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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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Underrecognized+Burden+of+Influenza+in+Young+Children.">The Underrecognized Burden of Influenza in Young Children.</a> New England Journal of Medicine. 355 (1), 31-40 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimates+of+Deaths+Associated+with+Seasonal+Influenza+---+United+States,+1976-2007.">Estimates of Deaths Associated with Seasonal Influenza --- United States, 1976-2007.</a> MMWR. 59 (33), 1057-1089 (2010).</li><li>Agrawal, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+real-time+PCR+and+conventional+RT-PCR+during+a+2+year+surveillance+for+influenza+and+respiratory+syncytial+virus+among+children+with+acute+respiratory+infections+in+Kolkata,+India,+reveals+a+distinct+seasonality+of+infection.">Comparative evaluation of real-time PCR and conventional RT-PCR during a 2 year surveillance for influenza and respiratory syncytial virus among children with acute respiratory infections in Kolkata, India, reveals a distinct seasonality of infection.</a> Journal of Medical Microbiology. 58 (12), 1616-1622 (2009).</li><li>Ginocchio, C. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+annual+impact+of+seasonal+influenza+in+the+US:+measuring+disease+burden+and+costs.">The annual impact of seasonal influenza in the US: measuring disease burden and costs.</a> Vaccine. 25 (27), 5086-5096 (2007).</li><li>Aoki, F. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+administration+of+oral+oseltamivir+increases+the+benefits+of+influenza+treatment.">Early administration of oral oseltamivir increases the benefits of influenza treatment.</a> Journal of Antimicrobial Chemotherapy. 51 (1), 123-129 (2003).</li><li>Noyola, D. E., Demmler, G. 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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">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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 ...

The overall goal of this assay is to diagnose or rule out influenza virus infection in patients presenting with respiratory systems. This method can help answer key questions in the clinical diagnostics field such as diagnosing key causes of respiratory infections. The main advantage of this technique is that it is a rapid method that can be used in appointive care setting.Demonstrating the procedure will be Sam Kaplan, a technician from our laboratory. To collect a nasal swab sample, insert the swab into the nostril that has the most visible drainage or that is most congested. With gentle rotation, push the swab into the nostril until resistance is met and rotate several times against the nasal wall before slowly removing from the nostril.Subsequently, store the swab and transport it in a vial containing three milliliters of viral transport media. Next bring the sample and the blue sample receiver to room temperature prior to testing. The orange test base can be tested without the need to warm to room temperature.Leave all the test pieces in the foil until immediately before use. Then turn on the instrument, enter the user ID and press OK.To begin the test process, touch Run Test on the instrument screen. Next enter the patient ID by using the on-screen keyboard or barcode scanner and press OK.After that open the lid and insert the orange test base tube into the orange test base holder.Confirm that the correct test is displayed on the screen and press OK.Then insert the blue sample receiver into the blue sample receiver holder and wait for the sample receiver to warm up. Afterward remove the foiled seal by placing two fingers on the edge of the sample receiver to ensure that it stays in place. Then place the patient's swab to be tested into the sample receiver.Vigorously mix the swab and the liquid for 10 seconds and press the swab head against the side of the sample receiver to help dislodge the specimen from the swab. If the testing swabs are stored in viral transport medium, vortex the viral transport media for 10 seconds and add 200 microliters of it to the sample receiver. Then press OK to proceed.After that press the white transfer cartridge into the blue sample receiver. Continue pressing on the sample receiver until the orange indicator rises. Then lift and connect the transfer cartridge to the test base.The orange indicator should descend once the cartridge transfer is attached correctly. Close the lid and do not open until the Results screen appears. Now touch Run QC test on the home screen.Select the QC Test to be run. Then confirm the test type to match the QC sample intended for testing by clicking OK and following the on-screen prompts to complete testing. After the test run is complete observe the results of the test displayed on the screen with interpretations for the presence of influenza A, B, or unknown subtypes and repeat the test for invalid results.After that clean the instrument and the surrounding bench area with 70%ethanol or 10%bleach solution. This figure shows the average CT values determined by the RP assay for the positive and negative samples for each influenza subtype. The results demonstrate that the false negative samples determined by the influenza A and B assay were due to low viral load samples.Once mastered, this technique can be done in 15 minutes if it is performed properly. While attempting this procedure it's important to remember that when initially setting up the assay, the transfer cartridge must be connected to the test base until they click into place. If these two pieces are not secured properly, the run will fail and will need to be repeated from the beginning.After watching this video you should have a good understanding of how to perform this diagnostic assay for influenza. Don't forget that working with patient samples potentially infected with influenza can be extremely hazardous and precautions, such as wearing appropriate personal protective equipment, should always be taken while performing this procedure.

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JoVE Journal

Immunology and Infection

15.5K visualizações.  Memorial Sloan Kettering Cancer Center. The overall goal of this assay is to diagnose or rule out influenza virus infection in patients presenting with respiratory systems. This method can help answer key questions in the clinical diagnostics field such as diagnosing key causes of respiratory infections. The main advantage of this technique is that it is a rapid method that can be used in appointive care setting.Demonstrating the procedure will be Sam Kaplan, a technician from our laboratory. To collect a nasal swab sample, ...