Molecular Epidemiology of Human Metapneumovirus in East Japan before and after COVID-19, 2017-2022.

Human metapneumovirus (hMPV) is genetically classified into two major subgroups, A and B, based on attachment glycoprotein (G protein) gene sequences. The A2 subgroup is further separated into three subdivisions, A2a, A2b (A2b1), and A2c (A2b2). Subgroup A2c viruses carrying 180- or 111-nucleotide duplications in the G gene (A2c 180nt-dup or A2c 111nt-dup ) have been reported in Japan and Spain. The coronavirus disease 2019 (COVID-19) pandemic disrupted the epidemiological kinetics of other respiratory viruses, including hMPV. In this study, we analyzed the sequences of hMPV isolates in Tokyo and Fukushima obtained from 2017 to 2022, i.e., before and after the COVID-19 pandemic. Subgroup A hMPV strains were detected from 2017 to 2019, and most cases were A2c 111nt-dup, suggesting ongoing transmission of this clade, consistent with global transmission dynamics. Subgroup B viruses, but not subgroup A viruses, were detected in 2022 after the COVID-19 peak. Phylogenetic analysis showed that the subgroup B viruses were closely related to strains detected in Yokohama from 2013 to 2016, and strains detected in Fukushima in 2019, suggesting the reappearance of local endemic viruses in East Japan.

Evidence of the simultaneous replications of active viruses in specimens positive for multiple respiratory viruses.

IMPORTANCE: Since the pandemic of coronavirus diseases 2019, the use of real-time PCR assay has become widespread among people who were not familiar with it in virus detection. As a result, whether a high real-time PCR value in one time test indicates virus transmissibly became a complicated social problem, regardless of the difference in assays and/or amplification conditions, the time and number of diagnostic test during the time course of infection. In addition, the multiple positives in the test of respiratory viruses further add to the confusion in the interpretation of the infection. To address this issue, we performed virus isolation using pediatric SARI (severe acute respiratory infections) specimens on air-liquid interface culture of human bronchial/tracheal epithelial cell culture. The result of this study can be a strong evidence that the specimens showing positivity for multiple agents in real-time PCR tests possibly contain infectious viruses.

Patients With Subacute Sclerosing Panencephalitis in Japan: A 2022 Nationwide Survey.

BACKGROUND: In Japan, the incidence of subacute sclerosing panencephalitis (SSPE) has reduced; however, the medical conditions and factors associated with disease progression remain unclear. METHODS: A nationwide survey of SSPE was conducted using a questionnaire in 2022. We conducted a descriptive analysis of the patients with SSPE in 2022 and Cox proportional hazards analyses for disease progression. We compared the patients with SSPE with those in a 2007 survey. RESULTS: A total of 37 surviving patients with SSPE were enrolled [median age: 32 years (range: 16-52 years)]. No new cases have been identified since 2017 in the survey. Jabbour stage IV was the most common stage (66.7%). The hazard ratios (95% confidence intervals) of male sex and age at the time of measles infection (years) were 2.56 (1.13-5.76) and 0.57 (0.34-0.93), respectively. Compared with those in 2007, the proportion of patients in hospitals decreased from 13.7% to 2.7%, whereas that of patients in nursing facilities increased from 17.6% to 29.7%. The proportions of patients prescribed inosine pranobex, interferon and ribavirin at the time of the survey decreased from 96.1% to 79.4%, 74.8% to 14.3% and 25.3% to 0%, respectively. The proportions of patients with gastrostomy, tracheostomy and ventilator use increased from 5.9% to 69.7%, 23.3% to 60.0% and 10.8% to 32.4%, respectively. CONCLUSIONS: Decreased measles cases in Japan reduced new SSPE cases. However, surviving patients in 2022 had advanced disease stages and needed medical care. Male sex and early measles infection were significantly associated with disease progression.

Development of a duplex real-time RT-PCR assay for the detection and identification of two subgroups of human metapneumovirus in a single tube.

Human metapneumovirus (hMPV) is a common cause of respiratory infections in children. Many genetic diagnostic assays have been developed, but most detect hMPV regardless of the subgroup. In this study, we developed a real-time RT-PCR assay that can detect and identify the two major subgroups of hMPV (A and B) in one tube. Primers and probes were designed based on the sequences of recent clinical isolates in Japan. The assay showed comparable analytical sensitivity to a previously reported real-time RT-PCR assay and specific reactions to hMPV subgroups. The assay also showed no cross-reactivity to clinical isolates of 19 species of other respiratory viruses. In a validation assay using post-diagnosed clinical specimens, 98% (167/170) positivity was confirmed for the duplex assay, and the three specimens not detected were of low copy number. The duplex assay also successfully distinguished the two major subgroups for all 12 clinical specimens, for which the subgroup had already been determined by genomic sequencing analysis. The duplex assay described here will contribute to the rapid and accurate identification and surveillance of hMPV infections.

Molecular Diversity of Human Respiratory Syncytial Virus before and during the COVID-19 Pandemic in Two Neighboring Japanese Cities.

Human respiratory syncytial viruses (HRSVs) are divided into subgroups A and B, which are further divided based on the nucleotide sequence of the second hypervariable region (HVR) of the attachment glycoprotein (G) gene. Understanding the molecular diversity of HRSV before and during the coronavirus disease 2019 (COVID-19) pandemic can provide insights into the effects of the pandemic on HRSV dissemination and guide vaccine development. Here, we analyzed HRSVs isolated in Fukushima Prefecture from September 2017 to December 2021. Specimens from pediatric patients were collected at two medical institutions in neighboring cities. A phylogenetic tree based on the second HVR nucleotide sequences was constructed using the Bayesian Markov chain Monte Carlo method. HRSV-A (ON1 genotype) and HRSV-B (BA9 genotype) were detected in 183 and 108 specimens, respectively. There were differences in the number of HRSV strains within clusters prevalent at the same time between the two hospitals. The genetic characteristics of HRSVs in 2021 after the COVID-19 outbreak were similar to those in 2019. HRSVs within a cluster may circulate within a region for several years, causing an epidemic cycle. Our findings add to the existing knowledge of the molecular epidemiology of HRSV in Japan. IMPORTANCE Understanding the molecular diversity of human respiratory syncytial viruses during pandemics caused by different viruses can provide insights that can guide public health decisions and vaccine development.

Sterilization efficacy of a new water-free breast milk pasteurizer.

BACKGROUND: Breast milk, nature's optimum source of nutrition for infants, can contain undesirable microorganisms that cause severe morbidity. After an outbreak of multidrug-resistant Escherichia coli among neonates receiving breast milk donated by another mother in our neonatal intensive care unit (NICU), we were motivated to develop a high-grade breast milk pasteurizer (BMP) designed to thaw and pasteurize breast milk at 63°C for 30 min in a sealed bag without having to open the bag or immerse it in water. METHODS: Pre-existing bacteria and spiked cytomegalovirus (CMV) were measured pre- and post-pasteurization in frozen breast milk donated by mothers of children admitted to the NICU. RESULTS: Among 48 breast milk samples (mean ± standard deviation [SD]), pre-existing bacterial counts of 5.1±1.1 × 104 colony forming units (cfu)/mL decreased to less than 10 cfu/mL (below detection level) in 45 samples after pasteurization for 30 min. In three samples, 10-110 cfu/mL persisted. As no CMV was detected in any of the 48 samples, CMV at ≥5 × 104 pfu/mL was spiked into 11 breast milk samples. After just 10 min of pasteurization, infectious CMV was not detected (threshold <50 pfu/mL) in any sample. CONCLUSION: A new BMP was shown to pasteurize milk effectively with more than a 3-log reduction of microorganisms. Compared to conventional pasteurizers, this device reduces the effort involved in pasteurizing breast milk, avoids various contamination risks, and may reduce the risk of infectious disease transmission via breast milk.

Serotypes and Antibiotic Resistance of Streptococcus pneumoniae before and after the Introduction of the 13-Valent Pneumococcal Conjugate Vaccine for Adults and Children in a Rural Area in Japan.

The increase in non-vaccine serotypes of Streptococcus pneumoniae and their multidrug resistance have become an issue following the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13). In this study, we investigated the serotypes and drug resistance of S. pneumoniae detected in adult and pediatric outpatients at a hospital in a rural area of Japan between April 2012 and December 2016. Serotypes of the bacterium were identified using the capsular swelling test and multiplex polymerase chain reaction testing of DNA extracted from the specimens. Antimicrobial susceptibility was determined using the broth microdilution method. The serotype 15A was classified using multilocus sequence typing. The results showed that the prevalence of non-vaccine serotypes increased significantly in children from 50.0% in 2012-2013 to 74.1% in 2016 (p ≤ 0.006) and in adults from 15.8% in 2012-2013 to 61.5% in 2016 (p ≤ 0.026), but no increase in drug-resistant isolates was evident. However, an increase in the drug-resistant serotypes 15A and 35B was observed in children. Although isolates of these two serotypes showed cefotaxime susceptibility, cefotaxime resistance was confirmed for the serotype 15A isolates. Future trends in the spread of these isolates should be monitored with caution.

Associations between fetal or infancy pet exposure and food allergies: The Japan Environment and Children's Study.

The hygiene hypothesis suggests that pet exposure is effective in preventing allergic disease, and some studies have reported the beneficial effects of dog exposure during fetal development or early infancy on food allergy. However, the effects of exposure to pets other than dogs on the kinds of food allergies remains unaddressed. This study aimed to explore the effect of exposure to various species of pets on the risk of food allergies. We obtained information on pet exposure and food allergy from the Japan Environment and Children's Study, a nationwide, prospective birth cohort study that included 97,413 mothers and their children. We examined the associations between exposure to various species of pets during fetal development or early infancy and the incidence risk of food allergies. We conducted logistic regression analysis for each pet species, causative food, and timing of exposure. Exposure to dogs or cats during fetal development or early infancy was estimated to reduce the incidence risk of food allergies until the age of 3 years. Dog exposure was estimated to reduce the incidence risk of egg, milk, and nut allergies, and cat exposure was estimated to reduce the incidence risk of egg, wheat, and soybean allergies. However, hamster exposure was estimated to increase the incidence risk of nut allergy. In conclusion, the association between pet exposure and food allergies might differ depending on the pet species and causative food. Continued dog and cat exposure from fetal development to infancy was estimated to reduce the incidence risk of food allergies. The findings of this study shall aid in the design of future studies.

Tobacco Exposure During Pregnancy and Infections in Infants up to 1 Year of Age: The Japan Environment and Children's Study.

BACKGROUND: Tobacco exposure during pregnancy is associated with several adverse outcomes in infants. We investigated the association between tobacco exposure during pregnancy (both active and second-hand) and various infections in infants up to 1 year. METHODS: This prospective cohort study used a fixed dataset (jecs-an-20180131) from the Japan Environment and Children's Study of registered births in Japan during 2011-2014 that included 104,065 fetal records from enrolled pregnant women. Based on the participants' responses to the questionnaire on smoking status, mothers were first divided into "never smoked," "quit smoking," and "current smoker" groups and then into "no second-hand smoking (SHS)" and "SHS" groups. Infectious diseases included central nervous system infection, otitis media (OM), upper respiratory tract infection (URTI), lower respiratory tract infection (LRTI), gastroenteritis (GI), and urinary tract infection. Adjusted odds ratios (aORs) and 95% confidence intervals (CIs) were calculated using logistic regression analysis and adjusted for maternal, socioeconomic, and postnatal confounding factors. RESULTS: Among the 73,205 newborns enrolled, multivariable analysis revealed that the aOR of LRTI and GI was 1.20 (95% CI, 1.07-1.33) and 1.18 (95% CI, 1.04-1.35), respectively, for the "current smoker with/without SHS" group compared with the "never smoked without SHS" group. "Quit smoking without SHS" was not associated with the risk of LRTI. SHS was associated with an increased risk of OM, URTI, LRTI, and GI, especially with LRTI and GI. CONCLUSION: Exposure to tobacco smoke during pregnancy was associated with an increased risk of OM, URTI, LRTI, and GI in infants during their first year of life.

Hydrophobic Alpha-Helical Short Peptides in Overlapping Reading Frames of the Coronavirus Genome.

In this study, we show that the coronavirus (CoV) genome may encode many functional hydrophobic alpha-helical peptides (HAHPs) in overlapping reading frames of major coronaviral proteins throughout the entire viral genome. These HAHPs can theoretically be expressed from non-canonical sub-genomic (sg)RNAs that are synthesized in substantial amounts in infected cells. We selected and analyzed five and six HAHPs encoded in the S gene regions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively. Two and three HAHPs derived from SARS-CoV-2 and MERS-CoV, respectively, specifically interacted with both the SARS-CoV-2 and MERS-CoV S proteins and inhibited their membrane fusion activity. Furthermore, one of the SARS-CoV-2 HAHPs specifically inhibited viral RNA synthesis by accumulating at the site of viral RNA synthesis. Our data show that a group of HAHPs in the coronaviral genome potentially has a regulatory role in viral propagation.

Nearly Complete Genome Sequences of 12 Types of Human Rhinoviruses Isolated from Pediatric Inpatients in Fukushima, Japan.

We reported nearly complete genomic sequences of 12 serotypes of human rhinoviruses (HRVs) isolated from pediatric inpatients in Fukushima, Japan using an air-liquid interface culture of human bronchial tracheal epithelial cells. We found that various serotypes of HRV circulated locally and simultaneously from 2018 to 2021.

Practical Validation of United States Centers for Disease Control and Prevention Assays for the Detection of Human Respiratory Syncytial Virus in Pediatric Inpatients in Japan.

The World Health Organization initiated a global surveillance system for respiratory syncytial virus (RSV) in 2015, and the pilot surveillance is ongoing. The real-time RT-PCR RSV assays (Pan-RSV and duplex assays) developed by the United States Centers for Disease Control and Prevention are applied as the standard assays. To introduce these as standard assays in Japan, their practicality was evaluated using 2261 specimens obtained from pediatric inpatients in Japan, which were collected from 2018 to 2021. Although the Pan-RSV and duplex assays had similar analytical sensitivities, they yielded 630 (27.9%) and 786 (34.8%) RSV-positive specimens, respectively (p < 0.001). Although sequencing analysis showed mismatches in the reverse primer used in the Pan-RSV assay, these mismatches did not affect its analytical sensitivity. The analysis of read numbers of RSV isolates from air−liquid interface culture of human bronchial/tracheal epithelial cells showed that the duplex assay had a greater number of reads than did the Pan-RSV assay. Therefore, the duplex assay has superior detection performance compared with the Pan-RSV assay, but the two assays have similar analytical sensitivities.

Ten Nearly Complete Genome Sequences of Human Orthorubulavirus 4 Isolated from Pediatric Inpatients in Fukushima, Japan.

We report 10 nearly complete genomic sequences of human orthorubulavirus 4, also called human parainfluenza virus 4 (HPIV4), isolated from pediatric inpatients with respiratory infections in Fukushima, Japan, by using an air-liquid interface culture of human bronchial and tracheal epithelial cells.

Diagnostic reference value of antibody levels measured using enzyme immunoassay for subacute sclerosing panencephalitis.

High measles-specific antibody titers in the cerebrospinal fluid (CSF) have important diagnostic significance for subacute sclerosing panencephalitis (SSPE), a progressive neurological disorder caused by measles virus variants. However, the diagnostic reference value of antibody levels and the usefulness of the CSF/serum ratio measured using enzyme immunoassays (EIAs) for SSPE diagnosis remain unclear. To facilitate SSPE diagnosis using EIAs, measles immunoglobulin G (IgG) titers in the CSF and serum of patients with and without SSPE were measured and their CSF/serum antibody ratios evaluated. Serum and CSF antibody levels were compared among three patients with SSPE (59 paired samples), 37 non-SSPE patients, and 2618 patients of unknown backgrounds. Of the 59 paired samples from three patients with SSPE, 56 paired samples (94.9%) showed CSF measles IgG levels ≥0.5 IU/mL and a CSF/serum ratio ≥0.05, whereas non-SSPE cases showed CSF measles IgG levels <0.1 IU/mL and a CSF/serum ratio <0.03. Of the 2618 CSF samples with unknown backgrounds, 951 showed measurable IgG levels with EIA, with a CSF/serum ratio peak of 0.005-0.02, with a 90th percentile of 0.05. Assuming the SSPE criteria as CSF measles IgG ≥0.5 IU/mL and a CSF/serum ratio ≥0.05, only 20 samples (0.8%) with unknown backgrounds were categorized as having SSPE. Conversely, assuming the non-SSPE criteria as CSF measles IgG <0.1 IU/mL and a CSF/serum ratio <0.03, 2403 samples (92%) with unknown backgrounds were categorized as not having SSPE. In conclusion, high CSF/serum ratios (≥0.05) and high measles CSF IgG levels (≥0.5 IU/mL) may be useful for diagnosing SSPE.

Neonatal reticulocytes among preterm infants of small for gestational age.

BACKGROUND: We previously reported that higher reticulocyte counts were observed in earlier preterm infants. Here we present an additional study that focused on reticulocyte counts among preterm infants of small for gestational age (SGA). To assess the relationship between SGA and perinatal variables during the early postnatal period. METHODS: A single-center study was undertaken at Ohta Nishinouchi Hospital between April 1, 2016 and June 30, 2021, using blood samples prospectively collected from infants admitted to the neonatal intensive care unit. These were assessed by univariate analysis and multivariate logistic regression analysis. RESULTS: A total of 467 preterm infants were analyzed, 367 (78.6%) non-SGA and 100 (21.4%) SGA. The median [interquartile range (IQR)] GA (weeks) for the two groups was 33 (31-35) and 34 (31-36), respectively (p = 0.058). The median (IQR) BW (g) for the two groups was 1888 (1480 to 2195) and 1381 (1019 to 1782), respectively (p < 0.001). There were significant relationships between SGA and BW (p < 0.001, OR, 0.997; 95% CI, 0.996 to 0.998), umbilical artery pH (p = 0.038, OR, 0.031; 95% CI, 0.010 to 0.827), Apgar at 5 min (p < 0.001, OR, 1.816; 95% CI, 1.301 to 2.536), nucleated erythrocyte count (p = 0.027, OR, 1.013; 95% CI, 1.001 to 1.024), reticulocyte count (p < 0.001, OR, 0.992; 95% CI, 0.988 to 0.995) and chorioamnionitis (p = 0.019, OR, 0.427; 95% CI, 0.210 to 0.868). CONCLUSION: These results suggest that preterm infants with SGA adapted more rapidly to the postnatal environment than did non-SGA preterm infants. Moreover, a lower reticulocyte count among preterm infants born SGA may be an indicator of good adaptation to the extra-uterine environment during the early postnatal period.

Changes in virus detection in hospitalized children before and after the severe acute respiratory syndrome coronavirus 2 pandemic.

The impact of strengthening preventive measures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the prevalence of respiratory viruses in children was examined. After the SARS-CoV-2 pandemic, the rate of multiple virus detection among hospitalized children decreased. Immediately after the SARS-CoV-2 pandemic, respiratory syncytial and parainfluenza viruses were rarely detected and subsequently reemerged. Human metapneumovirus and influenza virus were not consistently detected. Non-enveloped viruses (bocavirus, rhinovirus, and adenovirus) were detected to some extent even after the pandemic. Epidemic-suppressed infectious diseases may reemerge as susceptibility accumulates in the population and should continue to be monitored.

Epidemiological and clinical characteristics of infections with seasonal human coronavirus and respiratory syncytial virus in hospitalized children immediately before the coronavirus disease 2019 pandemic.

INTRODUCTION: Seasonal human coronavirus (HCoV)-229E, -NL63, -OC43, and -HKU1 are seasonal coronaviruses that cause colds in humans. However, the clinical characteristics of pediatric inpatients infected with HCoVs are unclear. This study aimed to compare and clarify the epidemiological and clinical features of HCoVs and respiratory syncytial virus (RSV), which commonly causes severe respiratory infections in children. METHODS: Nasopharyngeal swabs were collected from all pediatric inpatients with respiratory symptoms at two secondary medical institutions in Fukushima, Japan. Eighteen respiratory viruses, including RSV and four HCoVs, were detected via reverse transcription-polymerase chain reaction. RESULTS: Of the 1757 specimens tested, viruses were detected in 1272 specimens (72.4%), with 789 single (44.9%) and 483 multiple virus detections (27.5%). RSV was detected in 639 patients (36.4%) with no difference in clinical characteristics between RSV-A and RSV-B. HCoV was detected in 84 patients (4.7%): OC43, NL63, HKU1, and 229E in 25 (1.4%), 26 (1.5%), 23 (1.3%), and 16 patients (0.9%), respectively. Patients with HCoV monoinfection (n = 35) had a significantly shorter period from onset to hospitalization (median [interquartile range] days, 2 [1-4.5] vs. 4 [2-5]), significantly shorter hospitalization stays (4 [3-5] vs. 5 [4-6]), and more cases of upper respiratory infections (37.1% vs. 3.9%) and croup (17.1% vs. 0.3%) but less cases of lower respiratory infection (54.3% vs. 94.8%) than patients with RSV monoinfection (n = 362). CONCLUSION: Seasonal HCoV-infected patients account for approximately 5% of children hospitalized for respiratory tract infections and have fewer lower respiratory infections and shorter hospital stays than RSV-infected patients.

Thirteen Nearly Complete Genome Sequences of Human Bocavirus 1 Isolated from Pediatric Inpatients in Fukushima, Japan.

We report 13 genomic sequences of human bocavirus 1 isolated from pediatric inpatients in Fukushima, Japan, using an air-liquid interface culture of human bronchial tracheal epithelial cells. This work suggests the endemic circulation of a human bocavirus variant with a unique amino acid signature in Fukushima.

Can serum periostin predict bronchopulmonary dysplasia in premature infants?

BACKGROUND: Bronchopulmonary dysplasia (BPD) is the most common morbidity complicating preterm birth and affects long-term respiratory outcomes. The objectives of this study were to establish whether serum periostin at birth, day of life (DOL) 28, and corrected 36 weeks' gestational age could be potential biomarkers for BPD. METHODS: A total of 98 preterm Japanese infants born at <32 weeks and comparing 41 healthy controls born at term, were divided into BPD (n = 44) and non-BPD (n = 54) cohorts. Serum periostin levels were measured using an enzyme-linked immunosorbent assay. RESULTS: Among 98 preterm infants, the median serum periostin levels at birth were higher with BPD (338.0 ng/mL) than without (275.0 ng/mL, P < 0.001). Multivariate analysis revealed that serum periostin levels at birth were significantly associated with BPD (P = 0.013). Serum periostin levels at birth with moderate/severe BPD (345.0 ng/mL) were significantly higher than those with non-BPD/mild BPD (283.0 ng/mL, P = 0.006). CONCLUSIONS: Serum periostin levels were significantly correlated with birth weight and gestational age, and serum periostin levels at birth in BPD infants were significantly higher than that in non-BPD infants. IMPACT: This study found higher serum periostin levels at birth in preterm infants subsequently diagnosed with bronchopulmonary dysplasia. It also emerged that serum periostin levels at birth significantly correlated with gestational age and birth weight. The mechanism by which serum periostin is upregulated in BPD infants needs further investigation.