Performance Comparison of ResPlex II and xTAGTM RVP Fast for Detecting Respiratory Viruses

S.Chong1, , L. Louie2 , A. Simor2,3, A. Petrich1,4, M. Smieja1,5, J. Mahony1, 4

St. Josephs Healthcare Hamilton1, Sunnybrook Health Science Centre2, University of Toronto3, Toronto, ON, and the Departments of Pathology & Molecular Medicine4 and Medicine5, McMaster University, Hamilton, ON, Canada INTRODUCTION
Multiplex PCR tests have recently been introduced and these assays can detect upwards of 20 different respiratory virus types and subtypes with improved sensitivity over traditional methods. Two commercially available multiplex assays that employ the Luminex xMAP system include the xTAGTM RVP Fast assay (CE Mark/Health Canada approved), a second generation xTAGTM RVP assay with a faster assay time and the ResPlex II (v. 2.0) , an RUO assay from Qiagen. Currently, very little comparative data is available on these newer assays. While assessing individual sensitivities for each viral target (see Figure 1), we found the ResPlex II missed 7/35 rhinoviruses. In addition, ResPlex II had a problem differentiating between rhinovirus and enterovirus; 22/35 rhinoviruses were identified as enterovirus and 2 specimens identified as enterovirus were sequenced as rhinovirus. The ResPlex II also missed 10/39 (25.6%) pandemic H1N1 influenza A viruses. Of these 10 influenza A misses, 8 were DFA negative, but positive by real-time PCR with crossing thresholds of 27 to 32 cycles. RVP Fast was not as sensitive for parainfluenza viruses, missing 15/43 (34.8%).

OBJECTIVE
To compare the performance of the ResPlex II (v. 2.0) from Qiagen to the xTAGTM RVP Fast from Luminex Molecular Diagnostics.

Figure 1: Number of Confirmed Positives Detected by the Qiagen ResPlex II (v 2.0) and the Luminex xTAGTM RVP Fast

METHODS
Specimens. A total of 298 nasopharyngeal swab (FLOQSwabs; Copan, Spa Brescia Italy) specimens including 100 prospectively collected and 198 stored specimens submitted to the Regional Virology Laboratory at St. Josephs Healthcare (Hamilton, Ontario) during the 2007 to 2009 respiratory seasons were included in this study. Nucleic Acid Extraction. Total nucleic acid was extracted from 200
L of NP specimen spiked with 20
L of the bacteriophage MS2, using the bioMrieux easyMAG TM automated extractor and eluted in 60
L of buffer. Direct Fluorescent Antibody Testing (DFA). NP samples were spun at 2,000 rpm for 10 minutes. Cell pellets resuspended in PBS were spotted onto slides, fixed in acetone and stained with pooled monoclonal antibodies targeting influenza A, influenza B, parainfluenza 1 to 3, respiratory syncytial virus (RSV), adenovirus, and metapneumovirus from Diagnostic Hybrids (Athens, OH ). Influenza A Real-Time PCR. NP samples were additionally tested for influenza A with an in-house real-time PCR assay with specific primers targeting the matrix gene. The final reaction volume of 25
L contained 1x QuantiTect Multiplex RTPCR Master Mix (Qiagen, Mississauga, ON), 0.1
M to 0.2
M primers, 0.1
M to 0.2
M FAM-Iowa Black FQ probe, 0.25
L of QuantiTect Multiplex RT Mix (Qiagen, Mississauga, ON), uracil-N-glycosylase, and 5
L total nucleic acid. Amplification was performed using the Rotor-Gene instrument (Corbett Research, Sydney, Australia) under the following conditions: 50 C x 10 min, 95C x 10 min, 40 cycles of: 94C x 30 s, 60 C x 1 min with data acquisition.

Table 1: Characteristics of the Qiagen ResPlex II (v. 2.0) and the Luminex xTAGTM RVP Fast

Targets

Influenza A Influenza B RSV A and B Coronaviruses 229E, OC43, NL63,HKU-1 Parainfluenza 1, 2, 3, 4 hMPV Coxsackievirus/Echovirus, Rhinovirus Adenovirus B and E Bocavirus Internal control extraction and PCR inhibition control Positive (IDS) control in vitro transcribed RNA corresponding to a human genomic DNA sequence 30-45 minutes 5 hours

Influenza A (seasonal H1, H3 subtypes) Influenza B RSV Coronaviruses 229E, OC43, NL63, HKU-1; Parainfluenza 1, 2, 3, 4 hMPV Entero-Rhinovirus Adenovirus Bocavirus Internal control- MS2 to control for extraction and the reverse transcription steps Run control- Lambda to control for PCR amplification and detection steps 50 minutes 3.5 to 4 hours

Controls (included in the kit)

Hands-On Time (for 96 rxns) Uniplex RT-PCR. For discordant results between the 2 assays evaluated, and for viral targets that were not tested for by DFA, an in-house uniplex RT-PCR was performed. The final reaction volume of 50
L contained 1x OneStep RT-PCR Buffer (Qiagen, Mississauga, ON) with 2.5 mM MgCl2, 0.4 mM dNTP, primers at a final concentration of 0.6
M, 2
L of OneStep RT-PCR Enzyme Mix (Qiagen, Mississauga, ON) and 5
L total nucleic acid. Amplification was performed using the, PTC-200 thermocycler (MJ Research) under the following conditions: 50C x 30 min, 95 C x 15 min, 40 cycles of: 95 C x 30 s, 50 C or 52C x 30 s, 72C x 30 s, and a final extension at 72C for 2 min. Products were visualized on a 2% (w/v) agarose gel with ethidium bromide staining under UV detection. Total Turn Around Time (for 96 rxns, post-extraction) Regulatory Status

Number of Positives Detected

Qiagen ResPlex II (v. 2.0)

xTAGTM RVP Fast

Viral Targets RUO CE Mark/Health Canada Approved

RESULTS
Qiagen ResPlex II (v. 2.0). The assay was performed as per manufacturers instructions. A 10
L volume of extracted nucleic acid was amplified in a single multiplex RT-PCR reaction, performed in a GeneAmp 9700 thermocycler (Applied Biosystems Incorporated, Foster City, CA). A 5
L aliquot of the amplified products was added to the bead mix and incubated at 52C for 10 min in a 96-well microtitre plate, followed by addition of the streptavidin-phycoerythrin conjugate and another incubation for 5 min at 52C. Stopping buffer was added and the reactions were read on the Qiagen LiquiChip 200 system. Signals were generated for each bead population and expressed as median fluorescence intensity (MFI). Using the MDD software provided, the presence of absence of viral targets and/or controls in each sample was determined. Of the 298 specimens tested, 179 were positive for one virus, 27 were positive for two viruses and 2 were triple positives for a total of 239 positives. These included the following: 58 influenza A (9 seasonal H1N1, 39 pandemic H1N1, 10 seasonal H3N2), 9 influenza B, 14 parainfluenza 1, 18 parainfluenza 2, 11 parainfluenza 3, 29 respiratory syncytial virus A, 6 respiratory syncytial virus B, 12 adenovirus, 11 metapneumovirus, 35 rhinovirus, 5 enterovirus, 9 229E, 3 OC43, 8 NL63, 4 HKU-1 and 7 bocavirus. With a combined gold standard of positive by 2 or more tests, the overall sensitivity and specificity of ResPlex II and RVP Fast were similar at 92.1% (220/239), 99% (295/298), 90.4% (216/239), and 99.7% (297/298) respectively (Table 2). Table 2: Sensitivity and Specificity of the Qiagen ResPlex II (v 2.0) and the Luminex xTAG TM RVP Fast 3. 1.

SUMMARY
In evaluating the performance of the Qiagen ResPlex II (ver. 2.0) and the xTAGTM RVP Fast from Luminex Molecular Diagnostics, we found the assays to be comparable with similar sensitivities (92.1% vs 90.4%) and specificities (99.0% vs 99.87%) for this data set.

2.

Luminex xTAGTM RVP Fast. The assay was performed according to manufacturers protocol. Briefly, 10
L of nucleic acid was amplified in a single multiplex RT-PCR to generate amplicons ranging in size from 58 to 206 bp. A 2
L aliquot of the RT-PCR product was then added to a hybridization/detection reaction containing the universal array and the Streptavidin, RPhycoerythrin conjugate. Each bead population detects a specific viral target by virtue of a specific anti-tag/tag hybridization. Following the 20 minute incubation at 45C of the RT-PCR products with the bead mix and reporter, the reactions were read on the Luminex 100 IS instrument. Signal readings expressed as median fluorescence intensity (MFI), were generated for each bead population. These fluorescence values were analyzed with the TDAS RVP Fast software to determine the presence or absence of viral targets and/or controls in each sample.

The assays differed on their performance for individual targets. The ResPlex II (v 2.0) showed reduced sensitivity for influenza A, particularly the pandemic H1N1 subtype, as well as difficulty distinguishing rhinoviruses from enteroviruses. The RVP Fast had a lower sensitivity for parainfluenza viruses.

Qiagen ResPlex II (v. 2.0)

xTAGTM RVP Fast

Assay performance, ease-of-use, turn around time and regulatory status are all important factors to consider in choosing a multiplex PCR assay. These multiplex PCR assays should assist clinical laboratories in identifying respiratory virus infections in hospitalized patients, aid in patient management, and provide assistance for outbreak investigations.

Sensitivity Specificity

220/239 (92.1%) 295/298 (99.0%)

216/239 (90.4%) 297/298 (99.7%)