Bioluminescence-Based Neuraminidase Inhibition Assay for Monitoring Influenza Virus Drug Susceptibility in Clinical Specimens

Abstract

The QFlu prototype bioluminescence-based neuraminidase (NA) inhibition (NI) assay kit was designed to detect NA inhibitor (NAI)-resistant influenza viruses at point of care. Here, we evaluated its suitability for drug susceptibility assessment at a surveillance laboratory. A comprehensive panel of reference viruses (n ‫؍‬ 14) and a set of 90 seasonal influenza virus A and B isolates were included for testing with oseltamivir and/or zanamivir in the QFlu assay using the manufacturer-recommended protocol and a modified version attuned to surveillance requirements. The 50% inhibitory concentrations (IC 50 s) generated were compared with those of NI assays currently used for monitoring influenza drug susceptibility, the fluorescent (FL) and chemiluminescent (CL) assays. To provide proof of principle, clinical specimens (n ‫؍‬ 235) confirmed by real-time reverse transcription (RT)-PCR to contain influenza virus A(H1N1)pdm09 and prescreened for the oseltamivir resistance marker H275Y using pyrosequencing were subsequently tested in the QFlu assay. All three NI assays were able to discriminate the reference NA variants and their matching wild-type viruses based on the difference in their IC 50 s. Unless the antigenic types were first identified, certain NA variants (e.g., H3N2 with E119V) could be detected among seasonal viruses using the FL assays only. Notably, the QFlu assay identified oseltamivir-resistant A(H1N1)pdm09 viruses carrying the H275Y marker directly in clinical specimens, which is not feasible with the other two phenotypic assays, which required prior virus culturing in cells. Furthermore, The QFlu assay allows detection of the influenza virus A and B isolates carrying established and potential NA inhibitor resistance markers and may become a useful tool for monitoring drug resistance in clinical specimens.

Respiratory Viral Infections in Patients With Cancer or Undergoing Hematopoietic Cell Transplant

“…Emergence of universal resistance of IV A H1N1 and IV A H3N2 to adamantanes, as well as rapid emergence of oseltamivir resistant IV A H1N1 led to increased efforts to closely monitor drug resistance (Renaud et al, 2010, 2011a,b; Okomo-Adhiambo et al, 2014, 2015). Both phenotypic and genotypic assays have developed, but genotypic methods are most commonly used for routine testing to detect neuraminidase inhibitor resistance of influenza viruses in clinical specimens (Hirsch et al, 2013; Marjuki et al, 2013; Okomo-Adhiambo et al, 2013; Laplante and St George, 2014; Tamura et al, 2015).…”

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

“…iART utilises an advanced enzyme substrate that enables measurement of NA activity in virus isolates and in clinical specimens. Unlike the substrate used in the bioluminescence-based assay [ 14 ], the substrate used in iART is specific to influenza NA, making it more suitable for testing clinical specimens that may contain other pathogens. In this assay, the sample is divided between two wells of a disposable ( Figure ), one well containing substrate and the other well containing substrate and oseltamivir carboxylate.…”

A Review of the Antiviral Susceptibility of Human and Avian Influenza Viruses over the Last Decade

“…However, it is important to be aware that the absence of common resistance mutations does not indicate that the viruses are NAI-sensitive, as novel mutations may occur. An alternative to the FL and CL assays is a bioluminescence-based NA inhibition assay (QFlu) that has the main advantage of being sensitive enough to detect low levels of virus in clinical specimens compared to the FL and CL assay which require a cultured viral isolate [ 31 ].…”