Real-world comparison of two molecular methods for detection of respiratory viruses

S Asad Ali,Tressa Pappas,Marie R Griffin,Tebeb Gebretsadik,Wai Ming Lee,E Kathryn Miller,James E Gern,John V Williams,Tina V Hartert,Kathryn M Edwards

doi:10.1186/1743-422x-8-332

S Asad Ali, Tressa Pappas + Show 8 more

Open Access

https://doi.org/10.1186/1743-422x-8-332

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Abstract

BackgroundMolecular polymerase chain reaction (PCR) based assays are increasingly used to diagnose viral respiratory infections and conduct epidemiology studies. Molecular assays have generally been evaluated by comparing them to conventional direct fluorescent antibody (DFA) or viral culture techniques, with few published direct comparisons between molecular methods or between institutions. We sought to perform a real-world comparison of two molecular respiratory viral diagnostic methods between two experienced respiratory virus research laboratories.MethodsWe tested nasal and throat swab specimens obtained from 225 infants with respiratory illness for 11 common respiratory viruses using both a multiplex assay (Respiratory MultiCode-PLx Assay [RMA]) and individual real-time RT-PCR (RT-rtPCR).ResultsBoth assays detected viruses in more than 70% of specimens, but there was discordance. The RMA assay detected significantly more human metapneumovirus (HMPV) and respiratory syncytial virus (RSV), while RT-rtPCR detected significantly more influenza A. We speculated that primer differences accounted for these discrepancies and redesigned the primers and probes for influenza A in the RMA assay, and for HMPV and RSV in the RT-rtPCR assay. The tests were then repeated and again compared. The new primers led to improved detection of HMPV and RSV by RT-rtPCR assay, but the RMA assay remained similar in terms of influenza detection.ConclusionsGiven the absence of a gold standard, clinical and research laboratories should regularly correlate the results of molecular assays with other PCR based assays, other laboratories, and with standard virologic methods to ensure consistency and accuracy.

Highlights

Molecular polymerase chain reaction (PCR) based assays are increasingly used to diagnose viral respiratory infections and conduct epidemiology studies
The performance of molecular diagnostic methods, either RT-rtPCR or multiplex assays, typically has been evaluated by comparing them with conventional direct fluorescent antibody (DFA) or culture, with few direct comparisons reported between molecular assays
Restricting the analysis to the 11 viruses tested with both methods, at least one virus was detected in 174 (78.3%) samples by RMA and in 163 (73.4%) samples by RT-rtPCR

Summary

Introduction

Molecular polymerase chain reaction (PCR) based assays are increasingly used to diagnose viral respiratory infections and conduct epidemiology studies. Molecular assays have generally been evaluated by comparing them to conventional direct fluorescent antibody (DFA) or viral culture techniques, with few published direct comparisons between molecular methods or between institutions. Molecular detection techniques have allowed previously known viruses to be more reliably identified and new viruses to be discovered[1,2,3,4,5,6,7,8,9,10] Molecular techniques such as real-time RT-PCR (RT-rtPCR) can be performed for each individual virus, or they can be combined into a multiplex RT-rtPCR assay. The performance of molecular diagnostic methods, either RT-rtPCR or multiplex assays, typically has been evaluated by comparing them with conventional direct fluorescent antibody (DFA) or culture, with few direct comparisons reported between molecular assays. Wisconsin-Madison) with RT-rtPCR (Vanderbilt University) for the detection of 11 common respiratory viruses

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