Impact of the emergence of severe acute respiratory syndrome coronavirus 2 omicron variants on routine childhood immunization in Japan.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic disrupted routine childhood vaccination worldwide, especially at the beginning of the pandemic. After the emergence of variants of concern, particularly the Omicron variants, the number of COVID-19 cases significantly increased, especially in children. However, the impact of the Omicron variants on routine childhood vaccination is unknown. METHODS: This retrospective observational study collected data on vaccines included in the Japanese national immunization program (NIP) of children 15 years or younger between 2016 and 2022 in Niigata, Japan. During the study period, hepatitis B virus vaccine and rotavirus vaccines were introduced to the NIP in October 2016 and October 2020, respectively. The monthly number of vaccine doses per eligible child population between 2020 and 2022 (pandemic period) was compared to the average number of vaccine doses in the corresponding month between 2016 and 2019 (pre-pandemic period). The effect of 8 surges of COVID-19 patients on trends in routine childhood vaccination was also analyzed. RESULTS: The numbers of doses during the pre-Omicron pandemic period were comparable to the values administered before the pandemic. However, after the emergence of the Omicron variants, and especially after the surge of Omicron variant BA.5 in 2022, the number of vaccine doses declined. The decline was notable in second doses of MR vaccine at age 5-6 years by - 9.6 % and diphtheria-tetanus vaccine at ages 11-12 years by - 14.1 %. CONCLUSIONS: Routine childhood immunization was significantly affected by the emergence of the Omicron variants most likely due to the facts that more candidates and their guardians contracted COVID-19, or were required isolation for close contacts. The findings highlight the importance of continued monitoring to maintain high coverage of routine childhood immunizations for controlling vaccine preventable diseases. A follow-up study should investigate changes in the number of declined vaccine doses during Omicron variant predominance.

Duration of fever in children infected with influenza A(H1N1)pdm09, A(H3N2) or B virus and treated with baloxavir marboxil, oseltamivir, laninamivir, or zanamivir in Japan during the 2012-2013 and 2019-2020 influenza seasons.

We compared the duration of fever in children infected with A(H1N1)pdm09, A(H3N2), or influenza B viruses following treatment with baloxavir marboxil (baloxavir) or neuraminidase inhibitors (NAIs) (oseltamivir, zanamivir, or laninamivir). This observational study was conducted at 10 outpatient clinics across 9 prefectures in Japan during the 2012-2013 and 2019-2020 influenza seasons. Patients with influenza rapid antigen test positive were treated with one of four anti-influenza drugs. The type/subtype of influenza viruses were identified from MDCK or MDCK SIAT1 cell-grown samples using two-step real-time PCR. Daily self-reported body temperature after treatment were used to evaluate the duration of fever by treatment group and various underlying factors. Among 1742 patients <19 years old analyzed, 452 (26.0%) were A(H1N1)pdm09, 827 (48.0%) A(H3N2), and 463 (26.0%) influenza B virus infections. Among fours treatment groups, baloxavir showed a shorter median duration of fever compared to oseltamivir in univariate analysis for A(H1N1)pdm09 virus infections (baloxavir, 22.0 h versus oseltamivir, 26.7 h, P < 0.05; laninamivir, 25.5 h, and zanamivir, 25.0 h). However, this difference was not significant in multivariable analyses. For A(H3N2) virus infections, there were no statistically significant differences observed (20.3, 21.0, 22.0, and 19.0 h) uni- and multivariable analyses. For influenza B, baloxavir shortened the fever duration by approximately 15 h than NAIs (20.3, 35.0, 34.3, and 34.1 h), as supported by uni- and multivariable analyses. Baloxavir seems to have comparable clinical effectiveness with NAIs on influenza A but can be more effective for treating pediatric influenza B virus infections than NAIs.

Molecular Epidemiology of Respiratory Syncytial Virus during 2019-2022 and Surviving Genotypes after the COVID-19 Pandemic in Japan.

To evaluate the changes in respiratory syncytial virus (RSV) collected between 2019 and 2022, we analyzed RSV-A and RSV-B strains from various prefectures in Japan before and after the COVID-19 pandemic. RT-PCR-positive samples collected from children with rapid test positivity at outpatient clinics in 11 prefectures in Japan were sequenced for the ectodomain of the G gene to determine the genotype. Time-aware phylogeographic analyses were performed using the second hypervariable region (HVR) of the G gene from 2012 to 2022. Of 967 samples, 739 (76.4%) were found to be RSV-positive using RT-PCR. RSV peaked in September 2019 but was not detected in 2020, except in Okinawa. Nationwide epidemics occurred with peaks in July 2021 and 2022. The genotype remained the same, ON1 for RSV-A and BA9 for RSV-B during 2019-2022. Phylogeographic analysis of HVR revealed that at least seven clusters of RSV-A had circulated previously but decreased to two clusters after the pandemic, whereas RSV-B had a single monophyletic cluster over the 10 years. Both RSV-A and RSV-B were transferred from Okinawa into other prefectures after the pandemic. The RSV epidemic was suppressed due to pandemic restrictions; however, pre-pandemic genotypes spread nationwide after the pandemic.

Factors associated with viral RNA shedding and evaluation of potential viral infectivity at returning to school in influenza outpatients after treatment with baloxavir marboxil and neuraminidase inhibitors during 2013/2014-2019/2020 seasons in Japan: an observational study.

BACKGROUND: This study assessed the differences in daily virus reduction and the residual infectivity after the recommended home stay period in Japan in patients infected with influenza and treated with baloxavir (BA), laninamivir (LA), oseltamivir (OS), and zanamivir (ZA). METHODS: We conducted an observational study on children and adults at 13 outpatient clinics in 11 prefectures in Japan during seven influenza seasons from 2013/2014 to 2019/2020. Virus samples were collected twice from influenza rapid test-positive patients at the first and second visit 4-5 days after the start of treatment. The viral RNA shedding was quantified using quantitative RT-PCR. Neuraminidase (NA) and polymerase acidic (PA) variant viruses that reduce susceptibility to NA inhibitors and BA, respectively, were screened using RT-PCR and genetic sequencing. Daily estimated viral reduction was evaluated using univariate and multivariate analyses for the factors such as age, treatment, vaccination status, or the emergence of PA or NA variants. The potential infectivity of the viral RNA shedding at the second visit samples was determined using the Receiver Operator Curve based on the positivity of virus isolation. RESULTS: Among 518 patients, 465 (80.0%) and 116 (20.0%) were infected with influenza A (189 with BA, 58 with LA, 181 with OS, 37 with ZA) and influenza B (39 with BA, 10 with LA, 52 with OS, 15 with ZA). The emergence of 21 PA variants in influenza A was detected after BA treatment, but NA variants were not detected after NAIs treatment. Multiple linear regression analysis showed that the daily viral RNA shedding reduction in patients was slower in the two NAIs (OS and LA) than in BA, influenza B infection, aged 0-5 years, or the emergence of PA variants. The residual viral RNA shedding potentially infectious was detected in approximately 10-30% of the patients aged 6-18 years after five days of onset. CONCLUSIONS: Viral clearance differed by age, type of influenza, choice of treatment, and susceptibility to BA. Additionally, the recommended homestay period in Japan seemed insufficient, but reduced viral spread to some extent since most school-age patients became non-infectious after 5 days of onset.

Pilot Study of Evaluating Attitudes toward Childhood Immunization among Healthcare Workers in Japan.

Providing appropriate immunization information during the perinatal period is important for improving immunization rates among infants and children; however, the distribution of immunization information by healthcare workers (HCWs) is not standardized in Japan. We investigated HCWs' attitudes toward childhood immunization and factors related to vaccine hesitancy. We conducted a cross-sectional descriptive survey of HCWs involved in childhood immunization in Niigata City, Japan, from November 2017 to January 2018. We assessed contextual, individual and group, and vaccine/vaccination-specific influences. Of 290 HCWs, 139 (47.9%) returned completed questionnaires. Most HCWs (87/139, 64.9%) reported providing immunization information verbally to parents; 51/87 (58.6%) spent fewer than five minutes doing so. Pediatricians provided vaccines based on the parents' best interest, whereas public health nurses and midwives emphasized government policy. Nurses had greater hesitancy related to personal perceptions and social/peer factors than pediatricians (p < 0.001). Nurses were significantly more likely than pediatricians to suggest that children receive more shots than necessary (p < 0.01). Nurses tended to have more negative attitudes toward vaccination and little awareness of immunization promotion compared to pediatricians. Thus, all HCWs involved in childhood immunization should receive sufficient information to provide timely and appropriate immunization to infants and children.

Duration of fever and symptoms in influenza-infected children treated with baloxavir marboxil during the 2019-2020 season in Japan and detection of influenza virus with the PA E23K substitution.

Data on the clinical effectiveness of the novel anti-influenza drug baloxavir marboxil (baloxavir) in children remain limited. We conducted an observational study to compare the duration of fever and symptoms between baloxavir- and oseltamivir-treated children infected with influenza A and B. In total, 159 outpatients with influenza A(H1N1)pdm09 or B/Victoria-lineage infections, aged <19 years, during the 2019-2020 influenza season in Japan were enrolled and assessed the duration of fever and symptoms using the Kaplan-Meier method and a multivariate Cox proportional hazard regression model. Polymerase acidic (PA) variants were examined before and after baloxavir treatment. In the multivariable analysis, the duration of fever and symptoms was unaltered between the A(H1N1)pdm09 (n = 116) and B/Victoria-lineage (n = 43) groups. Conversely, the fever duration was marginally longer in the oseltamivir-treated group (n = 59) than in the baloxavir group (n = 100) (hazard ratio (HR) = 0.67, p = 0.05); however, the duration of symptoms was unaltered between the two groups (HR = 0.74, p = 0.11). No patient presented PA reduced susceptibility marker(s) before baloxavir treatment in the analyzed groups. The PA/E23K variant was detected in one case (1.5%, 1/66) of A(H1N1)pdm09 after baloxavir treatment. One case (2.0%, 1/50) of A(H1N1)pdm09 with an N295S substitution in neuraminidase was detected following oseltamivir treatment. These results suggested that the duration of fever was likely to be shorter with baloxavir than with oseltamivir, but the difference between influenza A (H1N1)pdm09 and B/Victoria-lineage was unclear. It is important to continue evaluating the clinical effectiveness of baloxavir and monitoring its drug susceptibility to the influenza virus.

Novel electrocardiographic criteria for short QT syndrome in children and adolescents.

AIMS: Although shortening of the corrected QT interval (QTc) is a key finding in the diagnosis of short QT syndrome (SQTS), there may be overlap of the QTc between SQTS patients and normal subjects in childhood and adolescence. We aimed to investigate electrocardiographic findings for differentiation of SQTS patients. METHODS AND RESULTS: The SQTS group comprised 34 SQTS patients <20 years old, including 9 from our institutions and 25 from previous reports. The control group comprised 61 apparently healthy subjects with an QTc of <360 ms who were selected from 13 314 participants in a school-based screening programme. We compared electrocardiographic findings, including QT and Jpoint-Tpeak intervals (QT and J-Tpeak, respectively), those corrected by using the Bazett's and Fridericia's formulae (cB and cF, respectively) and early repolarization (ER) between the groups. QT, QTc by using Bazett's formula (QTcB), QTc by using Fridericia's formula (QTcF), J-Tpeak, J-Tpeak cB, and J-Tpeak cF were significantly shorter in the SQTS group than in the control group. On receiver operating characteristic curve analysis, the area under the curve (AUC) was largest for QTcB (0.888) among QT, QTcB, and QTcF, with a cut-off value of 316 ms (sensitivity: 79.4% and specificity: 96.7%). The AUC was largest for J-Tpeak cB (0.848) among J-Tpeak, J-Tpeak cB, and J-Tpeak cF, with a cut-off value of 181 ms (sensitivity: 80.8% and specificity: 91.8%). Early repolarization was found more frequently in the SQTS group than in the control group (67% vs. 23%, P = 0.001). CONCLUSION: A QTcB <316 ms, J-Tpeak cB < 181 ms, and the presence of ER may indicate SQTS patients in childhood and adolescence.

Development of cycling probe based real-time PCR methodology for influenza A viruses possessing the PA/I38T amino acid substitution associated with reduced baloxavir susceptibility.

Baloxavir marboxil has been used for influenza treatment since March 2018 in Japan. After baloxavir treatment, the most frequently detected substitution is Ile38Thr in polymerase acidic protein (PA/I38T), and this substitution reduces baloxavir susceptibility in influenza A viruses. To rapidly investigate the frequency of PA/I38T in influenza A (H1N1)pdm09 and A (H3N2) viruses in clinical samples, we established a rapid real-time system to detect single nucleotide polymorphisms in PA, using cycling probe real-time PCR. We designed two sets of probes that were labeled with either 6-carboxyfluorescein (FAM) or 6-carboxy-X-rhodamine (ROX) to identify PA/I38 (wild type strain) or PA/I38T, respectively. The established cycling probe real-time PCR system showed a dynamic linear range of 101 to 106 copies with high sensitivity in plasmid DNA controls. This real-time PCR system discriminated between PA/I38T and wild type viruses well. During the 2018/19 season, 377 influenza A-positive clinical samples were collected in Japan before antiviral treatment. Using our cycling probe real-time PCR system, we detected no (0/129, 0.0%) influenza A (H1N1)pdm09 viruses with PA/I38T substitutions and four A (H3N2) (4/229, 1.7%) with PA/I38T substitution prior to treatment. In addition, we found PA/I38T variant in siblings who did not received baloxavir treatment during an infection caused by A (H3N2) that afflicted the entire family. Although human-to-human transmission of PA/I38T variant may have occurred in a closed environment, the prevalence of this variant in influenza A viruses was still limited. Our cycling probe-PCR system is thus useful for antiviral surveillance of influenza A viruses possessing PA/I38T.

Duration of fever and symptoms in children after treatment with baloxavir marboxil and oseltamivir during the 2018-2019 season and detection of variant influenza a viruses with polymerase acidic subunit substitutions.

We conducted a prospective, multicenter, non-randomized observational study to assess the duration of fever and symptoms of influenza A/H1N1pdm09 and A/H3N2 infected children < 19 years old treated with either baloxavir or oseltamivir. Additionally, these symptoms were investigated in association with pre- and post-baloxavir treatment-emergent polymerase acidic unit (PA) variants as compared to non-substituted viruses. Following receipt of informed consent, baloxavir was administered to 102 influenza A patients, and oseltamivir to 52 patients during the 2018-2019 influenza season in Japan. The average age was higher in the baloxavir treatment group compared to the oseltamivir treatment group (10.6 ± 2.7 versus 6.9 ± 2.9 years old, p < 0.01). The duration of fever and symptoms in baloxavir-treated A/H1N1pdm09 and A/H3N2-infected children did not differ from those in oseltamivir-treated groups (median 22.0, 11.8, 23.0, and 21.0 h, and median 114.5, 121.0, 123.0, and 122.0 h, respectively). One (1.2%) of 83 A/H3N2 patients possessed a PA/I38T substituted virus prior to treatment. The frequency of PA variants in post-treatment samples obtained 2-11 days after beginning of baloxavir was 12.5% (4/32) for A/H1N1pdm09 and 14.1% (9/64) for A/H3N2 when the total number of cases was used as the denominator, however, were 57.1% (4/7) and 33.3% (9/27) when PCR-positive cases at the time of second sampling was used as the denominator. The most frequent PA substitution was I38T (9), with E23K (1), I38K (1), I38M (1), and PA/I38S (1) also observed. The duration of fever and overall symptoms did not differ significantly following baloxavir treatment in individuals with PA variant viruses, non-substituted virus, or in those that were PCR negative at the second sampling (median 20, 24 and 11 h, and median 121, 115 and 121 h, respectively). Rebound of viral RNA load was observed in 13.5% (2/13) of PA variants but it was not associated with recurrence of fever and symptoms. Hence, prolonged fever or symptoms were not observed in children treated with baloxavir following emergence of PA variants, however, further studies are needed to evaluate the clinical impact of PA variants.

Effectiveness of the quadrivalent inactivated influenza vaccine in Japan during the 2015-2016 season: A test-negative case-control study comparing the results by real time PCR, virus isolation.

BACKGROUND: We estimated influenza vaccine effectiveness (VE) in 2015-2016 season against medically attended, laboratory-confirmed influenza, when quadrivalent inactivated vaccine (IIV4) was first introduced in Japan, using test-negative case-control design. Influenza A(H1N1)pdm09 cocirculated with B/Yamagata and B/Victoria during the study period in Japan. METHOD: We based our case definition on two laboratory tests, real-time reverse transcription polymerase chain reaction (RT PCR), and virus isolation and compared VEs based on these tests. In addition, VE was evaluated by rapid diagnostic test (RDT). Nasopharyngeal swabs were collected from outpatients who visited clinics with influenza-like illness (ILIs) in Hokkaido, Niigata, Gunma and Nagasaki prefectures. RESULTS: Among 713 children and adults enrolled in this study, 578 were influenza positive by RT PCR including, 392 influenza A and 186 influenza B, while 135 were tested negative controls. The adjusted VE by RT PCR for all ages against any influenza was low protection of 36.0% (95% confidence interval [CI], 3.1% to 58.6%), for influenza A was 30.0% (95% CI: -10.0% to 55.5%), and influenza B was moderate 50.2% (95% CI: 13.3% to 71.4%). Adjusted VE for virus isolation for A(H1N1)pdm09 was 37.1% (95% CI: 1.7% to 59.7%), Yamagata lineage 51.3% (95% CI: 6.4% to 74.7%) and Victoria lineage 21.3% (95% CI: -50.0% to 58.9%). VE was highest and protective in 0-5 years old group against any influenza and influenza A and B/Yamagata, but the protective effect was not observed for other age groups and B/Victoria. RDT demonstrated concordant results with RT PCR and virus isolation. Sequencing of hemagglutinin gene showed that all A(H1N1)pdm09 belong to clade 6B including 31 strains (88.6%), which belong to clade 6B.1 possessing S162N mutations that may alter antigenicity and affect VE for A(H1N1)pdm09. CONCLUSIONS: IIV4 influenza vaccine during 2015-2016 was effective against A(H1N1)pdm09 and the two lineages of type B. Younger children was more protected than older children and adults by vaccination.

Improved parental attitudes and beliefs through stepwise perinatal vaccination education.

This study examined the effects of providing vaccination education during the perinatal period on Japanese parents' knowledge, attitudes, and beliefs about childhood vaccination. A cluster-randomized controlled-trial method was used on a sample of 160 pregnant women recruited from 9 obstetrical sites in Niigata, Japan. The treatment group received a stepwise interactive education intervention, while the control group received a general vaccination leaflet. Changes in parental attitudes toward and beliefs about infant vaccination were assessed on the child's one-month and 6-month birthdays using paper questionnaires. Of the initial 188 participants, 160 (90.4%) completed the final post-survey questionnaire. Scores on injunctive social norms (a morally neutral perception of the behavior of the majority) and descriptive social norms (a moral perception of what individuals should do) significantly increased in the treatment group (p = .02 and p = .01, respectively). There was a significant difference between the 2 groups over time in terms of perceived benefit (efficacy of available preventive actions) (p = .03), but no significant differences in perceived severity (seriousness of a disease outcome), perceived susceptibility (likelihood of getting a disease), perceived benefits, perceived behavioral control, or descriptive social norms between the groups at any time point or in the patterns of change over time (p > .31). Thus, stepwise perinatal vaccination education was found to positively influence maternal attitudes and beliefs about infant vaccination. This study suggests the importance of vaccination education during the perinatal period.

Effect of stepwise perinatal immunization education: A cluster-randomized controlled trial.

BACKGROUND: Perinatal immunization education is important for improving the immunization outcomes of infants; however, the content of educational materials used at each perinatal period has not been carefully evaluated. We hypothesized that stepwise education offered at different perinatal periods would improve infant immunization status and enhance maternal immunization knowledge. METHODS: In this cluster-randomized controlled trial, pregnant women were recruited from nine obstetric sites in Niigata, Japan. The intervention group received a stepwise, interactive education intervention (prenatally, postnatally, and 1month after birth). The control group received a leaflet containing general information on immunization. Infant immunization status was evaluated at 6months of age, and maternal immunization knowledge was evaluated by a written survey after each intervention. RESULTS: Among 188 study participants, 151 (80.3%) replied to the final post-intervention survey. At 6months of age, the percentage of children who completed three doses of inactivated polio, diphtheria, tetanus toxoid, and acellular pertussis (DTaP-IPV) vaccine was higher in the intervention group than in the control (p=0.04); however, no differences between groups were observed for the Haemophilus influenzae type b (Hib) (p=0.67) or 13-valent pneumococcal conjugate (PCV13) vaccines (p=0.20). The duration to the completion of the third dose of the DTaP-IPV, Hib, and PCV13 vaccines was shorter in the intervention group than in the control (p=0.03, p<0.01, and p<0.01, respectively). Furthermore, maternal knowledge scores exhibited significantly greater improvement in the intervention group over time compared with those of the control group (p=0.02). CONCLUSIONS: Stepwise perinatal immunization education improved immunization schedule adherence for required vaccines and improved maternal immunization knowledge.

Community- and hospital-acquired infections with oseltamivir- and peramivir-resistant influenza A(H1N1)pdm09 viruses during the 2015-2016 season in Japan.

We report five cases of community- and hospital-acquired infections with oseltamivir- and peramivir-resistant A(H1N1)pdm09 viruses possessing the neuraminidase (NA) H275Y mutation during January-February 2016 in Japan. One case was hospitalized and was receiving oseltamivir for prophylaxis. The remaining four cases were not taking antiviral drugs at the time of sampling. These cases were geographically distant and epidemiologically unrelated. The five viruses showed ~300-fold rise in IC50 values against oseltamivir and peramivir, defined as highly reduced inhibition according to the WHO definition. Overall, the prevalence of the H275Y A(H1N1)pdm09 viruses was 1.8 % (5/282). The resistant viruses possessed the V241I, N369 K, and N386 K substitutions in the NA that have been previously reported among A(H1N1)pdm09 to alter transmission fitness. Analysis of Michaelis constant (Km) revealed that two of the isolates had reduced NA affinity to MUNANA, while the other three isolates displayed a slightly decreased affinity compared to the sensitive viruses. Further studies are needed to monitor the community spread of resistant viruses and to assess their transmissibility.

Current practices and needs regarding perinatal childhood immunization education for Japanese mothers.

BACKGROUND: Accurate, standardized information on childhood immunization is not available in Japan. We investigated current practices in perinatal childhood immunization education in the community and the needs and interest for such education among Japanese mothers. METHODS: This cross-sectional, descriptive study evaluated pregnant and postnatal women at four institutions in Niigata, Japan from May through July 2014. Data were collected using questionnaires inquiring about demographics, immunization education received, intent to receive childhood vaccines, and needs regarding information on childhood immunization. RESULTS: Questionnaires were distributed to 300 women, and 116 (38.6%) were returned; 70 (59.6%) of the respondents were pregnant women and 46 (40.3%) were postnatal women. Fourteen (20%) of the 70 pregnant women reported receiving some form of immunization education; in contrast, 34 (73.9%) of 46 postnatal women had received such education within 1 month of delivery. The rates of respondents who felt that the information was insufficient were high: 78.6% among pregnant women and 52.9% among postnatal women. Pregnant women reported that the most important information was general concepts of immunization; in contrast, postnatal women desired more-detailed information, e.g., on immunization scheduling. CONCLUSIONS: Japanese women do not receive sufficient perinatal immunization education. The information needed during the prenatal and postnatal periods differs. Thus, educational approaches may need to provide carefully targeted information.

Skin thickness in young infants and adolescents: Applications for intradermal vaccination.

As compared with standard intramuscular and subcutaneous vaccines, intradermal (ID) vaccines elicit a more potent immune response in both adults and children, with equivalent dosage or antigen dose sparing. Recently, various devices for ID injection have been developed; the length of needles ranges in 0.6-1.5 mm. However, skin thickness must be measured to determine optimal needle length for ID vaccines. Use of ID vaccines in infants and children is appealing because children require more vaccines than do adults; however, information on skin thickness in infants and children is limited. We used ultrasound echography to measure skin thickness in Japanese infants aged 2 months (n=78) and adolescents aged 13-15 years (n=82). Mean (range) deltoid and suprascapular skin thickness was 1.67 mm (1.16-2.39 mm) and 1.83 mm (1.24-2.60 mm), respectively, in infants and 1.81 mm (1.25-3.00 mm) and 2.43 mm (1.51-3.95 mm), respectively, in adolescents. Among infants who underwent re-measurement of skin thickness at age 6 months (n=11), mean deltoid skin thickness (1.84 mm) was significantly greater than at age 2 months (1.60 mm) (P<0.001). In contrast, no significant difference was observed in suprascapular skin thickness (1.79 mm vs. 1.67 mm, respectively; P=0.17). Gender was not associated with skin thickness in either age group. Skin thickness was positively correlated with body weight in adolescents (r=0.43, P<0.001 in deltoid region; r=0.30, P=0.01 in suprascapular region). In conclusion, this is the first study to evaluate skin thickness in different age groups of children, including at age 2 months. Skin thickness gradually increased from age 2 months to age 13-15 years, but no consistent trend was noted in analysis stratified by measurement site, gender, or age. These findings suggest that an appropriate length of ID device needle for infants and children is likely to be less than 1.2mm and a special device with shorter length of needle is warranted for infants and children.

Clinical effectiveness of neuraminidase inhibitors--oseltamivir, zanamivir, laninamivir, and peramivir--for treatment of influenza A(H3N2) and A(H1N1)pdm09 infection: an observational study in the 2010-2011 influenza season in Japan.

The clinical effectiveness of the newly released neuraminidase inhibitors (NAIs) laninamivir and peramivir has not been sufficiently evaluated in influenza-infected patients in clinical and practical settings. In this study, we analyzed the clinical data of 211 patients infected with influenza A virus subtype H3N2 (A(H3N2)) and 45 patients infected with influenza A virus subtype H1N1pdm (A(H1N1)pdm09) who received the NAIs oseltamivir, zanamivir, laninamivir, or peramivir during the 2010-2011 influenza season. The duration of fever from the first dose of the NAI to fever alleviation to <37.5 °C was evaluated as an indicator of the clinical effectiveness of the NAIs in the influenza-infected patients. For the A(H3N2)-infected patients, Kaplan-Meier analysis showed the peramivir treatment group had the fastest time of fever alleviation to <37.5 °C (median 17.0 h, 95 % confidence interval [CI] 7.2-26.8 h) of the four treatment groups. No significant difference was found in the time to fever alleviation among the other antivirals, oseltamivir, zanamivir, and laninamivir. Results of multivariate analysis, using a Cox proportional-hazards model (hazard ratio 3.321) adjusted for the factors age, sex, body weight, vaccination status, time from onset to the clinic visit, and body temperature showed significantly faster fever alleviation in the peramivir treatment group compared with the oseltamivir treatment group. For the A(H1N1)pdm09-infected patients, only the oseltamivir and zanamivir treatment groups were compared, and no significant difference in time to alleviation of fever was observed between the two groups. Based on a cycling probe real-time polymerase chain reaction (PCR) assay, none of the A(H1N1)pdm09 strains in this study had the H275Y mutation conferring oseltamivir resistance. Further evaluation of the clinical effectiveness of the newly released NAIs for influenza-infected patients, including those infected with A(H1N1)pdm09, is needed.

Involvement of the distal Arg residue in Cl⁻ binding of midge larval haemoglobin.

Monomeric haemoglobin component V (Hb V) from the larva of the midge Propsilocerus akamusi shows high Cl⁻ affinity under high salt concentrations at acidic pH. In order to understand the structural changes that depend on Cl⁻ binding, crystal structures of Hb V were determined under acidic high-salt conditions and the structural changes arising from different haem-bound ligands were simulated. Crystal structures of Hb V under acidic high-salt conditions indicated that the side chain of ArgE10 on the distal face of the haem contributes to stabilizing haem-bound Cl⁻. The conformation of the Arg side chain in the Cl⁻-bound form was almost identical to that in ligated Hb V at neutral pH but not to that in met Hb V under acidic salt-free conditions. Furthermore, preliminary molecular-dynamics simulations also indicated that the swinging of the Arg side chain into the haem pocket depends on Cl⁻ ligation. This result suggests that, like pH change, Cl⁻ binding affects the location of the distal Arg residue. Owing to the increased positive electrostatic potential observed in the haem pocket at acidic pH, it was concluded that electrostatic changes caused by pH change and anionic ligand binding may affect the behaviour of the polar Arg residue.

Identification of oseltamivir resistance among pandemic and seasonal influenza A (H1N1) viruses by an His275Tyr genotyping assay using the cycling probe method.

Neuraminidase inhibitors are agents used against influenza viruses; however, the emergence of drug-resistant strains is a major concern. Recently, the prevalence of oseltamivir-resistant seasonal influenza A (H1N1) virus increased globally and the emergence of oseltamivir-resistant pandemic influenza A (H1N1) 2009 viruses was reported. In this study, we developed a cycling probe real-time PCR method for the detection of oseltamivir-resistant seasonal influenza A (H1N1) and pandemic influenza A (H1N1) 2009 viruses. We designed two sets of primers and probes that were labeled with 6-carboxyfluorescein or 6-carboxy-X-rhodamine to identify single nucleotide polymorphisms (SNPs) that correspond to a histidine and a tyrosine at position 275 in the neuraminidase protein, respectively. These SNPs confer susceptibility and resistance to oseltamivir, respectively. In the 2007-2008 season, the prevalence of oseltamivir-resistant H1N1 viruses was 0% (0/72), but in the 2008-2009 season, it increased to 100% (282/282). In the 2009-2010 season, all of the pandemic influenza A (H1N1) 2009 viruses were susceptible to oseltamivir (0/73, 0%). This method is sensitive and specific for the screening of oseltamivir-resistant influenza A (H1N1) viruses. This method is applicable to routine laboratory-based monitoring of drug resistance and patient management during antiviral therapy.

Reduced effectiveness of oseltamivir in children infected with oseltamivir-resistant influenza A (H1N1) viruses with His275Tyr mutation.

BACKGROUND: Little is known about whether neuraminidase inhibitors are effective for children infected with oseltamivir-resistant influenza A(H1N1) viruses. METHODS: Children aged 15 years and younger having influenza-like illness and who visited outpatient clinics within 48 hours of fever onset were enrolled from 2006-2007 to 2008-2009 influenza seasons in Japan. Patients received oseltamivir, zanamivir, or no treatment after screening by a rapid antigen test. Nasopharyngeal swabs were collected before antiviral therapy and were used for virus isolation. Oseltamivir resistance was determined by detection of the H275Y mutation in neuraminidase, and susceptibility test using neuraminidase inhibition assay. Daily body temperature was evaluated according to drug type and susceptibility by univariate and multivariate analyses. RESULTS: Of 1647 patients screened, 238 oseltamivir-resistant H1N1 cases (87 oseltamivir-treated, 64 zanamivir-treated, and 87 nontreated) and 110 oseltamivir-susceptible cases (60 oseltamivir-treated and 50 nontreated) were evaluated. In oseltamivir-resistant cases, fever on days 4 to 5 after the start of treatment was significantly higher in oseltamivir-treated and nontreated than in zanamivir-treated patients (P < 0.05). In oseltamivir-susceptible cases, fever was significantly lower in oseltamivir-treated than nontreated on days 3 to 6 (P < 0.01). Similar findings were obtained for duration of the fever and proportion of recurrent fever. Reduced effectiveness of oseltamivir was more prominent in children 0 to 6 years old than in those 7 to 15 years old. Multiple logistic regression analysis showed that lower age, nontreatment, and oseltamivir treatment of oseltamivir-resistant patients were factors associated with the duration of the longer fever. CONCLUSIONS: Infection with oseltamivir-resistant viruses significantly reduced the effectiveness of oseltamivir, and this tendency was more apparent in younger children.

pH-dependent structural changes in haemoglobin component V from the midge larva Propsilocerus akamusi (Orthocladiinae, Diptera).

Haemoglobin component V (Hb V) from the midge larva Propsilocerus akamusi exhibits oxygen affinity despite the replacement of HisE7 and a pH-dependence of its functional properties. In order to understand the contribution of the distal residue to the ligand-binding properties and the pH-dependent structural changes in this insect Hb, the crystal structure of Hb V was determined under five different pH conditions. Structural comparisons of these Hb structures indicated that at neutral pH ArgE10 contributes to the stabilization of the haem-bound ligand molecule as a functional substitute for the nonpolar E7 residue. However, ArgE10 does not contribute to stabilization at acidic and alkaline pH because of the swinging movement of the Arg side chain under these conditions. This pH-dependent behaviour of Arg results in significant differences in the hydrogen-bond network on the distal side of the haem in the Hb V structures at different pH values. Furthermore, the change in pH results in a partial movement of the F helix, considering that coupled movements of ArgE10 and the F helix determine the haem location at each pH. These results suggested that Hb V retains its functional properties by adapting to the structural changes caused by amino-acid replacements.