Qflu™

hours of onset of illness. Thus, prompt diagnosis of influenza viral infections may aid physicians in undertaking appropriate preventative and therapeutic intervention. For Detection of Resistance to
Influenza Neuraminidase Inhibitors
Influenza types A and B virus possess surface glycoproteins with neuraminidase activity, which hydrolyzes substrates that contain alpha-ketosidically linked N-acetylneuraminic acid (Neu5Ac). The Cellex, Inc.
QFluTM NI Assay uses a substrate that enables the production of light signal in the presence of influenza viral neuraminidase. Thus, the QFluTM Flu NI Assay detects viral enzyme activity rather than the I. INTENDED USE
The QFluTM NI Assay is a rapid chemiluminescent assay for the The emitted light signal in the QFluTM assay is directly proportional direct and quantitative detection of influenza A and B viral to influenza viral neuraminidase activity, which in turn is proportional neuraminidase enzyme activity and resistance of antiviral drugs to virus concentration in the sample. This test can detect both influenza virus type A and B, but does not differentiate between these two influenza virus types, nor does the QFluTM NI Assay QFluTM NI Assay is intended for research use only. detect influenza type C, which does not possess neuraminidase. However, Type C influenza is not considered a significant clinical II. SUMMARY
Influenza illness is classically characterized by sudden onset of Since influenza viral neuraminidase is the target of oseltamivir fever, chills, headache, myalgias, and a non-productive cough. carboxylate and Zanamivir and a number of flu antiviral candidates, Epidemics of influenza typically occur during winter months with an the assay can be used to detect influenza virus that is resistant to estimated 114,000 hospitalizations and about 36,000 deaths per these drugs or drug candidates or used to screening drug year in the U.S. Influenza viruses can also cause pandemics, during which the numbers of illnesses and deaths from influenza-related complications can increase dramatically. The QFluTM NI Assay is simple and rapid, and requires no prior titration of the sample. When used as a neuraminidase inhibition Management of influenza includes use of antiviral therapy. assay, the assay involves 1) mixing the master mix with the sample Currently available antivirals target one of the two influenza viral (40:5 ratio), 2) dispensing 45 µL of the resulting mix into multiple components, the M2 ion channel or neuraminidase (for reviews, see wells (normally 8-10) containing various concentrations of inhibitor ref 1-3). Influenza viral strains resistant to the M2 channel inhibitors to a final concentration of 0 to 1000 nM, 3) incubating at room rimantadine and amantadine are prevalent during interpandemic temperature for 30 minutes, and 4) measuring the light signal using influenza. More strikingly, some H5N1 isolates are already resistant a luminometer that accommodates microwell plates. The entire to one or both M2 channel inhibitors, suggesting that these inhibitors process takes less than 10 min manual time. might not be effective antivirals during a pandemic (1-3). Thus, the neuraminidase inhibitors oseltamivir and zanamivir are the mainstay III. REAGENTS AND SUPPLIES
drugs for therapeutic intervention in cases of influenza virus infections. Since zanamivir is administered through inhalation, a cumbersome procedure, oseltamivir is the favored choice for Lyophilized master mix; each vial is sufficient for two epidemic influenza treatment or stockpiling in preparation of a Based on widespread resistance to the M2 channel blockers, it is not unreasonable to speculate that widespread use of neuraminidase inhibitors may eventually lead to emergence of their resistance in seasonal or potential pandemic influenza virus. A – Luminometer that accommodates microwell plates number of neuraminidase inhibitor resistant mutants have been – External positive and negative controls identified during preclinical or clinical studies. In vitro selection did – Microwell plates suitable for chemiluminescence assays lead to the isolation of NA gene mutations E119V, R292K, H274Y, and R152K, which confer resistance to oseltamivir (4-9). In a three- IV. APPROPRIATE SAMPLES
year surveillance (1999-2002), eight virus variants with a > 10-fold decrease in susceptibility to oseltamivir were isolated (10). These findings paint a worrisome picture for the use of influenza – Other sample types as determined by the user Indeed, widespread of seasonal influenza virus H1N1 resistant to Sample Storage Media: The following media and solutions have oseltamivir did appear during the 2008/2009 flu season in U.S., been tested and found to be compatible with the QFluTM NI Assay: which heightens the need to more closely monitor flu antiviral drug Earle’s Minimal Essential Medium (EMEM) Still, recent findings of high concentrations of oseltamivir carboxylate in sewage water (15) unveiled another avenue – the avian population – from which oseltamivir resistant influenza virus strains may emerge because wild ducks live in environmental water systems and are the natural hosts of influenza virus. Although it may be a coincidence, it was reported that a resistant H5N1 strain carrying the H274Y mutation caused viremia in two patients who subsequently died from avian influenza (16). Incompatible Solvents and Solutions: The following solvent and solutions were tested and found to be incompatible with the QFluTM Patients who are suspected of having influenza may benefit from treatment with an antiviral agent especially if given within the first 48 Table 1: The Raw Data (RLU) from the Precision Study V. INHIBITION
PROTOCOL
The following protocol is designed to estimate the IC50 value for the virus in a sample. Other user-specific protocol should be developed by the user accordingly. For example, drug candidate screening Step 1 – Prepare the neuraminidase inhibitors at various concentrations. The followings are examples: 0 (buffer), 5, 25, 50, 100, 250, 500, 750, 1000, 5000 and 10,000 nM. 874529 85420 819245 855301 880671 935644 Dispense 5 µL each of the inhibitor to a microwell. The mean, standard deviation (SD) and coefficient of variation (CV) Step 2 – Add 5 µL of sample to each well of the signal (RLU) were calculated and are presented in Table 2. Except for the background counts, the test results for all the Step 3 – Dissolve the lyophilized master mix in 4 or 8 mL of samples containing influenza virus were quite reproducible with CV deionized water for 1 plate or 2 plate kits, respectively. Place the dissolved reagent on ice if not used immediately. Table 2: Statistical Profile of the Data from the Precision Study Step 4 – Dispense 40 µL of the detection mix from Step 3 to the microwells. Incubate at room temperate for 30 minutes. The relatively long incubation duration is based on the observation that influenza neuraminidase inhibitor binding is a slow reaction (ref 17), but it can be optimized by the user. In our hands, short incubation time (e.g., 5-10 min) results IC50 values that are similar to those with longer Step 5 – Place the microwell plate in the luminometer and measure The reactions were initiated by adding influenza virus to the detection mix as described in the Inhibition Assay Protocol except A linearity study was performed to assess the linearity and linear that there is no inhibitor in the reactions; instead, increasing range of the assay. Sample containing 0, 10, 100, 200, 400, 600, amounts of virus were added to the reactions. The signal over a 1000, 10,000, 100,000, or 200,000 TCID50 units of cell culture- period of 60 minutes was recorded using a Bethold Sirius adapted influenza virus, strain A/WS/33, was mixed with the luminometer. The light signal was measured once every 10 detection mix and incubated at room temperature for 15 minutes, seconds. The numbers in the column in Fig. 1 are the input followed by measurement of the emitted light signal using a Bethold influenza virus amounts in TCID50 units. In the presence of influenza virus, the signal increased rapidly in the The net RLU from each sample was plotted against the input TCID50 first 10 minutes, after which the signal reached a plateau. units of the virus. As shown in Fig 2, the assay was linear throughout the entire tested spectrum (10 to 200,000 TCID50 units) with a correlation coefficient (R2) of 0.997 (95% confidence interval: Fig 2 I Relationship between Signal Output and Virus Input 20000000
A precision study was performed to assess run-to-run and day-to- day variability of the assay. The light signal was measured at 15 minutes after addition of a sample to the detection mix using the CLX 2000 luminometer. The study was performed over a three day The inhibition assay was carried out by following the inhibition assay period (not consecutive days), during which duplicate samples for protocol using oseltamivir carboxylate as the inhibitor, which was each concentration were tested each day. The raw data are used in final concentrations shown in Fig 3. Cultured influenza virus (A/WS/33) was used as the sample. The reactions were immediately placed in a luminometer for signal measurement. The light signal was collected over a period of 60 minutes. Handel A, Longini IM Jr, Antia R. Neuraminidase Inhibitor Fig 3 | Oseltamivir Carboxylate Inhibition Kinetics Resistance in Influenza: Assessing the Danger of Its Generation and Spread. PLoS Comput Biol 3: e240. Lipsitch M, Cohen T, Murray M, Levin BR. Antiviral Resistance and the Control of Pandemic Influenza. PLoS Barnett JM., Cadman A, Gor D, Dempsey M, Walters M, Candlin A, Tisdale M, Morley PJ, Owens IJ, Fenton RJ, Lewis AP, Claas ECJ, Rimmelzwaan GF, De Groot R, Osterhaus ADME. Zanamivir susceptibility monitoring and characterization of influenza virus clinical isolates obtained during phase II clinical efficacy studies. l In 100000
Antimicrob. Agents Chemother. 44:78–87. (2000) Gubareva LV, M. N. Matrosovich, M. K. Brenner, R. C. Bethell, and R. G. Webster. Evidence for zanamivir resistance in an immunocompromised child infected with influenza B virus. J. Infect. Dis. 178:1257–1262 (1998). 10. Monto AS, McKimm-Breschkin JL, Macken C, Hampson AW, Hay A, Klimov A, Tashiro M, Webster RG, Aymard M, Hayden FG, Zambon M. Detection of influenza viruses Time (Min)
resistant to neuraminidase inhibitors in global surveillance during the first 3 years of their use. Antimicrob. Agents 11. Rungrotmongkol T, Intharathep P, Malaisree M, Nunthaboot N, Kaiyawet N, Sompornpisut P, Payungporn S, Poovorawan Y, Hannongbua S.: Susceptibility of The following bacterial species were tested at a concentration antiviral drugs against 2009 influenza A (H1N1) virus. of 2x109 colony forming units (CFU)/mL and found not to have Biochem Biophys Res Commun. 385(3):390-394 (2009) cross reactivity in negative samples, or inhibition in positive Moscona A.N Engl J Med. 360:953-6 (2009) 13. Dharan NJ, Gubareva LV, Meyer JJ, Okomo-Adhiambo M, Acinetobacter calcoaceticus anitratus, Enterobacter cloacae, McClinton RC, Marshall SA, St George K, Epperson S, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Brammer L, Klimov AI, Bresee JS, Fry AM; Oseltamivir- Pseudomonas aeruginosa, Salmonella choleraesuis, Serratia marcescens, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Yersinia pseudotuberculosis, and Candida albicans.. VI. WARNING
PRECAUSIONS
15. Fick J, Lindberg RH, Tysklind M, Haemig PD, Waldenström J, et al. Antiviral Oseltamivir Is not 2. Freezing of neuraminidase samples tend to reduce Removed or Degraded in Normal Sewage Water Treatment: Implications for Development of Resistance by VII. LIMITATIONS
16. deJong MD, Tran TT, Truong HK, Vo MH, Smith GJD, 1.This version of QFluTM NI Assay is not specific for influenza Nguyen VC, Bach VC, Phan TQ, Do QH, Yi G, Peiris JGM, neuraminidase. A version specific for influenza neuraminidase will Tran TH, Farrar J. Oseltamivir resistance during treatment of influenza A (H5N1) infection. N. Engl. J. Med. 17. Kati WM, Saldivar AS, Mohamadi F, Sham HJ, Laver WG, REFERENCES
Kohlbrenner WK. GS4071 Is a Slow-Binding Inhibitor of Influenza Neuraminidase from Both A and B Strains. Hoffmann C, Korsman S, Kamps BS. Treatment and Bioch Biophy Res Comm. 244: 408-413 (1998) MORE INFORMATION
Kamps BS, Hoffmann C. Drug Profile. In: Influenza Report McKimm-Breschkin JL. Resistance of influenza viruses to neuraminidase inhibitors—a review. Antivir. Res. 47:1–17 Hatakeyama S, Sugaya N, Ito M, Yamazaki M, Ichikawa M, Kimura K, Kiso M, Shimizu H, Kawakami C, Koike K, Mitamura K, Kawaoka Y. Emergence of influenza B viruses with reduced sensitivity to neuraminidase and pandemic influenza. J Infect Dis. 194 Suppl 2:S119-26 (2006)

Source: http://www.cellex.us/files/usermanual1.pdf