Name: rapid molecular detection and differentiation of influenza viruses a and b
Uploaded: 2017-01-30T14:00:00
Description: Watch this Scientific Journal Video about Rapid Molecular Detection and Differentiation of Influenza Viruses A and B at JoVE.com

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).

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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M., Elkan, M., Leet, M., McGowan, K. L., Hodinka, R. 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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...

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 death6,7.
Influenza infection is unique from other respiratory viruses in that prompt administration of antiviral therapy within 48 h of symptom onset can reduce the disease severity and length8. Rapid identification of influenza has also been shown to reduce the use of unnecessary antibiotics9,10. Further, hospitalized patients with influenza infections must be placed in isolated rooms with appropriate infection control precautions. However, respiratory illnesses caused by non-influenza viruses can be difficult to clinically distinguish from influenza. For this reason, rapid and accurate diagnostic testing for influenza is very important for clinical patient management.
Several assays are available for the detection and identification of influenza viruses. Rapid influenza antigen detection tests (RIDTs) are widely used in clinical practice as point-of-care tests because they are simple to use and provide results within 15 to 30 min11,12; however, their sensitivities vary widely (10-80%) depending on the manufacturer and the population being tested, and the influenza type and subtype13,14,15. Direct fluorescence assays (DFAs) provide superior sensitivities over RIDTs, but the processing time is greater (~3 hr) and must be completed by skilled technologists16,17. Viral culture has been the gold standard for influenza diagnostics and has improved sensitivity over both RIDTs and DFAs18. However, influenza viral culture can take anywhere from 2-14 d to complete, diminishing its utility in aiding patient management19. Lastly, nucleic acid amplification tests (NAAT) have replaced culture techniques as the new gold standard in influenza diagnostics. NAAT are considered to have the greatest sensitivity for detecting influenza in a few hours. However, NAAT are the most expensive assays and require specialized equipment and technologists to perform5,20,21,22,23,24,25.
The influenza A and B assay described here is the first CLIA-waived molecular rapid flu test that is readily available. This assay works by employing a Nicking Endonuclease Amplification Reaction (NEAR) that uses isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. This assay differentiates influenza A from B, requires 2 min to set up and process one sample, and requires a total of 15 min to complete.
Here, we present the protocol for the Influenza A &#38; B assay. In addition, we provide a sample data set comparing the performance of the Influenza A and B assay on archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) to another respiratory pathogen panel assay.

<table border="0" cellpadding="0" cellspacing="0" width="660">
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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">Alere i Instrument</td>
			<td style="width:64px;">Alere</td>
			<td style="width:303px;">NAT-000 (Global), NAT-024 (US)</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alere i Influenza A &#38; B 24 Test Kit</td>
			<td>Alere</td>
			<td style="width:303px;">425-000 (Global), 425-024 (US)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alere i Barcode Scanner</td>
			<td>Alere</td>
			<td>EQ001001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alere Universal Printer</td>
			<td>Alere</td>
			<td style="width:303px;">55115 (Global), alereiprinter&#160;(US)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">200 &#181;L precision pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">200 &#181;L disposable pipette tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Viral transport medium</td>
			<td>Remel</td>
			<td>M4-RT</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid molecular detection and differentiation of influenza viruses a and b

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.

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...

Watch this Scientific Journal Video about Rapid Molecular Detection and Differentiation of Influenza Viruses A and B at JoVE.com

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

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 ...

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