Vol.8 No.2 2015

Research paper : Detection of influenza viruses with the waveguide mode sensor (K. AWAZU et al.)−98−Synthesiology - English edition Vol.8 No.2 (2015) may occur.[6]-[8]Influenza is a serious problem for animals as well as humans. When infection of poultry or cattle by avian flu virus is confirmed, the current measure is to conduct extensive culling and disposal of farm animals according to the Animal Infectious Diseases Control Law. For example, in 2010, a subtype H5N1 was detected in a poultry farm of Shimane Prefecture, and 21,549 birds were culled.[9] In addition to culling, inspection of the poultry farms in the 10-kilometer radius range, epidemic prevention work, and traffic blocks of the roads to poultry houses are needed, hence generating enormous economic damage. Therefore, if the source of the avian flu is found immediately, and the disease can be contained with minimum culling, wide-range measures do not have to be taken. It also becomes possible to prevent the development of a pandemic, and the economic effect is large. With this lesson in mind, when a highly pathogenic avian flu broke out in a poultry farm of Kumamoto Prefecture on April 13, 2014 (later this flu was determined to be H5N8), the spread of damage was kept to a minimum due to prompt early response.The issues for types H5N1 and H7N9 are totally different even if they are similar avian flus that are currently at issue. Type H5N1 is hypervirulent and causes fatal infections to many birds including chickens, but the infection from birds to human is thought to be rare. In contrast, H7N9 is hypovirulent, and no symptoms occur in infected birds, and therefore, monitoring of birds’ temperature is meaningless. However, although hypovirulent, infection from birds to humans does occur, and people develop serious conditions because humans have no immunity.Type H5N1 and H7N9 avian flus are no longer problems of animals only. Both viruses can be transmitted from animals to humans. Moreover, as shown in Table 1, the fatality is extremely high. There is also concern for a complication of the problem. By frequent infection of humans, there is the possibility that the flu virus undergoes better adaptive mutations and the infection may spread from human to human efficiently. At this point, both viruses are thought to be transmitted from chickens to humans, and then the humans develop symptoms. However, no one can predict when the situation may change and an epidemic may occur among humans. 2 BenchmarkNormally, for the identification of pathogenic microorganisms, the identification of genes or antigenic proteins is necessary. When the polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR) are used for gene identification, there is a high possibility of producing false positive results due to the aerosol contamination that may be caused by a leakage of just one positive sample. Although dramatic progresses have been made in the last few years in the digital PCR and the real-time PCR such as qRT-PCR and qPCR that are gene amplification methods in sealed tubes, the possibility of aerosol contamination of the PCR products cannot be denied. Once this occurs, PCR false positive response becomes normal. The manufacturers’ guidebooks spend several pages on the methods to avoid contamination, and recommend the preliminary and post treatments to be done in separate rooms, and to prepare exclusive reagent sets, pipettes, and disposable equipment for each room.[10] The PCR analysis method works well only when done by skilled personnel in a well-equipped research facility, but is not suitable for detection and identification in ordinary facilities such as general clinics, airport quarantine stations, meeting halls, or schools. In case of the avian flu virus, detection must be done in the field such as poultry houses, poultry markets, or slaughtering facilities, but PCR is not effective in such environments. If PCR is conducted in the field, even if the negative control is negative in the first test, the possibility of contamination by PCR products increases dramatically in the second measurement at the same place. Another disadvantage is that time is needed for detection and the procedure is complicated. In contrast, the detection by antibodies is a simple method with short detection time and does not require special procedures or gene amplification, although the detection sensitivity does not come close to the genetic test. There is almost no possibility of contamination of the antigenic proteins, because there is no process of amplification of the antigenic proteins.The immuno-chromatography is the most common method for testing using antibodies. Because it has high National Health Insurance (NHI) points, the immuno-chromatography is widely used for diagnosing types A and B influenza. However, in a recent study in 2009, Hurt et al. conducted a sensitivity test using three types of immuno-chromatography and five types of influenza A H1N1 and H3N2.[11] As a result, it was found that the detection limits differed by test kits and target viruses, and the sensitivity was 103~105 TCID 50/mL. Yamaguchi et al. conducted sensitivity comparison using the virus positive control attached to the immuno-chromatography.[12] As a result, in the case of high infectivity titer of 0.7-1.4 × 103 TCID 50/mL, the hit rate was relatively high at 98.8 % for skilled clinical lab technicians and 85.4 % for lab personnel (assistant, nurse, or nursing school faculty). However, in the case of low infectivity titer 3.5-7.0 × 102 TCID 50/mL, the hit rate was extremely low at 60.7 % for H7N9H5N1393/667118/393Number of deaths / Number of infected peopleTable 1. Number of deaths by avian flu virusAs of June 27, 2014[7][8]


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