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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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230 235 240 245 250 255 detected 0.1 CFU of L. pneumophila serotype 1 in 100µl lysis buffer in 4/4 and 2/4 aliquots, respectively. When real-time mono and multiplex NASBA were applied to the same nucleic acid extract from L. pneumophila serotypes 2, 3, 4, 5, 6, and 10 as well as to L. longbeachae 4a, and L. micdadei spp the difference in sensitivity between the real-time mono and multiplex NASBA was 1 log with the former being the more sensitive assay. For L. longbeachae 4b and L. bozemanii the difference in sensitivity was 2 logs. The sensitivity on spiked respiratory specimens with M. pneumoniae, C. pneumoniae and L. pneumophila as a single target are shown in Tables 3A, 3B and 3C, respectively. Multiple targets: With inputs of 330 or 3300 molecules of in vitro generated RNA of each organism, all three organisms could be detected by the real-time multiplex NASBA. With an input of 33 molecules of in vitro generated RNA of each organism, a positive result was always obtained for C. pneumoniae whereas the assay failed to detect Legionella and M. pneumoniae in some but not all samples (Table 5). In a mixture of 3300 molecules of both M. pneumoniae and C. pneumoniae in vitro generated RNA and 330 molecules of Legionella in vitro generated RNA an input of 330 molecules of Legionella in vitro generated RNA, was not detected by the real-time ACCEPTED multiplex NASBA. Negative specimens: Out of 149 M. pneumoniae, C. pneumoniae and L. pneumophila PCR negative respiratory specimens, 3, 2 and 2 specimens were positive by real-time mono NASBA for M. pneumoniae, C. pneumoniae, and Legionella, respectively. One out of these three M. pneumoniae positive specimens was also positive by real-time multiplex NASBA. Two specimens originated from the same patient. Reproducibility of the real-time multiplex NASBA: The intrarun variability coefficients for the detection of 500 and 5000 CCU of M. pneumoniae were 13.5 and 13.5 whereas the interrun variation coefficients for the same inputs were 16.7 and 16.8. 10

260 265 270 275 280 285 The intrarun variability coefficients for the detection of 0.1, 1, 10 and 100 IFU of C. pneumoniae were 13.3, 10.3, 10.7, 13.0 by real-time multiplex NASBA. The interrun variation coefficients for the same inputs were 20.8, 11.0, 13.7, and 15.4. For L. pneumophila serotype 1, the intrarun variability coefficients were 13.4 and 13.1 for an input of 100 and 1000 CFU respectively. The interrun variation coefficients for the same inputs were 10.6 and 11.6. Archived specimens from PCR positive patients: From the 51 archived respiratory specimens collected from 33 patients who had at least one specimen positive for M pneumoniae by PCR, 40 were PCR positive. Twenty-one specimens were M. pneumoniae positive by the three detection procedures: PCR, real- time mono and multiplex NASBA. Four specimens were M. pneumoniae positive by PCR and real-time mono NASBA. Four M. pneumoniae PCR negative specimens were positive by real-time mono NASBA only. Two of these were also positive in the multiplex assay. Seven specimens were M. pneumoniae negative by all three techniques (Table 2). Fifteen M. pneumoniae PCR positive specimens were negative by both real- time mono and multiplex NASBA. However seven of these specimens were U1A negative and thus contained no RNA (nos. 19, 20, 23, 24, 29, 32, and 33. Table 2). For 3 ACCEPTED patients (nrs. 23, 25, and 26. Table 2), the PCR-result was confirmed by a positive IgM result, one of whom (no. 23. Table 2) had a second specimen positive for M. pneumoniae, by both real-time mono and multiplex NASBA. For patients no. 19, 20, and 22, the PCR result was confirmed by a second specimen positive for M. pneumoniae by real-time NASBA, and for patient 24 by culture, (Table 2). The M. pneumoniae PCR positive results on the specimens from the remaining 4 patients (nos. 27, 28, 30, and 31. Table 2) and from 3 patients with a negative U1A result (29, 32, and 33. Table 2) could not be confirmed by serology or culture. C. pneumoniae positive specimens: all 3 archived C. pneumoniae PCR positive nasopharyngeal aspirates were found positive in the NASBA assays. L. pneumophila positive specimens: 5/5 lung biopsy specimens and 5/5 water samples were positive in the three assays, the 4 sputum specimens were positive by PCR and real- time mono NASBA (Table 2b). 11

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