Application of a 7-target pyrosequencing assay to improve the detection of neuraminidase inhibitor resistant influenza A(H3N2) viruses.

Tamura D1, Okomo-Adhiambo M2, Mishin VP2, Guo Z2, Xu X2, Villanueva J2, Fry AM2, Stevens J2, Gubareva LV3.

Author information

Abstract

National U.S. influenza antiviral surveillance incorporates data generated by neuraminidase (NA) inhibition (NI) testing of isolates supplemented with NA sequence analysis; and pyrosequencing analysis of clinical specimens. Lack of established correlates for clinically relevant resistance to NA inhibitors (NAIs) hinders interpretation of NI assay data. Nonetheless, A(H3N2) viruses are commonly monitored for highly reduced or reduced inhibition in the NI assay and/or presence of NA markers, E119V, R292K and N294S. In 2012-2013, three drug resistant A(H3N2) viruses were detected by NI assay among isolates (n=1424); all showed highly reduced inhibition by oseltamivir and had E119V. In addition, one R292K variant was detected among clinical samples (n=1024) by a 3-target pyrosequencing assay. Overall, frequency of NAI resistance was low, 0.16% (4 of 2448). To screen for additional NA markers previously identified in viruses from NAI-treated patients, the pyrosequencing assay was modified to include Q136K, I222V, del245-248 and del247-250. The 7-target pyrosequencing assay detected NA variants carrying E119V, Q136 and del245-248 in an isolate from an oseltamivir-treated patient. Next, this assay was applied to clinical specimens collected from hospitalized patients and submitted for NI testing, but failed cell culture propagation. Of the 27 clinical specimens tested, 4 (15%) contained NA changes: R292K (n=2), E119V (n=1) and del247-250 (n=1). Recombinant NAs with del247-250 and del245-248, respectively, conferred highly reduced inhibition by oseltamivir, reduced inhibition by zanamivir, and normal inhibition by peramivir and laninamivir. Our results demonstrated the benefits of the 7-target pyrosequencing assay in conducting A(H3N2) antiviral surveillance and testing for clinical care.