Coronavirus Infections in Pediatric Outpatients with Febrile Respiratory Tract Infections in Hiroshima, Japan, over a 3-Year Period.

Previously, we conducted a 3-year prospective study to determine the viral causes of acute respiratory tract infections among 495 febrile pediatric outpatients. We collected 495 nasopharyngeal aspirate specimens, and used both real-time PCR assays and viral culture to test each for respiratory viruses other than coronavirus. Here, we used real-time PCR to test the 495 archival specimens for four human coronavirus strains. We identified 15 coronavirus-positive specimens: eight with OC43, 5 with NL63, 2 with HKU1, and none with 229E. Of the 15 children (5 boys) infected with human coronavirus, the mean age was 3.5 years, and the age range was 1.1 to 5.8 years; one child was diagnosed with lower respiratory infection; the other 14 were diagnosed with upper respiratory infection. Of these 15 patients, none were hospitalized, 5 were infected with coronavirus alone, 8 were co-infected with another virus, and 2 were co-infected with 2 other viruses. The multi-virus infections involved 6 adenoviruses, 3 respiratory syncytial viruses, 2 parainfluenza viruses, and 1 rhinovirus. In conclusion, the burden of human coronaviruses was relatively light among this cohort of 495 pediatric outpatients, and the incidence of these infections was low.

Comparison of throat swab and nasopharyngeal aspirate specimens for rapid detection of adenovirus.

Nasopharyngeal aspirate (NPA) and throat swab (TS) specimens from individual patients were compared with regard to usefulness for adenovirus detection. In 153 adenovirus-infected patients, rapid test sensitivities with NPAs (90.8%) were nearly equivalent to those with TSs (91.5%) based on real-time polymerase chain reaction standards, indicating that NPAs are equally useful.

Three-year study of viral etiology and features of febrile respiratory tract infections in Japanese pediatric outpatients.

BACKGROUND: For most febrile respiratory tract infections (RTIs) in children, the causative pathogen is never identified. We sought to identify the causative pathogen in individual cases of pediatric outpatient with RTIs and to determine whether particular clinical features of RTIs are associated with particular viruses. METHODS: Over 3 years, we prospectively collected nasopharyngeal aspirate specimens from individual pediatric outpatients with an RTI accompanied by persistent fever (>3 days, ≥38.0°C) and peak temperature ≥39.0°C. Two methods-(1) viral culture for respiratory viruses and (2) real-time polymerase chain reaction (PCR) assays identifying 9 different respiratory viruses and 2 respiratory bacteria-were used to test specimens. RESULTS: For 495 specimens, viral culture and real-time PCR assays together identified at least 1 pathogen in 83.0% and ≥1 viruses alone in 79.4%. These 2 methods identified 138 children with respiratory syncytial virus, 66 with human metapneumovirus, 73 with parainfluenza viruses, 124 with adenovirus, 23 with rhinovirus, 38 with enterovirus, 11 with influenza type C virus, 15 with Mycoplasma pneumoniae and 3 with Chlamydophila pneumoniae; the coinfection rate was 19.7% among all infections. Among the patients with single-pathogen infections, the rate of lower RTI was 37.6% for respiratory syncytial virus, 40.7% for human metapneumovirus, 18.2% for parainfluenza viruses and 2.2% for adenovirus (P < 0.01). CONCLUSIONS: Viral culture and real-time PCR assays were used together to identify causative pathogens in 83% of febrile outpatient children with RTI; specific viruses were associated with particular clinical diagnoses.

[Simultaneous multiple assay (Luminex xTAG respiratory viral panel FAST assay) efficacy in human respiratory virus detection].

Luminex xTAG respiratory viral panel FAST (RVP FAST) assay detects 17 human respiratory virus strains per measurement. Studying RVP FAST efficacy in detecting respiratory viruses in 67 aspirate samples from the nasal cavities of children with acute respiratory infection, we compared RVP FAST results to those of conventional nucleic acid amplification tests (NAT), e.g., real-time PCR, targeting 8 strains. RVP FAST assay detected 13 strains (98 isolates) in 59 of 67 samples. Of these, 8--influenza virus (Inf.V)-AH1, Inf. V-AH3, novel Inf.V-AH1, and Inf.V-B, and adenovirus, RS virus, metapneumovirus, and bocavirus--were compared to NAT results. RVP FAST showed higher sensitivity (83.3-100%) and specificity (98.2-100%) than NAT. RVP FAST also detected coronavirus (CoV) 229E, OC43, NL63, and HKU1 from 10 virus strain samples and enterovirus and/or rhinovirus from 35. RVP FAST assay thus comprehensively detects clinically important viruses in a single measurement, making RVP FAST assay useful in detecting causative respiratory tract viruses.

[Rapid detection of novel influenza A virus and seasonal influenza A (H1N1, H3N2) viruses by reverse transcription-loop-mediated isothermal amplification (RT-LAMP)].

Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay we developed detects novel influenza A (H1N1) of swine origin and seasonal influenza A (H1N1 and H3N2) viruses. Individual primer sets targeting the HA gene for novel H1N1, H1N1, and H3N2 were newly designed to specifically detect these subtypes. No cross-reactions occurred among novel H1N1, H1N1, and H3N2, and 7 respiratory viruses-influenza B virus, influenza C virus, adenovirus, respiratory syncytial virus, metapneumovirus, parainfluenza virus, and rhinovirus-had no reaction to 3 RT-LAMP assays. RT-LAMP is assayed at 63 degrees C for 40 min. In our RT-LAMP assay, Eriochrome Black T was added to the reaction mixture as an amplification indicator to detect virus genomes without using real-time turbidimetry. Positive reactions were indicated in blue and negative reactions remained purple. Of 139 samples from suspected novel H1N1 subjects tested by both RT-LAMP and real-time RT-PCR assay, 110 were positive in both assays. Two samples with low copy numbers were positive only in real-time RT-PCR assay. Of 27 novel negative H1N1 samples, 4 were positive for H3N2 on viral isolation and conventional RT-PCR assay. RT-LAMP assay for detecting H3N2 obtained the same findings. Our RT-LAMP assay is thus potentially useful in rapidly detecting influenza A virus such as novel H1N1, H1N1, and H3N2.

Rapid diagnosis of adenovirus respiratory tract infections using the chromatographic immunoassay test in the pediatric outpatient setting.

To assess the usefulness of a new rapid chromatographic immunoassay test for the detection of adenovirus, a prospective 3-year study was conducted in 587 febrile outpatient children suspected of adenovirus infection. A total of 332 children were diagnosed with this infection, using a viral culture. The sensitivity and specificity of the rapid test were 89.2% and 98.0%, respectively.

Human metapneumovirus infection in febrile children with lower respiratory diseases in primary care settings in Hiroshima, Japan.

Human metapneumovirus (hMPV) has been shown to be a leading cause of viral lower respiratory tract infections in children. Nevertheless, few reports regarding hMPV infections over consecutive years in children in primary care settings are available. We carried out virologic and clinical studies to determine the role of hMPV in febrile lower respiratory infections in children at a primary care clinic over 3 years and 5 months. Nasopharyngeal aspirates obtained from children with acute respiratory tract infections accompanied by high-grade fever (> or = 39 degrees C) and productive cough were studied for hMPV by reverse transcription-polymerase chain reaction and for other respiratory viruses by viral cultures and immunoassays. Of 379 patients tested, 202 were positive for at least 1 virus, including 98 with hMPV, 69 with respiratory syncytial virus, 18 with adenovirus, 12 with enterovirus, 8 with parainfluenza virus, 3 with rhinovirus, 2 with influenza virus type C, and 1 with herpes simplex virus. The male:female ratio of hMPV-infected children was 0.96:1 with an overall mean age of 3.5 years (range, 2 months to 9 years). These infections occurred predominantly from February to July, and the hospitalization rate was 4%. Of 93 patients infected with hMPV alone, 52 (56%) showed evidence of a lower respiratory tract infection.

A nationwide epidemic of influenza C virus infection in Japan in 2004.

During the period from January to July 2004, a total of 131 influenza C viruses were detected by cell culture or reverse transcription-PCR (RT-PCR) from specimens that were obtained from children with acute respiratory symptoms in 10 prefectures across Japan. Influenza C virus was identified most frequently in the Miyagi (1.4%, 45 of 3,226 specimens) and Yamagata (2.5%, 31 of 1,263 specimens) prefectures, and the frequency in this year was the highest since 1990. Phylogenetic analysis of the hemagglutinin esterase gene of the 13 strains isolated in nine prefectures revealed that genetically similar strains belonging to the Kanagawa/1/76-related lineage dominantly spread throughout Japan. During the 2004 influenza season, influenza C virus coexisted with epidemics of influenza A virus (H3 strain), and 12 cases were identified from patients who had been diagnosed with influenza-like illness (7 were detected by RT-PCR, and 5 were detected by culture). A comparison of specimens that were found positive by culture with those found positive only by RT-PCR shows that the amount of virus in PCR-positive specimens tended to be lower than in isolation-positive specimens. Although the mean peak temperature in patients in the PCR-positive group was slightly lower, there were no significant differences in characteristics between specimens (i.e., kind of specimen, period from onset to specimen collection, age distribution of patients, and severity of illness). These results suggest that an epidemic of influenza C virus occurred on a national scale during this period and that RT-PCR can be an effective supplemental tool for the evaluation of clinical and epidemiological information.

Rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay.

In this study, we developed a one-step, single-tube genogroup-specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of norovirus (NoV) genomes targeting from the C terminus of the RNA-dependent RNA polymerase gene to the capsid N-terminal/shell domain region. This is the first report on the development of an RT-LAMP assay for the detection of NoV genomes. Because of the diversity of NoV genotypes, we used 9 and 13 specially designed primers containing mixed bases for genogroup I (GI) and II (GII), respectively. The RT-LAMP assay had the advantages of rapidity, simplicity, specificity, and selectively and could obtain results within 90 min, generally even within 60 min, under isothermal conditions at 62 degrees C. The detection limits for NoV genomes were between 10(2) and 10(3) copies/tube for GI and GII with differentiation by genotype, and no cross-reactions among NoV GI and GII and other gastroenteritis viruses, such as sapovirus, human astrovirus, adenovirus type 40 and 41, and group A and C rotavirus, were found. In the evaluation tests with fecal specimens obtained from gastroenteritis outbreaks, the sensitivity and specificity of the RT-LAMP assay with regard to RT-PCR were 100 and 94% for GI and 100 and 100% for GII, respectively. These findings establish that the RT-LAMP assay is potentially useful for the rapid detection of NoV genomes from fecal specimens in outbreaks of food-borne and person-to-person-transmitted gastroenteritis.