A variety of clinical diagnostic assays utilizing different methods and targets for RV assessment have been reported, but cell culture is still considered the "gold standard". However, this method has drawn criticism for difficulties such as maintaining cell cultures, instability of cultured cells, and a long turnaround cycle, from 1 to as many as 14 days [23
]. The prolonged turnaround time of this diagnostic method for confirming RVs in clinical samples is an obstacle to rapid initiation of antiviral therapy, isolation of infected patients, and/or cessation of the unnecessary use of antibiotics [27
]. The rapid diagnosis of viral infections has depended mainly on viral antigen detection; however, the sensitivities of these assays vary (50-90%), depending on the assay method and the virus of interest [28
]. For these reasons, rapid, easy-to-perform, sensitive, specific, and cost-effective diagnostic techniques are increasingly needed in the clinical microbiology laboratory.
Multiplex real-time RT-PCR is a recognized technique offering a faster and effective technology for the rapid detection of viruses than the conventional diagnostic methods of virus culture, antigen tests, or direct immunofluorescence [29
]. In addition, multiplex RT-PCR assays that are able to target several types of respiratory pathogens have been reported previously [30
]. In this study, we evaluated the performance of the one-step AdvanSure multiplex real-time PCR assay for common respiratory viral pathogens, which include HCoV-229E/NL63, HCoV-OC43, PIV-1, PIV-2, PIV-3, FluA, FluB, hRSV-A, hRSV-B, ADVs, and hRVs and compared it to that of a conventional multiplex RT-PCR assay. The one-step AdvanSure assay provides a faster turnaround time because it avoids additional nested amplification or hybridization steps needed for identification of viral products. Moreover, the AdvanSure assay minimizes the possibility of contamination as it eliminates the need for additional post-PCR processing of the samples and the 11 viral targets could be detected simultaneously.
The analytical sensitivity of the AdvanSure assay demonstrated its clinical potential, in particular for diagnosis of HCoV-229E, HCoV-NL63, PIV-3, FluA, hRSV-A, ADVs, and hRVs (1-10 PFU/mL). These LOD levels for RVs are similar to values previously reported by other investigators [30
]. The LOD levels for each of the viral targets have also been reported for FDA-approved RV assay kits [19
]. Although the AdvanSure assay had higher LOD values for FluB and hRVs than those of the FDA-approved RV assay kits, it had lower LOD values for ADVs. For other viral targets, the LODs from the AdvanSure assay proved to be comparable to those associated with the FDA-approved RV assay kits. In general, the multiplex RT-PCR assay for the detection of RVs supports the proper primer against the RVs and their mutations and the sensitivity of the RT-PCR assays depend on the degree of sequence identity/similarity between the primers and the genomes of the tested viruses [33
]. In this study, the higher LODs obtained for FluB and hRVs determinations likely reflect unsuitable or sub-optimal primer sequences for these viruses in the AdvanSure assay kit against the tested standard viruses.
Our evaluation of specificity or cross-reactivity in the AdvanSure assay against 23 reference strains indicated that none of the samples gave a false positive reaction. These outstanding results obtained from our trial of the AdvanSure assay clearly demonstrate its clinical potential compared with the specificities recently reported for alternative detection methods [33
To determine the clinical performance of the one-step AdvanSure multiplex real-time PCR assay for the RVs, we compared the results of the AdvanSure assay to those of a conventional multiplex RT-PCR assay by using 320 collected clinical samples. The sensitivity of the conventional multiplex RT-PCR was 44.06% (141/320), while the sensitivity of the AdvanSure assay was 44.68% (143/320). The main advantage of the AdvanSure assay is the ability to identify 2 additional RVs not detected by the conventional RT-PCR assay, including hMPVs and HBoV. Although, the 2 kinds of multiplex RT-PCR assays showed equivalent positive rates overall, 6 samples that had different results with the 2 assays were identified. Two samples that were positive for FluA, 1 for HCoV-NL63, and 1 for hRVs by the AdvanSure assay were identified as negative by the conventional RT-PCR assay. Two samples that were positive for PIV-2 by the conventional multiplex RT-PCR assay were identified as negative by the AdvanSure assay. For resolving the discrepancies between the 2 assays for these 6 samples, nested real-time PCR and gene sequencing were performed to confirm that the results were indeed true positive and not false-positive. The results of nested RT-PCR and gene sequencing indicated that the samples classified as positive by either assay were indeed positive, and that both assays had, therefore, yielded false-negative results for these samples. This discrepancy may result from the limitations of multiplex PCR assays. One of the major limitations is the assay principle of multiplex PCRs, which could produce false results if a primer region has nucleotide variations and is unable to detect new types or strains of a virus. This is the reason that direct antigen tests or virus cultures cannot be completely replaced by multiplex PCR assays [33
]. Another limitation is the false positive result as a consequence of exposure to amplified PCR products during a conventional 2-step RT-PCR [28
]. Although the conventional multiplex RT-PCR kit used in this study featured a dual priming oligonucleotide system, it still yielded higher false-positive results than the one-step AdvanSure assay, and these findings agree with those of a previous publication [37
In conclusion, the AdvanSure RV real-time PCR assay demonstrated excellent overall sensitivity and specificity. Furthermore, the results of this assay may be obtained within 4 h, requiring minimal technician time. It offers clinical laboratories a valuable option for detection of RVs potentially improving clinical management with earlier diagnosis and treatment.