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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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290 295 300 305 310 Statistical analysis: Statistical analysis using the X2-test was done for each organisms on the total number of samples, spiked and real clinical samples, tested for each organism. The differences between real-time mono and multiplex NASBA were not significant for C. pneumoniae and M. pneumoniae, p=0.11 and p= 0.24 respectively. A significant difference was observed for L. pneumophila positive specimens, p=0.04. 1.3 DISCUSSION The aim of this study was to develop a real-time multiplex NASBA assay for the detection of M. pneumoniae, C. pneumoniae and Legionella spp. in respiratory specimens based on 16S rRNA. The P1 primers and the beacons used were described previously (18, 19, 20). To reach a satisfactory sensitivity for all three pathogens, a generic P2 primer was designed to allow the simultaneous amplification of M. pneumoniae, C. pneumoniae and as many Legionella spp. and as many serogroups as possible. The specificity of the test was maintained in the multiplex format. The sensitivity of the real-time multiplex NASBA is between 5-50 CCU, 0.1-1 IFU and ACCEPTED 1-10 CFU/100µl of M. pneumoniae, C. pneumoniae, and L. pneumophila serotype 1 per 100µl, respectively. The only significant difference between the real-time mono-and multiplex NASBA reactions is for Legionella species. However, there is a tendency in all other instances for the multiplex format to be consistently, (be it not significantly), less sensitive than the monoplex reaction. All 3 individual primer pairs used in the real-time mono NASBAs were optimized for the absence of any cross-reactivity among the pathogens and were combined in a multiplex NASBA together with the differently colored molecular beacons. Unfortunately, a substantial decrease in sensitivity was observed for the detection of Mycoplasma pneumoniae and the Legionella species in this multiplex assay. This appeared to be due to primer interferences in the complex cocktail of P1 and P2 primers (results not shown). From the beginning this was known as a possible threat for NASBA multiplex assays and, therefore, the original P2 primers were chosen in the same segment of the 16S ribosomal RNA gene sequence and, as such, were combined into a single generic P2 primer for all three targets. Although several attempts 12

315 320 325 330 335 340 345 were necessary, finally a generic P2 primer could be designed that abolished the observed primer interferences and revealed good sensitivity for all three targets when using in vitro generated RNA. The spiking experiments with in vitro generated RNA from multiple targets in 1 tube showed that double infections might not be detected by the real-time multiplex NASBA. Since the number of organisms present in clinical specimens of patients is not known, it is impossible to judge the clinical value of the NASBA reactions studied on the basis of the in vitro sensitivities presented. Until now, in the literature, M. pneumoniae and C. pneumoniae double infections have been seldomly reported and if reported then they were detected by means of serology which has well known specificity and sensitivity problems for the detection of both organisms. Although it is very unlikely that double infections will occur the possibility should be taken into account. Comparison between mono-and multiplex assays has rarely been performed (6, 7, 9, 10, 27, 34). Greijer et al. reported a somewhat lower sensitivity of a real-time multiplex NASBA compared with the mono NASBAs, for the quantification of HCMV IE1 mRNA (9). Corsaro et al. detected 2 CFU of M. pneumoniae in clinical samples by both duplex PCR as well as mono PCR (7). In the study of Welti et al. (34) there was no significant ACCEPTED difference in sensitivity between the multiplex and monoplex PCR assays when tested on dilutions of DNA of C. pneumoniae, L. pneumophila and M. pneumoniae cloned in plasmids. However Tong et al. (27) found that the sensitivity of three PCRs applied in a triplex format decreased by about 1 log as compared with the individual tests. In this study the lower sensitivity of the real-time multiplex NASBA for the detection of M. pneumoniae and Legionella species on spiked respiratory samples was confirmed when both real-time mono- and multiplex NASBA were applied to dilutions of the in wild type in vitro generated RNA and to archived L. pneumophila positive specimens: none of the 4 L. pneumophila positive sputum samples was positive by the multiplex assay. However, it is not clear if this is due to a low number of bacteria present in the samples or to a higher sensitivity of the real-time multiplex NASBA to inhibition. For C. pneumoniae, only three PCR positive specimens could be tested. From the 51 specimens from 33 M pneumoniae PCR positive patients, 40 were PCR positive upon re-extraction-amplification. Within this group 15 specimens were negative 13

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