Respiratory Virus Plus*

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Literature Cited

" Simultaneous detection of influenza A and its subtypes (H1, H3, 2009 H1N1), influenza B, and RSV A and B in respiratory specimens on an automated, random access, molecular platform"
Clinical Microbiology and Infection
04 May 2011
Full data
B. Buchan, N. Anderson, P. Jannetto, N. Ledeboer
Volume 17: Issue Supplement s4
Abstract
Introduction: Multiplexed molecular detection of influenza A and B and RSV is becoming increasingly available with a number of commercial assays on the market. However, there are no ‘sample-to-result’ platforms that detect these viruses along with influenza A and RSV subtypes.
Numerous studies have demonstrated increased sensitivity of these techniques compared to culture and antigen detection methods. This study evaluated the sensitivity and specificity of Nanosphere’s Verigene System, with the Processor SP, for the detection of these respiratory
viruses.
Methods: Nasopharyngeal specimens were collected at multiple sites during the 2008−9 and 2009−10 respiratory seasons. The presence of influenza A/B and RSV was determined using culture/DFA. Influenza A and RSV subtypes were determined using bi-directional sequencing. Discrepant results were resolved using bi-directional sequencing. The culture/DFA and bi-directional sequencing results for the samples were blinded to the investigators until the conclusion of the study.
Results: Of the 323 specimens enrolled in the clinical trial, 121 were culture/DFA positive for Influenza A, 14 were positive for influenza B, 33 were positive for RSV, and 155 specimens were culture/DFA negative. In comparison to culture/DFA for influenza A, the RV+ assay was 100% sensitive and 97.0% specific. For influenza B, the RV+ assay was 100% sensitive and 100% specific. For RSV, the RV+ assay was 97.0% sensitive and 99.3% specific. There were 9 discrepant specimens between RV+ and culture/DFA. Bidirectional sequencing revealed that the RV+ assay resulted in 8 additional true positives which were otherwise negative by culture/DFA, and 1 additional true negative which was resulted as positive by culture/DFA. All positive influenza A samples were subtyped as 2009 H1N1 by sequencing (n = 127). The RV+ assay performance for this subtype was 99.2% sensitivity and 100% specificity. For the H1 and H3 subtypes, the specificities ranged from 99.7–100%. For the RSV A and B subtypes, the sensitivities and specificities were 100% and 100%, respectively, for both subtypes.
Conclusion: The RV+ assay for detection and subtyping of respiratory viruses offers a rapid result time of ~2.5 hours with sensitivity and specificity equal to or greater than traditional culture-based detection methods. Additionally, the fully automated platform allows simple, on demand assay set-up which permits workflow flexibility and requires minimal hands on time.
Link to poster
Buchan et al ECCMID 2011 Poster.pdf
"Random Access, Simultaneous Detection of Influenza A and its Subtypes (H1, H3, H1N1 2009), Influenza B, and RSV Subtypes A and B in Respiratory Specimens Using Nanoparticle Probes"
10 May 2011
Full data
Blake W. Buchan, Paul J. Jannetto, Neil W. Anderson, Jane S. Gibson, Roy W. Tarnuzzer, Tina Schmeisser, Ray Mills, Sandra Kindig, Stephen Young, and Nathan A. Ledeboer
Abstract
Introduction:  Multiplexed molecular detection of influenza A, influenza B, and respiratory syncytial virus (RSV) is becoming increasingly available with a number of commercial assays on the market. However, there are no ‘sample-to-result’ platforms available that detect these 3 respiratory virus types along with influenza A and RSV subtypes (H1, H3, H1N12009) and (A and B), respectively. Numerous studies have demonstrated increased sensitivity of nucleic acid-based techniques compared to culture and antigen-based detection methods. This study evaluated the sensitivity and specificity of the Verigene Respiratory Virus Plus Nucleic Acid Test (RV+) (Nanosphere, Northbrook, IL) for the detection of these respiratory viruses and subtypes.
Methods:  A total of 1022 nasopharyngeal specimens were collected prospectively at three geographically distinct sites during the 2008-9 and 2009-10 respiratory seasons. The presence of influenza A/B and RSV was determined at the time of collection using culture/DFA techniques.  Subtyping for influenza A and RSV was accomplished using bidirectional sequencing.  Residual frozen samples were divided among 3 test sites which conducted testing on each specimen using the RV+.  The culture/DFA and bidirectional sequencing results were blinded to the study investigators until the conclusion of the study.  Discrepant results were resolved using bi-directional sequencing.
Results:  Across all 3 test sites, 988 of 1022 specimens generated a definitive result after initial analysis (initial call rate 96.7%).  All but 2 of these samples generated a definitive result upon retest (final call rate 99.8%).  Of the 1022 specimens enrolled in the clinical trial, 315 (30.8%) were culture/DFA positive for influenza A, 43 (4.2%) were positive for influenza B, 107 (10.5%) were positive for RSV.  In comparison to culture/DFA for influenza A, the RV+ assay was 98.7% sensitive and 93.2% specific. Upon resolution of 52 discrepant results, the RV+ sensitivity and specificity were 100%.  Notably, this included the detection of an additional 44 influenza A positive specimens missed by culture/DFA.  For influenza B, the RV+ assay was 100% sensitive and 99.7% specific. Specificity was 100% following discrepant resolution.  For RSV, the RV+ assay was 99.1% sensitive and 100% specific. Of the 354 sequence confirmed influenza A specimens, 207 were subtyped as 2009 H1N1. The RV+ assay identified 206 of the 207 2009 H1N1 specimens, indicating 99.5% sensitivity and 100% specificity for this subtype. For the H1 (n=39) and H3 (n=108) subtypes, the sensitivity and specificity was ≥ 99.9%.  The RV+ assay was also 100% sensitive and specific for identifying RSV A (n=57) and B (n=53) subtypes.
Conclusion:  The RV+ assay for detection and subtyping of respiratory viruses offers a rapid result time of ~2.5 hours with sensitivity and specificity equal to or greater than traditional culture-based detection methods.  Additionally, the fully automated platform allows simple, on demand assay set up which permits workflow flexibility and requires minimal hands on time.
Link to Poster
Buchan et al CVS 2011 Poster.pdf
"Analytical Reactivity of the Nanosphere Verigene® Respiratory Virus Plus Nucleic Acid Test (RV+) Utilizing a Panel of Heat Inactivated H5 and H7 Influenza Reference Strains "
10 May 2011
Full data
Roy W. Tarnuzzer, Jane S Gibson, Tina Schmeisser, Jennifer Rannings, Sudhakar Marla, and Teresa Raich
Abstract
Introduction: The Verigene^® Respiratory Virus Plus Nucleic Acid Test (RV+) is an FDA cleared sample-to-answer, nucleic acid amplification, array hybridization, nanoparticle probe-based assay for the detection of influenza and RSV.  The RV+ assay detects and differentiates influenza A, influenza A subtypes (H1, H3, and 2009 H1N1), influenza B, and RSV subtypes (A and B).  The purpose of this study was to determine the range of analytical reactivity of the RV+ assay inclusive of influenza H5 and H7 subtypes.
Methods: For this analytical reactivity study, three (3) H5N1 and two (2) H7N7 influenza A viral subtypes were prepared as follows.  Viruses were grown, titered, heat inactivated, and confirmed sterile prior to use. Qualification of the heat inactivated virus stocks was performed using real-time RT-PCR on an ABI 7500^®utilizing primers and probes specific for the Influenza A M2 gene. Additionally, subtypes were confirmed using primers and probes specific for H5 and H7 gene markers. Relative titers (TCID₅₀/mL) of the inactivated virus were calculated based on the differences in the Ct values between RNA amplified from live virus prior to inactivation and the subsequent inactivated virus stocks.  A known positive Influenza A clinical sample was included in each qualification run both as a positive control and as a benchmark for typical clinical titer. Upon qualification, dilutions of 1:1000, 1:10,000 and 1:100,000 for each inactivated virus subtype were made fresh prior to each run.   Two hundred microliters of each dilution was run in triplicate on the RV+ test.
Results: Heat inactivation had a slight effect on relative virus titer as detected at the nucleic acid level.  On average, there was a 1 log loss (approximately 3 Ct) in relative titer due to the inactivation of the 5 virus subtypes.  The qualified inactivated viruses were tested in three (3) concentrations with relative titers ranging from 5.3 E⁰ to 2.8 E⁶ (TCID₅₀).  The H5 and H7 strains gave a ‘Detected’ result for Influenza A and a ‘Not Detected’ result for H1, H3, and 2009 H1N1. As expected, these strains were unsubtypeable in the RV+ assay. No cross-reactivity with H1, H3, or 2009 H1N1 was observed at any of the dilutions, including higher concentrations, further demonstrating good assay specificity. In addition, all extracts produced from the RV+ assay were confirmed as either H5 or H7 and relative virus titers confirmed by real-time RT-PCR.
Conclusion: All dilutions of the H5 and H7 heat inactivated virus samples produced positive results and appropriate dose responses in the RV+ assay.  Based on these results, the Nanosphere Verigene^® RV+ test has the ability to detect a wide range of influenza A subtypes with high sensitivity and specificity.
Link to Poster
Tarnuzzer et al CVS 2011 Poster.pdf
"Real-time detection of influenza A, influenza B, and respiratory syncytial virus A and B in respiratory specimens by use of nanoparticle probes"
Journal of Clinical Microbiology
01 November 2010
Full data
Jannetto PJ, Buchan BW, Vaughan KA, Ledford JS, Anderson DK, Henley DC, Quigley NB, Ledeboer NA
48(11):3997-4002
Abstract
Seasonal epidemics of influenza and respiratory syncytial virus are responsible for significant morbidity and mortality worldwide. Infrequently, novel or reemergent strains of influenza A virus have caused rapid, severe global pandemics resulting in millions of fatalities. The ability to efficiently and accurately detect and differentiate respiratory viruses is paramount for effective treatment, infection control, and epidemiological surveillance. We evaluated the ability of two FDA-cleared nucleic acid-based tests, the semiautomated respiratory virus nucleic acid test (VRNAT) and the fully automated respiratory virus nucleic acid test SP (RVNAT(SP)) (Nanosphere Inc., Northbrook, IL) to detect influenza A virus, influenza B virus, and respiratory syncytial virus A and B (RSV A/B) from clinical nasopharyngeal swab specimens. Detection of viral RNA in both tests is based on nucleic acid amplification followed by hybridization to capture probes immobilized on a glass slide. A novel technology utilizing gold nanoparticle-conjugated probes is utilized to detect the presence of captured target DNA. This microarray-based approach to detection has proven to be more sensitive than the traditional culture/direct fluorescent-antibody assay (DFA) method for detecting RSV and influenza viruses in clinical specimens, including the novel 2009 H1N1 strain. Specifically, we report 98.0% sensitivity and 96.5% specificity for the VRNAT compared to culture/DFA. Further, the VRNAT detected virus in an additional 58% of specimens that were culture negative. These data were confirmed using bidirectional sequencing. Evaluation of the fully automated RVNAT(SP), which is built on the same detection technology as the VRNAT but contains an updated processor enabling complete automation, revealed the two tests to be functionally equivalent. Thus, the RVNAT(SP) is a fully automated sample-to-result test capable of reliable detection of select respiratory viruses directly from clinical specimens in 3.5 h.
Link to Article
"The Clinical Significance of a Rapid and Sensitive Test for Detection of Influenza Virus Infection"
16 November 2011
Full data
Nakao A, Hisata K, et al
Abstract
In Japan, immunochromatography is the diagnostic test of choice in the diagnosis of influenza virus infection. However, its sensitivity is affected by the time between onset of disease and when testing is performed. On the other hand, PCR has significantly higher sensitivity; however, testing is performed in the laboratory and results are not available on a timely basis to have clinical impact. During the period December 2010 to April 2011, three hospitals affiliated with Juntendo University assessed a bed-side, high sensitivity molecular assay (Verigene RV+, Nanosphere, USA) against immunochromatography with respect to impact on patient management. A total of 103 pediatric patients with symptoms suggesting influenza infection were tested by both immunochromatography (Poctem InfluenzaAB, Sysmex Corporation, Japan) and Verigene RV+ (RV+). Our results show that the RV+ sensitivity was significantly higher than immunochromography; furthermore, the most significant difference was seen during the 3 hour period between early onset of fever and testing. This finding has implications for infection control in the pediatric population during triaging at admissions. Furthermore, the timely availability of a high sensitive molecular assay suggests that the RV+ assay will have a positive impact on outpatient pediatric patient management
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"Modular near-patient PCR for Influenza & RSV provides rapid and accurate results to guide management decisions"
06 June 2011
Full data
Rodrigues F, Muir P, Vipond B, et al
Abstract
Background and aims: We evaluated a new modular near-patient PCR system in 2 centres between January and March 2011
Methods: 146 children with fever and respiratory symptoms presenting to the emergency service at Coimbra(C) had nasopharyngeal samples and clinical data collected prospectively with consent. 67 samples collected routinely at Bristol(B) during the same period were studied. At each centre, sample sets were analysed using the Verigene modular PCR system for Influenza A(H1, H1 2009,H3),B, RSV A&B & Oseltamivir resistance mutation H275Y. The B set was also analysed using established PCR assays.
Results: By Verigene, in C, 15,0&48/146 were positive for InflA,B,RSV(24A,24B) (4 double positives(dp)) and 87 were negative. The corresponding B results were 27,22&8(5A,3B)/67 (2dp) &12 negative. In C-B 14-25 InflA were H1 2009, 0-1 H3, 1-1 untyped (none H275Y+ in C, 14/25 & 2 indeterminate in B). Results by established assays in B were concordant except the untyped H1 sample which was positive for H1 2009, the indeterminate samples which were H275Y+ & 2 H275Y negatives which were weakly positive. In C, 6/15 InflA+ ( 1 received Oseltamivir) and 16/48 RSV+ children were admitted.
Conclusions: The Verigene results matched those from established assays closely. The small number of discrepant results generally related to weakly reactive or indeterminate samples. Evident clinical benefit of rapid-near patient testing included RSV bed-management and cohorting decisions and guidance of Oseltamivir use in seriously sick children. The system would also permit tracking of local outbreak epidemiology in centres without ready access to viral PCR testing
 
ESPID 2011 Rodrigues et al.pdf
"Beta testing evaluation of the Verigene RV+ nucleic acid test for rapid detection and typing of influenza and respiratory syncytial viruses"
13 September 2011
Full data
Abstract
INTRODUCTION
Timely and sensitive diagnosis of influenzavirus (IF) and respiratory syncytial virus (RSV) infections in hospitalised patients is important for infection control and clinical management. PCR testing offers greater sensitivity that traditional methods, but methodologies for rapid and flexible automated testing have only recently become available. We performed a beta test evaluation of the Verigene Respiratory Virus Plus nucleic acid test (RV+, Nanosphere Inc.), which detects and types RSV and IF, including detection of oseltamivir resistance in seasonal influenza H1N1 and influenza H1N1 (2009).
METHODS
Clinical specimens submitted to HPA Microbiology Services Bristol for investigation of suspected respiratory virus infection were tested for IF and RSV by real-time PCR (1,2). Samples testing positive for influenza H1N1 (2009) were further tested for genotypic evidence of oseltamivir resistance using a real-time PCR to discriminatea major single nucleotide polymorphism encoding a point mutation the neuraminidase gene (H275Y) which confers oseltamivir resistance(3). Most of these samples were also tested by pyrosequencing analysis of N1 sequences to determine the proportions of sensitive and resistant virus (4). A panel of samples testing positive or negative for these viruses was selected from samples with sufficient residual volume after completion of routine diagnostic investigations for evaluation of RV+. Fully automated nucleic acid extraction, multiplex reverse transcription PCR amplification and microarray hybridization was performed using a Verigene Processor SP(Figure 1a). Microarrays were then transferred to a Verigene Reader to analyse and report results (Figure 1b). Results of RV+ testing were then compared with those of the comparator laboratory tests.
RESULTS
The RV+ assay was found to be technically straightforward, and delivered unequivocal results in 2.5 hours with approximately 5 minutes hands-on time per sample. The results of the detection of IF and RSV types by RV+ in samples previously characterised by real-time PCR for these viruses is summarised in Table 1. Further details of results of genotypic detection of oseltamivir resistance in IFA H1N1 2009 positive samples by RV+, real-time PCR and pyrosequencing are shown in Table 2 (discordant results shaded magenta).
DISCUSSION
We have compared the RV+ assay with highly sensitive and specific molecular diagnostic assays, and not with less sensitive virus culture or antigen detection methods, as is common in commercial test evaluations. Nevertheless, the RV+ showed good concordance with results of the comparator test. RV+ did not detect or report oseltamivir resistance in 3/21 samples in which resistant virus was detected by pyrosequencing; a mixture of sensitive and resistant virus was detected in two of these. Samples with <50% resistant virus detected by pyrosequencing are not officially reported as oseltamivir resistant. The diagnostic and prognostic significance of detecting minor populations of resistant virus requires further investigation.
CONCLUSIONS
RV+ is suitable for rapid diagnosis and typing of IF and RSV infections. Its speed, flexibility and ease of use makes this assay suitable for:
—Urgent and out-of-hours testing
—Testing in laboratories without specialist molecular diagnostic capability
—Near patient testing.
Provision of oseltamivirresistance results within the primary screen will be useful for:
—Guiding antiviral therapy decisions
—Monitoring virologicresponse and emergence of resistance in patients on treatment
—Recruitment of patients for clinical trials of antiviral therapy.
 
Muir et al 2011.pdf
"Analytical Performance of the Nanosphere Verigene RV+ Assay and the Focus Simplexa Flu A/B & RSV Kit"
Clinical Virology Symposium 2012
23 April 2012
Full data
Alby K, Popowitch EB, Miller MB
Abstract
Introduction:  Viral respiratory infections are a leading cause of morbidity and mortality.  Influenza and respiratory syncytial viruses (RSV) are responsible for the majority of severe respiratory illness during the winter months.  Infections with influenza affect people of all ages, are easily spread from person to person and result in hundreds of thousands of hospitalizations every year.  RSV infections primarily affect very young children as well as the elderly.  These infections result in upwards of 100,000 hospitalizations of children every year.  The purpose of this study was to compare the performance characteristics of two FDA cleared assays for detecting influenza A/B and RSV.
Methods:  Two hundred retrospective samples were analyzed on two systems according to the manufacturers’ package inserts.  The two systems evaluated in this study were the Verigene RV+ assay (Nanosphere, Northbrook, IL) and the Simplexa Flu A/B & RSV kit for the Integrated Cycler (Focus Diagnostics, Cypress, CA).   Laboratory-developed PCR tests (LDT) for influenza A/B and RSV served as the standard for comparison and resolution of discordant samples.
Results:  Of the 200 samples analyzed in this study, 182 samples (91%; 81 negative, 101 positive) were concordant among all three methods.  There was only one false positive by the Verigene RV+ assay (influenza A + RSV) that was negative upon repeat.  There were no false positives by the Simplexa test.  The breakdown of the sensitivity by assay and virus is as follows:
	Influenza A	Influenza B	RSV	Total
Verigene RV+	96.4% (53/55)	100% (21/21)	100% (42/42)	98.3% (116/118)
Simplexa	81.8% (45/55)	76.2% (16/21)	95.2% (40/42)	85.6% (101/118)
 
Taken together our results indicate a sensitivity and specificity of 98.3% and 98.8% for the Verigene RV+ assay and a sensitivity and specificity of 85.6% and 100% for the Simplexa assay.
Conclusions:  We undertook a side by side evaluation of two FDA cleared assays for detecting influenza A/B and RSV.  Assay turn around time for the two systems was similar.  Hands on time for the RV+ assay was significantly less than the Simplexa assay, about 15 minutes as compared to 45 minutes, though only one sample could be run on a module at a time.  In terms of analytical performance, the Verigene RV+ assay showed superior sensitivity and a comparable specificity to the Simplexa assay.
CVS 2012