An efficient cell culture and infection system for hepatitis C virus (HCV) facilitates analyses of the complete virus life cycle. Human hepatic Huh7.5.1 cells and an HCV-JFH1 strain have been widely employed in infection experiments. In the present study, cultured Huh7.5.1 cells exhibited heterogeneous phenotypes of HCV infection. Using single-cell cloning of Huh7.5.1 cells, we isolated a clone highly permissive to HCV (Huh7.5.1-8) and a CD81-defective clone nonpermissive to HCV (Huh7.5.1-5). Expression of CD81 in Huh7.5.1-5 cells restored permissiveness to HCV, indicating that CD81 is essential for HCV infection and a defect in CD81 causes nonpermissiveness to HCV in Huh7.5.1-5 cells. Huh7.5.1-8 cells had approximately 10-fold higher HCV replication rates, with cellular HCV RNA copy numbers of >109 copies/μg of cellular RNA and viral titers of >106 infectious units/ml of culture supernatant. Permissiveness of Huh7.5.1-8 cells to HCV infection was phenotypically very stable because there was no difference in permissiveness after more than 100 passages (1-year culture). This efficient cell culture system for HCV using Huh7.5.1-8 cell provides a powerful tool for studying the HCV life cycle and constructing antiviral strategies.
This study aimed to establish a spinal tuberculosis model by implanting Mycobacterium tuberculosis strain H37Rv into the lumbar vertebral body of New Zealand white rabbits. A hole was first drilled into the top of the 6th lumbar vertebra of each rabbit, which was then filled with a gelatin sponge to adsorb 0.2 ml of M. tuberculosis suspension (107 CFU /ml) for the infection group or normal saline for the control group. The holes were then closed with sutures. CT findings demonstrated that 5 and 10 rabbits developed spinal tuberculosis at 4 and 8 weeks, respectively, after this procedure. MRI examinations revealed that 7 and 15 rabbits had positive results at 4 and 8 weeks, respectively, after this procedure. HE staining of the vertebral body and paravertebral soft tissue biopsies of infected rabbits indicated inflammatory cell infiltration or necrosis in 15 rabbits. M. tuberculosis was cultured in 67% of the abscesses. The modeling success rate was 68.1%. By implanting an appropriate dosage of M. tuberculosis strain H37Rv into a local lumbar vertebral body of New Zealand white rabbits, we successfully established a spinal tuberculosis model, the pathological changes of which are similar to those of human spinal tuberculosis.
This article reviews Japanese HIV/AIDS surveillance data from 1985 to 2011. It revealed that heterosexual males are more prone to be detected as “AIDS cases,” whereas male homosexuals and females are more prone to be detected as “HIV cases,” irrespective of the gender, age, infection route, residential area, and nationality. The probability of being detected as an “AIDS case” increased with advanced age, irrespective of the gender and infection route. Interpretation of the data requires further information on the clinical latency of AIDS that could differ depending on differences in infection routes, gender, age, nature of the acute-phase syndrome and factors enhancing it, e.g., route and dose of infection, and mucosal immunity involved in sexually transmitted HIV/AIDS infection and the influence of age and gender on it.
Our aims were to determine the seroprevalence rates for the most common types of zoonosis among the population of Extremadura (southwestern Spain) and to identify the associated risk factors. We conducted a seroepidemiological survey to collect information on family background and the habits of people residing in Extremadura between 2002 and 2003. Antibodies to Brucella were determined by Rose Bengal staining and a standard tube agglutination test; a titer of 1/80 was considered to be positive. Antibody titers for spotted fever, leishmaniasis, echinococcosis, and toxoplasmosis were determined by enzyme-immunoassays. Independent risk factors identified were age (younger age for brucellosis), male gender (brucellosis, spotted fever, and toxoplasmosis), occupation and contact with animals (brucellosis and spotted fever for those in contact with goats, hydatidosis for those in contact with sheep, leishmaniasis for those in contact with dogs, and toxoplasmosis for those in contact with cats and pigs), and consuming contaminated food (brucellosis by eating fresh cheese, hydatidosis by eating homemade sausages, and toxoplasmosis by eating pork). Except for leishmaniasis, the other zoonoses were more prevalent in rural areas, and, with the exception of brucellosis, they were all more prevalent in Badajoz. The distribution of zoonoses in Extremadura was strongly influenced by keeping livestock and eating habits. Thus, brucellosis was more prevalent in Caceres (associated with cheese consumption), while toxoplasmosis (pork consumption) and spotted fever (from hunting) were more common in Badajoz.
The first human cases of infection with avian influenza A(H7N9) virus were reported in March 2013 in China. The number of confirmed cases continues to increase, although almost all the cases are limited to China. In this study, a one-step real-time RT-PCR assay was developed for detecting the novel A(H7N9) virus. This assay was shown to have high specificity, good linearity, and high sensitivity to a broad range of Eurasian H7 viruses. The assay is useful both for diagnostic purposes in cases of suspected human infection with the influenza A(H7N9) virus and in the surveillance of both avian and human influenza viruses. A diagnostic system using this assay was prepared at 74 prefectural and municipal public health institutes and 16 quarantine stations in Japan early into the human H7N9 infection outbreaks, enabling potential diagnoses of H7N9 infection across Japan.
Raw fish consumption is increasing worldwide. Since around the year 2000, western regions of Japan have reported a foodborne disease of unknown cause that occurred after the consumption of flounder. In October 2010, a particularly large outbreak was reported in these regions among individuals who consumed flounder fish that had been raised in aquaculture systems. The median incubation period was 5 h (range, 4–19 h), and the most frequently reported symptom was diarrhea (80%). The risk estimate of the consumption of flounder was significantly higher than that of the development of symptoms (odds ratio = 9.50; 95% confidence interval, 1.59–∞). According to a trace-back investigation, all of the flounder responsible for the outbreak were raised in aquaculture systems. Microscopic examination revealed that the median amount of Kudoa septempunctata present in the muscle of flounder fish from the aquaculture farm was 4.5 × 103 spores/g (range, 1.0 × 103−9.6 × 106 spores/g). The number of K. septempunctata spores required for the development of illness, as estimated using the Monte Carlo simulation, was 7.2 × 107 spores/g; therefore, thus this might be the minimum ingestion threshold for the development of gastrointestinal symptoms. As a public health measure, the current study results should be referred to for the prevention of the gastrointestinal symptoms related to the consumption of flounder; the national public health authority has disseminated these results. We concluded that K. septempunctata-contaminated flounder fish were associated with the gastrointestinal symptoms of this recent outbreak.
The relationship between blood groups and some infections such as norovirus, cholera, and malaria has been reported. Despite the importance of brucellosis, there is a lack of data on the relationship between blood groups and brucellosis. Thus, in this study, we examined the relationship between blood groups and brucellosis. In this case-control study, the blood groups of 100 patients with brucellosis and 200 healthy individuals were studied. Exclusion criteria for the control group consisted of a positive Coombs Wright test or a history of brucellosis. The chi-square test was used to compare qualitative variables between the two groups. The variables that met inclusion criteria for the regression model were entered into the logistic regression model. A total of 43% patients were female and 57% male; 27% were urban and 73% rural. Regression analysis showed that the likelihood of brucellosis infection was 6.26 times more in people with blood group AB than in those with blood group O (P < 0.001). However, Rh type was not associated with brucellosis infection. Thus, there is a relationship between blood group and brucellosis. People with blood group AB were susceptible to brucellosis, but no difference was observed for brucellosis infection in terms of blood Rh type.
Skin and soft tissue infection (SSTI) due to Alcaligenes faecalis is very rare and has never been studied. The aim of the present study was to investigate the clinical and microbiological characteristics of this infection. We conducted a retrospective review of 5 cases that occurred at our institution over a period of 6 years. All patients had underlying diseases, and infection was secondary to vascular disease or recent surgery in 4 of them. The most common clinical presentations were vascular ulcer infection and surgical site infection. The clinical outcome was uniformly good after treatment, except in 1 patient. In conclusion, A. faecalis should be considered a potential pathogen of SSTI, particularly in patients with vascular diseases or after surgery. The history of contact with water or aqueous solutions should be investigated in all cases. The clinical outcome is usually good, but treatment can be difficult in some cases due to the high level of resistance to commonly used antibiotics.
Here, we report a case of a Bulgarian patient with imported falciparum malaria that manifested 6 days after his arrival in Bulgaria, which was complicated by bloody diarrhea 2 days later. Blood smear revealed high parasitemia, with annular forms and gametocytes of Plasmodium falciparum. In addition, RNA of the Crimean-Congo hemorrhagic fever (CCHF) virus was detected in the blood sample by real-time reverse transcription (RT)-PCR and nested RT-PCR. The obtained sequence was found to be clustered within the Europe 1 lineage close to the other Bulgarian strains. Notably, the two infectious diseases may appear with many similar symptoms that are difficult to distinguish.
Human cytomegalovirus (HCMV) is a common pathogen that causes persistent infections in immune deficient patients and results in significant morbidity and mortality, particularly among transplant recipients and children. Different HCMV glycoprotein H (gH) genotypes may cause different diseases and affect the severity of these diseases. To develop a sensitive quantitative real-time PCR assay that could rapidly distinguish between two HCMV gH genotypes, primers were designed to target the conserved region of the gH gene. gH1 and gH2 probes were designed to target the two variable regions. Standard HCMV strains (AD169 and TOWNE) and 203 clinical urine samples from HCMV infected children were used for the present study. Based on the primer-probe set used to detect the target gH gene segment of HCMV, our quantitative real-time PCR assay specifically discriminated between HCMV gH1 and gH2 with a detection limit of approximately 102 viral copies/ml. Among the 203 clinical urine samples tested, 145 were gH1 positive, 56 were gH2 positive, and 2 were positive for both. Thus, we developed a gH gene-based real time-PCR method that could rapidly, stably, and specifically distinguish between two HCMV gH genotypes. We found HCMV gH1 to be common among children examined in Zhejiang, China.
The available literature on human coronaviruses (HCoVs) in Japan is limited to epidemiological studies conducted over a maximum of 1 year. We conducted a 4-year study of HCoVs by analyzing 4,342 respiratory specimens obtained in Yamagata, Japan, between January 2010 and December 2013. A pan-coronavirus reverse transcription-PCR screening assay was performed, and all HCoV-positive specimens were subsequently confirmed by sequencing of the PCR products. We detected in 332 (7.6%) HCoV strains during the study period, comprising 133 (3.1%) HCoV-NL63, 83 (1.9%) HCoV-HKU1, 78 (1.8%) HCoV-OC43, and 38 (0.9%) HCoV-229E strains. HCoV detection per year ranged from 3.5% to 9.7%. HCoVs were detected mainly in winter, with January (28.5%) and February (25.3%) 2011 and December 2012 (14.6%) being the only months in which HCoV-NL63 detection per month exceeded 10.0%. HCoV-HKU1 displayed clear biennial peaks in January (18.3%) and February (10.7%) 2010 and in February (18.8%) and March (14.7%) 2012. The peak detection of HCoV-OC43 was 13.6% in November 2010, while that of HCoV-229E was 10.8% in March 2013. Our results indicated that there may be annual variations in the circulation of individual HCoV strains. Further long-term surveillance is necessary to clarify HCoV prevalence and circulation patterns in Japan.
Fourteen patients were laboratory-confirmed cases of imported infectious diseases at the Narita Airport Quarantine Station in 2013. Blood tests were performed on 283 subjects suspected of having imported infectious diseases. Of these, 11 were diagnosed as having dengue fever (dengue) and 3 as having chikungunya fever (chikungunya) using real-time RT-PCR. The possible countries from which dengue virus infections were contracted were Thailand, Laos, Sri Lanka, and some other countries in Southeast Asia and South Asia. The 3 chikungunya cases were also diagnosed in individuals that returned from Southeast Asia. Most of the patients with dengue had a fever of over 38℃. The other symptoms were generalized fatigue, dull headache, pain behind the eyes, arthralgia, and digestive symptoms. Four of the patients were unaware of any mosquito bites. The information obtained from the confirmed cases showed that it is important to consider both the destination to which individuals travelled and the clinical symptoms, regardless of whether the subjects were aware of mosquito bites. The detection rate of chikungunya at the Quarantine Station was higher than that of dengue in all reported cases in Japan.
Kudoa septempunctata is a newly identified causative agent of foodborne diseases associated with consuming raw olive flounder. Qualitative PCR and quantitative real-time PCR have been used as notification methods to identify K. septempunctata in Japan. However, these methods require expensive equipment and are time-consuming (2–3 h for screening). To address these problems, in this study, we developed new rapid and simple methods using real-time loop-mediated isothermal amplification (LAMP) and nucleic acid sequence based amplification-nucleic acid chromatography (NASBA-NAC). Using these methods, the total procedure required approximately 45 min and did not require any expensive equipment. With regard to validating these new methods in comparison with the notification methods used in Japan, we performed an inter-laboratory study of 5 laboratories using samples that included olive flounders infected with 4 different amounts of K. septempunctata. These results demonstrated that the sensitivity of NASBA-NAC was equivalent to that of qualitative PCR, and that the sensitivity of real-time LAMP was equivalent to that of quantitative real-time PCR, which indicated that these new methods were acceptable screening methods for identifying K. septempunctata.
Interleukin (IL)-17A affects the immune system of the lung. Legionella infection can potentially lead to severe pneumonia. The present study aimed to evaluate the role of IL-17A in Legionella pneumonia. Serum IL-17A levels were quantified in both patients with Legionella pneumonia and control subjects; IL-17 was detected in sera from 4 out of 31 patients with Legionella pneumonia but in any controls. There were no differences in peripheral white blood cell counts or other serum biomarkers (C-reactive protein, and lactate dehydrogenase) between IL-17A-positive and IL-17A-negative patients. All IL-17A-positive patients in this cohort survived, where 8 of 27 IL-17A-negative patients did not. IL-17A was detected in available bronchoalveolar (BA) fluid samples from 7 patients with Legionella pneumonia within our cohort. However, the IL-17A and IFN-γ concentrations in BA fluids did not correlate with each other. IL-17A might play a significant role in some cases of Legionella pneumonia.
This study reports the epidemiological characteristics of hospitalized cases of influenza A(H1N1)pdm09 infection analyzed on the basis of surveillance data collected from July 24, 2009, the date on which the hospital-based surveillance of influenza cases was implemented in Japan, to September 5, 2010. During the study period, 13,581 confirmed cases were reported. Among those cases with information regarding the reason for hospitalization, 39% were admitted to hospitals for non-therapeutic purposes such as medical observation and laboratory testing. The overall hospitalization rate was 5.8 cases per 100,000 population when cases hospitalized for non-therapeutic purposes were excluded. While those aged under 20 years accounted for over 85% of hospitalized cases, the largest proportion of fatal cases was observed in those aged over 65 years. The overall case fatality rate for all hospitalized cases was 1.5%. The year-round surveillance for hospitalized influenza-like illness cases was launched in 2011, and it was expected that this surveillance system could add value by monitoring changes in the epidemiological characteristics of hospitalized cases of seasonal influenza.
A large rubella outbreak has been observed since June 2012 in Tokyo, Japan, and a rapid increase in the number of congenital rubella syndrome (CRS) cases have also been reported in Japan since October 2012. All the clinically diagnosed and laboratory-confirmed rubella cases reported in Tokyo from January 2012 to December 2013 and all the laboratory-confirmed CRS cases from January 2012 to March 2014 were analyzed. In total, 4,116 rubella cases were reported in Tokyo. Of these, 77.2% (n = 3,176) were male; the highest number of cases occurred in males aged 35–39 years and in females aged 20–24 years. Complications included arthralgia/arthritis (19.4%), thrombocytopenic purpura (0.5%), hepatic dysfunction (0.3%), and encephalitis (0.1%). The circulating rubella virus in Tokyo was genotype 2B. The most possible site of transmission was the workplace. Because of the rubella epidemic, 16 CRS cases were reported in Tokyo from March 2013 to February 2014. Domestic infection with rubella was proven for all mothers of 16 cases. This situation suggests that Japan is still working to achieve rubella elimination.