Infect. 3:95-101. 3. Bernet, C., M. Garret, B. de Barbeyrac, C. Bébéar, and J. Bonnet. 1989. Detection of Mycoplasma pneumoniae by using the polymerase chain reaction. J. Clin. Microbiol. 27:2492- 2496. 4. Boman J., A. Allard, K. Persson, M. Lundborg, P. Juto, and G. Wadell. 1997. Rapid diagnosis of respiratory Chlamydia pneumoniae infection by nested touchdown polymerase chain reaction compared with culture and antigen detection by EIA. J. Infect. Dis. 175:1523-1526. 5. Boom, R., C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. E. Wertheim-van Dillen, and J. van der Noordaa. 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28:495-503. 6. Cadieux, N., P. Lebek, and R. Brousseau. 1993. Use of a triplex polymerase chain reaction for the detection and differentiation of Mycoplasma pneumoniae and Mycoplasma genitalium in the presence of human DNA. J. Gen. Microbiol. 139:2431-2437. ACCEPTED 7. Corsaro, D., M. Valassina, D. Venditti, V. Venard, A. Le Faou, and P. E. Valensin. 1999. Multiplex PCR for rapid and differential diagnosis of Mycoplasma pneumoniae and Chlamydia pneumoniae in respiratory infections. Diagn. Microbiol. Infect. Dis. 35:105-108. 8. de Baar, M. P., M. W. Van Dooren, E. De Rooij, M. Bakker, B. Van Gemen, J. Goudsmit, and A. De Ronde. 2001. Single rapid real-time monitored isothermal RNA amplification assay for quantification of human immunodeficiency virus type 1 isolates from groups M, N, and O. J. Clin. Microbiol. 39:1378-1384. 9. Greijer, A. E., H. M. A. Adriaanse, C. A. J. Dekkers, and J. M. Middeldorp. 2002. Multiplex real-time NASBA for monitoring expression dynamics of human cytomegalovirus encoded IE1 and pp67 RNA. J. Clin. Virol. 24:57-66. 10. Gröndahl, B., W. Puppe, A. Hoppe, I. Kühne, J. A. I. Weigl, and H. -J. Schmitt. 1999. Rapid identification of nine microorganisms causing acute respiratory tract infections by single tube multiplex reverse transcription PCR: Feasibility study. J. Clin. Microbiol. 37:1-7. 11. Helbig, J. H., T. Engelstädter, M. Maiwald, S. A. Uldum, W. Witzleb, and P. C. Lück. 1999. Diagnostic relevance of the detection of Legionella DNA in urine samples by the polymerase chain reaction. Eur. J. Clin. Microbiol. Infect. Dis. 18:716-722. 15

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