1. NI Assays: Troubleshooting & Analysis of Curve Fitting Graph 1: Standard clinical isolate Good NA activity S shaped curve (observed points) IC50 in expected range for susceptible B Good fit of calculated curve to observed points Conclusion: VALID sensitive isolate

2. NI Assays: Troubleshooting & Analysis of Curve Fitting Graph 2: Known resistant mutant Low NA activity Known resistant virus High IC50 S shaped curve, but displaced right because of drug inhibition Good fit curve to observed points Conclusion: VALID resistant isolate

3. NI Assays: Troubleshooting & Analysis of Curve Fitting Graph 3: Clinical isolate with low IC50 Low NA activity Poor fit observed points to calculated curve IC50 value low Conclusion: Probably isolate with normal IC50 → REPEAT

4. NI Assays: Troubleshooting & Analysis of Curve Fitting Graph 4: Clinical isolate with high IC50 Low NA activity Poor fit observed points to curve IC50 displaced to right High IC50 Conclusion: Probably resistant isolate REPEAT to check

5. NI Assays: Troubleshooting & Analysis of Curve Fitting Graph 5: Clinical isolate with low IC50 Good NA activity Poor fit observed values to curve especially at high drug concentrations Non sigmoidal shape of observed curve Low IC50 Conclusion: Probable error drug dilution for normal iC50 invalid → REPEAT

6. NI Assays: Troubleshooting & Analysis of Curve Fitting Graph 6: Clinical isolate with high IC50 Low NA activity Non sigmoidal observed points High IC50 Bimodal distribution observed points Poor fit between observed points and curve Conclusion: Possible resistant but technical errors → REPEAT

7. NI Assays: Troubleshooting & Analysis of Curve Fitting Graph 7: Clinical isolate with normal IC50 Good NA activity Increase in drug concentration associated with increase NA activity (Biologically implausible) Non sigmoidal observed points Poor fit observed points and curve Conclusion: Invalid analysis but probably normal IC50. Technical errors → REPEAT

8. NI Assays: Troubleshooting & Analysis of Curve Fitting Graph 8: Clinical isolate with low IC50 Low NA activity Increase NA activity with drug concentration (Biologically implausible) Bimodal distribution observed points Poor fit observed points to curve Low IC50 Conclusion: INVALID analysis → REPEAT as probably normal IC50

9. NI Assays: Troubleshooting & Analysis of Curve Fitting Graph 9: Clinical isolate with low IC50 Good NA activity NA activity increases with drug concentration Bimodal distribution observed points Poor fit curves and observed points Low IC50 Conclusion: Invalid analysis → REPEAT as probably normal IC50