Unlike sequence-based assays, the NA inhibition (NAI) assay enables the detection of potentially drug-resistant viruses regardless of underlying genetic change(s). It is the gold standard method for assessing susceptibility to NA inhibitors [9,10]. Interpretation of the NAI assay is based on the determined IC₅₀, a drug concentration needed to inhibit 50% of the NA enzyme activity. Depending on the fold increase of IC₅₀ compared with a control, results are reported as normal (NI), reduced (RI) or highly reduced (HRI) inhibition. In the absence of established laboratory correlates of clinically-relevant oseltamivir resistance, all viruses displaying RI/HRI are considered to be potentially drug resistant and as such are monitored [9,10]. Although useful, this method is labour intensive, complex, and requires propagation of the viruses in cell culture. 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].

In this study, we investigated whether the influenza Antiviral Resistance Test (iART), a rapid prototype assay developed by Becton Dickinson R and D for research use only, could be used to improve oseltamivir resistance surveillance by providing a simpler and faster testing method. 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 simple device is used to measure the chemiluminescent signal generated from each well of the disposable. The built-in software calculates the ratio of signal intensity between the wells (R-factor), which appears on the device’s display along with the final result: ‘resistant’ or ‘nonresistant’. The threshold between nonresistant and resistant is different for type A and type B viruses, with R-factors of 0.7 and 2.2, respectively. If the NA activity is too low or absent, the message ‘insufficient signal’ appears on the display.

Clinical specimens (n=149) were either applied to the gravity-fed column as is, or were diluted fivefold using viral transport medium (VTM). Virus isolates (n=76) were diluted 100- or 1,000-fold using VTM to meet the assay requirement (40,000 < signal < 6,000,000 luminescent units).

In the first experiment, the international reference panel for NAI assay was tested using iART and the United States Centers for Disease Control and Prevention (US CDC) standardised fluorescence-based NAI assay [13] (Table 1). Viruses identified as resistant by iART, displayed RI or HRI by NAI assay; viruses with NI were identified as nonresistant, indicating good agreement between the two assays (Table 1).

Monitoring the spread of A(H1N1)pdm09 viruses exhibiting HRI by oseltamivir and carrying H275Y is a priority for surveillance. To evaluate the ability of iART to detect oseltamivir-resistance conferred by H275Y, 13 virus isolates with this mutation, which had been collected between 2009 and 2016 were tested. All these H275Y viruses exhibited HRI by NAI assay and were also identified as resistant by iART with R-factor of 5.3 ± 0.76 (Table 2).

In some instances, a sample may contain the drug-resistant and wild-type viruses (mix), but still be detected as normally inhibited in the NAI assay. To assess the ability of iART to detect oseltamivir resistance in such samples, we next tested samples with increasing proportions of H275Y (as determined by pyrosequencing [15]). Notably, isolates containing ≥ 24% of the H275Y variant were identified as resistant by iART, whereas NAI assay required ≥ 52% of the H275Y variant to detect RI, suggesting that iART was more efficient at this task (Table 3).

Next, we assessed iART ability to detect influenza viruses harbouring NA mutations other than H275Y and displaying RI/HRI against oseltamivir (Table 2): A(H1N1)pdm09 viruses that displayed RI by oseltamivir carrying the S247R (n = 3) or I223R (n = 1) were identified as resistant with high R-factors for S247R and an R-factor of 1.99 ± 0.30 for I223R. One virus carrying I223K was detected as nonresistant with an R-factor substantially below the resistance threshold (0.42 ± 0.03). The virus carrying D199G displayed NI (eightfold) by NAI assay and was identified as resistant by iART (Table 2). A(H3N2) viruses that display HRI by NAI assay were all identified as resistant by iART. The R-factor of the R292K virus was much greater than those harbouring either E119V or a four-amino acid deletion (del245–248). Three B/Victoria/2/87-lineage viruses – harbouring E117G, N294S or A200T – that displayed RI against oseltamivir were all identified as resistant by iART (Table 2). B/Yamagata/16/98-lineage viruses harbouring E117A, R150K or R374K, that displayed HRI by NAI assay were identified as resistant; and two viruses carrying H273Y and one carrying G407 presenting borderline NI/RI were identified as nonresistant by iART (Table 2). Finally, a group of viruses from both B/lineages – carrying D197N, K152N and I221V – showed borderline NI/RI by NAI assay (4–8-fold), and these viruses were identified as resistant by iART. These results demonstrate that iART may detect some influenza viruses harbouring NA changes in the enzyme active site (e.g. D199G in A(H1N1)pdm09 and I221V in type B) that otherwise would be classified as NI by oseltamivir using NAI assay. Notably, the criteria to separate viruses exhibiting NI from those with RI is arbitrary [9], and can be refined as more data become available. The interpretation of results obtained for viruses displaying borderline IC₅₀ should be made cautiously.