Monitoring Influenza C and D Viruses in Patients With Respiratory Diseases in Japan, January 2018 to March 2023.

BACKGROUND: Influenza viruses can cause zoonotic infections that pose public health risks. Surveillance of influenza A and B viruses is conducted globally; however, information on influenza C and D viruses is limited. Longitudinal monitoring of influenza C virus in humans has been conducted in several countries, but there has been no long-term monitoring of influenza D virus in humans. The public health risks associated with the influenza D virus therefore remain unknown. METHODS: We established a duplex real-time RT-PCR to detect influenza C and D viruses and analyzed respiratory specimens collected from 2144 patients in Japan with respiratory diseases between January 2018 and March 2023. We isolated viruses and conducted hemagglutination inhibition tests to examine antigenicity and focus reduction assays to determine susceptibility to the cap-dependent endonuclease inhibitor baloxavir marboxil. RESULTS: We detected three influenza C viruses belonging to the C/Kanagawa- or C/Sao Paulo-lineages, which recently circulated globally. None of the specimens was positive for the influenza D virus. The C/Yokohama/1/2022 strain, isolated from the specimen with the highest viral RNA load and belonging to the C/Kanagawa-lineage, showed similar antigenicity to the reference C/Kanagawa-lineage strain and was susceptible to baloxavir. CONCLUSIONS: Our duplex real-time RT-PCR is useful for the simultaneous detection of influenza C and D viruses from the same specimen. Adding the influenza D virus to the monitoring of the influenza C virus would help in assessing the public health risks posed by this virus.

A mixed methods study on the readiness of dental, medical, and nursing students for interprofessional learning.

BACKGROUND: Interprofessional education (IPE) is crucial in dentistry, medicine, and nursing. However, scant mixed methods studies have compared the IPE outcomes across these disciplines to develop evidence-based IPE. This study explored the differences in the readiness of dental, medical, and nursing students for interprofessional learning before and after IPE workshops and elucidated reasons for this disparity. METHODS: Data were obtained from dental, medical, and nursing students who participated in IPE workshops conducted at Tokyo Medical and Dental University in Japan in 2019 and 2020. The participants filled the validated Japanese version of the Readiness for Interprofessional Learning Scale (RIPLS) before and after attending the workshops (n = 378). Paired t-tests were performed to assess differences between the pre- and post- workshop RIPLS scores. Welch's t-tests were deployed to evaluate interdisciplinary differences in their scores. Qualitative analyses were conducted using an explanatory sequential design with focus group discussions (FGDs) held with 17 dental students to explain the quantitative results. RESULTS: Total RIPLS scores increased significantly for every discipline after the workshops (p < 0.001). Dental students scored significantly lower pre- and post- workshop aggregates than medical and nursing students, respectively (p < 0.001). The FGDs yielded three principal themes in the explanations tendered by dental students on their lower scores: 1) dental students rarely felt the need for interprofessional collaborations, 2) dentists often worked without the need for interprofessional collaborations, and 3) dental students believed their contribution to the workshop was insufficient. CONCLUSIONS: The results revealed divergences in the readiness of dental, medical, and nursing students for interprofessional learning, and the study illuminated possible reasons for these disparities. These outcomes will help develop evidence-based IPE by indicating approaches to place a higher value on interprofessional collaborations in educational environments, ameliorate the awareness of educators, and enhance the workshop facilitation style.

Highly specific monoclonal antibodies and epitope identification against SARS-CoV-2 nucleocapsid protein for antigen detection tests.

The ongoing coronavirus disease 2019 (COVID-19) pandemic is a major global public health concern. Although rapid point-of-care testing for detecting viral antigen is important for management of the outbreak, the current antigen tests are less sensitive than nucleic acid testing. In our current study, we produce monoclonal antibodies (mAbs) that exclusively react with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and exhibit no cross-reactivity with other human coronaviruses, including SARS-CoV. Molecular modeling suggests that the mAbs bind to epitopes present on the exterior surface of the nucleocapsid, making them suitable for detecting SARS-CoV-2 in clinical samples. We further select the optimal pair of anti-SARS-CoV-2 nucleocapsid protein (NP) mAbs using ELISA and then use this mAb pair to develop immunochromatographic assay augmented with silver amplification technology. Our mAbs recognize the variants of concern (501Y.V1-V3) that are currently in circulation. Because of their high performance, the mAbs of this study can serve as good candidates for developing antigen detection kits for COVID-19.

Influenza vaccine effectiveness against influenza A in children based on the results of various rapid influenza tests in the 2018/19 season.

During influenza epidemics, Japanese clinicians routinely conduct rapid influenza diagnostic tests (RIDTs) in patients with influenza-like illness, and patients with positive test results are treated with anti-influenza drugs within 48 h after the onset of illness. We assessed the vaccine effectiveness (VE) of inactivated influenza vaccine (IIV) in children (6 months-15 years old, N = 4243), using a test-negative case-control design based on the results of RIDTs in the 2018/19 season. The VE against influenza A(H1N1)pdm and A(H3N2) was analyzed separately using an RIDT kit specifically for detecting A(H1N1)pdm09. The adjusted VE against combined influenza A (H1N1pdm and H3N2) and against A(H1N1)pdm09 was 39% (95% confidence interval [CI], 30%-46%) and 74% (95% CI, 39%-89%), respectively. By contrast, the VE against non-A(H1N1)pdm09 influenza A (presumed to be H3N2) was very low at 7%. The adjusted VE for preventing hospitalization was 56% (95% CI, 16%-77%) against influenza A. The VE against A(H1N1)pdm09 was consistently high in our studies. By contrast, the VE against A(H3N2) was low not only in adults but also in children in the 2018/19 season.

Increased risk of rhinovirus infection in children during the coronavirus disease-19 pandemic.

BACKGROUND: Coronavirus disease (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first detected in Japan in January 2020 and has spread throughout the country. Previous studies have reported that viral interference among influenza virus, rhinovirus, and other respiratory viruses can affect viral infections at the host and population level. METHODS: To investigate the impact of COVID-19 on influenza and other respiratory virus infections, we analyzed clinical specimens collected from 2244 patients in Japan with respiratory diseases between January 2018 and September 2020. RESULTS: The frequency of influenza and other respiratory viruses (coxsackievirus A and B; echovirus; enterovirus; human coronavirus 229E, HKU1, NL63, and OC43; human metapneumovirus; human parainfluenza virus 1, 2, 3, and 4; human parechovirus; human respiratory syncytial virus; human adenovirus; human bocavirus; human parvovirus B19; herpes simplex virus type 1; and varicella-zoster virus) was appreciably reduced among all patients during the COVID-19 pandemic except for that of rhinovirus in children younger than 10 years, which was appreciably increased. COVID-19 has not spread among this age group, suggesting an increased risk of rhinovirus infection in children. CONCLUSIONS: Rhinovirus infections should be continuously monitored to understand their increased risk during the COVID-19 pandemic and viral interference with SARS-CoV-2.

Ideal Test Time for Coronavirus Disease 2019 Contact Tracing.

Background: Epidemiological contact tracing is a powerful tool to rapidly detect SARS-CoV-2 infection in persons with a close contact history with COVID-19-affected patients. However, it remains unclear whom and when should be PCR tested among the close contact subjects. Methods: We retrospectively analyzed 817 close contact subjects, including 144 potentially SARS-CoV-2-infected persons. The patient characteristics and contact type, duration between the date of the close contact and specimen sampling, and PCR test results in PCR positive and negative persons were compared. Results: We found that male gender {adjusted odds ratio 1.747 [95% confidence interval (CI) 1.180-2.608]}, age ≥ 60 [1.749 (95% CI 1.07-2.812)], and household contact [2.14 (95% CI 1.388-3.371)] are independent risk factors for close contact SARS-CoV-2 infection. Symptomatic subjects were predicted 6.179 (95% CI 3.985-9.61) times more likely to be infected compared to asymptomatic ones. We could observe PCR test positivity between days 1 and 17 after close contact. However, no subject could be found with a Ct-value <30, considered less infective, after day 14 of close contact. Conclusions: Based on our results, we suggest that contact tracing should be performed on the high-risk subjects between days 3 and 13 after close contacts.

Clinical usefulness of a rapid molecular assay, ID NOW™ influenza A & B 2, in adults.

INTRODUCTION: ID NOW™ Influenza A & B 2 (ID NOW 2) is a rapid molecular assay that combines two characteristics, namely the rapidness of rapid antigen detection test (RADT) and the high sensitivity of molecular assay. METHODS: The clinical performance of ID NOW 2 compared with real-time RT-PCR was evaluated in adults. RESULTS: The sensitivity of ID NOW 2 over multiple seasons from 2016/2017 to 2019/2020 was 97.3% (95% CI: 90.7-99.7) for Type A, 100% (95% CI: 81.9-100) for Type B, and 97.8% (95% CI: 92.2-99.7) for influenza (Type A + Type B), and it was significantly higher than the sensitivity of RADT, which was 80.0% (95% CI: 69.2-88.4) for Type A, 73.3% (95% CI: 44.9-92.2) for Type B, and 78.9% (95% CI: 69.0-86.8) for influenza. The sensitivity of RADT tended to be lower in patients in the particularly early period, within 12 h from disease onset; however, the sensitivity of ID NOW 2 remained high, increasing the difference between the sensitivity of RADT and ID NOW 2. The viral loads were low within 12 h from onset, and it was considered this affected the sensitivity of RADT due to its low analytical sensitivity. The specificity of ID NOW 2 was 98% or greater in all groups. CONCLUSIONS: Since ID NOW 2 has a high sensitivity and specificity in adults, it is anticipated to be used in clinical practice, particularly in patients who require early and accurate diagnosis.

Early combination treatment with baloxavir and peramivir for hospitalized adults with influenza A in Yokohama, Japan.

Baloxavir marboxil is a new anti-influenza drug, but data on the clinical efficacy of a combination treatment of baloxavir and peramivir is scarce. We conducted a single-center retrospective analysis comparing the mortality of a combination of baloxavir and peramivir (B & P, n = 10) and peramivir without baloxavir (P-mono, n = 132) in hospitalized adults with influenza A between 2011 and 2019 in Yokohama City, Japan. Sequencing analysis was conducted in the B & P group to check the I38 mutation in polymerase acidic protein which is associated with baloxavir resistance. The 30-day mortality rates were 0 (0%) in the B & P group and 6 (4.5%) in the P-mono group, respectively, which was not statistically significant. The I38 mutation was not detected before and after the combination treatment. A combination treatment of baloxavir and peramivir might be more effective than peramivir without baloxavir and prevent the emergence of baloxavir resistance in hospitalized adults with influenza A.

Comparison of the Pathogenicity in Mice of A(H1N1)pdm09 Viruses Isolated between 2009 and 2015 in Japan.

The A(H1N1)pdm09 virus emerged in 2009 and continues to circulate in human populations. Recent A(H1N1)pdm09 viruses, that is, A(H1N1)pdm09 viruses circulating in the post-pandemic era, can cause more or less severe infections than those caused by the initial pandemic viruses. To evaluate the changes in pathogenicity of the A(H1N1)pdm09 viruses during their continued circulation in humans, we compared the nucleotide and amino acid sequences of ten A(H1N1)pdm09 viruses isolated in Japan between 2009 and 2015, and experimentally infected mice with each virus. The severity of infection caused by these Japanese isolates ranged from milder to more severe than that caused by the prototypic pandemic strain A/California/04/2009 (CA04/09); however, specific mutations responsible for their pathogenicity have not yet been identified.

Clinical evaluation of ID NOW influenza A & B 2, a rapid influenza virus detection kit using isothermal nucleic acid amplification technology - A comparison with currently available tests.

In this study, we evaluated the performance of ID NOW Influenza A & B 2 (ID NOW 2), a rapid molecular point-of-care test for influenza within 13 min, in comparison with currently available tests. A total of 254 nasopharyngeal swabs (NPS) and 271 nasopharyngeal aspirates (NPA) collected from 373 children and 152 adults with influenza-like illness were tested using ID NOW 2, viral culture, rapid antigen detection test, and loop-mediated isothermal amplification test to evaluate the sensitivity and specificity compared with real-time reverse transcription polymerase chain reaction as the reference method. The sensitivities of ID NOW 2 for influenza A were 95.9% and 95.7% in NPS and NPA, respectively, and for influenza B were 100% and 98.7% in NPS and NPA, respectively. The specificity was 100% for both influenza A and influenza B in NPS and NPA. Sensitivity of each test method reflected the difference of analytical sensitivity among the tests, and ID NOW 2 was not affected by time after illness onset and patient age. In conclusion, ID NOW 2 demonstrated a high sensitivity and specificity that is useful for diagnosis of influenza in the clinical setting and infection control.

Predominant Detection of the Subgroup A2b Human Metapneumovirus Strain with a 111-Nucleotide Duplication in the G gene in Yokohama City, Japan in 2018.

Human metapneumovirus (HMPV) has been a major causative agent of acute respiratory infections in humans. Recently, two types of variant A2b subtype HMPV strains possessing a 111- or 180-nucleotide duplication (nt-dup) in the G gene (HMPV A2b180nt-dup and HMPV A2b111nt-dup, respectively) were detected in Japan, Spain, Vietnam, and China. Our surveillance for infectious agents in Yokohama City, Japan revealed that the HMPV A2b111nt-dup strain became predominant in Yokohama City in 2018. In contrast, no classic HMPV A2b strain was detected after 2017. These data indicate a beneficial role of the 111nt-dup in the G gene for the transmission of HMPV.

A humanized MDCK cell line for the efficient isolation and propagation of human influenza viruses.

Here, we developed hCK, a Madin-Darby canine kidney (MDCK) cell line that expresses high levels of human influenza virus receptors and low levels of avian virus receptors. hCK cells supported human A/H3N2 influenza virus isolation and growth much more effectively than conventional MDCK or human virus receptor-overexpressing (AX4) cells. A/H3N2 viruses propagated in hCK cells also maintained higher genetic stability than those propagated in MDCK and AX4 cells.

Influenza A(H3N2) virus exhibiting reduced susceptibility to baloxavir due to a polymerase acidic subunit I38T substitution detected from a hospitalised child without prior baloxavir treatment, Japan, January 2019.

In January 2019, two influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic subunit (PA), which confers reduced susceptibility to baloxavir, were detected from epidemiologically unrelated hospitalised children in Japan. The viruses exhibited reduced susceptibility to baloxavir but were susceptible to neuraminidase inhibitors. Only one of the two children had been treated with baloxavir. An epidemiological analysis suggests possible transmission of the PA I38T mutant A(H3N2) virus among humans.

Genetic and antigenic characterisation of influenza A(H3N2) viruses isolated in Yokohama during the 2016/17 and 2017/18 influenza seasons.

BACKGROUND: Influenza A(H3N2) virus rapidly evolves to evade human immune responses, resulting in changes in the antigenicity of haemagglutinin (HA). Therefore, continuous genetic and antigenic analyses of A(H3N2) virus are necessary to detect antigenic mutants as quickly as possible. AIM: We attempted to phylogenetically and antigenically capture the epidemic trend of A(H3N2) virus infection in Yokohama, Japan during the 2016/17 and 2017/18 influenza seasons. METHODS: We determined the HA sequences of A(H3N2) viruses detected in Yokohama, Japan during the 2016/17 and 2017/18 influenza seasons to identify amino acid substitutions and the loss or gain of potential N-glycosylation sites in HA, both of which potentially affect the antigenicity of HA. We also examined the antigenicity of isolates using ferret antisera obtained from experimentally infected ferrets. RESULTS: Influenza A(H3N2) viruses belonging to six clades (clades 3C.2A1, 3C.2A1a, 3C.2A1b, 3C.2A2, 3C.2A3 and 3C.2A4) were detected during the 2016/17 influenza season, whereas viruses belonging to two clades (clades 3C.2A1b and 3C.2A2) dominated during the 2017/18 influenza season. The isolates in clades 3C.2A1a and 3C.2A3 lost one N-linked glycosylation site in HA relative to other clades. Antigenic analysis revealed antigenic differences among clades, especially clade 3C.2A2 and 3C.2A4 viruses, which showed distinct antigenic differences from each other and from other clades in the antigenic map. CONCLUSION: Multiple clades, some of which differed antigenically from others, co-circulated in Yokohama, Japan during the 2016/17 and 2017/18 influenza seasons.

Detection of influenza A(H3N2) viruses exhibiting reduced susceptibility to the novel cap-dependent endonuclease inhibitor baloxavir in Japan, December 2018.

The novel cap-dependent endonuclease inhibitor baloxavir marboxil was approved for the treatment of influenza virus infection in Japan in February 2018. Two influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic subunit (PA) were detected in baloxavir-treated children in December 2018. This mutation is known to confer reduced susceptibility to baloxavir, and the two mutant viruses exhibited 76- and 120-fold reduced susceptibility to baloxavir.

Diversity of antigenic mutants of influenza A(H1N1)pdm09 virus escaped from human monoclonal antibodies.

Since the 2017 Southern Hemisphere influenza season, the A(H1N1)pdm09-like virus recommended for use in the vaccine was changed because human, but not ferret, sera distinguish A(H1N1)pdm09 viruses isolated after 2013 from the previously circulating strains. An amino acid substitution, lysine to glutamine, at position 166 (H3 numbering) in the major antigenic site of HA was reported to be responsible for the antigenic drift. Here, we obtained two anti-A(H1N1)pdm09 HA monoclonal antibodies that failed to neutralize viruses isolated after 2013 from a vaccinated volunteer. Escape mutations were identified at position 129, 165, or 166 in the major antigenic site of HA. Competitive growth of the escape mutant viruses with the wild-type virus revealed that some escape mutants possessing an amino acid substitution other than K166Q showed superior growth to that of the wild-type virus. These results suggest that in addition to the K166Q mutation that occurred in epidemic strains, other HA mutations can confer resistance to antibodies that recognize the K166 area, leading to emergence of epidemic strains with such mutations.

Reactivity and sensitivity of commercially available influenza rapid diagnostic tests in Japan.

Seasonal influenza virus routinely causes epidemic infections throughout the world. Sporadic infections by H5N1, H5N6, and H7N9 viruses are also reported. To treat patients suffering from such viral infections, broadly reactive and highly sensitive influenza rapid diagnostic tests (IRDTs) are required. Here, we examined the reactivity and sensitivity of 25 IRDTs available in Japan for the detection of seasonal H1N1pdm09, H3N2, and type B viruses, as well as highly pathogenic H5 and H7 viruses. All of the IRDTs tested detected the seasonal viruses and H5 and H7 viruses albeit with different sensitivities. Several IRDTs detected the H5 and H7 viruses and the seasonal viruses with similar (high) sensitivity.

A novel 111-nucleotide duplication in the G gene of human metapneumovirus.

In 2017, novel human metapneumovirus (HMPV) A2b subgroup strains with a 111-nucleotide duplication in the G gene was detected by the present team. These strains were related to previously identified HMPV A2b strains with a 180-nucleotide duplication; however, they appeared to be different strains, produced by an independent duplication event. The recent evolution of HMPV suggests that careful monitoring of this virus is required.

180-Nucleotide Duplication in the G Gene of Human metapneumovirus A2b Subgroup Strains Circulating in Yokohama City, Japan, since 2014.

Human metapneumovirus (HMPV), a member of the family Paramyxoviridae, was first isolated in 2001. Seroepidemiological studies have shown that HMPV has been a major etiological agent of acute respiratory infections in humans for more than 50 years. Molecular epidemiological, genetic, and antigenetic evolutionary studies of HMPV will strengthen our understanding of the epidemic behavior of the virus and provide valuable insight for the control of HMPV and the development of vaccines and antiviral drugs against HMPV infection. In this study, the nucleotide sequence of and genetic variations in the G gene were analyzed in HMPV strains prevalent in Yokohama City, in the Kanto area, Japan, between January 2013 and June 2016. As a part of the National Epidemiological Surveillance of Infectious Diseases, Japan, 1308 clinical specimens (throat swabs, nasal swabs, nasal secretions, and nasal aspirate fluids) collected at 24 hospitals or clinics in Yokohama City were screened for 15 major respiratory viruses with a multiplex reverse transcription-PCR assay. HMPV was detected in 91 specimens, accounting for 7.0% of the total specimens, and the nucleotide sequences of the G genes of 84 HMPV strains were determined. Among these 84 strains, 6, 43, 10, and 25 strains were classified into subgroups A2a, A2b, B1, and B2, respectively. Approximately half the HMPV A2b subgroup strains detected since 2014 had a 180-nucleotide duplication (180nt-dup) in the G gene and clustered on a phylogenic tree with four classical 180nt-dup-lacking HMPV A2b strains prevalent between 2014 and 2015. The 180nt-dup causes a 60-amino-acid duplication (60aa-dup) in the G protein, creating 23-25 additional potential acceptor sites for O-linked sugars. Our data suggest that 180nt-dup occurred between 2011 and 2013 and that HMPV A2b strains with 180nt-dup (A2b180nt-dup HMPV) became major epidemic strains within 3 years. The detailed mechanism by which the A2b180nt-dup HMPV strains gained an advantage that allowed their efficient spread in the community and the effects of 60aa-dup on HMPV virulence must be clarified.