Molecular Epidemiology of Rubella Virus Strains Detected Around the Time of the 2012-2013 Epidemic in Japan.

A nationwide rubella epidemic occurred from 2012 to 2013 in Japan, resulting in around 17,000 rubella cases and the birth of 45 infants with congenital rubella syndrome. The aim of this study was to genetically characterize the rubella viruses (RVs) circulating around the time of the epidemic in Japan. In total, 221 RV strains detected from 14 prefectures in Japan between 2010 and 2014 were sequenced in the 739 nucleotide-window region within the E1 gene. The virus strains were chronologically and geographically characterized into groups based on phylogenetic analysis. Among the 221 strains analyzed, 192 (87%), 26 (12%), and 3 (1%) strains were classified into genotypes 2B, 1E, and 1J, respectively. The majority (n = 184) of the genotype 2B strains belonged to lineage 2B-L1 and shared nucleotide homology with the strains detected in Southeast and East Asian countries. Phylogenetic analyses demonstrated that at least six distinct clusters of RV strains (clusters 1-6) induced outbreaks in Japan between 2010 and 2014. Among them, strains from clusters 3, 4, and 6 circulated almost simultaneously during 2012-2013. The cluster 3 strains circulated locally, whereas strains from cluster 4 spread nationwide. The findings suggest that RVs were introduced into Japan many times from neighboring countries. The 2012-2013 epidemic was a complex of outbreaks induced by at least three clusters of RV strains.

Genetic evidence for containment of viruses in the first outbreak of influenza A pandemic (H1N1) 2009 in Kobe, Japan.

BACKGROUND: On 16 May 2009, a high school student in Kobe with no history of overseas travel was reported as the first case of influenza A pandemic (H1N1) 2009 virus infection in Japan. Subsequently, it was revealed that the infection had spread to some cities in the Kansai region and most patients were high school students. The number of patients decreased rapidly within a week; however, it began to increase in the middle of July. METHODS: We phylogenetically analyzed viral characteristics using 27 viruses isolated from patients living in Kobe. RESULTS AND CONCLUSIONS: We demonstrated that viruses isolated in the early phase of the outbreak were distinguishable from those after the reappearance of patients. These findings provide genetic evidence for the effectiveness of public health containment measures in the Kansai region in preventing the progression of the outbreak.

Reevaluation of laboratory methods for diagnosis of measles.

The purpose of this study is to reevaluate the sensitivities of different methods used in the diagnosis of measles including virus isolation, RT-PCR, and measurement of IgM. Sixty-three throat swabs, 84 peripheral blood mononuclear cell (PBMC) samples, and 85 plasma samples were collected from 85 cases of suspected measles. The sensitivity of virus isolation using throat swabs and PBMC in comparison with RT-PCR was 58.1 and 93.5%, respectively. We defined laboratory-confirmed cases as those in which at least one of the methods was positive. The percentage of positive results by the different methods was compared among 49 laboratory-confirmed cases. The percentage of positive results from PBMC by RT-PCR and virus isolation was 100 and 91.7%, respectively. The percentage of positive results from throat swabs by RT-PCR and virus isolation was 91.2 and 52.8%, respectively. The percentage of IgM positive (79.6%) was significantly lower than that of PBMC by RT-PCR. Ten of 27 plasma samples collected within 5 days of the onset of fever were IgM negative. In contrast, all of the 21 plasma samples collected 6 days after the onset of fever were IgM positive. In conclusion, the detection of measles virus RNA in PBMC by RT-PCR was the most effective method for diagnosis of measles.

Detection of measles virus RNA on SYBR green real-time reverse transcription-polymerase chain reaction.

BACKGROUND: As the coverage rate of the measles vaccine increases, not all patients present the typical symptoms of measles after exposure to the measles virus (MV). The virus loads in clinical specimens from patients with vaccine-modified non-typical measles are expected to be low compared with those of primary MV infection. A rapid and sensitive laboratory procedure is required for diagnosis of measles. METHODS: SYBR Green (TaKaRa) and TaqMan (ABI) real-time reverse transcription-polymerase chain reaction (RT-PCR) assays were developed to detect MV-RNA. For the real-time RT-PCR, primer sets were designed from a region of the MV H gene of the Edmonston strain (genotype A). A TaqMan probe specific for the H gene of genotype D MV was used. The minimum detectable level of MV-RNA in the SYBR Green and TaqMan real-time RT-PCR assays was evaluated using synthetic MV-RNA. The sensitivity of real-time RT-PCR was compared with that of nested RT-PCR and the virus isolation method using throat swabs and peripheral blood samples from patients with measles. RESULTS: The minimum detectable level of RNA was 10 and 10(2) copies for SYBR Green RT-PCR and TaqMan RT-PCR, respectively. Ten-10(6) copies of standard RNA were linearly detected on SYBR Green RT-PCR. The sensitivity of SYBR Green RT-PCR was equal to that of nested RT-PCR. MV-RNA was detected in virus isolation-negative throat swabs on SYBR Green RT-PCR. CONCLUSION: SYBR Green RT-PCR is a highly sensitive, rapid, and useful diagnostic procedure for the detection of MV.

[Psittacosis outbreak at an avian exhibition].

Psittacosis outbreak due to Chlamydophila psittaci occurred among staff members at an avian exhibition of nearly 1,000 birds in Kobe, Japan, in December 2005. Staff members not trained about zoonosis or psittacosis used little protective attire such as masks and gloves when caring for their discharges. Two of 67 staff members contracted psittacosis pneumonia. Additional two suffered from pneumonia and 19 reported symptoms such as fever and cough, although none were diagnosed with psittacosis. The roughly 970 birds were kept without quarantine and identified by leg bands. Doxycycline administrated in drinking water and food failed to eradicate chlamydia, so all birds were captured, identified by leg band, and tested for chlamydia by PCR. Six were found to carry large amounts of chlamydia. Major outer membrane protein (MOMP) DNA sequence of chlamydia in a patient's bronchoalveolar lavage fluid (BALF) was identical to that derived from a channel-billed toucan kept in a closed aviary, and staff members may have been infected by inhaling excrement while working in the aviary. The MOMP DNA sequence was useful in comparing strains. We review the difficulty of diagnosing psittacosis and the knowledge and infection control measures required against it.

Detection of adenovirus DNA in clinical samples by SYBR Green real-time polymerase chain reaction assay.

BACKGROUND: Adenoviruses are associated with a variety of diseases including upper respiratory tract infections, acute conjunctivitis, cystitis and gastroenteritis. Adenoviruses can also cause fatal disseminated infections in patients undergoing stem cell transplantation. Measurement of adenovirus load in clinical samples from localized adenovirus infections or disseminated adenovirus infections may provide important information for analyzing the pathogenesis of various adenovirus infections. The purpose of the present study was to develop and optimize a highly sensitive real-time polymerase chain reaction (PCR) assay to detect a wide range of adenoviruses and to detect adenovirus DNA in clinical samples from immunocompetent children. METHODS: Clinical samples of throat swabs and blood were collected from 111 patients suspected of having adenovirus infection. The copy number of adenovirus DNA was measured by real-time PCR assay. RESULTS: SYBR Green real-time PCR assay is able to detect 10-10(6) copies of standard adenovirus DNA per run. Adenovirus DNA was detected in all culture-positive samples serotyped as 1, 2, 3, 4, 5, 6, 8 and 11. Viral loads on throat swabs from immunocompetent children with adenovirus infection ranged from 10(5) to 10(11) copies/mL. Adenovirus DNA was detected in 60% of blood samples and copy number ranged from 10(3) to 10(5) copies/mL. CONCLUSION: SYBR Green real-time PCR is a useful quantitative tool for analysis of adenovirus DNA. The present results for immunocompetent children with adenovirus infections provided basic data for comparison with data obtained from immunocompromised patients.

Clinical spectrum in hospitalized children with echovirus type 13 infection.

BACKGROUND: The aim of this study was to investigate the clinical spectrum of echovirus type 13 (E13) infection in hospitalized children. METHODS: From April to August 2002, prospective viral surveillance was performed for hospitalized patients (aged 10 days to 14 years) irrespective of their presenting symptoms and severity. Medical records of laboratory-confirmed echovirus 13 infection were reviewed. RESULTS: Of the 41 patients analyzed, the median age was 3.4 years and 30% of them were less than 1 year of age. The male:female ratio was 1.6:1. The main clinical features were non-specific febrile illness (nine patients), gastroenteritis (seven), bronchitis (seven), aseptic meningitis (16) and idiopathic thrombocytopenic purpura (two). Each age group had their representative symptoms: less than 1 year of age, non-specific febrile illness; from 1 to 6 years of age, enterocolitis and bronchitis; more than 6 years of age, aseptic meningitis. CONCLUSION: The representative symptoms of E13 infection in hospitalized patients were variable but strongly associated with age distribution. It was of interest to note that two patients developed idiopathic thrombocytopenic purpura along with the infection.