40 results on '"Nagakubo Y"'
Search Results
2. Development of MO imaging plate for MO color imaging
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Nagakubo, Y., primary, Baba, Y., additional, Liu, Q., additional, Lou, G., additional, and Ishibashi, T., additional
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- 2017
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3. Development of a cryo-FIB technique for the 3D structural characterization of liquid samples
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Tsuchiya, M, primary, Iwahori, T, additional, Morikawa, A, additional, Sato, T, additional, and Nagakubo, Y, additional
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- 2015
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4. In-situ SEM/TEM observation of platinum catalysts on carbon support in a gaseous atmosphere using a 300 kV CFE TEM/SEM
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Matsumoto, H., primary, Sato, T., additional, Nakano, K., additional, Yaguchi, T., additional, Nagaoki, I., additional, and Nagakubo, Y., additional
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- 2013
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5. Development of a high temperature-atmospheric pressure environmental cell for high-resolution TEM
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Yaguchi, T., primary, Suzuki, M., additional, Watabe, A., additional, Nagakubo, Y., additional, Ueda, K., additional, and Kamino, T., additional
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- 2011
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6. Three-Dimensional Evaluation of a Multi-Walled Carbon Nanotube Probe by Using High-Resolution Transmission Electron Microscope
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Tanigaki, T, primary, Nagakubo, Y, additional, and Hidaka, K, additional
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- 2010
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7. An Analysis of Li Ion Secondary Battery Materials by Using Focused Ion Beam Micro-sampling Technique and Cold Field Emission Scanning Transmission Electron Microscope
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Tanigaki, T, primary, Ito, K, additional, Nakamura, K, additional, Nagakubo, Y, additional, Azuma, J, additional, and Kanemura, T, additional
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- 2010
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8. Development of an environmental cell for the H-9500 in-situ TEM and its application
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Yaguchi, T, primary, Watabe, A, additional, Nagakubo, Y, additional, Ueda, K, additional, Fukui, M, additional, Kamino, T, additional, and Kawaski, T, additional
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- 2010
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9. An in situ heating TEM analysis method for an interface reaction
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Tanigaki, T., primary, Ito, K., additional, Nagakubo, Y., additional, Asakawa, T., additional, and Kanemura, T., additional
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- 2009
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10. Development of a Multifunctional Specimen Heating Holder and its Application
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Kamino, T, primary, Yaguchi, T, additional, Nagakubo, Y, additional, Ukiana, M, additional, and Watabe, A, additional
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- 2007
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11. Deep trench isolated CMOS devices.
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Rung, R.D., Momose, H., and Nagakubo, Y.
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- 1982
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12. Fast Programmable 256K Read Only Memory with On-Chip Test Circuits.
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Atsumi, S., Tanaka, S., Shinada, K., Yoshikawa, K., Makita, K., Nagakubo, Y., and Kanzaki, K.
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- 1985
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13. A programmable 256K CMOS EPROM with on-chip test circuits
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Tanaka, S., primary, Atsumi, S., additional, Momodomi, M., additional, Shinada, K., additional, Yoshikawa, K., additional, Nagakubo, Y., additional, and Kanzaki, K., additional
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- 1984
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14. Fast programmable 256K read only memory with on-chip test circuits
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Atsumi, S., primary, Tanaka, S., additional, Shinada, K., additional, Yoshikawa, K., additional, Makita, K., additional, Nagakubo, Y., additional, and Kanzaki, K., additional
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- 1985
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15. Comparison of diagnostic performance between Oncomine Dx target test and AmoyDx panel for detecting actionable mutations in lung cancer.
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Nagakubo Y, Hirotsu Y, Yoshino M, Amemiya K, Saito R, Kakizaki Y, Tsutsui T, Miyashita Y, Goto T, and Omata M
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- Humans, Female, Male, ErbB Receptors genetics, Middle Aged, Proto-Oncogene Proteins c-ret genetics, Biomarkers, Tumor genetics, Aged, Proto-Oncogene Proteins c-met genetics, Protein-Tyrosine Kinases genetics, Proto-Oncogene Proteins genetics, Multiplex Polymerase Chain Reaction methods, Lung Neoplasms genetics, Lung Neoplasms diagnosis, Mutation, High-Throughput Nucleotide Sequencing methods
- Abstract
Companion diagnostic (CDx) tests play important roles in identifying oncogenic driver genes and tailoring effective molecularly targeted therapies for lung cancer patients. In Japan, the Oncomine Dx target test (ODxTT) and the AmoyDx pan lung cancer PCR panel (AmoyDx) are prominent CDx tests and only one of these tests is covered by the domestic insurance system. However, these CDx tests cover different target regions and apply different technologies (ODxTT is amplicon-based next-generation sequencing and AmoyDx is multiplex PCR-based assay), which may lead to missing of actionable mutations affecting patient prognosis. Here, we performed a direct comparison analysis of 1059 genetic alterations of eight driver genes from 131 samples and evaluated the concordance between two CDx tests for detecting actionable variants and fusions. When excluding the eight uncovered variants (ODxTT: two variants, AmoyDx: six variants), the overall percent agreement was 97.6% (1026/1051) with 89.0% of overall positive percent agreement (89/100) and 98.5% of overall negative percent agreement (937/951). Of the 25 discordant genetic alterations, two were undetected despite being covered in the AmoyDx (one EGFR variant and one ROS1 fusion). Furthermore, there were potential false positives in the ODxTT (nine MET exon 14 skippings) and in the AmoyDx (five variants, six ROS1 and three RET fusions). These potential false positives in the AmoyDx likely due to non-specific amplification, which was validated by the unique molecular barcoding sequencing. The ODxTT missed two uncovered EGFR rare variants, which was visually confirmed in the raw sequencing data. Our study provides insights into real-world performance of CDx tests for lung cancer and ensures reliability to advance precision medicine., (© 2024. The Author(s).)
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- 2024
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16. Brief Report: Tepotinib as a Treatment Option in MET Exon 14 Skipping-Positive Lung Cancers-Investigating Discordance Between ArcherMET and the Oncomine Dx Target Test.
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Miyashita Y, Hirotsu Y, Nagakubo Y, Kobayashi H, Kawaguchi M, Hata K, Saito R, Kakizaki Y, Tsutsui T, Oyama T, and Omata M
- Abstract
Introduction: NSCLC is a leading cause of cancer-related mortality worldwide. Specific genetic alterations, such as MET exon 14 ( MET ex14) skipping, have been identified in NSCLC, allowing targeted therapy. Tepotinib, a highly selective MET inhibitor, has displayed promise in patients with advanced NSCLC. Nevertheless, challenges arise when identifying treatment strategies for patients with discordant results regarding MET ex14 skipping detection between diagnostic tests., Methods: We investigated patients with NSCLC and discordant results for MET ex14 skipping between the Oncomine Dx Target Test (ODxTT) and ArcherMET. Clinical response, adverse events, and the duration of tepotinib treatment were assessed, and statistical analysis was performed., Results: Among the 19 patients deemed MET ex14 skipping positive by ODxTT, only 10 had concordant results with ArcherMET. The number of MET ex14 skipping reads detected by ODxTT was significantly lower in discordant cases. Of the 19 patients, 14 received tepotinib, and comparable response and disease control rates were observed in both concordant and discordant cases. The duration of treatment did not significantly differ between the two groups., Conclusions: Our findings suggest that tepotinib has comparable therapeutic effects in patients with MET ex14 skipping-positive NSCLC irrespective of the concordance of results between ODxTT and ArcherMET. Tepotinib is a possible treatment option for patients with MET ex14 skipping, even in patients with discordant test results., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)
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- 2024
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17. Changes in Viral Dynamics Following the Legal Relaxation of COVID-19 Mitigation Measures in Japan From Children to Adults: A Single Center Study, 2020-2023.
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Hirotsu Y, Nagakubo Y, Maejima M, Shibusawa M, Hosaka K, Sueki H, Mochizuki H, and Omata M
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- Child, Adult, Humans, Middle Aged, Aged, Japan epidemiology, Pandemics prevention & control, Rhinovirus, SARS-CoV-2, COVID-19 epidemiology, COVID-19 prevention & control, Viruses, Respiratory Tract Infections epidemiology, Respiratory Tract Infections prevention & control, Paramyxoviridae Infections epidemiology
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Introduction: Respiratory infections are an ongoing global health challenge. The COVID-19 pandemic triggered global nonpharmacological measures that reshaped public health. In Japan, the shift from legal to individual discretion in pandemic management started on May 8, 2023. However, it still unknown how the relaxation of measures affects respiratory pathogens across age groups., Methods: We collected 16,946 samples from 13,526 patients between February 2020 and September 2023, analyzing the circulating respiratory pathogen dynamics using FilmArray respiratory panel., Results: Our analysis revealed significant increases in the positivity rates of respiratory pathogens across multiple age groups after relaxation. The pathogens including adenovirus, Bordetella pertussis, parainfluenza 2 and parainfluenza 4 showed increased positivity predominantly in children aged under 10 years. Conversely, some pathogens including human metapneumovirus, rhinovirus/enterovirus, and respiratory virus (RSV) increased in broad range of age groups. SARS-CoV-2 positivity rates decreased in children under 10 years but increased in those aged over 60 years., Discussion: Age-stratified analysis reveals a dynamic pattern of circulating pathogen in each age group after relaxation measures. This study provides essential epidemiologic data that can guide strategies to protect different age groups and effectively respond to respiratory infections in post-COVID-19 era., (© 2024 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)
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- 2024
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18. Simulation analysis of EGFR mutation detection: Oncomine Dx target test and AmoyDx panel impact on lung cancer treatment decisions.
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Hirotsu Y, Nakagomi T, Nagakubo Y, Goto T, and Omata M
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- Humans, Mutation, Exons, ErbB Receptors genetics, ErbB Receptors therapeutic use, Lung Neoplasms drug therapy, Lung Neoplasms genetics, Lung Neoplasms diagnosis, Carcinoma, Non-Small-Cell Lung drug therapy
- Abstract
Lung cancer is a leading cause of cancer-related deaths worldwide. Epidermal growth factor receptor (EGFR) driver mutations are crucial for treatment decisions for patients with non-small cell lung cancer (NSCLC). This study aimed to assess the differences in EGFR mutation detection between two companion diagnostic (CDx) tests-the Oncomine Dx Target Test (ODxTT) and the AmoyDx Pan Lung Cancer PCR Panel-and their impact on treatment applicability. To this end, we used an in-house targeted sequencing dataset of 282 samples from 127 EGFR-mutated NSCLC patients to simulate the concordance between the EGFR variants targeted by the ODxTT and AmoyDx panel, the oncogenicity of the variants, and their therapeutic potential. Of the 216 EGFR mutations identified by the in-house panel, 51% were detectable by both CDx tests, 3% were specific to ODxTT, and 46% were not targeted by either test. Most non-targeted mutations did not have oncogenicity and were located outside exons 18-21. Notably, 95% of the mutations detectable by both tests had potential oncogenicity. Furthermore, among the 96 patients harboring actionable EGFR mutations, 97% had mutations detectable by both CDx tests and 1% by ODxTT, while 2% had mutations not covered by either test. These findings suggest that while both CDx tests are effective in detecting almost all actionable EGFR mutations, ODxTT provides slightly broader coverage. These results emphasize the importance of selecting appropriate CDx tests to inform treatment decisions for EGFR-positive NSCLC patients., (© 2024. The Author(s).)
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- 2024
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19. Influence of formalin fixation duration on RNA quality and quantity from formalin-fixed paraffin-embedded hepatocellular carcinoma tissues.
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Amemiya K, Hirotsu Y, Nagakubo Y, Mochizuki H, Oyama T, and Omata M
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- Humans, Formaldehyde, Tissue Fixation methods, RNA, Paraffin Embedding methods, Carcinoma, Hepatocellular genetics, Liver Neoplasms genetics
- Abstract
Analyzing RNA samples from formalin-fixed paraffin-embedded (FFPE) tissues is essential for precision medicine. We investigated RNA quantity and quality from FFPE tumor tissues fixed in formalin for various times and compared sequencing metrics from next-generation sequencing (NGS). Hepatocellular carcinoma (HCC) tissues were fixed in 10% neutral buffered formalin (1-240 h) and FFPE blocks were prepared. Total RNA was extracted, and the quantity and quality were assessed using the NanoDrop, Qubit and Bioanalyzer. After preparing sequencing libraries, NGS was performed on the Oncomine Dx Multi-CDx system. Total RNA yields of all samples met the threshold required for NGS, but longer fixation times resulted in decreased total RNA and long RNA fragment (>200 nt) yields. NGS analysis showed fewer sequencing reads of internal control genes from RNA with longer fixation times. RNA extracted from FFPE blocks stored for 500 days had reduced RNA yield and quality compared with RNA obtained from FFPE blocks prepared immediately. In conclusion, short and over-fixation should be avoided because of their negative impact on sequencing quality. Fixation process should be finished promptly within recommended guidelines (6-72 h) for cancer patients., (© 2023 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)
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- 2023
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20. Nucleic Acid Quality Assessment is Critical to the Success of the Oncomine Dx Target Test for Lung Cancer.
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Nagakubo Y, Hirotsu Y, Amemiya K, Mochizuki H, Tsutsui T, Kakizaki Y, Miyashita Y, Higuchi R, Nakagomi T, Goto T, Oyama T, and Omata M
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- Humans, RNA, DNA, Nucleic Acids, Lung Neoplasms diagnosis, Lung Neoplasms genetics, Lung Neoplasms pathology, Adenocarcinoma of Lung diagnosis, Adenocarcinoma of Lung genetics
- Abstract
Background and Objective: The Oncomine Dx Target Test (ODxTT) has been used as a companion diagnostic test for lung cancer. Here, we evaluated whether the amount of nucleic acid and the degree of RNA degradation are related to the success of the ODxTT., Methods: This study included 223 samples from 218 patients with lung cancer. For all samples, DNA and RNA concentrations were quantified using Qubit, and the degree of RNA degradation was evaluated using the Bioanalyzer., Results: Of the 223 samples, 219 samples were successfully analyzed in the ODxTT and four were not. DNA analysis failed in two samples, which were attributed to low DNA concentrations and both were cytology specimens. Meanwhile, RNA analysis failed in the other two samples. These samples had sufficient amounts of RNA, but it was highly degraded with DV200 (the percentage of RNA fragments > 200 base pairs) less than 30. Compared with RNA samples with DV200 ≥ 30, analysis of RNA with DV200 < 30 yielded significantly fewer reads for the internal control genes. This test showed actionable mutations were identified in 38% (83/218) of all patients and in 46.6% (76/163) of patients with lung adenocarcinoma., Conclusions: DNA concentration and degree of RNA degradation are key factors determining the success of diagnostic testing by the ODxTT., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)
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- 2023
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21. Impact of the FilmArray Rapid Multiplex PCR Assay on Clinical Outcomes of Patients with Bacteremia.
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Okamoto M, Maejima M, Goto T, Mikawa T, Hosaka K, Nagakubo Y, Hirotsu Y, Amemiya K, Sueki H, and Omata M
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Bacteremia is a serious disease with a reported mortality of 30%. Appropriate antibiotic use with a prompt blood culture can improve patient survival. However, when bacterial identification tests based on conventional biochemical properties are used, it takes 2 to 3 days from positive blood culture conversion to reporting the results, which makes early intervention difficult. Recently, FilmArray (FA) multiplex PCR panel for blood culture identification was introduced to the clinical setting. In this study, we investigated the clinical impact of the FA system on decision making for treating septic diseases and its association with patients' survival. Our hospital introduced the FA multiplex PCR panel in July 2018. In this study, blood-culture-positive cases submitted between January and October 2018 were unbiasedly included, and clinical outcomes before and after the introduction of FA were compared. The outcomes included (i) the duration of use of broad-spectrum antibiotics, (ii) the time until the start of anti-MRSA therapy to MRSA bacteremia, and (iii) sixty-day overall survival. In addition, multivariate analysis was used to identify prognostic factors. In the FA group, overall, 122 (87.8%) microorganisms were concordantly retrieved with the FA identification panel. The duration of ABPC/SBT use and the start-up time of anti-MRSA therapy to MRSA bacteremia were significantly shorter in the FA group. Sixty-day overall survival was significantly improved by utilizing FA compared with the control group. In addition, multivariate analysis identified Pitt score, Charlson score, and utilization of FA as prognostic factors. In conclusion, FA can lead to the prompt bacterial identification of bacteremia and its effective treatment, thus significantly improving survival in patients with bacteremia.
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- 2023
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22. Simple IHC reveals complex MMR alternations than PCR assays: Validation by LCM and next-generation sequencing.
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Amemiya K, Hirotsu Y, Nagakubo Y, Watanabe S, Amemiya S, Mochizuki H, Oyama T, Kondo T, and Omata M
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- Humans, DNA Mismatch Repair genetics, Microsatellite Instability, Immunohistochemistry, Polymerase Chain Reaction, Neoplasms, Colorectal Neoplasms pathology
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Evaluation of the status of mismatch repair (MMR) in tumors is crucial for determining the application of immune checkpoint inhibitors (ICIs). Conventional PCR (MSI-PCR) is the gold standard for confirming the MMR status. However, it requires visual confirmation and presents difficulties in determining MMR status. Immunohistochemistry (IHC) is a simple method and can confirming MMR protein expression in the whole tumor. We aim to investigate IHC is more suitable for evaluating MMR status in the tumor. We compared MSI-PCR and IHC by testing 319 samples from 284 patients across 14 cancer types. In discordant cases, we performed laser-capture microdissection and microsatellite instability assay by next-generation sequencing (MSI-NGS). The concordance rate between IHC and MSI-PCR testing was 98.1% (313/319). Two reasons for these discrepancies were ambiguous MSI-PCR results and heterogeneous MSI status within the tumor. Among six cases (1.9%), three were judged as MSI-H by MSI-PCR but with proficient MMR by IHC. The results of MSI-NGS revealed microsatellite stable in these three cases. The remaining three cases, two of three were MSI-H and one was MSS in whole tumor in MSI-PCR. IHC showed a "mosaic" pattern containing both proficient MMR and deficient MMR portions by IHC in all three cases. We performed microdissection and MSI-PCR and found intratumoral heterogeneity of MMR status. These results indicated the advantages of IHC and performed expanded samples (n = 1082) and two additional mosaic cases were identified. Our results clearly indicated that simple IHC is the best choice for determining MMR alterations in critical cases for ICIs treatment., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)
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- 2022
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23. Classification of Omicron BA.1, BA.1.1, and BA.2 sublineages by TaqMan assay consistent with whole genome analysis data.
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Hirotsu Y, Maejima M, Shibusawa M, Natori Y, Nagakubo Y, Hosaka K, Sueki H, Mochizuki H, Tsutsui T, Kakizaki Y, Miyashita Y, and Omata M
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- Humans, COVID-19, SARS-CoV-2 genetics
- Abstract
Objectives: Recently, the Omicron strain of SARS-CoV-2 has spread and replaced the previously dominant Delta strain. Several Omicron sublineages (BA.1, BA.1.1, and BA.2) have been identified, with in vitro and preclinical reports showing that the pathogenicity and therapeutic efficacy differs between BA.1 and BA.2. We sought to develop a TaqMan assay to identify these subvariants., Methods: A TaqMan assay was constructed for rapid identification and genotyping of Omicron sublineages with 171 samples. We analyzed three characteristic mutations of the spike gene, Δ69-70, G339D, and Q493R, by TaqMan assay. The accuracy of the TaqMan assay was examined by comparing its results with the results of whole genome sequencing (WGS) analysis., Results: A total of 171 SARS-CoV-2 positive samples were analyzed by WGS and TaqMan assay. The 127 samples determined as BA.1/BA.1.1 by WGS were all positive for Δ69-70, G339D and Q493R by TaqMan assay. A total of 42 samples, determined as BA.2 by WGS, were negative for Δ69-70 but positive for G339D and Q493R by TaqMan. Two samples with G339N were determined to be inconclusive by the TaqMan method. Except for these two samples, the concordance rate between WGS and the TaqMan assay was 100% (169/169)., Conclusion: TaqMan assays targeting characteristic mutations are useful for identification and discrimination of Omicron sublineages., Competing Interests: Conflict of interest The authors have no competing interests to declare., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
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- 2022
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24. SARS-CoV-2 Omicron sublineage BA.2 replaces BA.1.1: Genomic surveillance in Japan from September 2021 to March 2022.
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Hirotsu Y, Maejima M, Shibusawa M, Natori Y, Nagakubo Y, Hosaka K, Sueki H, Mochizuki H, Tsutsui T, Kakizaki Y, Miyashita Y, and Omata M
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- Genome, Viral, Genomics, Humans, Japan epidemiology, COVID-19, SARS-CoV-2 genetics
- Abstract
Competing Interests: Declaration of Competing Interest None.
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- 2022
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25. Direct comparison of Xpert Xpress, FilmArray Respiratory Panel, Lumipulse antigen test, and RT-qPCR in 165 nasopharyngeal swabs.
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Hirotsu Y, Maejima M, Shibusawa M, Natori Y, Nagakubo Y, Hosaka K, Sueki H, Amemiya K, Hayakawa M, Mochizuki H, Tsutsui T, Kakizaki Y, Miyashita Y, and Omata M
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- COVID-19 Testing, Humans, Nasopharynx, SARS-CoV-2, Sensitivity and Specificity, COVID-19 diagnosis
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Background: The nucleic acid amplification test (NAAT) and antigen test are approved diagnostic tests for COVID-19. In this study, we aimed to investigate the assay performance of two NAATs (Xpert Xpress SARS-CoV-2 and FilmArray Respiratory Panel) and a quantitative antigen test (Lumipulse)., Methods: One hundred and sixty-five nasopharyngeal swabs were subjected to Xpert, FilmArray, Lumipulse, and RT-qPCR assays., Results: Of 165 samples, RT-qPCR showed 100 positives and 65 negatives. The Xpert had an overall agreement of 99.4% (95% confidence interval [CI]: 96.7-99.4%), sensitivity of 99% (95% CI: 96.8-99%), and specificity of 100% (95% CI: 96.6-100%). FilmArray had an overall agreement of 98.8% (95% CI: 95.9-98.8%), sensitivity of 98% (95% CI: 95.6-98%), and specificity of 100% (95% CI: 96.3-100%). Lumipulse had an overall agreement of 95.5% (95% CI: 91.8-95.5%), sensitivity of 92.3% (95% CI: 89.2-92.3%), and specificity of 100% (95% CI: 95.5-100%). The κ coefficient showed excellent agreement between each test and RT-qPCR. There was a high correlation between Xpert Ct values, RT-qPCR Ct values, viral loads and antigen level., Conclusions: Xpert Xpress and FilmArray Respiratory Panel exhibited an equivalent performance. The Lumipulse antigen test was slightly less sensitive than the NAATs, but showed high assay performance except for samples with low viral load. The Xpert Xpress, FilmArray Respiratory Panel and Lumipulse antigen tests offer rapid sample-to-answer data, allowing random access detection on automated devices., (© 2022. The Author(s).)
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- 2022
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26. Non-pharmaceutical interventions during the COVID-19 epidemic changed detection rates of other circulating respiratory pathogens in Japan.
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Nagakubo Y, Hirotsu Y, Maejima M, Shibusawa M, Hosaka K, Amemiya K, Sueki H, Hayakawa M, Mochizuki H, Tsutsui T, Kakizaki Y, Miyashita Y, and Omata M
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- Adenoviruses, Human classification, Adolescent, Adult, Aged, Aged, 80 and over, COVID-19 diagnosis, COVID-19 prevention & control, COVID-19 virology, Child, Child, Preschool, Enterovirus classification, Female, Hand Disinfection methods, Humans, Infant, Infant, Newborn, Japan epidemiology, Male, Masks supply & distribution, Middle Aged, Multiplex Polymerase Chain Reaction, Nasopharynx virology, Prevalence, Quarantine organization & administration, Respiratory Tract Infections diagnosis, Respiratory Tract Infections prevention & control, Respiratory Tract Infections virology, Rhinovirus classification, SARS-CoV-2 pathogenicity, Adenoviruses, Human genetics, COVID-19 epidemiology, Enterovirus genetics, Respiratory Tract Infections epidemiology, Rhinovirus genetics, SARS-CoV-2 genetics
- Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has circulated worldwide and causes coronavirus disease 2019 (COVID-19). At the onset of the COVID-19 pandemic, infection control measures were taken, such as hand washing, mask wearing, and behavioral restrictions. However, it is not fully clear how the effects of these non-pharmaceutical interventions changed the prevalence of other pathogens associated with respiratory infections. In this study, we collected 3,508 nasopharyngeal swab samples from 3,249 patients who visited the Yamanashi Central Hospital in Japan from March 1, 2020 to February 28, 2021. We performed multiplex polymerase chain reaction (PCR) using the FilmArray Respiratory Panel and singleplex quantitative reverse transcription PCR targeting SARS-CoV-2 to detect respiratory disease-associated pathogens. At least one pathogen was detected in 246 (7.0%) of the 3,508 samples. Eleven types of pathogens were detected in the samples collected from March-May 2020, during which non-pharmaceutical interventions were not well implemented. In contrast, after non-pharmaceutical interventions were thoroughly implemented, only five types of pathogens were detected, and the majority were SARS-CoV-2, adenoviruses, or human rhinoviruses / enteroviruses. The 0-9 year age group had a higher prevalence of infection with adenoviruses and human rhinoviruses / enteroviruses compared with those 10 years and older, while those 10 years and older had a higher prevalence of infection with SARS-CoV-2 and other pathogens. These results indicated that non-pharmaceutical interventions likely reduced the diversity of circulating pathogens. Moreover, differences in the prevalence of pathogens were observed among the different age groups., Competing Interests: The authors have declared that no competing interests exist.
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- 2022
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27. A cryo-TSEM with temperature cycling capability allows deep sublimation of ice to uncover fine structures in thick cells.
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Usukura J, Narita A, Matsumoto T, Usukura E, Sunaoshi T, Watanabe S, Tamba Y, Nagakubo Y, Mizuo T, Azuma J, Osumi M, Nimura K, Tamochi R, and Ose Y
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- Capsid ultrastructure, Cell Membrane ultrastructure, Cytoskeleton, Equipment Design, Frozen Sections, Ice, Image Processing, Computer-Assisted, Ribosomes ultrastructure, Temperature, Tobacco Mosaic Virus ultrastructure, Cryoelectron Microscopy methods, Endoplasmic Reticulum ultrastructure, Microscopy, Electron, Transmission methods
- Abstract
The scanning electron microscope (SEM) has been reassembled into a new type of cryo-electron microscope (cryo-TSEM) by installing a new cryo-transfer holder and anti-contamination trap, which allowed simultaneous acquisition of both transmission images (STEM images) and surface images (SEM images) in the frozen state. The ultimate temperatures of the holder and the trap reached - 190 °C and - 210 °C, respectively, by applying a liquid nitrogen slush. The STEM images at 30 kV were comparable to, or superior to, the images acquired with conventional transmission electron microscope (100 kV TEM) in contrast and sharpness. The unroofing method was used to observe membrane cytoskeletons instead of the frozen section and the FIB methods. Deep sublimation of ice surrounding unroofed cells by regulating temperature enabled to emerge intracellular fine structures in thick frozen cells. Hence, fine structures in the vicinity of the cell membrane such as the cytoskeleton, polyribosome chains and endoplasmic reticulum (ER) became visible. The ER was distributed as a wide, flat structure beneath the cell membrane, forming a large spatial network with tubular ER., (© 2021. The Author(s).)
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- 2021
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28. The dynamic change of antibody index against Covid-19 is a powerful diagnostic tool for the early phase of the infection and salvage PCR assay errors.
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Omata M, Hirotsu Y, Sugiura H, Maejima M, Nagakubo Y, Amemiya K, Hayakawa M, Tsutsui T, Kakizaki Y, Mochizuki H, and Miyashita Y
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- Adult, Africa, Aged, Aged, 80 and over, Antibodies, COVID-19 Testing methods, Disease Outbreaks, Early Diagnosis, Female, Humans, Japan, Male, Middle Aged, Ships, Young Adult, Antibodies, Viral, COVID-19 diagnosis, COVID-19 immunology, COVID-19 Serological Testing methods, Diagnostic Errors, Polymerase Chain Reaction methods, SARS-CoV-2 isolation & purification
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Background: Currently, PCR assay is a golden standard for diagnosis of Covid-19. However, it needs nasopharyngeal swabs, expensive instruments and expertise. It even causes PCR errors., Methods: We validated the antibody assay (Roche) in 36 followed patients and 1879 controls (medical staffs)., Results: Of 1879 medical staffs, only two (0.11%) were positive by Cut off Index (COI; 1.0) (mean ± SD, 0.094 ± 0.047). Thirty six patients were composed of three groups; Group A,4 from Diamond Princess cruise ship, Group B, 2 infected in Africa, and Group C, 30 infected in Japan. PCR assays were conducted at outside laboratories before and repeated in house after hospitalized. Of 36 at admission, positive antibody was seen in 4/4 from the ship, 0/2 from Africa, and 5/30 from Japan. Two from Africa showed the increase of COI and became positive on days 8 and 13. Thirty Japanese was divided in two groups, e.g., 23 showed dynamic increase of COI up to 84.4 within 3 days while active virus replication present (Group C). In remaining 7 (7/30, 23%) (Group C'), no rise of antibody nor positive in house PCR assays, indicative of false positive results of PCR at the beginning., Conclusion: This antibody testing has a wide dynamic ranges of COI and, thus, could be utilized in the early infection phase. This may also compliment and even help to avoid possible PCR errors. Therefore, this can serve as a powerful diagnostic tool, needed in the frontline of the clinic and hospitals., Competing Interests: Declaration of competing interest None., (Copyright © 2021. Published by Elsevier B.V.)
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- 2021
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29. Actionable driver DNA variants and fusion genes can be detected in archived cytological specimens with the Oncomine Dx Target Test Multi-CDx system in lung cancer.
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Amemiya K, Hirotsu Y, Nagakubo Y, Mochizuki H, Higuchi R, Tsutsui T, Kakizaki Y, Miyashita, Oyama T, and Omata M
- Subjects
- Diagnostic Tests, Routine, ErbB Receptors, Gene Fusion, Humans, Mutation, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins B-raf, RNA, Neoplasm genetics, DNA, Neoplasm genetics, Lung Neoplasms genetics
- Abstract
Background: Molecular testing is critical for identifying actionable variants in lung cancer for precision medicine. When tumor tissue samples are unavailable, archived cytological specimens (ACSs) can be used. The authors examined whether oncogenic variants could be accurately detected in ACSs versus paired formalin-fixed, paraffin-embedded (FFPE) tumor tissues with in vitro diagnostic tests., Methods: The authors collected 18 ACSs and 15 FFPE tissues from 15 patients with lung cancer and investigated genomic profiles with the Oncomine Dx Target Test Multi-CDx system, which is an integrated next-generation sequencing platform that comprehensively examines 4 companion diagnostic target genes (epidermal growth factor receptor [EGFR]; B-Raf proto-oncogene, serine/threonine kinase [BRAF]; anaplastic lymphoma kinase [ALK]; and ROS proto-oncogene 1, receptor tyrosine kinase [ROS1]). They compared the quantity and quality of extracted nucleic acids, the sequencing quality control (QC), and the detected variants between ACSs and FFPE tissues., Results: The total amount of DNA and RNA obtained from 1 slide was higher in FFPE tissues than ACSs. The RNA integrity number was higher in ACSs. There were no differences in sequencing QC between ACSs and FFPE tissues. A total of 21 variants, including EGFR mutations and ALK and ROS1 fusion genes, were detected in both ACSs and FFPE tissues with 100% concordance., Conclusions: ACSs can be a feasible alternative with which to identify actionable mutations and fusion genes via the Oncomine Dx Target Test Multi-CDx system., (© 2021 American Cancer Society.)
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- 2021
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30. Prospective study of 1308 nasopharyngeal swabs from 1033 patients using the LUMIPULSE SARS-CoV-2 antigen test: Comparison with RT-qPCR.
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Hirotsu Y, Maejima M, Shibusawa M, Amemiya K, Nagakubo Y, Hosaka K, Sueki H, Hayakawa M, Mochizuki H, Tsutsui T, Kakizaki Y, Miyashita Y, and Omata M
- Subjects
- Humans, Prospective Studies, Retrospective Studies, COVID-19 diagnosis, COVID-19 Nucleic Acid Testing, COVID-19 Serological Testing, Nasopharynx virology, SARS-CoV-2 immunology
- Abstract
Background: Reverse transcription polymerase chain reaction (RT-PCR) is the gold standard for detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Previously, the accuracy of the quantitative LUMIPULSE SARS-CoV-2 antigen test was demonstrated using samples collected retrospectively. In this study, the LUMIPULSE antigen test was clinically validated using prospective samples., Methods: In total, 1033 nasopharyngeal swab samples were collected from 1033 individuals, and an additional 275 follow-up samples were collected from 43 patients who subsequently tested positive for coronavirus disease 2019 (COVID-19). All 1308 samples were subjected to quantitative RT-PCR (RT-qPCR) and the antigen test. The antibody response was investigated for patients with discordant results to clarify if seroconversion had occurred., Results: RT-qPCR identified 990 samples as negative and 43 as positive, while the antigen test identified 992 samples as negative, 37 as positive and four as inconclusive. The overall concordance rate was 99.7% (1026/1029). Sensitivity, specificity, positive predictive value and negative predictive value of the antigen test were 92.5% (37/40), 100% (989/989), 100% (37/37) and 99.7% (989/992), respectively, after exclusion of the four inconclusive results. The kappa coefficient was 0.960 (95% confidence interval 0.892-0.960), suggesting excellent agreement between the two tests. Seropositivity in five of seven patients with discordant results suggested that the discrepancy was caused by samples collected during the late phase of infection. Using follow-up samples, correlation was observed between the antigen level and the viral load or cycle threshold value. The concordance rate between these test results tended to be high among samples collected 0-9 days after symptom onset, but this decreased gradually in samples collected thereafter., Conclusions: This prospective study demonstrated that the LUMIPULSE antigen test is a highly accurate diagnostic test for SARS-CoV-2., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
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- 2021
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31. Analysis of a persistent viral shedding patient infected with SARS-CoV-2 by RT-qPCR, FilmArray Respiratory Panel v2.1, and antigen detection.
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Hirotsu Y, Maejima M, Shibusawa M, Amemiya K, Nagakubo Y, Hosaka K, Sueki H, Hayakawa M, Mochizuki H, Tsutsui T, Kakizaki Y, Miyashita Y, and Omata M
- Subjects
- Antigens, Viral analysis, Humans, Multiplex Polymerase Chain Reaction methods, Nasopharynx virology, RNA, Viral analysis, Reverse Transcriptase Polymerase Chain Reaction methods, SARS-CoV-2 immunology, Sensitivity and Specificity, Viral Load, COVID-19 diagnosis, COVID-19 Testing methods, SARS-CoV-2 isolation & purification, Virus Shedding
- Abstract
Various diagnostic tests utilizing different principles are currently under development for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, these tests can occasionally produce discrepant results, causing confusion in their interpretation. Here, we evaluated the performance and features of three diagnostic assays: quantitative reverse transcription polymerase chain reaction (RT-qPCR), FilmArray Respiratory Panel (RP) v2.1, and the LUMIPULSE antigen test. Twenty-seven serial nasopharyngeal swabs were collected from a prolonged viral shedding patient who had been hospitalized for 51 days. We examined the SARS-CoV-2 detection rates of the three tests. The overall agreement rate was 81% between RT-qPCR and FilmArray RP v2.1, 63% between the antigen test and FilmArray RP v2.1, and 59% between the antigen test and RT-qPCR. We obtained concordant results in samples with high viral loads (low threshold cycle values) by all three tests. RT-qPCR and FilmArray RP v2.1 accurately detected SARS-CoV-2 at the early to intermediate phases of infection, but the results varied at the late phase. The antigen test also produced a positive result at the early phase but varied at the intermediate phase and consistently produced negative results at late phase of infection. These results demonstrated FilmArray RP v2.1 could detect SARS-CoV-2 with accuracy comparable to RT-qPCR. Further, there were discrepant results using different types of diagnostic tests during the clinical course of prolonged viral shedding patient. We provided insights into how to utilize different types of kits to assess and manage SARS-CoV-2 infections., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2021
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32. Pooling RT-qPCR testing for SARS-CoV-2 in 1000 individuals of healthy and infection-suspected patients.
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Hirotsu Y, Maejima M, Shibusawa M, Nagakubo Y, Hosaka K, Amemiya K, Sueki H, Hayakawa M, Mochizuki H, Tsutsui T, Kakizaki Y, Miyashita Y, and Omata M
- Subjects
- Betacoronavirus genetics, Betacoronavirus pathogenicity, COVID-19 Testing, Clinical Laboratory Techniques standards, Coronavirus Infections diagnosis, Coronavirus Infections epidemiology, Humans, Limit of Detection, Mass Screening standards, Reproducibility of Results, Respiratory Mucosa virology, Reverse Transcriptase Polymerase Chain Reaction standards, SARS-CoV-2, Clinical Laboratory Techniques methods, Mass Screening methods, Reverse Transcriptase Polymerase Chain Reaction methods
- Abstract
Severe acute respiratory coronavirus 2 (SARS-CoV-2) testing reagents are expected to become scarce worldwide. However, little is known regarding whether pooling of samples accurately detects SARS-CoV-2. To validate the feasibility of pooling samples, serial dilution analysis and spike-in experiments were conducted using synthetic DNA and nucleic acids extracted from SARS-CoV-2-positive and -negative patients. Furthermore, we studied 1000 individuals, 667 of whom were "healthy" individuals (195 healthcare workers and 472 hospitalized patients with disorders other than COVID-19 infection), and 333 infection-suspected patients with cough and fever. Serial dilution analysis showed a limit of detection of around 10-100 viral genome copies according to the protocol of the National Institute of Infectious Diseases, Japan. Spike-in experiments demonstrated that RT-qPCR detected positive signals in pooled samples with SARS-CoV-2-negative and -positive patients at 5-, 10-, 20-fold dilutions. By screening with this pooling strategy, by the end of April 2020 there were 12 SARS-CoV-2-positive patients in 333 infection-suspected patients (3.6%) and zero in 667 "healthy" controls. We obtained these results with a total of 538 runs using the pooling strategy, compared with 1000 standard runs. In a prospective study, we successfully detected SARS-CoV-2 using 10- to 20-fold diluted samples of nasopharyngeal swabs from eighteen COVID-19 patients with wide ranges of viral load. Pooling sample is feasible for conserving test reagents and detecting SARS-CoV-2 in clinical settings. This strategy will help us to research the prevalence infected individuals and provide infected-status information to prevent the spread of the virus and nosocomial transmission.
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- 2020
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33. Reply to 'Analysis of Covid-19 and non-Covid-19 viruses, including influenza viruses, to determine the influence of intensive preventive measures in Japan'.
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Hirotsu Y, Maejima M, Shibusawa M, Amemiya K, Nagakubo Y, Hosaka K, Sueki H, Mochizuki H, Tsutsui T, Kakizaki Y, Miyashita Y, and Omata M
- Subjects
- Betacoronavirus, COVID-19, Humans, Japan epidemiology, SARS-CoV-2, Coronavirus Infections epidemiology, Orthomyxoviridae, Pandemics, Pneumonia, Viral epidemiology
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- 2020
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34. Comparison of automated SARS-CoV-2 antigen test for COVID-19 infection with quantitative RT-PCR using 313 nasopharyngeal swabs, including from seven serially followed patients.
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Hirotsu Y, Maejima M, Shibusawa M, Nagakubo Y, Hosaka K, Amemiya K, Sueki H, Hayakawa M, Mochizuki H, Tsutsui T, Kakizaki Y, Miyashita Y, Yagi S, Kojima S, and Omata M
- Subjects
- Betacoronavirus, COVID-19, COVID-19 Testing, Humans, Immunoenzyme Techniques, Luminescent Measurements, Nasal Cavity virology, Pandemics, SARS-CoV-2, Sensitivity and Specificity, Viral Load, Clinical Laboratory Techniques methods, Coronavirus Infections diagnosis, Pneumonia, Viral diagnosis, Real-Time Polymerase Chain Reaction methods
- Abstract
In routine clinical practice, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is determined by reverse-transcription PCR (RT-PCR). In the current pandemic, a more rapid and high-throughput method is in growing demand. Here, we validated the performance of a new antigen test (LUMIPULSE) based on chemiluminescence enzyme immunoassay. A total of 313 nasopharyngeal swabs (82 serial samples from 7 infected patients and 231 individual samples from 4 infected patients and 215 uninfected individuals) were analyzed for SARS-CoV-2 with quantitative RT-PCR (RT-qPCR) and then subjected to LUMIPULSE. We determined the cutoff value for antigen detection using receiver operating characteristic curve analysis and compared the performance of the antigen test with that of RT-qPCR. We also compared the viral loads and antigen levels in serial samples from seven infected patients. Using RT-qPCR as the reference, the antigen test exhibited 55.2% sensitivity and 99.6% specificity, with a 91.4% overall agreement rate (286/313). In specimens with > 100 viral copies and between 10 and 100 copies, the antigen test showed 100% and 85% concordance with RT-qPCR, respectively. This concordance declined with lower viral loads. In the serially followed patients, the antigen levels showed a steady decline, along with viral clearance. This gradual decline was in contrast with the abrupt positive-to-negative and negative-to-positive status changes observed with RT-qPCR, particularly in the late phase of infection. In summary, the LUMIPULSE antigen test can rapidly identify SARS-CoV-2-infected individuals with moderate to high viral loads and may be helpful for monitoring viral clearance in hospitalized patients., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2020
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35. Analysis of Covid-19 and non-Covid-19 viruses, including influenza viruses, to determine the influence of intensive preventive measures in Japan.
- Author
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Hirotsu Y, Maejima M, Shibusawa M, Amemiya K, Nagakubo Y, Hosaka K, Sueki H, Mochizuki H, Tsutsui T, Kakizaki Y, Miyashita Y, and Omata M
- Subjects
- Alphacoronavirus isolation & purification, COVID-19, Coinfection virology, Humans, Incidence, Japan, Metapneumovirus isolation & purification, Multiplex Polymerase Chain Reaction, Nasopharynx virology, Orthomyxoviridae isolation & purification, Prospective Studies, Reverse Transcriptase Polymerase Chain Reaction, Rhinovirus isolation & purification, Coinfection epidemiology, Communicable Disease Control methods, Coronavirus Infections epidemiology, Coronavirus Infections prevention & control, Disease Transmission, Infectious prevention & control, Pandemics prevention & control, Pneumonia, Viral epidemiology, Pneumonia, Viral prevention & control, Respiratory Tract Infections epidemiology
- Abstract
Background: Severe acute respiratory coronavirus 2 (SARS-CoV-2) has spread and caused death worldwide. Preventive measures and infection control are underway, and some areas show signs of convergence. Other viruses in addition to SARS-CoV-2 cause cold-like symptoms and spread in the winter. However, the extent to which SARS-CoV-2, influenza viruses and other causative viruses have prevailed since implementing preventive measures is unclear., Objectives: We aim to investigate the incidence of causative viruses and pathogens in patients., Study Design: We collected 191 nasopharyngeal swabs from patients with cold-like symptoms in Japan. All samples were subjected to multiplex PCR with the FilmArray Respiratory Panel and reverse transcription PCR (RT-PCR) to detect SARS-CoV-2., Results: FilmArray Respiratory Panel analysis detected at least one virus in 32 of 191 patients with cold-like symptoms (21 %). Of these, we frequently identified human rhinoviruses/enteroviruses (5.8 %, n=11), human metapneumovirus (3.7 %, n=7), coronavirus 229E (2.1 %, n=4) and coronavirus OC43 (1.6 %, n=3); while no influenza viruses were detected. RT-PCR analysis detected SARS-CoV-2 (4.2 %, n=8) in patients who were not infected with the aforementioned respiratory viruses., Conclusions: Co-infection with SARS-CoV-2 and other viruses was not observed. Causative viruses remain prevalent after implementing preventive measures. SARS-CoV-2 differs from influenza viruses in its infectivity., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)
- Published
- 2020
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36. Genome analysis of peeling archival cytology samples detects driver mutations in lung cancer.
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Kunimasa K, Hirotsu Y, Amemiya K, Nagakubo Y, Goto T, Miyashita Y, Kakizaki Y, Tsutsui T, Otake S, Kobayashi H, Higuchi R, Inomata K, Kumagai T, Mochizuki H, Nakamura H, Nakatsuka SI, Nishino K, Imamura F, Kumagai T, Oyama T, and Omata M
- Subjects
- Adult, Aged, Aged, 80 and over, Alleles, Anaplastic Lymphoma Kinase analysis, Exome genetics, Female, Gene Expression Profiling methods, High-Throughput Nucleotide Sequencing methods, Humans, Lung Neoplasms pathology, Male, Middle Aged, Oncogene Fusion, Paraffin Embedding, Protein-Tyrosine Kinases genetics, Proto-Oncogene Proteins genetics, Genome, Human, Lung Neoplasms genetics, Mutation
- Abstract
Introductions: When tumor tissue samples are unavailable to search for actionable driver mutations, archival cytology samples can be useful. We investigate whether archival cytology samples can yield reliable genomic information compared to corresponding formalin-fixed paraffin-embedded (FFPE) tumor samples., Patients and Methods: Pretreatment class V archival cytology samples with adequate tumor cells were selected from 172 lung cancer patients. The genomic profiles of the primary lung tumors have been analyzed through whole-exome regions of 53 genes. We compared the genomic profiles based on the oncogenicity and variant allele frequency (VAF) between the archival cytology and the corresponding primary tumors. We also analyzed the genomic profiles of serial cytological samples during the treatment of EGFR-TKI., Results: A total of 43 patients were analyzed with the paired samples for DNA mutations and other three patients were analyzed for their fusion genes. A total of 672 mutations were detected. Of those, 106 mutations (15.8%) were shared with both samples. Sixty of seventy-seven (77.9%) shared mutations were oncogenic or likely oncogenic mutations with VAF ≧10%. As high as 90% (9/10) actionable driver mutations and ALK and ROS1 fusion genes were successfully detected from archival cytology samples. Sequential analysis revealed the dynamic changes in EGFR-TKI-resistant mutation (EGFR p.T790M) during the course of treatment., Conclusion: Archival cytology sample with adequate tumor cells can yield genetic information compared to the primary tumors. If tumor tissue samples are unavailable, we can use archival cytology samples to search for actionable driver mutations., (© 2020 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)
- Published
- 2020
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37. Measurement of complex refractive index with tunable extreme ultraviolet high harmonic source.
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Hirano D, Nagakubo Y, Omachi J, Yoshioka K, and Kuwata-Gonokami M
- Abstract
We report a broadband refractive index measurement method based on a higher harmonic generation tabletop coherent extreme ultraviolet source. We measured the complex refractive index of a sample material by measuring the interference pattern produced by a bare double slit and comparing this with the pattern produced by another double slit with one slit covered by the sample material. We validated the method by measuring the complex refractive index of aluminum in the photon energy range of 63-78 eV using a neon gas jet. The measurement system had errors of less than 0.02%.
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- 2020
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38. Microsatellite instability status is determined by targeted sequencing with MSIcall in 25 cancer types.
- Author
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Hirotsu Y, Nagakubo Y, Amemiya K, Oyama T, Mochizuki H, and Omata M
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- Algorithms, Cohort Studies, Computational Biology, Humans, Microsatellite Instability, Neoplasms pathology, Polymerase Chain Reaction, High-Throughput Nucleotide Sequencing, Neoplasms genetics
- Abstract
Background: Microsatellite instability (MSI) occurs in solid tumors and is a predictive biomarker for remarkable response to immune checkpoint inhibitors. Detection of MSI status has been conventionally conducted by PCR-electrophoresis-based assay (MSI-PCR) and immunohistochemistry (IHC) of mismatch repair proteins. However, these approaches require visual confirmation and involve some difficulties in determining MSI statuses from equivocal results., Methods: We performed amplicon-based targeted sequencing of 76 microsatellite loci (MSI-NGS) in 184 formalin-fixed paraffin-embedded (FFPE) tumor tissues and baseline control samples. A bioinformatics tool, MSIcall, was used to calculate the quantitative values based on the aligned sequence reads and evaluated MSI status. Furthermore, we examined the concordance between the results from MSI-NGS and MSI-PCR/IHC. Diagnostic accuracy, sensitivity, and specificity were estimated by receiver operating characteristic (ROC) curve analysis. For validation cohort, we studied additional 50 tumor samples to determine the MSI status., Results: Of 184 tumor samples, MSI-PCR and IHC analysis classified 161 tumors as MSS/pMMR and 23 as MSI-H/dMMR. Using MSI-NGS combined with MSIcall, we predicted MSI status with high accuracy (98.9%), specificity (91.3%), and sensitivity (100%) in 25 types of cancers. This method achieved an area under the ROC curve (AUC) value of 0.9986. Furthermore, we achieved the 100% concordant results using additional 50 samples for validation., Conclusion: We demonstrated newly developed MSI-NGS with MSIcall accurately determines the MSI status of FFPE tumor tissues thorough sequencing of tumor samples alone without patient-matched normal controls. This approach can be applied to all types of solid tumors to determine responders to immune-oncology therapy., Competing Interests: Declaration of Competing Interest The authors declare no potential conflicts of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)
- Published
- 2020
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39. Accurate detection of KRAS, NRAS and BRAF mutations in metastatic colorectal cancers by bridged nucleic acid-clamp real-time PCR.
- Author
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Nagakubo Y, Hirotsu Y, Amemiya K, Oyama T, Mochizuki H, and Omata M
- Subjects
- Colorectal Neoplasms pathology, DNA chemistry, DNA isolation & purification, DNA metabolism, High-Throughput Nucleotide Sequencing, Humans, Mutation, Neoplasm Metastasis, Sequence Analysis, DNA, Colorectal Neoplasms genetics, GTP Phosphohydrolases genetics, Membrane Proteins genetics, Proto-Oncogene Proteins B-raf genetics, Proto-Oncogene Proteins p21(ras) genetics, Real-Time Polymerase Chain Reaction methods
- Abstract
Background: Patients with metastatic colorectal cancer can benefit from anti-EGFR therapy, such as cetuximab and panitumumab. However, colorectal cancers harboring constitutive activating mutations in KRAS, NRAS and BRAF genes are not responsive to anti-EGFR therapy. To select patients for appropriate treatment, genetic testing of these three genes is routinely performed., Methods: We applied bridged nucleic acid-clamp real-time PCR (BNA-clamp PCR) to detect somatic hotspot mutations in KRAS, NRAS and BRAF. PCR products from BNA-clamp PCR were subsequently analyzed Sanger sequencing. We then compared results with those from the PCR-reverse sequence-specific oligonucleotide probe (PCR-rSSO) method, which has been used as in vitro diagnostic test in Japan. To validate the mutation status, we also performed next generation sequencing using all samples., Results: In 50 formalin-fixed paraffin-embedded tissues, KRAS mutations were detected at frequencies of 50% (25/50) and 52% (26/50) by PCR-rSSO and BNA-clamp PCR with Sanger sequencing, respectively, and NRAS mutations were detected at 12% (6/50) and 12% (6/50) by PCR-rSSO and BNA-clamp PCR with Sanger sequencing, respectively. The concordance rate for detection of KRAS and NRAS mutations between the two was 94% (47/50). However, there were three discordant results. We validated these three discordant and 47 concordant results by next generation sequencing. All mutations identified by BNA-clamp PCR with Sanger sequencing were also identified by next generation sequencing. BNA-clamp PCR detected BRAF mutations in 6% (3/50) of tumor samples., Conclusions: Our results indicate that BNA-clamp PCR with Sanger sequencing detects somatic mutations in KRAS, NRAS and BRAF with high accuracy.
- Published
- 2019
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40. Design of a 300-kV gas environmental transmission electron microscope equipped with a cold field emission gun.
- Author
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Isakozawa S, Nagaoki I, Watabe A, Nagakubo Y, Saito N, Matsumoto H, Zhang XF, Taniguchi Y, and Baba N
- Abstract
A new in situ environmental transmission electron microscope (ETEM) was developed based on a 300 kV TEM with a cold field emission gun (CFEG). Particular caution was taken in the ETEM design to assure uncompromised imaging and analytical performance of the TEM. Because of the improved pumping system between the gun and column, the vacuum of CFEG was largely improved and the probe current was sufficiently stabilized to operate without tip flashing for 2-3 h or longer. A high brightness of 2.5 × 10(9) A/cm(2) sr was measured at 300 kV, verifying the high quality of the CFEG electron beam. A specially designed gas injection-heating holder was used in the in situ TEM study at elevated temperatures with or without gas around the TEM specimen. Using this holder in a 10 Pa gas atmosphere and specimen temperatures up to 1000°C, high-resolution ETEM performance and analysis were achieved., (© The Author 2016. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2016
- Full Text
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