Few outbreaks of the serious enterovirus 71 (EV71) infections, which affect the central nervous system (CNS), had been reported in Japan before 2000. During June through August 2000, a patient died of pulmonary edema caused by brainstem encephalitis accompanied by EV71‐induced hand, foot, and mouth disease (HFMD), and many patients complicated by serious CNS disease, including paralysis, were hospitalized in a restricted area in Hyogo Prefecture, Japan (K‐area). During the same period, endemics of HFMD were reported in other areas in Hyogo Prefecture, where EV71 was isolated from HFMD patients, but few patients developed aseptic meningitis. The isolations of EV71 from K‐area patients were difficult with the use of Vero cells, so the strains were isolated by use of GL37 cells; Vero cells, however, could isolate EV71 strains from other areas in Hyogo Prefecture. We sequenced VP4 coding regions of these EV71 isolates and found that the isolates from K‐area had the same sequence, which, except for one isolate, was different from the sequences of EV71 strains isolated from other areas of Hyogo Prefecture. Although these results were not enough to state that EV71 from K‐area was a virulent strain, it seemed reasonable to conclude that serious CNS diseases in K‐area were caused by EV71 because it was the only infectious agent detected in the inpatients of K‐area.

An immunochromatography (IC) kit for human adenovirus (HAdV) was evaluated with 138 patient nasopharyngeal samples. The samples were collected at a sentinel clinic in Japan from January through June 2003. Patients were diagnosed by clinical manifestation of pharyngoconjunctival fever (n ‫؍‬ 38) or exudative tonsillitis (n ‫؍‬ 100). The IC kit was positive for 84% (116 of 138) of patients diagnosed at bedside. The remaining extract solution of the IC kit test was transferred into maintenance medium and tested via laboratory diagnoses. The IC kit had 95% sensitivity (116 of 122 patients) with HAdV isolation (isolation) as the standard and 91% sensitivity (116 of 128 patients) with PCR as the standard. All of the IC kit-positive samples were isolation and PCR positive. Similarly, all the isolation-positive samples were PCR positive. Twenty-two IC kit-negative samples were evaluated by real-time PCR. Six samples were IC kit negative and isolation positive and contained 3.8 ؋ 10 7 to 2.5 ؋ 10 9 copies of the HAdV genome/ml. Five samples that were only PCR positive contained 3.0 ؋ 10 4 to 3.8 ؋ 10 5 copies of the HAdV genome/ml, but one sample was real-time PCR negative. We conclude that the IC kit is a useful bedside diagnostic tool for HAdV infections because it has 95% sensitivity (compared to isolation), but a negative result does not always rule out HAdV infection.

We examined 976 sika deer serum samples, 159 liver tissue samples and 88 stool samples collected from 16 prefectures in Japan, and performed ELISA and RT-PCR assays to detect antibodies to HEV and HEV RNA, respectively. Although 25 (2.6%) of 976 samples were positive for anti-HEV IgG, the antibody titers were very low. The OD values ranged between 0.018 and 0.486, forming a single distribution rather than a bimodal distribution, suggesting that the antibody detected in this study was not induced by HEV infection, or that deer have low sensitivity to HEV. HEV RNA was not detected in these samples, also suggesting that deer may not play a role as an HEV reservoir.

BackgroundIn 2009, a pandemic (pdm) influenza A(H1N1) virus infection quickly circulated globally resulting in about 18,000 deaths around the world. In Japan, infected patients accounted for 16% of the total population. The possibility of human-to-human transmission of highly pathogenic novel influenza viruses is becoming a fear for human health and society.MethodologyTo address the clinical need for rapid diagnosis, we have developed a new method, the “RT-SmartAmp assay”, to rapidly detect the 2009 pandemic influenza A(H1N1) virus from patient swab samples. The RT-SmartAmp assay comprises both reverse transcriptase (RT) and isothermal DNA amplification reactions in one step, where RNA extraction and PCR reaction are not required. We used an exciton-controlled hybridization-sensitive fluorescent primer to specifically detect the HA segment of the 2009 pdm influenza A(H1N1) virus within 40 minutes without cross-reacting with the seasonal A(H1N1), A(H3N2), or B-type (Victoria) viruses.Results and ConclusionsWe evaluated the RT-SmartAmp method in clinical research carried out in Japan during a pandemic period of October 2009 to January 2010. A total of 255 swab samples were collected from outpatients with influenza-like illness at three hospitals and eleven clinics located in the Tokyo and Chiba areas in Japan. The 2009 pdm influenza A(H1N1) virus was detected by the RT-SmartAmp assay, and the detection results were subsequently compared with data of current influenza diagnostic tests (lateral flow immuno-chromatographic tests) and viral genome sequence analysis. In conclusion, by the RT-SmartAmp assay we could detect the 2009 pdm influenza A(H1N1) virus in patients' swab samples even in early stages after the initial onset of influenza symptoms. Thus, the RT-SmartAmp assay is considered to provide a simple and practical tool to rapidly detect the 2009 pdm influenza A(H1N1) virus.

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