Comparison of the Luminex xTAG Respiratory Viral Panel with In-House Nucleic Acid Amplification Tests for Diagnosis of Respiratory Virus Infections

Abstract

Detection of respiratory viruses using sensitive real-time nucleic acid amplification tests (NATs) is invaluable for patient and outbreak management. However, the wide range of potential respiratory virus pathogens makes testing using individual real-time NATs expensive and laborious. The objective of this study was to compare the detection of respiratory virus targets using the Luminex xTAG respiratory viral panel ( The sensitivity and specificity of respiratory virus detection have improved considerably with the advent of nucleic acid amplification tests (NATs). There have been several recent reviews of the use of NATs for detection of respiratory viruses that outline the methodologies, advantages, and future directions (4, 5, 8). The fluorogenic probe-based real-time assays are particularly useful for detection of a small number of targets. However, multiplexing real-time NATs can be technologically challenging and can result in a loss of sensitivity. The reagents for these assays are expensive, and setup for individual targets is time-consuming. Multiplex assays for amplification and detection of a panel of respiratory viruses using suspension microarrays may provide a practical solution. Currently, up to 100 different spectrally distinct fluorescence-labeled beads are available for multiplex target detection on the Luminex suspension microarray platform (3). A different nucleic acid probe can be conjugated to each bead, and a mixture of beads is used for detection and differentiation of products amplified in a multiplex PCR. The technology is flexible since more probes can be added or replaced by mixing individual beads, making it ideal for complex analyses, such as those needed for respiratory virus detection.The development of kits for the detection of a panel of respiratory viruses using suspension microarrays has been described (1, 2, 9, 11-13, 16). These assays show good (and in some cases enhanced) performance compared with traditional antigen and culture methods for respiratory virus detection. Based on these studies, it was thought that these assays may be a suitable replacement for in-house NATs with good sensitivity, specificity, and savings in cost and time. Array approaches also have the added benefit of broad viral detection compared with our in-house NAT panel. The Luminex xTAG respiratory viral panel (RVP) used in this evaluation allows detection of a panel of 20 respiratory virus targets with differentiation between 19 of these (enteroviruses and rhinoviruses are detected by the generic picornavirus probe) (14). Here we report the results of a prospective and retrospective evaluation of the RVP assay compared with a panel of in-house real-time NATs.

Comparison of three multiplex PCR assays for detection of respiratory viruses: Anyplex II RV16, AdvanSure RV, and Real‐Q RV

“…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.…”

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

“…A previous article 115 describing the human RVP c assay used a kappa score analysis to assess the level of agreement between real-time PCR and the BBMA where a kappa score of 1.0 indicates almost perfect agreement. 143 Of the 6 viral targets that were compared by real-time PCR and BBMA, the kappa scores were 0.97-1.0 for 4 targets but were lower for 2 viral targets (i.e., adenoviruses, kappa score = 0.72; RSV, kappa score = 0.91).…”

“…Bead-based multiplex assay strategies for detecting human respiratory viruses has been reviewed, 81 and these assays are performed routinely in some hospitals. 115 Another commercially available BBMA e has been used for the detection of 6 respiratory bacterial pathogens including Streptococcus pneumoniae, Neisseria meningitidis, encapsulated or nonencapsulated Haemophilus influenzae, Legionella pneumophila, Mycoplasma pneumoniae, and Chlamydia pneumoniae. Although this assay had less sensitivity when compared to real-time PCR, it was useful for surveillance purposes 15 and, when combined with a commercial assay including viral respiratory pathogens f for a 21-plex assay, significant coinfection rates could be established.…”

Comparison of the Eragen Multi-Code Respiratory Virus Panel with Conventional Viral Testing and Real-Time Multiplex PCR Assays for Detection of Respiratory Viruses

“…An exciting recent advance is the development of multiplex molecular assays that allow the simultaneous detection of multiple targets in the same reaction (12,13,15,16). One such test is the xTAG Respiratory Virus Panel, produced by Luminex Molecular Diagnostics (Toronto, ON, Canada).…”