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 samples.
<ol>
	<li>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. Store the swabs and transport them in vials containing 3 ml of viral transport media (VTM). 
	NOTE: Careful attention to appropriate specimen collection must be taken as inadequate specimen collection may yield erroneous results. 
	NOTE: Swab specimens should be tested as soon as possible after collection. However, they may be stored at room temperature for up to 2 hr or refrigerated at 2-8 &#176;C for up to 24 hr if testing is not immediately available. Freshly collected specimens are ideal for optimal test performance. Improper sample handling, storage, and/or transport may yield erroneous results. 
	NOTE: As a precautionary measure, individuals performing this test should treat all specimens as potentially infectious by following universal precautions when handling specimens.</li>
	<li>Bring all samples to room temperature prior to testing. 
	NOTE: All test components are for a single use only and should not be used to perform multiple tests. Do not mix kit components from different lot numbers. Do not use kit reagents past their expiration date.</li>
	<li>Bring 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.</li>
	<li>Leave all test pieces in the foil wrapping until immediately before use. Turn on the instrument by pressing the power button on the side of the instrument. Enter the user-ID and press &#39;OK.&#39;</li>
</ol>
2. Running the Test
<ol>
	<li>To begin the test process, touch &#39;Run Test&#39; on the instrument screen. Enter the patient ID by using on-screen keyboard or barcode scanner and press &#39;OK.&#39; Open the lid and insert the Orange test base into the Orange test base holder.</li>
	<li>Confirm that the correct test is displayed on the screen and press &#39;OK.&#39; Insert the blue sample receiver into the blue sample receiver holder. 
	NOTE: Do not open the sample receiver before placing in the instrument as this will prevent the elution buffer from reaching the correct operating temperature and may impact test performance.</li>
	<li>Wait for the sample receiver to warm up. When prompted by the instrument, remove the foil seal and place the patient swab to be tested into the sample receiver. 
	NOTE: When removing the foil, place two fingers on the edge of the Sample Receiver to ensure that it stays in place.</li>
	<li>Vigorously mix the swab in the liquid for 10 sec, pressing the swab head against the side the sample receiver as you mix it to help dislodge the specimen from the swab. Press &#39;OK&#39; to proceed. If testing swabs stored in viral transport medium, vortex the VTM for 10 sec then add 200 &#181;l to the sample receiver.</li>
	<li>Press the white transfer cartridge into the blue sample receiver. Continue pressing on the sample receiver until the orange indicator rises.</li>
	<li>Lift and then connect the transfer cartridge to the test base. Observe the orange indicator descend once the cartridge transfer is attached correctly. Close the Lid and do not open until the &#34;Test Complete&#34; message appears on the screen.</li>
</ol>
3. Quality Control
<ol>
	<li>Touch &#39;Run QC Test&#39; on the home screen. Select the QC Test to be Run. Confirm the test type to match the QC sample intended for testing by touching &#39;OK&#39; and following the on screen prompts to complete testing.</li>
</ol>
4. Result Interpretation
<ol>
	<li>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, unknown subtypes. For invalid results repeat the test.</li>
</ol>
5. Maintenance and Cleaning
<ol>
	<li>Clean the instrument and the surrounding bench area daily with 70% ethanol or 10% bleach solution. Spray the cleaning solution on a damp, lint-free cloth to clean. Do not pour the solutions directly on the instrument.</li>
</ol>

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Prevention control of seasonal influenza with vaccines. Recommendations of the Advisory Committee on Immunization Practices--United States, 2013-2014. , 1-43 (2013).</li><li>Poehling, K. 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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengths+and+Weaknesses+of+FDA-Approved/Cleared+Diagnostic+Devices+for+the+Molecular+Detection+of+Respiratory+Pathogens.">Strengths and Weaknesses of FDA-Approved/Cleared Diagnostic Devices for the Molecular Detection of Respiratory Pathogens.</a> Clinical Infectious Diseases. 52, 312-325 (2011).</li><li>Grohskopf, L. 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=Prevention+and+control+of+seasonal+influenza+with+vaccines:+recommendations+of+the+Advisory+Committee+on+Immunization+Practices-United+States,+2013-2014.">Prevention and control of seasonal influenza with vaccines: recommendations of the Advisory Committee on Immunization Practices-United States, 2013-2014.</a> MMWR Recomm Rep. 62 (07), 1-43 (2013).</li><li>Molinari, N. -. A. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+rapid+diagnosis+on+management+of+influenza+A+infections.">Effect of rapid diagnosis on management of influenza A infections.</a> The Pediatric infectious disease journal. 19 (4), 303-307 (2000).</li><li>Sharma, V., Dowd, M., Slaughter, A. J., Simon, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EFfect+of+rapid+diagnosis+of+influenza+virus+type+a+on+the+emergency+department+management+of+febrile+infants+and+toddlers.">EFfect of rapid diagnosis of influenza virus type a on the emergency department management of febrile infants and toddlers.</a> Archives of Pediatrics &amp; Adolescent Medicine. 156 (1), 41-43 (2002).</li><li>Dale, S. E., Mayer, C., Mayer, M. C., Menegus, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+and+clinical+sensitivity+of+the+3M+rapid+detection+influenza+A+B+assay.">Analytical and clinical sensitivity of the 3M rapid detection influenza A+B assay.</a> J Clin Microbiol. 46 (11), 3804-3807 (2008).</li><li>van Doorn, H. R., 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=Clinical+validation+of+a+point-of-care+multiplexed+in+vitro+immunoassay+using+monoclonal+antibodies+(the+MSD+influenza+test)+in+four+hospitals+in+Vietnam.">Clinical validation of a point-of-care multiplexed in vitro immunoassay using monoclonal antibodies (the MSD influenza test) in four hospitals in Vietnam.</a> J Clin Microbiol. 50 (5), 1621-1625 (2012).</li><li>Hurt, A. C., Alexander, R., Hibbert, J., Deed, N., Barr, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+six+influenza+rapid+tests+in+detecting+human+influenza+in+clinical+specimens.">Performance of six influenza rapid tests in detecting human influenza in clinical specimens.</a> Journal of Clinical Virology. 39 (2), 132-135 (2007).</li><li>Fiore, A. E., 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=Antiviral+agents+for+the+treatment+and+chemoprophylaxis+of+influenza+---+recommendations+of+the+Advisory+Committee+on+Immunization+Practices+(ACIP).">Antiviral agents for the treatment and chemoprophylaxis of influenza --- recommendations of the Advisory Committee on Immunization Practices (ACIP).</a> MMWR Recomm Rep. 60 (1), 1-24 (2011).</li><li>Harper, S. 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=Seasonal+influenza+in+adults+and+children--diagnosis,+treatment,+chemoprophylaxis,+and+institutional+outbreak+management:+clinical+practice+guidelines+of+the+Infectious+Diseases+Society+of+America.">Seasonal influenza in adults and children--diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the Infectious Diseases Society of America.</a> Clin Infect Dis. 48 (8), 1003-1032 (2009).</li><li>Hannoun, C., Tumova, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+on+influenza+laboratory+diagnostic+and+surveillance+methods+in+Europe.">Survey on influenza laboratory diagnostic and surveillance methods in Europe.</a> European Journal of Epidemiology. 16 (3), 217-222 (2000).</li><li>Leonardi, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+of+2009+H1N1+influenza+A+virus+using+fluorescent+antibody+methods.">Rapid identification of 2009 H1N1 influenza A virus using fluorescent antibody methods.</a> Am J Clin Pathol. 134 (6), 910-914 (2010).</li><li>Chartrand, C., Leeflang, M. M. G., Minion, J., Brewer, T., Pai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+Rapid+Influenza+Diagnostic+TestsA+Meta-analysis.">Accuracy of Rapid Influenza Diagnostic TestsA Meta-analysis.</a> Annals of Internal Medicine. 156 (7), 500-511 (2012).</li><li>Lee, G. C., 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=Evaluation+of+a+rapid+diagnostic+test,+NanoSign(R)+Influenza+A/B+Antigen,+for+detection+of+the+2009+pandemic+influenza+A/H1N1+viruses.">Evaluation of a rapid diagnostic test, NanoSign(R) Influenza A/B Antigen, for detection of the 2009 pandemic influenza A/H1N1 viruses.</a> Virol J. 7, 244 (2010).</li><li>Tang, Y. W., 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=Clinical+accuracy+of+a+PLEX-ID+flu+device+for+simultaneous+detection+and+identification+of+influenza+viruses+A+and+B.">Clinical accuracy of a PLEX-ID flu device for simultaneous detection and identification of influenza viruses A and B.</a> J Clin Microbiol. 51 (1), 40-45 (2013).</li><li>Bandt, D., 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=Economic+high-throughput-identification+of+influenza+A+subtypes+from+clinical+specimens+with+a+DNA-oligonucleotide+microarray+in+an+outbreak+situation.">Economic high-throughput-identification of influenza A subtypes from clinical specimens with a DNA-oligonucleotide microarray in an outbreak situation.</a> Molecular and Cellular Probes. 26 (1), 6-10 (2012).</li><li>Pierce, V. M., Elkan, M., Leet, M., McGowan, K. L., Hodinka, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+Idaho+Technology+FilmArray+system+to+real-time+PCR+for+detection+of+respiratory+pathogens+in+children.">Comparison of the Idaho Technology FilmArray system to real-time PCR for detection of respiratory pathogens in children.</a> J Clin Microbiol. 50 (2), 364-371 (2012).</li><li>Teo, J., 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=VereFlu&#8482;:+an+integrated+multiplex+RT-PCR+and+microarray+assay+for+rapid+detection+and+identification+of+human+influenza+A+and+B+viruses+using+lab-on-chip+technology.">VereFlu&#8482;: an integrated multiplex RT-PCR and microarray assay for rapid detection and identification of human influenza A and B viruses using lab-on-chip technology.</a> Archives of Virology. 156 (8), 1371-1378 (2011).</li><li>Babady, N. E., 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=Comparison+of+the+Luminex+xTAG+RVP+Fast+assay+and+the+Idaho+Technology+FilmArray+RP+assay+for+detection+of+respiratory+viruses+in+pediatric+patients+at+a+cancer+hospital.">Comparison of the Luminex xTAG RVP Fast assay and the Idaho Technology FilmArray RP assay for detection of respiratory viruses in pediatric patients at a cancer hospital.</a> J Clin Microbiol. 50 (7), 2282-2288 (2012).</li><li>Chidlow, G., 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=Duplex+real-time+reverse+transcriptase+PCR+assays+for+rapid+detection+and+identification+of+pandemic+(H1N1)+2009+and+seasonal+influenza+A/H1,+A/H3,+and+B+viruses.">Duplex real-time reverse transcriptase PCR assays for rapid detection and identification of pandemic (H1N1) 2009 and seasonal influenza A/H1, A/H3, and B viruses.</a> J Clin Microbiol. 48 (3), 862-866 (2010).</li><li>Bell, J. J., Selvarangan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Alere+I+influenza+A&amp;B+nucleic+acid+amplification+test+by+use+of+respiratory+specimens+collected+in+viral+transport+medium.">Evaluation of the Alere I influenza A&amp;B nucleic acid amplification test by use of respiratory specimens collected in viral transport medium.</a> J Clin Microbiol. 52 (11), 3992-3995 (2014).</li><li>Nie, 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=Evaluation+of+Alere+i+Influenza+A&amp;B+for+Rapid+Detection+of+Influenza+Viruses+A+and+B.">Evaluation of Alere i Influenza A&amp;B for Rapid Detection of Influenza Viruses A and B.</a> Journal of Clinical Microbiology. 52 (9), 3339-3344 (2014).</li><li>Chapin, K. C., Flores-Cortez, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+the+Molecular+Alere+i+Influenza+A&amp;B+Test+Compared+to+That+of+the+Xpert+Flu+A/B+Assay.">Performance of the Molecular Alere i Influenza A&amp;B Test Compared to That of the Xpert Flu A/B Assay.</a> Journal of Clinical Microbiology. 53 (2), 706-709 (2015).</li></ol>

The authors have nothing to disclose.

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 study and protocol is a CLIA-waived, rapid molecular test for Influenza A and B. The technology is based on an isothermal-amplification for the detection and differentiation of influenza viruses A and B. We show representative results to demonstrate the performance of the influenza A and B assay on a panel of archived, frozen NPS VTM samples was evaluated as compared to another molecular-based respiratory panel assay.
The samples that were determined to be &#34;equivocal&#34; or &#34;untypeable&#34; by the RP test had the lowest sensitivity by the Influenza A and B assay. In addition, discordant results were observed more frequently for specimens with higher CT values in RP assays, suggesting that the lower detection rate by the assay was associated with the lower viral titers. The assay had an improved performance for the detection on influenza virus B over influenza virus A detection with a 90.2% sensitivity and 98.1% specificity. The results here are consistent with previously published literature26,27,28.
This study has two important limitations. First, samples were stored in VTM for testing on the platform. However, at the time the samples were tested, the platform did not have FDA approval for this sample type. Therefore, the dilution into VTM, may have reduced the detection of low-titer VTM samples. Second, the specimens that were tested with the Influenza A and B assay had been stored at -70 &#176;C for 6 to 9 months prior to testing whereas the RP testing was done on fresh specimens immediately after they were received. It is possible that freezing the specimens further reduced influenza detection in the samples.
The influenza A &#38; B assay has few steps and is simple to perform. However, there are two key steps that must be completed appropriately and that users can troubleshoot if they experience errors. First, 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 appropriately, the run will fail and will need to be repeated from the beginning. Second, the instrument needs to warm up for 2 min prior to adding the sample. After this warm up time, users have 30 sec to add the sample, connect the transfer cartridge to the test base, and close the lid. If the time passes before this step is complete, the instrument will fail the run and the sample set up will have to be repeated from the beginning.
The limitations of the assay include slightly increased hands-on time and lower sensitivity compared to the respiratory panel and does not subtype influenza A. Further, the only specimen types that can be used with the assay are nasopharyngeal swabs or the NPS that are stored in viral transport media; the performance of the assay for any other specimen type, such as lower respiratory specimens, has not been evaluated. It is important to note that all of the steps in this protocol are critical. Any modifications or deviation from the protocol as written may result in lower performance or of the assay and must be assessed by the user. The benefit of the technology is that the instrument has a small footprint, only takes 15 min to perform and, even though the technology is based on molecular methods, does not require special training or qualifications to perform. The Influenza A and B assay evaluated in this study has the potential to serve as an alternative to RIDTs for the diagnosis of influenza. The system has the advantage of a short total test time, has a small footprint and is easy to use. Overall, the design of the instrument is suitable for point-of-care testing.

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 submitted to the MSKCC clinical laboratory for testing. Of those specimens, 45, 425, 37, and 77 tested positive for influenza viruses A-1, A-3, A-un-subtypable (A-u), and B, respectively with the RP. A total of 360 positive specimens, 40 samples from each of the influenza subtypes, and 37 A-u. In addition, 1-2 negative samples that arrived to the laboratory immediately before or after each of the positive specimens were chosen for subsequent testing with the influenza A and B assay.
In total, the Influenza A and B assay detected 79 influenza virus A, 37 influenza virus B, 240 negative, and 4 invalid results (failure of internal controls) (Table 1). There were 46 discrepant results with 44 samples positive on RP only and two positive on the Influenza A and B assay only. A third molecular-based influenza assay was performed on the 44 discordant specimens which detected 33/43 (8 influenza virus A, 21 A-u, and 2 influenza virus B) that were positive by the RP and 0/1 positive (influenza virus A) by the Influenza A and B assay. An agreement between two or more assays was considered an accurate identification.
The overall Influenza A and B assay sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5). The sensitivity for the influenza A subtypes was A-1, 84.6% (CI, 58.5-100), A-3, 90.2% (CI, 62.8-100), and A-u, 21.9% (CI, 19.6-24.1). The overall specificity for the influenza virus A 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.
Overall, the mean CT values in specimens with discordant results were significantly higher than those obtained for concordant results (16.4 &#177; 0.48 vs. 25.7 &#177; 0.51; p &#62;0.000) (Figure 1).
<img alt="Figure 1" src="/files/ftp_upload/54312/54312fig1.jpg" /> 
Figure 1: Average CT values determined by RP assay for samples that were positive and negative, respectively by the assay for each influenza subtype. Error bars represent standard deviation. **, p &#60;0.01; ***, p &#60;0.001. <a href="http://cloudflare.jove.com/files/ftp_upload/54312/54312fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/54312/54312table1.jpg" /> 
Table 1: Performance of the Influenza A&#38;B on VTM specimens compared to reference results.

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 ...

rapid-molecular-detection-differentiation-influenza-viruses

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

15.5K Views.  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.

Video: Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...