key: cord-0998037-b70sfktd authors: Stevens, Bryan A.; Hogan, Catherine A.; Mfuh, Kenji O.; Khan, Ghazala; Sahoo, Malaya K.; Huang, ChunHong; Garamani, Natasha; Zehnder, James; Kurzer, Jason; Pinsky, Benjamin A. title: Combined SARS-CoV-2 Nucleic Acid Amplification Testing and Respiratory Virus Panel RT-PCR on the Hologic Panther Fusion System date: 2021-03-10 journal: J Clin Virol DOI: 10.1016/j.jcv.2021.104792 sha: 13905cf4c7d9377a0668290f15316f900bd9582f doc_id: 998037 cord_uid: b70sfktd BACKGROUND: Significant overlap exists between the symptoms of SARS-CoV-2 and other respiratory viruses. This poses a serious challenge to clinical diagnosis, laboratory testing, and infection control programs. OBJECTIVES: To evaluate the performance of the Hologic Panther Fusion Respiratory Assays (RA) compared to the GenMark ePlex Respiratory Pathogen Panel (RPP) and to assess the ability of the Panther Fusion to perform parallel testing of SARS-CoV-2 and other respiratory viruses from a single sample. STUDY DESIGN: A diagnostic comparison study was carried out using 375 clinical nasopharyngeal specimens. Assay performance was assessed by overall, positive, and negative percent agreement and Cohen’s kappa coefficient. RESULTS: Overall agreement between the Fusion RA and ePlex RPP was 97.3% (95% CI 96.3-98.0), positive percent agreement was 97.2% (95% CI 93.0-99.2), negative percent agreement was 97.3% (95% CI 96.3-98.0), and the kappa coefficient was 0.85 (95% CI 0.81-0.89). Forty additional viruses in 30 specimens were detected by Fusion that were not detected by ePlex. The maximum specimen throughput for parallel testing of the Fusion Respiratory Assays with SARS-CoV-2 was 275 samples in 20.7 hours for Fusion SARS-CoV-2 and 350 samples in 20.0 hours for Aptima Transcription Mediated Amplification SARS-CoV-2. CONCLUSION: Fusion RA demonstrated substantial agreement compared to the ePlex RPP. However, the Fusion detected respiratory viruses not identified by ePlex, consistent with higher clinical sensitivity. Workflows for parallel testing of respiratory pathogens and SARS-CoV-2 demonstrate that the Panther Fusion instrument provides a flexible, moderate to high throughput testing option for pandemic and seasonal respiratory viruses. This poses a serious challenge to clinical diagnosis, laboratory testing, and infection control programs. To evaluate the performance of the Hologic Panther Fusion Respiratory Assays (RA) compared to the GenMark ePlex Respiratory Pathogen Panel (RPP) and to assess the ability of the Panther Fusion to perform parallel testing of SARS-CoV-2 and other respiratory viruses from a single sample. A diagnostic comparison study was carried out using 375 clinical nasopharyngeal specimens. Assay performance was assessed by overall, positive, and negative percent agreement and Cohen's kappa coefficient. Overall agreement between the Fusion RA and ePlex RPP was 97.3% (95% CI 96. 3 The clinical presentation of COVID-19 ranges from mild upper respiratory illness to severe respiratory failure, and overlaps significantly with influenza and other common respiratory viruses [1] . In the Northern Hemisphere, the onset of the pandemic occurred during the conventional respiratory virus season, emphasizing the challenge of differentiating between viral etiologies of acute respiratory infections based on signs, symptoms, and epidemiologic data. Diagnostic testing for non-SARS-CoV-2 respiratory viruses is critical, particularly for high-risk groups such as the immunocompromised, those with underlying heart and/or lung disease, and individuals over 65 years of age. Distinguishing COVID-19 from the clinical presentation of other respiratory virus infections, and identifying individuals with SARS-CoV-2 co-infections [2] [3] [4] , will help inform hospital admissions, isolation and quarantine policies, and therapeutic options. The SARS-CoV-2 pandemic has revealed significant deficiencies in the supply chain for critical assay reagents and consumables [5, 6] . Clinical laboratories have therefore been required to validate and implement multiple platforms and workflows to ensure that SARS-CoV-2 diagnostic testing proceeds uninterrupted. In anticipation of similar challenges for all respiratory virus reagents, we performed a diagnostic comparison study of the Hologic Panther Fusion RT-PCR Respiratory assays (Fusion RA) with the GenMark ePlex Respiratory Pathogen Panel Table 1 (Table 1) . Fusion RA detected an additional 26 viral targets in 20 NP samples in which the ePlex RPP was negative for all targets, including 6 influenza B, 10 RSV, 4 hMPV, 3 RV, and 3 Table 1 ). The median CT of the additional targets was 37.5 (IQR: 35.9 -39.3). XT8 RVP confirmed 22/26 (84.6%) additional viral targets detected by Fusion RA; 6/6 influenza B, 10/10 RSV, 2/4 hMPV, 1/3 RV, and 3/3 PIV. In addition, XT8 RVP detected 5 additional targets, 2 RSV and 2 RV, not detected by either Fusion RA or ePlex RPP. Seegene Allplex Respiratory Panels [8] [9] [10] [11] . The GenMark ePlex also exhibited similar performance when compared to a laboratory-developed RT-PCR panel and BioFire Film Array (v. 1.7), respectively [12, 13] . In this study, we observed substantial agreement between Fusion RA and ePlex RPP for targets common to both tests. Importantly, Fusion RA does not detect seasonal coronaviruses and therefore, these viruses were not included in the analysis. This workflow may be continued iteratively until testing is complete. J o u r n a l P r e -p r o o f Clinical Characteristics of Coronavirus Disease 2019 in China Rates of Co-infection Between SARS-CoV-2 and Other Respiratory Pathogens Co-infection with SARS-CoV-2 and Influenza A Virus in Patient with Pneumonia Clinical characteristics of critically ill patients co-infected with SARS-CoV-2 and the influenza virus in Wuhan, China Under-allocation: Critical Supply Chain Hurdles Negatively Impact the Ability of Community Hospitals To Perform Repeat SARS-CoV-2 Testing American Association for Clinical Chemistry Comparison of the Panther Fusion and a Laboratory-developed Test Targeting the Envelope gene for Detection of SARS-CoV-2 Evaluation of Performance Characteristics of Panther Fusion Assays for Detection of Respiratory Viruses from Nasopharyngeal and Lower Respiratory Tract Specimens Panther Fusion(R) Respiratory Virus Assays for the detection of influenza and other respiratory viruses Comparison of the Panther Fusion respiratory panels to routine methods for detection of viruses in upper and lower respiratory tract specimens Comparison of the Panther Fusion and Allplex assays for the detection of respiratory viruses in clinical samples Multicenter Evaluation of the ePlex Respiratory Pathogen Panel for the Detection of Viral and Bacterial Respiratory Tract Pathogens in Nasopharyngeal Swabs Comparison of ePlex Respiratory Pathogen Panel with Laboratory-Developed Real-Time PCR Assays for Detection of Respiratory Pathogens Performance Characteristics of a High-Throughput Automated Transcription-Mediated Amplification Test for SARS-CoV-2 Detection High-Throughput Transcription-mediated amplification on the Hologic Panther is a highly sensitive method of detection for SARS-CoV-2 Comparison of Four Molecular In Vitro Diagnostic Assays for the Detection of SARS-CoV-2 in Nasopharyngeal Specimens Analytical and Clinical Comparison of Three Nucleic Acid Amplification Tests for SARS-CoV-2 Detection 95% confidence interval; PPA, positive percent agreement; NPA, negative percent agreement; RSV, respiratory syncytial virus; AdV, adenovirus; hMPV 95% CI: 95% confidence interval PPA, positive percent agreement; NPA, negative percent agreement; LDT, laboratory developed test; EUA, emergency use authorization TMA, transcription-mediated amplification *One specimen was invalid via TMA and was excluded from the analysis The authors would like to thank the hard-working clinical laboratory scientists and laboratory assistants of the Stanford Healthcare Clinical Virology Laboratory, who continually demonstrate professional excellence and are the backbone of the COVID-19 laboratory response.J o u r n a l P r e -p r o o f