Verification of a Novel Multiplex PCR Respiratory Virus Panel in a US Biocontainment Unit

Abstract Emerging infectious diseases carry unique logistical, financial, and clinical ramifications. Rapid diagnostic testing methods can alleviate some of these challenges by providing definitive diagnoses earlier in the clinical course, leading to appropriate targeted therapy, cost savings, and improved patient outcomes. The BioFire FilmArray Respiratory Panel 2 plus (RP2plus; bioMérieux, Marcy l’Etoile, France) is a multiplexed nucleic acid test for detection of Middle East respiratory syndrome coronavirus (MERS-CoV) and 14 common viral and 4 bacterial respiratory pathogens in nasopharyngeal swabs obtained from those meeting MERS-CoV epidemiological criteria. The aim of this study was to verify the FilmArray RP2plus for use in our biocontainment unit. Of note, the RP2plus is FDA approved but not currently available for sale in the United States. Eight patient samples were tested with known results (GenMark Respiratory Virus Panel [RVP] or Cepheid Xpert Flu/RSV). We had concordant results between the platforms for samples containing influenza A, respiratory syncytial virus (RSV), parainfluenza virus 2, rhinovirus, and a negative sample. We evaluated two influenza B samples from two different patients. The FilmArray RP2plus did not detect influenza B in one of the patient samples. The sample was exhausted and repeat testing could not be performed. A second rhinovirus sample was not detected by the RP2plus, but Coronavirus 229E was detected in this sample, a virus not detected by the RVP. The sample was repeated and again did not detect rhinovirus. Further investigation into this discrepancy revealed that rhinovirus was originally detected by RVP at a signal of 34.4 nA (repeat of 46.9 nA). The concordant rhinovirus sample had a signal of 226.7 nA by RVP, which was much higher than the discrepant sample. Because of the low signal by RVP in the discrepant sample, perhaps the viral load was below the limit of detection of the RP2plus. All other quality control sample pools passed verification testing, including day-to-day and operator variance. It is not uncommon for a person under investigation (PUI) for a highly communicable disease to be evaluated in our facility. The performance of the RP2plus test on clinical samples showed acceptable concordance with our current means of testing for respiratory pathogens. The RP2plus will eliminate challenges implicated in storing and transporting specimens to an off-site lab, facilitate quicker turnaround time, and streamline the often cumbersome, complex protocols and practices required to work up a serious communicable disease.

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The Panther Fusion System with Open Access Functionality for Laboratory-Developed Tests for Influenza A Virus Subtyping

Journal of Clinical Microbiology ◽

10.1128/jcm.00188-20 ◽

2020 ◽

Vol 58 (6) ◽

Author(s):

Kathleen A. Stellrecht ◽

Jesse L. Cimino ◽

Vincente P. Maceira

Keyword(s):

Open Access ◽

Influenza A ◽

Limit Of Detection ◽

Turnaround Time ◽

Nucleic Acid Amplification ◽

Influenza B ◽

Fusion System ◽

Valuable Complement ◽

Syncytial Virus

ABSTRACT Nucleic acid amplification tests, such as PCR, are the method of choice for respiratory virus testing, due to their superior diagnostic accuracy and fast turnaround time. The Panther Fusion (Fusion; Hologic) system has an array of highly sensitive in vitro diagnostic (IVD) real-time PCR assays for respiratory viruses, including an assay for influenza A (FluA) virus, influenza B (FluB) virus, and respiratory syncytial virus (RSV) (FFABR assay). The Fusion system has Open Access functionality to perform laboratory-developed tests (LDTs) alongside IVD assays. We developed two LDTs for FluA virus strain typing on the Panther Fusion instrument, enabling side-by-side testing with the FFABR assay. The LDT-FAST assay uses proprietary primers and probes designed by Hologic for the Prodesse ProFAST+ (PFAST) assay. The exWHO-FAST assay is an expanded redesign of the WHO-recommended reverse transcriptase PCRs (RT-PCRs). To evaluate the performance of these two LDTs, 110 FluA virus-positive samples were tested. Of these, 104 had been subtyped previously; 54 were H3, 46 were 09H1, and 4 were fsH1. All were appropriately subtyped by both LDTs. Of the untyped FluA virus samples, three were subtyped as H3 by both LDTs and two were subtyped as H3 by the LDT-FAST assay only. The sample not subtyped by either LDT was retested with the FFABR assay and was now negative. Limit-of-detection (LOD) analyses were performed with five FluA virus strains. The LDT-FAST LODs were similar to the FFABR assay LODs, while the exWHO-FAST LODs were higher for two H3N2 strains, findings that were explained by analysis of primer/probe homology. In conclusion, either FluA virus typing assay would be a valuable complement to the Panther Fusion respiratory menu given the performance of these LDTs, the system’s full automation, and the ability to split eluates for both IVD and LDT testing.

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Performance of a Novel Point-of-Care Molecular Assay for Detection of Influenza A and B Viruses and Respiratory Syncytial Virus (Enigma MiniLab) in Children with Acute Respiratory Infection

Journal of Clinical Microbiology ◽

10.1128/jcm.02887-15 ◽

2015 ◽

Vol 54 (1) ◽

pp. 212-215 ◽

Cited By ~ 11

Author(s):

Sam T. Douthwaite ◽

Charlotte Walker ◽

Elisabeth J. Adams ◽

Catherine Mak ◽

Andres Vecino Ortiz ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Influenza A ◽

Respiratory Virus ◽

Point Of Care ◽

Influenza B Virus ◽

Influenza B ◽

Molecular Assay ◽

B Virus ◽

Syncytial Virus ◽

Virus Influenza

The performance of the Enigma MiniLab assay for influenza A and B viruses and respiratory syncytial virus (RSV) was compared to a centralized laboratory respiratory virus panel. The positive and negative percent agreement for influenza A virus, influenza B virus, and RSV were 79.2% (95% confidence interval [95% CI], 57.8 to 92.9%) and 99.4% (95% CI, 98.4 to 99.9), 100% (95% CI, 47.8 to 100%) and 100% (95% CI, 99.3 to 100%), 98.5% (95% CI, 94.6 to 99.8%) and 94.5% (95% CI, 91.9 to 96.4%), respectively.

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Evaluation of Performance Characteristics of Panther Fusion Assays for Detection of Respiratory Viruses from Nasopharyngeal and Lower Respiratory Tract Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.00787-18 ◽

2018 ◽

Vol 56 (8) ◽

Cited By ~ 10

Author(s):

Soya S. Sam ◽

Angela M. Caliendo ◽

Jessica Ingersoll ◽

Deborah Abdul-Ali ◽

Charles E. Hill ◽

...

Keyword(s):

Respiratory Tract ◽

Positive Result ◽

Lower Respiratory Tract ◽

Influenza A ◽

Performance Characteristics ◽

Influenza B Virus ◽

Influenza B ◽

Respiratory Pathogens ◽

Percent Agreement ◽

Syncytial Virus

ABSTRACT Accurate and rapid diagnosis is needed for timely intervention and clinical management of acute respiratory infections. This study evaluated performance characteristics of the Panther Fusion assay for the detection of influenza A virus (Flu A), influenza B virus (Flu B), respiratory syncytial virus (RSV), parainfluenza viruses 1 to 3 (Para 1 to 3), human metapneumovirus (hMPV), rhinovirus (RV), and adenovirus (Adeno) targets in comparison to those of the eSensor and Lyra assays using 395 nasopharyngeal (NP) and 104 lower respiratory tract (LRT) specimens. Based on the consensus positive result established (positive result in 2 of the 3 assays), the NP specimens for the Fusion and eSensor assays had 100% positive percent agreement (PPA) for all the analytes and the Lyra assays had 100% PPA for Flu A and Adeno analytes. A 100% negative percent agreement (NPA) was observed for all the Lyra analytes, whereas those for the Fusion targets ranged from 98.4 to 100% and those for the eSensor ranged from 99.4 to 100% for all the analytes except RV. For the LRT specimens, Fusion had 100% PPA and 100% NPA for all the targets except hMPV. There was a 100% PPA for eSensor analytes; the NPA ranged from 98 to 100%, except for RV. For the Lyra assays, the PPA ranged between 50 and 100%, while the NPA was 100% for all the targets except Adeno. The Fusion assay performed similarly to the eSensor assay for majority of the targets tested and provides laboratories with a fully automated random-access system to test for a broad array of viral respiratory pathogens.

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Coinfection with Respiratory Pathogens in COVID-19 in Korea

10.1101/2020.12.18.20248449 ◽

2020 ◽

Author(s):

Kyoung Ho Roh ◽

Yu Kyung Kim ◽

Shin-Woo Kim ◽

Eun-Rim Kang ◽

Yong-Jin Yang ◽

...

Keyword(s):

Influenza A ◽

Respiratory Virus ◽

Respiratory Viruses ◽

Diagnostic Value ◽

Respiratory Pathogens ◽

Respiratory Specimen ◽

Upper Respiratory ◽

Viral Coinfection ◽

Syncytial Virus ◽

Respiratory Specimens

AbstractDetection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in upper and lower respiratory specimens and coinfection with other respiratory pathogens in patients with coronavirus disease 2019 (COVID-19) were investigated. From the study subjects (N = 258) retrospectively enrolled when confirmed as SARS-CoV-2 positive, nasopharyngeal (NPS), oropharyngeal swabs (OPS), and sputum specimens were restored for retesting SARS-CoV-2 and detecting respiratory pathogens. Majority of the study subjects (95.7%, N = 247) were confirmed as SARS-CoV-2 positive using NPS/OPS specimens, suggesting that the upper respiratory specimen is most valuable in detecting SARS-CoV-2. Coinfection rates in COVID-19 patients (N = 258) with respiratory pathogens were 9.7% (N = 25); 8.5% (N = 22) respiratory viruses and 1.2% (N = 3) Mycoplasma pneumoniae, an atypical bacterium. Of the respiratory virus coinfection cases (N = 22), 20 (90.9%) were co-infected with a single respiratory virus and 2 (0.8%) (metapneumovirus/adenovirus and rhinovirus/bocavirus 1/2/3/4) with two viruses. Respiratory viruses in single viral coinfection cases with SARS-CoV-2 were as follows: non-SARS-CoV-2 coronaviruses (229E, NL63, and OC43, N = 5, 1.9%), rhinovirus (N = 4, 1.6%), metapneumovirus (N = 3, 1.2%), influenza A (N = 3, 1.2%), respiratory syncytial virus A and B (N = 3, 1.2%), and adenovirus (N = 2, 0.8%). No mixed coinfections with respiratory viruses and M. pneumoniae were found. In conclusion, the diagnostic value of utilizing NPS/OPS specimen is excellent, and, as the first report in Korea, coinfection with respiratory pathogens were detected at a rate of 9.7% in patients with COVID-19.

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Comparative Seasonal Respiratory Virus Epidemic Timing in Utah

Viruses ◽

10.3390/v12030275 ◽

2020 ◽

Vol 12 (3) ◽

pp. 275

Author(s):

Zayne Y. Callahan ◽

Trevor K. Smith ◽

Celeste Ingersoll ◽

Rebecca Gardner ◽

E. Kent Korgenski ◽

...

Keyword(s):

Influenza A ◽

Respiratory Virus ◽

Local Level ◽

Late Summer ◽

Influenza B ◽

Viral Interference ◽

Early Fall ◽

Syncytial Virus ◽

Time Specific ◽

Epidemic Timing

Previous studies have found evidence of viral interference between seasonal respiratory viruses. Using laboratory-confirmed data from a Utah-based healthcare provider, Intermountain Health Care, we analyzed the time-specific patterns of respiratory syncytial virus (RSV), influenza A, influenza B, human metapneumovirus, rhinovirus, and enterovirus circulation from 2004 to 2018, using descriptive methods and wavelet analysis (n = 89,462) on a local level. The results showed that RSV virus dynamics in Utah were the most consistent of any of the viruses studied, and that the other seasonal viruses were generally in synchrony with RSV, except for enterovirus (which mostly occurs late summer to early fall) and influenza A and B during pandemic years.

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Mortality Following Isolation of Various Respiratory Viruses in Nursing Home Residents

Infection Control and Hospital Epidemiology ◽

10.1086/501589 ◽

1999 ◽

Vol 20 (12) ◽

pp. 812-815 ◽

Cited By ~ 33

Author(s):

Paul J. Drinka ◽

Stefan Gravenstein ◽

Elizabeth Langer ◽

Peggy Krause ◽

Peter Shult

Keyword(s):

Nursing Home ◽

Influenza A ◽

Skilled Nursing Facility ◽

Nursing Home Residents ◽

Respiratory Illness ◽

Respiratory Viruses ◽

Influenza B ◽

Respiratory Pathogens ◽

Syncytial Virus ◽

Viral Respiratory

AbstractObjective:To compare mortality following isolation of influenza A to mortality following isolation of other respiratory viruses in a nursing home.Setting:The Wisconsin Veterans Home, a 688-bed skilled nursing facility for veterans and their spouses.Participants:All residents with respiratory viral isolates obtained between 1988 and 1999.Design:Thirty-day mortality was determined following each culture-proven illness.Results:Thirty-day mortality following isolation of viral respiratory pathogens was 4.7% (15/322) for influenza A 5.4% (7/129) for influenza B; 6.1% (3/49) for parainfluenza type 1; 0% (0/26) for parainfluenza types 2,3, and 4; 0% (0/26) for respiratory syncytial virus (RSV); and 1.6% (1/61) for rhinovirus.Conclusions:Mortality following isolation of certain other respiratory viruses may be comparable to that following influenza A (although influenza A mortality might be higher without vaccination and antiviral agents). The use of uniform secretion precautions for all viral respiratory illness deserves consideration in nursing homes.

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Magnetofluidic platform for rapid multiplexed screening of SARS-CoV-2 variants and respiratory pathogens

10.1101/2021.05.10.21256995 ◽

2021 ◽

Author(s):

Alexander Y Trick ◽

Fan-En Chen ◽

Liben Chen ◽

Pei-Wei Lee ◽

Alexander C Hasnain ◽

...

Keyword(s):

Influenza A ◽

Respiratory Symptoms ◽

Limit Of Detection ◽

Respiratory Viruses ◽

Nasopharyngeal Swab ◽

Influenza B ◽

Respiratory Pathogens ◽

Multiplexed Detection ◽

New Challenges ◽

Sensitivity Specificity

The rise of highly transmissible SARS-CoV-2 variants brings new challenges and concerns with vaccine efficacy, diagnostic sensitivity, and public health responses in the fight to end the pandemic. Widespread detection of variant strains will be critical to inform policy decisions to mitigate further spread, and post-pandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. In this work, we have developed a portable, magnetofluidic cartridge platform for automated PCR testing in <30 min. Cartridges were designed for multiplexed detection of SARS-CoV-2 with either distinctive variant mutations or with Influenza A and B. The platform demonstrated a limit of detection down to 2 copies/μL SARS-CoV-2 RNA with successful identification of B.1.1.7 and B.1.351 variants. The multiplexed SARS-CoV-2/Flu assay was validated using archived clinical nasopharyngeal swab eluates (n = 116) with an overall sensitivity/specificity of 98.1%/95.2%, 85.7%/100%, 100%/98.2%, respectively, for SARS-CoV-2, Influenza A, and Influenza B. Further testing with saliva (n = 14) demonstrated successful detection of all SARS-CoV-2 positive samples with no false-positives.

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Post-pandemic influenza A/H1N1pdm09 is associated with more severe outcomes than A/H3N2 and other respiratory viruses in adult hospitalisations

Epidemiology and Infection ◽

10.1017/s095026881900195x ◽

2019 ◽

Vol 147 ◽

Cited By ~ 5

Author(s):

C. A. Minney-Smith ◽

L. A. Selvey ◽

A. Levy ◽

D. W. Smith

Keyword(s):

Clinical Outcomes ◽

Influenza A ◽

Respiratory Virus ◽

Respiratory Illness ◽

Respiratory Viruses ◽

Influenza B ◽

Virus Infections ◽

Icu Admission ◽

Hospitalised Patients ◽

Syncytial Virus

Abstract This study compares the frequency and severity of influenza A/H1N1pdm09 (A/H1), influenza A/H3N2 (A/H3) and other respiratory virus infections in hospitalised patients. Data from 17 332 adult hospitalised patients admitted to Sir Charles Gairdner Hospital, Perth, Western Australia, with a respiratory illness between 2012 and 2015 were linked with data containing reverse transcription polymerase chain reaction results for respiratory viruses including A/H1, A/H3, influenza B, human metapneumovirus, respiratory syncytial virus and parainfluenza. Of these, 1753 (10.1%) had test results. Multivariable regression analyses were conducted to compare the viruses for clinical outcomes including ICU admission, ventilation, pneumonia, length of stay and death. Patients with A/H1 were more likely to experience severe outcomes such as ICU admission (OR 2.5, 95% CI 1.2–5.5, P = 0.016), pneumonia (OR 3.0, 95% CI 1.6–5.7, P < 0.001) and lower risk of discharge from hospital (indicating longer lengths of hospitalisation; HR 0.64 95% CI 0.47–0.88, P = 0.005), than patients with A/H3. Patients with a non-influenza respiratory virus were less likely to experience severe clinical outcomes than patients with A/H1, however, had similar likelihood when compared to patients with A/H3. Patients hospitalised with A/H1 had higher odds of severe outcomes than patients with A/H3 or other respiratory viruses. Knowledge of circulating influenza strains is important for healthcare preparedness.

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Multicenter Evaluation of the QIAstat-Dx Respiratory Panel for Detection of Viruses and Bacteria in Nasopharyngeal Swab Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.00155-20 ◽

2020 ◽

Vol 58 (5) ◽

Cited By ~ 5

Author(s):

Amy L. Leber ◽

Jan Gorm Lisby ◽

Glen Hansen ◽

Ryan F. Relich ◽

Uffe Vest Schneider ◽

...

Keyword(s):

Influenza A ◽

Parainfluenza Virus ◽

Nasopharyngeal Swab ◽

Influenza B ◽

Respiratory Pathogens ◽

Content Type ◽

Percent Agreement ◽

Influenza A H1n1 ◽

Syncytial Virus

ABSTRACT The QIAstat-Dx Respiratory Panel (QIAstat-Dx RP) is a multiplex in vitro diagnostic test for the qualitative detection of 20 pathogens directly from nasopharyngeal swab (NPS) specimens. The assay is performed using a simple sample-to-answer platform with results available in approximately 69 min. The pathogens identified are adenovirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, human metapneumovirus A and B, influenza A, influenza A H1, influenza A H3, influenza A H1N1/2009, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, rhinovirus/enterovirus, respiratory syncytial virus A and B, Bordetella pertussis, Chlamydophila pneumoniae, and Mycoplasma pneumoniae. This multicenter evaluation provides data obtained from 1,994 prospectively collected and 310 retrospectively collected (archived) NPS specimens with performance compared to that of the BioFire FilmArray Respiratory Panel, version 1.7. The overall percent agreement between QIAstat-Dx RP and the comparator testing was 99.5%. In the prospective cohort, the QIAstat-Dx RP demonstrated a positive percent agreement of 94.0% or greater for the detection of all but four analytes: coronaviruses 229E, NL63, and OC43 and rhinovirus/enterovirus. The test also demonstrated a negative percent agreement of ≥97.9% for all analytes. The QIAstat-Dx RP is a robust and accurate assay for rapid, comprehensive testing for respiratory pathogens.

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Using routine testing data to understand circulation patterns of influenza A, respiratory syncytial virus and other respiratory viruses in Victoria, Australia

Epidemiology and Infection ◽

10.1017/s0950268819001055 ◽

2019 ◽

Vol 147 ◽

Cited By ~ 6

Author(s):

O. H. Price ◽

S. G. Sullivan ◽

C. Sutterby ◽

J. Druce ◽

K. S. Carville

Keyword(s):

Respiratory Syncytial Virus ◽

Influenza A ◽

Respiratory Virus ◽

Respiratory Viruses ◽

Influenza B ◽

Routine Testing ◽

Circulation Patterns ◽

Cross Correlations ◽

Time Series Analyses ◽

Syncytial Virus

Abstract Several studies have reported evidence of interference between respiratory viruses: respiratory viruses rarely reach their epidemic peak concurrently and there appears to be a negative association between infection with one respiratory virus and co-infection with another. We used results spanning 16 years (2002–2017) of a routine diagnostic multiplex panel that tests for nine respiratory viruses to further investigate these interactions in Victoria, Australia. Time series analyses were used to plot the proportion positive for each virus. The seasonality of all viruses included was compared with respiratory syncytial virus (RSV) and influenza A virus using cross-correlations. Logistic regression was used to explore the likelihood of co-infection with one virus given infection with another. Seasonal peaks were observed each year for influenza A and RSV and less frequently for influenza B, coronavirus and parainfluenza virus. RSV circulated an average of 6 weeks before influenza A. Co-infection with another respiratory virus was less common with picornavirus, RSV or influenza A infection. Our findings provide further evidence of a temporal relationship in the circulation of respiratory viruses. A greater understanding of the interaction between respiratory viruses may enable better prediction of the timing and magnitude of respiratory virus epidemics.

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