Table 3. Results from neuraminidase inhibition (NAI) and iART assays on mixtures of influenza A(H1N1)pdm09 viruses containing different proportions of mutants with H275Y in the neuraminidase (n = 22).
| Virus | Pyrosequencing (%)a | NAI assay | iART | |||
|---|---|---|---|---|---|---|
| H275 | H275Y | IC50, nM (Fold)b | Interpretationc | R-factor | Resultd | |
| A/Louisiana/08/2013 | 0 | 100 | 190.84 (1,004) | HRI | 5.97 | Resistant |
| A/Mississippi/11/2013 | 3 | 97 | 177.62 (935) | HRI | 6.67 | Resistant |
| A/North Carolina/04/2014 | 3 | 97 | 199.91 (1,052) | HRI | 6.17 | Resistant |
| A/Michigan/73/2016 | 3 | 97 | 157.39 (828) | HRI | 5.89 | Resistant |
| A/Texas/09/2014 | 7 | 93 | 131.02 (690) | HRI | 5.39 | Resistant |
| A/Texas/100/2013 | 9 | 91 | 150.21 (791) | HRI | 5.03 | Resistant |
| A/Massachusetts/06/2016 | 10 | 90 | 121.85 (641) | HRI | 6.03 | Resistant |
| A/Pennsylvania/18/2014 | 11 | 89 | 127.1 (669) | HRI | 6.20 | Resistant |
| A/Florida/10/2014 | 14 | 86 | 111.35 (586) | HRI | 6.46 | Resistant |
| A/Colorado/07/2014 | 16 | 84 | 110.24 (580) | HRI | 6.22 | Resistant |
| A/Brazil/0257 S2/2016 | 25 | 75 | 97.73 (514) | HRI | 4.92 | Resistant |
| A/Brazil/9061/2014 | 32 | 68 | 39.32 (207) | HRI | 3.47 | Resistant |
| A/Quebec/RV1424/2016 | 48 | 52 | 4.14 (22) | RI | 1.93 | Resistant |
| Mix #1e | 63 | 37 | 1.37 (8) | NI | 1.09 | Resistant |
| A/Utah/10/2013 | 68 | 32 | 0.98 (5) | NI | 1.25 | Resistant |
| A/North Carolina/21/2013 | 72 | 28 | 0.95 (5) | NI | 1.28 | Resistant |
| Mix #2 | 76 | 24 | 0.73 (4) | NI | 0.71 | Resistant |
| Mix #3 | 84 | 16 | 0.49 (3) | NI | 0.46 | Nonresistant |
| A/Michigan/36/2016 | 89 | 11 | 0.57 (3) | NI | 0.43 | Nonresistant |
| Mix #4 | 92 | 8 | 0.37 (2) | NI | 0.28 | Nonresistant |
| Mix #5 | 96 | 4 | 0.35 (2) | NI | 0.12 | Nonresistant |
| A/Maryland/08/2013 | 100 | 0 | 0.22 (1) | NI | 0.18 | Nonresistant |
iART: influenza Antiviral Resistance Test; IC50: inhibitory concentration 50%; R-factor: ratio of chemiluminescent signal intensity generated by viral neuraminidase activity on the substrate, with and without inhibitor (i.e. oseltamivir carboxylate).
a Proportion of H275 and H275Y virus subpopulations was determined by a single-nt polymorphism (SNP) pyrosequencing analysis in allele quantification mode (AQ) as described in reference [15].
b Fold increase calculated using the median oseltamivir IC50 for influenza A(H1N1)pdm09 viruses circulating during 2015/16 influenza season.
c Interpretation of NAI assay results based on the fold increase in IC50 value: normal (< 10-fold), reduced (10–100-fold) and highly reduced (> 100-fold) inhibition; NI, normal inhibition; RI, reduced inhibition; HRI, highly reduced inhibition.
d Output result as shown on the device’s display; result is based on the pre-set cutoffs for influenza A (≥ 0.7) and influenza B (≥ 2.2) viruses.
e H275 and H275Y mixes were prepared by combining the two virus isolates A/Maryland/08/2013 and A/Louisiana/08/2013, at different ratios.