Reactivity and sensitivity of commercially available influenza rapid diagnostic tests in Japan

Seasonal influenza virus routinely causes epidemic infections throughout the world. Sporadic infections by H5N1, H5N6, and H7N9 viruses are also reported. To treat patients suffering from such viral infections, broadly reactive and highly sensitive influenza rapid diagnostic tests (IRDTs) are required. Here, we examined the reactivity and sensitivity of 25 IRDTs available in Japan for the detection of seasonal H1N1pdm09, H3N2, and type B viruses, as well as highly pathogenic H5 and H7 viruses. All of the IRDTs tested detected the seasonal viruses and H5 and H7 viruses albeit with different sensitivities. Several IRDTs detected the H5 and H7 viruses and the seasonal viruses with similar (high) sensitivity.

Influenza is one of the most prevalent infectious diseases in the world. Seasonal influenza viruses, including H1N1pdm09, H3N2, and type B viruses, are responsible for high morbidity and mortality especially among the elderly and immunocompromised individuals. Despite the availability of influenza vaccines, seasonal influenza viruses cause epidemics every year. Moreover, other subtypes of influenza A virus from other animal species have sporadically transmitted to humans. For example, highly pathogenic avian influenza H5N1 viruses are circulating among poultry in eastern Asia and Egypt and transmit to humans 1 . Reassortant viruses (H5N2, H5N6, and H5N8 viruses) that possess the hemagglutinin (HA) segment of a highly pathogenic avian H5N1 virus and the neuraminidase (NA) segment of another subtype have emerged because of the sustained circulation of highly pathogenic avian H5N1 viruses among birds. H5N6 viruses also cause sporadic infection in humans 2 , and H5N2 virus replicates well in mammalian hosts 3,4 . In addition to these H5 viruses, human infections with avian influenza H7N9 virus were first reported in 2013 5 . Since then, the H7N9 virus has infected humans every influenza season, with the fifth wave occurring in the 2016-17 season 6 . During the fifth wave, highly pathogenic H7N9 viruses possessing HA with multi-basic amino acids at the cleavage site were isolated from avian and human cases 7,8 . It is difficult to prepare vaccines against these viruses in a timely manner. Therefore, the first line of defense against H5 and H7 virus infections is antiviral drugs, such as NA inhibitors.
For optimum efficacy, the NA inhibitors (oseltamivir, zanamivir, peramivir, and laninamivir) should be administered within 2 days of symptom onset 9,10 . Healthcare providers therefore need a rapid, easy, and sensitive diagnosis test. For influenza diagnosis, basic virologic approaches such as virus isolation and RT-PCR have been used, but these methods require time and specialized techniques, so they are not suitable in the clinical setting.
To overcome this constraint, influenza rapid diagnostic tests (IRDTs) have been developed and are now widely used even at the local, small clinic level in Japan. However, conventional IRDTs fail to detect influenza viruses at early time points after onset 11,12 . Recently, some manufacturers developed analyzers to increase the sensitivity of IRDTs. These analyzers are able to evaluate the results instead of relying on the human eye. Here, we examined the sensitivity of 25 IRDTs (4 IRDTs that used analyzers and 21 conventional IRDTs) for various isolates of seasonal influenza A and B viruses as well as for human and avian H5 and H7 viruses, which possess the potential to transmit to humans 13 .

Results and Discussion
We evaluated the sensitivity of 25 IRDTs commercially available in Japan in 2017 (Table 1). These IRDTs are optimized to detect seasonal influenza, including H1N1pdm09, H3N2, and type B viruses, by using mouse monoclonal antibodies against the influenza A and B virus nucleoproteins (NPs), which are conserved among the influenza A or B viruses. Because the epitopes on NP are conserved among type A viruses, it is stated that 20 of the 25 IRDTs (the exceptions being QuickNavi Flu, QuickNavi-Flu+RSV, Nanotrap Flu A•B, BD Veritor System Flu, and Rapiim Flu-AB) can detect several avian influenza A viruses, across subtypes H1 through H15. The major determinant of the sensitivity of the IRDTs is the reactivity of the monoclonal antibody against the NP used in the IRDT. In addition, the composition of the lysis buffer, the proportion of sample in the analyte, and the method used to visualize the results can affect the sensitivity. The 25 IRDTs can be divided into two formats: the test strip format and the well format. The well format can be further subdivided into two groups based on how the result is evaluated: BD Veritor System Flu, Fuji dri-chem immuno AG cartridge FluAB, Spotchem FLORA FluAB, and Rapiim Flu-AB require a specific analyzer to evaluate the results, whereas the other well format types are assessed by the human eye. These analyzers can only read one sample at a time; although BD Veritor System Flu and Spotchem FLORA FluAB require less than one minute to read, Fuji dri-chem immuno AG cartridge FluAB and Rapiim Flu-AB require 10-15 min and 7.5 minutes, respectively. Therefore, patients wait times for results are extended when many influenza patients come to a clinic that has only one analyzer. In contrast, human eye-judged IRDTs can be used to test many samples in parallel. Mechanistically, 23 of the IRDTs employ an     Table 3. Sensitivity of IRDTs for seasonal influenza A and B viruses a . a Ten-fold serial dilutions of the indicated viruses (10 1 -10 6 TCID 50 per 100 μl) were examined with each IRDT according to the manufacturers' instructions. The minimum viral titers required for a positive reaction were determined in two independent experiments. The average titers are shown.
immunochromatographic method, whereas Immunotrap Influenza A•B utilizes magnetic energy for the movement of the immune-complexes, and Rapiim Flu-AB detects the immune-complexes by light scattering. All 25 IRDTs take between 1 and 15 min to complete each test. We examined the sensitivity of each IRDT for influenza viruses of various subtypes isolated between 2013 and 2017 (see Table 2). The detection limit for seasonal influenza A viruses, such as H1N1pdm09 and H3N2 viruses, of the tested IRDTs ranged from 10 2.5 to 10 6 TCID 50 per 100 μl ( Table 3). The sensitivity for H1N1pdm09 viruses tended to be higher than that for H3N2 viruses. A similar trend was observed in our previous report 14 . The most sensitive IRDT for seasonal H1N1pdm09 and H3N2 viruses was Prorast Flu One. The detection limit for influenza B viruses, including both lineages, of the tested IRDTs ranged from 10 3 to 10 6 TCID 50 per 100 μl. All tested IRDTs possessed similar or reduced sensitivity for influenza B viruses compared with that for seasonal influenza A viruses (H1N1pdm09 and H3N2 viruses) ( Table 3). The most sensitive IRDT for seasonal type B viruses was Fuji dri-chem immuno AG cartridge FluAB.
We next examined the sensitivity of the 25 IRDTs against H5N1, H5N2, and H5N6 viruses. These H5 viruses are circulating in avian species and have the potential to transmit to humans [15][16][17][18][19] . The detection limit of the tested IRDTs ranged from 10 2 to 10 6 TCID 50 per 100 μl for H5N1 viruses and H5N2 viruses and from 10 4 to 10 7 TCID 50 per 100 μl for H5N6 viruses (Table 4). This finding indicates that the sensitivity of the IRDTs for H5N6 viruses (H5-5, -6, and -7) was 10-100 lower than that for H5N1 and H5N2 viruses (H5-1, -2, -3, and -4). The detection limits of each IRDT for H5N1 viruses were lower than those in our previous experiments 14,20 . For H7 viruses, we used highly pathogenic H7N9 isolates (H7-2 and -3) from humans that emerged in the 2016-17 season in China 7,8 , and a prototype H7N9 virus (H7-1) 5 . H7N2 virus (H7-4), which caused an outbreak in cats 21 , was also examined. The detection limits of the tested IRDTs for these H7 viruses ranged from 10 3.5 to 10 7 TCID 50 per 100 μl. All tested H7 isolates were detected by the IRDTs with varying sensitivity and the sensitivity was comparable to or slightly lower than that for type B viruses. The most sensitive IRDT for H5 and H7 viruses was Fuji dri-chem immuno AG cartridge FluAB.
For H1N1pdm09, H3N2, and type B viruses, which are the main targets for all IRDTs, the sensitivity of the analyzer-based IRDTs was similar to or better than that of the conventional IRDTs. In the case of seasonal viruses, virus titers usually peak at 10 2 -10 6 TCID 50 per 100 μl of nasopharyngeal wash during the first 24-72 h of illness 22 . Therefore, most IRDTs tested could accurately detect influenza virus in patients during this period. However, for H5 and H7 viruses, the analyzer-based IRDTs tended to show greater sensitivity than the conventional IRDTs. In particular, Fuji dri-chem immuno AG cartridge FluAB detected H5 and H7 viruses at a sensitivity level comparable to that for seasonal influenza A and B viruses; the detection limits were 10 2 -10 5 and 10 3 -10 4 TCID 50 per 100 μl, respectively. IRDTs possessing high sensitivity for potentially zoonotic H5 and H7 viruses are thus available to diagnose influenza caused by such viruses.

Materials and Methods
Diagnostic tests. The IDRTs listed in Table 1 were purchased from the manufacturers and evaluated for reactivity and sensitivity according to the manufacturers′ procedures. Rapiim™ Flu-AB requires an analyzer to read the test results and only a rental analyzer was available. Test samples were adjusted to 10 1 to 10 6 TCID 50 per 100 μl with Eagle's minimal essential medium (EMEM) containing 0.3% bovine serum albumin (BSA). The minimum virus titres required for a positive reaction were determined in duplicate examinations. The average virus titre for a positive reaction of two examinations is shown in the tables. Table 2 were propagated in MDCK cells or chicken embryonated eggs.

Viruses. The influenza viruses listed in
Their virus titres (TCID 50 ) were determined using MDCK cells.

Biosafety statements.
All experiments with H5N1, H5N2, H5N6, and H7N9 viruses were performed in biosafety level 3 (BSL3) laboratories at the University of Tokyo, which are approved for such use by the Ministry of Agriculture, Forestry, and Fisheries, Japan.
Data availability. All data analyzed during this study are included in this published article.