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Lower respiratory tract infections (LRTIs) are the most frequent cause of hospitalization among children worldwide (Ahn K M, et al., 1999; Garbino J, et al., 2004; Ruuskanen O, et al., 2011; Sung C C, et al., 2011; Thompson W W, et al., 2003; van Woensel J B, et al., 2003). A large proportion of LRTIs are caused by respiratory viruses including influenza virus A (FluA) and B (FluB), parainfluenza viruses 1-4 (PIV1-4), respiratory syncytial virus (RSV), rhinoviruses (RhV), enteroviruses (EnV), and adenoviruses (AdV) (Juven T, et al., 2000; Legg J P, et al., 2005; Weigl J A, et al., 2005). Over the past decade, the viral pathogen list has been expanded to several newly discovered viruses including human metapneumovirus (hMPV) (van den Hoogen B G, et al., 2001), some coronaviruses (NL63, HKU1, SARS) (Pyrc K, et al., 2007), human bocaviruses (hBoV) (Allander T, et al., 2005), and pandemic influenza A/H1N1 2009 virus (H1N1-p) (Anonymous, 2009). Various viruses with different shedding levels may result in a large disease severity range from common bronchitis to fatal pneumonia (Li C C, et al., 2010; Martin E T, et al., 2012; Takeyama A, et al., 2012; Torres J P, et al., 2010).
Rapid and accurate etiologic diagnosis of LRTIs is essential to patient management. Several multiplex RTPCR-based devices are commercially available for detection and differentiation of a panel of respiratory viral pathogens (Zhang S, et al., 2011). They are being increasingly used in the clinical setting as a cornerstone technique in the clinical virology laboratory as they possess sensitivities greater than rapid viral antigen testing and test turnaround time shorter than standard respiratory virus culture (Balada-Llasat J M, et al., 2011; Kim S R, et al., 2009; Rand K H, et al., 2011; Schindera C, et al., 2010). Varying sensitivities have been reported between different devices for specific viral pathogens in different studies (Balada-Llasat J M, et al., 2011; Kim S R, et al., 2009; Rand K H, et al., 2011; Schindera C, et al., 2010). In this study, we evaluated the ResPlex Ⅱ V2.0 kit (Qiagen, Germany), which uses a target enriched multiplexing RT-PCR amplification coupled with a suspension array detection, for detection and identification of a panel of respiratory specimens in pediatric inpatients with LRTIs. Clinical accuracy of the ResPlex Ⅱ assay was validated on a panel of prospectively collected consecutive nasopharyngeal swab (NPS) specimens in comparison to viral culture and a monoplex real-time TaqMan RT-PCR. We also correlated the ResPlex Ⅱ assay sensitivity with viral loads determined by the quantitative, monoplex real-time TaqMan RT-PCR.
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A total of 438 qualified NPS specimens collected during a one full year study period were included in the final testing and analysis. The male ratio was 77.6%. The mean age of patients was 1.4 years, ranging from one month to 11 years. Among the total specimens tested, 263 (60.0%), 134 (30.6%) and 41 (9.4%) were from children of < 1, 1-3 and > 3 years old, respectively.
Among the total 438 NPS specimens, one or more viral pathogens were detected in 274 (62.6%) and 201(45.9%) specimens by monoplex TaqMan RT-PCR and multiplex ResPlex RT-PCR, respectively. Viral culture was positive for 56 (12.8%) specimens including 17 FluA, 3 FluB, 14 PIV1, and 22 PIV3. Among these culture positive specimens, monoplex real-time TaqMan RT-PCR results were fully concordant while ResPlex results were negative for 13 specimens including six FluA, four PIV1 and three PIV3. When results from monoplex RT-PCR or cell culture were used as the reference standard, the multiplex PCR possessed specificities ranging from 92.9% to 100.0% for the viruses tested. The sensitivities of multiplex PCR varied with high sensitivity observed for PIV3, hMPV, PIV1 and hBoV (55.6-73.1%) and low sensitivity for FluA, EnV, OC43, RSV and H1N1-p (11.1-40.0%) (Table 1).
Virus No. detected Sensitivity Specificity PPV NPV M+R+ M+R- M-R+ M-R- (%) (%) (%) (%) RSV 46 92 6 294 33.3 98 88.5 76.2 FluA 4 32 2 400 11.1 99.5 66.7 92.6 hMPV 21 9 3 405 70 99.3 87.5 97.8 PIV3 19 7 5 407 73.1 98.9 79.2 98.4 H1N1-p** 10 15 0 289 40 100 100 95.1 PIV1 12 6 3 417 66.7 99.3 80 98.6 hBoV 10 8 0 420 55.6 100 100 98.1 OC43 4 11 1 422 26.7 99.8 80 97.5 EnV 2 12 30 394 14.3 92.9 6.3 97 ADV 4 3 0 431 57.1 100 100 99.3 NL63 3 3 1 431 50 99.8 75 99.3 229E 3 2 1 432 60 99.8 75 99.5 FluB 2 2 0 434 50 100 100 99.5 PIV4 2 1 1 434 66.7 99.8 66.7 99.8 PIV2 0 3 0 435 NA 100 NA 99.3 HKU1 0 0 0 438 NA NA NA NA * RhV was not evaluated as it was not detected by the monoplex real-time RT-PCR. PPV, positive predictive value; NPV, negative predictive value. ** The Resplex Ⅱ Panel v2.0 was replaced by a ResPlex Ⅱ Plus Panel PRE to enhance H1N1-P coverage during the study. A total of 314 samples were tested by the ResPlex Ⅱ Plus Panel PRE version. Table 1. The comparison of ResPlex (R) and monoplex real-time TaqMan RT-PCR (M) for 16 respiratory viruses *
Rhinoviruses were detected from 36 specimens (8.3%) by the ResPlex assay including 22 rhinovirus alone and 14 co-detected with other viruses including RhV/EnV (n=10), RhV/RSV (n=2), RhV/hMPV (n=1), and RhV/229E (n=1). Since rhinoviruses were not detected by the monoplex TaqMan RT-PCR, these data were not further analyzed.
We further correlated viral load information determined by the TaqMan RT-PCR with the ResPlex diagnostic sensitivities for seven viruses/genotypes detected with higher frequencies (positive rate > 4% detected by monoplex RT-PCR). The ResPlex multiplex PCR sensitivities correlated significantly with viral loads for RSV, FluA and H1N1-p (p=0.011-0.000). Significant correlation was not observed for PIV1, PIV3, hBoV, and hMPV (p > 0.05) (Table 2).
Monoplex
copies/mLNo. of ResPlex-PCR positive/No. of Monoplex PCR positive RSV (%) FluA (%) hMPV (%) PIV3 (%) H1N1-p (%) PIV1 (%) hBoV (%) ≥106 6/9 (66.7) 1/1 (100) 3/4 (75.0) 2/3 (66.7) 4/5 (80.0) 0/0 (0) 9/11 (81.8) 1-9.9×105 34/45 (75.5) 2/5 (40.0) 10/14 (71.4) 8/8 (100.0) 5/6 (83.3) 5/5 (100.0) 1/3 (33.3) 1-9.9×104 5/38 (13.2) 1/7 (14.3) 6/9 (66.7) 4/5 (80.0) 1/6 (16.7) 6/7 (85.7) 0/2 (0) 1-9.9×103 1/33 (3.0) 0/12 (0) 2/3 (66.7) 2/4 (50.0) 0/6 (0) 1/3 (33.3) 0/1 (0) 1-9.9×102 0/13 (0) 0/11 (0) 0/0 (0) 3/6 (50.0) 0/2 (0) 0/3 (0) 0/1 (0) Negative 6/300 (2.0) 2/402 (0.5) 3/408 (0.7) 5/412 (1.2) 0/289 (0) 3/420 (0.7) 0/420 (0) z -6.859 -5.119 -1.267 -1.011 -2.538 -1.068 -1.511 p 0.000 0.000 0.205 0.312 0.011 0.285 0.131 Table 2. Relationship of sensitivity of ResPlex PCR and viral loads determined by monoplex PCR
During the study, a new version of ResPlex, ResPlex Ⅱ Plus Panel PRE, was manufactured to increase coverage of H1N1-p. There were 124 and 314 specimens which were tested by ResPlex Ⅱ Panel and ResPlex Ⅱ Plus Panel PRE, respectively (Table 1). For the seven non-H1N1-p viruses/genotypes detected with higher frequencies, the sensitivities dropped (range from 3.6% to 43.7%) for five viruses including FluA, RSV, hBoV, PIV1 and PIV3. The sensitivity increased only for hMPV (Table 3). None of these sensitivity differences reached statistical significance by χ2 or Fisher's exact test (p > 0.05). When the MantelHaenszel χ2 test was used for the combined strata analysis, no significant difference was observed (χM-H2=2.676, p=0.102).
Virus Resplex Ⅱ Panel
No. of specimensSensitivity
(%)ResPlex Ⅱ Plus Panel PRE
No. of specimensSensitivity
(%)χ2
valueP
valueM+R+ M+R- M-R+ M-R- M+R+ M+R- M-R+ M-R- RSV 14 25 3 82 35.9 32 67 3 212 32.3 0.161 0.688 FluA 2 2 2 118 50.0 2 30 0 282 6.3 6.891 0.053 hMPV 4 4 2 114 50.0 17 5 1 291 77.3 2.078 0.195 PIV3 6 0 2 116 100.0 13 7 3 291 65.0 2.874 0.146 PIV1 3 0 2 119 100.0 9 6 1 298 60.0 1.80 0.515 hBoV 5 1 0 118 83.3 5 7 0 302 41.7 2.813 0.152 * R, ResPlex Ⅱ Panel; M, monoplex real-time TaqMan RT-PCR. Table 3. Sensitivity of two kinds of ResPlex Ⅱ kit for common viruses *