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Immunology and Infection

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Published: January 30th, 2017



1Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.

The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.

This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

Influenza virus infections result in a significant amount of morbidity and mortality each year1,2,3. Uncomplicated influenza is characterized by constitutional and respiratory symptoms such as fever, myalgia, headache, and non-productive cough4,5. Older individuals, young children, immunocompromised patients, and patients with underlying comorbidities are at a higher risk for serious complications such as pneumonia, myocarditis, central nervous system disease, or death6,.css-f1q1l5{display:-webkit-box;display:-webkit-flex;display:-ms-flexbox;display:flex;-webkit-align-items:flex-end;-webkit-box-align:flex-end;-ms-flex-align:flex-end;align-items:flex-end;background-image:linear-gradient(180deg, rgba(255, 255, 255, 0) 0%, rgba(255, 255, 255, 0.8) 40%, rgba(255, 255, 255, 1) 100%);width:100%;height:100%;position:absolute;bottom:0px;left:0px;font-size:var(--chakra-fontSizes-lg);color:#676B82;}

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ETHICS STATEMENT: The use of left-over clinical specimens is approved and follows the guidelines of the Memorial Sloan Kettering Cancer Center Institutional Review Board.

1. Before Running the Assay

NOTE: The Influenza A and B assay is approved for nasopharyngeal swab specimens and for nasopharyngeal swabs stored in viral transport media. Swabs are included in the kit and should be used for optimal performance. However, rayon, foam, flocked swabs or polyester nasal swabs can also be used to collect nasal swab.......

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In this study, archived NPS specimens were collected from inpatients presenting with influenza-like symptoms at Memorial Sloan-Kettering Cancer Center (MSKCC) during an influenza outbreak between December 15, 2012 and March 1, 2013. The NPS specimens were submitted in 3 ml of VTM and tested as part of routine clinical practice with a molecular assay that detects a panel of respiratory viruses (RP), including Influenza A, A-1, A-3, and B. During the study period, 3,675 NPS specimens were s.......

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Influenza viruses are significant world-wide causes of morbidity and mortality. Rapid and accurate diagnosis of influenza is one of the major keys to managing flu outbreaks during respiratory season. Other antigen-based tests are rapid and easy to perform; however, they have low sensitivities13. On the other hand, traditional molecular tests have improved sensitivity, but require more experienced laboratory technologists to perform and are more costly. The Influenza A and B assay described in this.......

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We thank the Clinical Microbiology Service staff of the Memorial Sloan-Kettering Cancer Center for help in collecting clinical specimens. This study was supported in part by a research agreement between MSKCC and Alere Scarborough (SK2013-0262).


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Name Company Catalog Number Comments
Alere i Instrument Alere NAT-000 (Global), NAT-024 (US)
Alere i Influenza A & B 24 Test Kit Alere 425-000 (Global), 425-024 (US)
Alere i Barcode Scanner Alere EQ001001
Alere Universal Printer Alere 55115 (Global), alereiprinter (US)
200 µL precision pipette
200 µL disposable pipette tips
Viral transport medium Remel M4-RT

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