key: cord-1045291-eyogf7xh authors: Yun, Seung Gyu; Kim, Min Young; Choi, Jong Moon; Lee, Chang Kyu; Lim, Chae Seung; Cho, Yunjung; Suh, In Bum title: Comparison of three multiplex PCR assays for detection of respiratory viruses: Anyplex II RV16, AdvanSure RV, and Real‐Q RV date: 2017-04-11 journal: J Clin Lab Anal DOI: 10.1002/jcla.22230 sha: d9e24a56fd02e643de973aab5753dbf7accc1e90 doc_id: 1045291 cord_uid: eyogf7xh BACKGROUND: Due to its great sensitivity, the nucleic acid amplification test (NAAT) is widely used for detection of respiratory viruses (RV). However, few reports have described a direct comparison between multiplex RT‐PCR assays for RV. The objective of this study was to perform a direct comparison of three multiplex RT‐PCR assays for the detection of respiratory viruses. METHODS: A total of 201 respiratory samples (161 nasopharyngeal swab samples and 40 sputum samples) were tested with three commercial RV assays: Seegene Anyplex II RV16 (AP), LG AdvanSure RV (AD), and Biosewoom Real‐Q RV (RQ). The additional tests for the discrepant results were conducted by repeat RV assay or monoplex PCR coupled direct sequencing. Data analysis using percent agreement, kappa, and prevalence‐adjusted and bias‐adjusted kappa (PABAK) values was performed for comparisons among the three RV assays. RESULTS: Of the 201 samples, AP, AD, and RQ detected 105 (52.2%), 99 (49.3%), and 95 (47.3%) positive cases respectively. The overall agreement, kappa, and PABAK values for the three assays ranged between 97%‐98%, 0.76‐0.86, and 0.93‐0.96 respectively. The performance of the three assays was very similar, with 94%‐100% agreement for all comparisons, each virus types. The additional testing of samples showed discrepant results demonstrating that AD assay had the highest rate of concordance with original results. CONCLUSIONS: We suggest that all multiplex assay would be suitable for the detection of for respiratory viruses in clinical setting. available faster than those of viral cultures. Speed to result allows medical professionals to prescribe antiviral agents and perform appropriate infection control more rapidly. Lastly, NAAT does not need a strict transportation protocol for maintaining viability of RV. 3 In addition, the application of multiplexing technology in NAAT allows better detection than classical methods of a broad range of viruses. For all of these reasons NAAT has replaced classical methods. There are a number of commercial multiplex kits that can detect between 12 and 23 virus types. 3, 4 Performance evaluation studies for newly developed multiplex RV kits are weak, do not establish the reference standard method and therefore do not sufficiently calculate sensitivity and specificity of each test. Instead, some studies have suggested the reference test with in-house multiplex real-time PCR or commercial duplex PCR tests. [5] [6] [7] [8] We performed a direct comparison of three commercial multiplex assays and produced the values of the agreement and kappa instead of sensitivity and specificity with the reference tests. The objective of this study was to perform a direct comparison of three multiplex RT-PCR assays for the detection of respiratory viruses. swab samples (n=161) and sputum samples (n=40) and were stored at −70°C until this study was conducted. Nasopharyngeal swab samples were obtained using flocked swabs and transported in 3 mL universal transport medium (COPAN Diagnostics, Murrieta, CA, USA). Sputum samples were received in sterile plastic containers and treated in order to homogenize samples using a 1:1 ratio dithiothreitol, which was diluted 1:100 with distilled water because of it viscosity. This study was approved by the Ethics Committee of Armed Forces Medical Command (AFMC-16-IRB-023). The nucleic acid extraction system was used for each RV assay. For Three commercial multiplex RV assays were performed based on manufacturers' protocols. Characteristics of the three multiplex assays are briefly reported in Table 1 . The AP assay is composed of two-step RT-PCR. Before multiplex target PCR was performed, com- The RQ assay is also a one-step RT-PCR method. Briefly, the assay was multiplexed into five tubes targeting 14 RVs and 1 IC gene, which was human RNase P, like in the AD assay. RQ was conducted in a 22 μL mixture containing 5 μL of nucleic acid, 12.5 μL of 2X PCR reaction mixture, 1 μL of RT-PCR enzyme, and 3.5 μL of water with CFX96 Real-time PCR detection system (Bio-rad). The RT-PCR reaction was performed with the following conditions for the RT step: 50°C for 30 minutes; 95°C for 15 minutes, followed by 45 cycles of 95°C for 15 seconds and 62°C for 45 seconds. A flow diagram of discrepant analysis is described in Figure 1 . In summary, 167 samples (83.1%) with positive results from at least two RV assays or negative results from all three assays were not tested with the additional assay. However, the other 34 samples (16.9%), containing 36 discordant results showing a single positive result in any assay, were tested with the following additional assay. A single positive result from AP and RQ assay was retested individually with RV assay. Whereas a single positive result from AD assay was performed with monoplex PCR followed by direct sequencing. If the results between the original and additional tests were concordant, they were considered to be a consensus of positive results. Conversely, if the results between original and additional tests showed a discordance, they were considered to be a negative consensus. Because both AD and RQ assay cannot detect PIV4 and EV, the results about PIV4 and EV were excluded from the discrepant analysis. Statistical methods of inter-rater agreement, including percent agreement and kappa statistics, were calculated to compare the three RV assays. In addition, prevalence-adjusted and bias-adjusted kappa (PABAK) was calculated to compensate for underestimation of kappa values caused by low prevalence of each respiratory type. 9 The kappa value can be interpreted as follows: <0.20 as poor; 0.21-0.40 as fair; 0.41-0.6 as moderate; 0.61-0.8 as good and 0.81-1 as very good agreement. 10 Statistical analyses were performed using Microsoft T A B L E 1 Summary of characteristics in multiplex assays for detection of respiratory viruses Statistical analysis using percent agreement between RV assays in each virus type were high, ranging from 94% to 100%. Overall agreement between three RV assays ranged from 97% to 98%. Overall distribution of respiratory viruses from 3 RV assays is presented in Table 4 . There are indications of the predominance of INFA, ADV, HRV, and MPV. Excluding these four major types, the rest of RV types accounted for only 0.8% to 8.9% of total viruses identified. The current study was performed for direct comparison between commercial multiplex RT-PCR for detection of respiratory viruses. A high degree agreement was found between AP, AD, and RQ assays. MPV, RSV, and ADV ranged from 94% to 100% ( against conventional multiplex RT-PCR. 16 Similarly, the performance evaluation of AD vis-à-vis a composite standard method revealed the most sensitive performance compared with viral culture and Seeplex RV15. 17 The performance study of RQ assay was not found in the pubmed database, but evaluation of a previous version (1-step RV real-time PCR) against direct sequencing as a standard method reported 94.1% sensitivity and 96.6% specificity. 18 We demonstrated that the agreement between three assays was excellent. Moreover, the end-user in clinical laboratories selecting the multiplexed RT-PCR for RV will need to consider the benefits of each assay in terms of both performance and user-friendliness. In the analysis of discrepant samples, 18 ples. This could skew results of RV detection based on storage conditions and the defrosting procedure. However, since all specimens were stored at the same storage condition and were processed by one skilled technician, we presume that comparison results between three assays was not significantly influenced by this characteristic of the sample. In conclusion, the agreement of the three assays were very good, with 94%-100% agreement for all comparisons. We suggest that all multiplex assay would be suitable for the detection of for respiratory viruses in clinical setting. 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