Standardizing the influenza neuraminidase inhibition assay among United States public health laboratories conducting virological surveillance

Abstract

Okomo-Adhiambo, M.; Mishin, V.P.; Sleeman, K.; Saguar, E.; Guevara, H.; Reisdorf, E.; Griesser, R.H.; Spackman, K.J.; Mendenhall, M.; Carlos, M.P.; Healey, B.; St. George, K.; Laplante, J.; Aden, T.; Chester, S.; Xu, X.; and Gubareva, L.V., "Standardizing the influenza neuraminidase inhibition assay among United States public health laboratories conducting virological surveillance" (2016 a b s t r a c tBackground: Monitoring influenza virus susceptibility to neuraminidase (NA) inhibitors (NAIs) is vital for detecting drug-resistant variants, and is primarily assessed using NA inhibition (NI) assays, supplemented by NA sequence analysis. However, differences in NI testing methodologies between surveillance laboratories results in variability of 50% inhibitory concentration (IC 50 ) values, which impacts data sharing, reporting and interpretation. In 2011, the Centers for Disease Control and Prevention (CDC), in collaboration with the Association for Public Health Laboratories (APHL) spearheaded efforts to standardize fluorescence-based NI assay testing in the United States (U.S.), with the goal of achieving consistency of IC 50 data. Methods: For the standardization process, three participating state public health laboratories (PHLs), designated as National Surveillance Reference Centers for Influenza (NSRC-Is), assessed the NAI susceptibility of the 2011e12 CDC reference virus panel using stepwise procedures, with support from the CDC reference laboratory. Next, the NSRC-Is assessed the NAI susceptibility of season 2011e12 U.S. influenza surveillance isolates (n ¼ 940), with a large subset (n ¼ 742) tested in parallel by CDC. Subsequently, U.S. influenza surveillance isolates (n ¼ 9629) circulating during the next three influenza seasons (2012e15), were independently tested by the three NSRC-Is (n ¼ 7331) and CDC (n ¼ 2298).Results: The NI assay IC 50 s generated by respective NSRC-Is using viruses and drugs prepared by CDC were similar to those obtained with viruses and drugs prepared in-house, and were uniform between laboratories. IC 50 s for U.S. surveillance isolates tested during four consecutive influenza seasons (2011 e15) were consistent from season to season, within and between laboratories. Conclusion: These results show that the NI assay is robust enough to be standardized, marking the first time IC 50 data have been normalized across multiple laboratories, and used for U.S. national NAI susceptibility surveillance.Published by Elsevier B.V.

Antiviral Drug–Resistant Influenza B Viruses Carrying H134N Substitution in Neuraminidase, Laos, February 2016

“…We propagated the viruses and then used the CDC standardized NA inhibition assay to assess their susceptibility to NAIs ( 4 ). Compared with the median 50% inhibitory concentration (IC 50 ) values for B-Victoria lineage viruses, IC 50 values for 2 of the 24 B-Victoria lineage viruses, B/Laos/0406/2016 and B/Laos/0525/2016, were elevated for zanamivir (129- to 158-fold), oseltamivir (4-fold), peramivir (72- to 74-fold), and laninamivir (41- to 42-fold) (Table 1).…”

“…This interpretation is useful but obscures the higher median oseltamivir IC 50 value (9.67 nmol/L vs. 0.42–1.47 nmol/L for other NAIs; Table 1) and the lower potency of oseltamivir in inhibiting NA activity of influenza B viruses ( 4 , 7 ). Moreover, reports from clinical studies indicate a lesser susceptibility of influenza B viruses to oseltamivir than to zanamivir ( 7 –9).…”

“…A) Thermostability of neuraminidase (NA) determined after viruses were incubated for 15 min at 4°C or at 30°C–57°C. NA enzyme activity was determined by a fluorescence-based assay ( 4 ). B) Replication kinetics of influenza B viruses in fully differentiated human primary NHBE cells that were inoculated with the designated viruses (multiplicity of infection 0.001).…”

Monitoring influenza virus susceptibility to oseltamivir using a new rapid assay, iART

“…Additionally, the viral NA sequence from both the isolate and matching clinical specimen should be compared to rule out culturing artefacts [9-11]. Due to the complexity of the assay and data interpretation, testing is mainly performed by specialised surveillance laboratories [10,12,13]. …”

Detection of influenza B viruses with reduced sensitivity to neuraminidase inhibitor in Morocco during 2014/15 season

“…Fluorescence was detected on the Victor X3 (WSLH. Series Multilabel Plate Readers PerkinElmer, Inc. Waltham, MA 02451 USA) equipped with filters for excitation (λ = 365 nm) and emission (λ = 450 nm) (14).…”