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.

How adolescent Japanese girls arrive at human papilloma virus vaccination: A semistructured interview study.

We examined the human papilloma virus (HPV) vaccination process in adolescent Japanese girls, including protective and obstructive factors to develop and practice enlightenment activities and educational interventions for promoting HPV vaccination to adolescent Japanese girls and their families. We conducted semistructured interviews with 20 adolescent Japanese girls who lived in the wider Tokyo area. To analyze the interview data, we adopted the modified grounded-theory approach. We identified three stages in the vaccination process: first encounter with cervical cancer and HPV vaccine, thoughts about vaccination, and adjustment with parents toward vaccination. The girls "knew" their knowledge and information on cervical cancer and the HPV vaccine from experts, parents, and friends, "considered and discussed" HPV vaccination in their own way or with parents, and "arranged" actual vaccination. This process was influenced by the promoting/obstructive factors in each stage. Healthcare providers should understand the experiences and feelings of adolescent girls who were confronted with HPV vaccination in the context of their vaccination process and conduct enlightenment activities to promote vaccination, keeping the promoting and obstructive factors suggested in this study in mind.

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.

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.

Demonstration and partial characterization of a bacterial growth enhancer in sera.

During our research into the pathogenesis of Vibrio parahaemolyticus, we noticed that the concentration of serum added to the tissue culture medium (Dulbecco's modified Eagle's medium: DMEM) greatly affected its growth. Using gel filtration column chromatography, we clearly demonstrated that serum contains not only a bacterial growth inhibitor (BGI) but also a bacterial growth enhancer (BGE) for Vibrio parahaemolyticus. Our data indicate that the BGI is transferrin, whereas the BGE seems to be an undescribed small molecule (molecular weight of 1,000-3,000 Da) and is associated with magnesium and molybdenum ions. BGE activity was not decreased by heat treatment (at 60 or 100°C for 30 min) and affected the growth rate of a wide range of Gram-negative and Gram-positive bacteria. The addition of EDTA into DMEM lowered the growth rate, whereas the addition of BGE restored the growth activity. This study suggests that sera contain a previously undescribed small BGE molecule.

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.

Quality and safety issues related to traditional animal medicine: role of taurine.

BACKGROUND: Calculus Bovis (:C.Bovis) is one of the most precious and commonly-used medicinal materials in Japan and China. As the natural occurrence is very rare, a source of supply for C. Bovis is far behind the actual need and great efforts have been taken for some substitutes of natural C. Bovis. Unfortunately, very little information is available on the quality and/or clinical efficacy of medication based on C. Bovis. To ensure sustainable use of traditional therapeutic agents derived from C. Bovis, we felt that several issues needed to be addressed: 1) the source of the C. Bovis materials and quality control; 2) the role of taurine in the efficacy of C. Bovis. METHODS: Nine samples of natural C. Bovis and its substitutes were collected. ICP-MS was used for elemental analysis and the characterization was performed by principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) as multivariate approaches. The efficacy of C. Bovis was evaluated for morphology, viability and beating pattern on cultured cardiac myocytes and/or fibroblasts. RESULTS: PCA and multi-elemental focus was effective in discriminating C. Bovis samples derived from different habitats. A satisfactory classification using SIMCA was obtained among Australia C. Bovis, other habitats and the substitutes. Australian samples had better batch uniformity than other habitats and were composed of fewer elements. We have used Australian C. Bovis for assessment on its bioactive compounds. Rat cardiac cells incubated with C. Bovis extract (0.01-0.1 mg/ml) maintained normal morphology, viability and beating pattern. Cardiac myocytes and fibroblasts treated for 48 h with CA (0.5 mM) or DCA (0.1 mM) caused cell injury, as reflected by changes in appearance and a reduction of viability detected by the MTS assay. In cardiomyocytes, 0.5 h exposure of CA (0.5 mM) markedly decreased the velocity ratio of beating, whereas the simultaneous addition of 1 mM taurine largely prevented the decrease. CONCLUSIONS: The multi-elemental focus provided some references for the quality control and the efficacy of C. Bovis. Taurine partly attenuated the harmful actions of bile acids. It is plausible that the relationship between taurine and the bile acids contributes to therapeutic effect of C. Bovis.

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.

Isolation and identification of the major plasma fibronectin cDNA from Japanese catfish Silurus asotus.

Major plasma fibronectin from Japanese catfish was isolated using affinity chromatography, and the fibronectin was digested with thermolysin. Peptide sequences of the fragments were obtained by peptide sequencer. Complete fibronectin cDNA was obtained from Japanese catfish liver cells using 5'-rapid amplification of cDNA end (RACE) and 3'-RACE based on the peptide sequences. It consists of a 6885 bp open reading frame, which is putatively translated to a protein of 2295 amino acids resides. The catfish fibronectin has 12 type-I modules, 2 type-II modules and 15 type-III modules, and variable sites V and lacks both EIIIA and EIIIB sites. Homology of the entire amino acids residues of catfish fibronectin with those of mammals (Homo sapiens, Rattus norvegicus, Bos taurus) is only 47-48% and 57% with that of Danio rerio. However, amino acid sequence of type-I module 3 and type-I module12 are highly conserved and homology exceeds 80% with corresponding regions of the mammals, Xenopus laevis and fish species (Silurus asotus and D. rerio). Phylogenetic analysis indicates that only type-I module 4 shows a different pattern of phylogenetic tree. One major fibronectin mRNA was detected in whole liver and hepatocytes by northern hybridization, however, five to six other bands were also detected in both samples.

Estimation of hydrogen sulfide removal efficiency with granulated coal ash applied to eutrophic marine sediment using a simplified simulation model.

Hydrogen sulfide generated in eutrophic marine sediment is harmful for living organisms. It is therefore necessary to remove hydrogen sulfide from the sediment to restore benthic ecosystems. Previous studies revealed that granulated coal ash, which is a by-product of coal thermal electric power stations, could remove and oxidize hydrogen sulfide. In this study, we propose a simplified simulation model to estimate the hydrogen sulfide removal efficiency of granulated coal ash. Hydrogen sulfide concentrations in eutrophic marine sediment pore water with and without the application of granulated coal ash were calculated by the proposed model, and the outputs were compared with semi-field or field observation data. The model outputs reproduced the observed data well. Using the proposed model outputs, we suggest an optimum application dosage of granulated coal ash for remediating eutrophic marine sediment.

Establishment of a methodology for identifying Paeoniae Radix based on metallomic analysis.

We aimed to establish a methodology for identifying Paeonia samples based on metallomic analysis. We prepared 66 batches of samples (16 batches of crude drugs and 50 batches of cultivars, comprising 64 batches of Paeonia lactiflora and 2 batches of P. veitchii) collected from Japan and China (Inner Mongolia and elsewhere) between 1996 and 2008. P. lactiflora samples were genetically classified into white peony root (WPR) type and red peony root (RPR) type. Up to 47 elements were measured by inductively coupled plasma-mass spectrometry, and RPR type crude drug samples contained up to five times as much calcium as the others. Principal component analysis (PCA) of the multi-element fingerprints obtained suggested that P. veitchii, which grows wild, were distinguishable from the other cultivated P. lactiflora samples. This was confirmed perfectly by soft independent modeling of class analogy (SIMCA). The PCA of the fingerprints of P. lactiflora crude drug samples also suggested that it was possible to classify them by production area (Japan, Inner Mongolia, and China excluding Inner Mongolia) and genetic type (RPR and WPR types). They were also classified 100 % to the predicted class by SIMCA in both cases. These analyses were successful among the samples whose collection dates varied. This simple metallomic method is an efficient approach for verifying the complex origin of Paeoniae Radix.

Imaging plant leaves to determine changes in radioactive contamination status in Fukushima, Japan.

The chemical composition of plant leaves often reflects environmental contamination. The authors analyzed images of plant leaves to investigate the regional radioactivity ecology resulting from the 2011 accident at the Fukushima No. 1 nuclear power plant, Japan. The present study is not an evaluation of the macro radiation dose per weight, which has been performed previously, but rather an image analysis of the radioactive dose per leaf, allowing the capture of various gradual changes in radioactive contamination as a function of elapsed time. In addition, the leaf analysis method has potential applications in the decontamination of food plants or other materials.

Application of terahertz absorption spectroscopy to evaluation of aging variation of medicine.

The absorption spectra of three kinds of medicines both before and after the expiration date: Amlodin OD(®) (5 mg), Basen OD(®) (0.2 mg) and Gaster D(®) (10 mg) have been measured by terahertz time domain spectroscopy (THz-TDS). All the medicines show some differences in the THz absorption spectra between medicines before and after the expiration dates. X-Ray powder diffraction (XRD) studies of all medicines suggest that the polymorph of the main effective compound is not changed before and after the expiration date. Therefore, the differences in the THz spectra between medicines before and after the expiration dates arise from aging variation of diluting agents and/or from modifications of intermolecular interaction between the effective compounds and diluting agents.

The relation between clinical effects of Tokishakuyakusan and the identity of Paeonia lactiflora materials.

AIM OF THE STUDY: To investigate the relation between the clinical effects and the quality of crude drugs, we focused on Tokishakuyakusan (TS), consisted of 6 crude drugs. MATERIALS AND METHODS: We prepared two kinds of TS containing either medicinal cultivar of Paeonia lactiflora (MTS) or ornamental one (OTS). Other components were the same. First, we assessed the clinical effects of two TS formulations by cross-over study among the anemia patients. Second, we investigated the chemical differences between them by using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Mössbauer analysis. RESULTS: The clinical effects of these formulations (3 g/day for 8 weeks) were tested in the cross-over study consisted of 12 women patients who were diagnosed as having anemia (Hb ≤ 11 g/dl) and consented to participate to this study. Both TS formulations were effective for anemia symptoms as shown by the improvement of several hematological parameters, whereas their comprehensive effects were distinguishable by Genetic Algorithm Partial Least Squares (GA-PLS) analysis. There were no significant differences in organic ingredients and Fe content measured by ultra performance liquid chromatography (UPLC) and ICP-MS, respectively. Interestingly, Mössbauer spectra of Fe ion were remarkably different between two formulations. Fe ion in MTS was only one form, but that in OTS was at least two forms. CONCLUSIONS: This study suggested that clinical effects of TS formulation reflect the quality of Paeoniae Radix.

Terahertz absorption spectra of original and generic ceftazidime.

The absorption spectra of ceftazidime and its generic versions (Modacin, Mosyl, and Mobenzocin) have been measured by terahertz time domain spectroscopy (THz-TDS). Differences in the THz absorption were observed between the original and generic versions. The results show small, but significant differences in the states of ceftazidime hydrate between the original and generic versions. THz-TDS can be used to evaluate the stability of medicines as well as to control their quality.

Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) arising in the small intestine with monoclonal cryoglobulinemia.

A case of small intestinal extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) with monoclonal cryoglobulinemia is described. The patient was a woman in her mid-sixties with purpura of the bilateral lower legs and abdominal pain. An immunoserological investigation showed expression of IgM-kappa type monoclonal cryoglobulin. A renal biopsy specimen revealed proliferative glomerulonephritis with cryoglobulin deposition. Physical examination disclosed a stenosis, edematous changes and ascariasis in the small intestine. In aspiration cytology of the ascites, proliferation of the atypical lymphoid cells with plasmacytoid differentiation was observed. These cells were positive for B-lineage antigens in immunocytochemistry, and showed an immunoglobulin heavy-chain gene rearrangement in Southern blotting and chromosomal alteration in G-banded karyotype analysis. Although medicinal treatment was used, the patient died of general prostration. The diagnosis of intestinal MALT lymphoma was made at autopsy. Expression of API2-MALT1 fusion transcripts was detected by reverse transcription-polymerase chain reaction analysis using formalin-fixed, paraffin-embedded tissue. Intestinal MALT lymphomas with API2-MALT1 expression have distinctive forms of infiltration compared with those without translocation. Therefore, detection of API2-MALT1 fusion transcripts is useful for evaluating the prognosis and clinical behavior of the disease.