Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.scitotenv.2023.169334

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.scitotenv.2023.169375

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.scitotenv.2024.170921

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.scitotenv.2024.170845

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.scitotenv.2024.170764

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.scitotenv.2023.168453

DOI： 10.1016/j.scitotenv.2023.168453

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.scitotenv.2023.167844

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.scitotenv.2023.165926

Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.watres.2023.120644

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.scitotenv.2023.165229

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.scitotenv.2023.165270

Authorship：Lead author,　Last author,　Corresponding author   Language：Japanese   Publishing type：Research paper (research society, symposium materials, etc.)  

Authorship：Last author   Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.scitotenv.2023.164001

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.scitotenv.2023.163487

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.coesh.2023.100458

Other Link： https://doi.org/10.1016/j.coesh.2023.100458

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

DOI： 10.1016/j.scitotenv.2022.160952

Other Link： https://doi.org/10.1016/j.scitotenv.2022.160952

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

DOI： 10.3390/w15061018

Other Link： https://doi.org/10.3390/w15061018

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

Wastewater-based epidemiology (WBE) complements the clinical surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants' distribution in populations. Many developed nations have established na-tional and regional WBE systems; however, governance and budget constraints could be obstacles for low-and middle -income countries. An urgent need thus exists to identify hotspots to serve as sentinel sites for WBE. We hypothesized that representative wastewater treatment plants (WWTPs) in two international gateway cities, Bangkok and Phuket, Thailand, could be sentineled for SARS-CoV-2 and its variants to reflect the clinical distribution patterns at city level and serve as early indicators of new variants entering the country. Municipal wastewater samples (n = 132) were col-lected from eight representative municipal WWTPs in Bangkok and Phuket during 19 sampling events from October 2021 to March 2022, which were tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using the US CDC N1 and N2 multiplex and variant (Alpha, Delta, and Omicron BA.1 and BA.2) singleplex assays. The variant detection ratios from Bangkok and Phuket followed similar trends to the national clinical testing data, and each variant's viral loads agreed with the daily new cases (3-d moving average). Omicron BA.1 was detected in Phuket wastewater prior to Bangkok, possibly due to Phuket's WWTPs serving tourist communities. We found that the Omi-cron BA.1 and BA.2 viral loads predominantly drove the SARS-CoV-2 resurgence. We also noted a shifting pattern in the Bangkok WBE from a 22-d early warning in early 2021 to a near real-time pattern in late 2021. The potential application of tourist hotspots for WBE to indicate the arrival of new variants and re-emerging or unprecedented infec-tious agents could support tourism-dependent economies by complementing the reduced clinical regulations while maintaining public health protection via wastewater surveillance.

DOI： 10.1016/j.scitotenv.2022.160317

Web of Science

Other Link： https://doi.org/10.1016/j.scitotenv.2022.160317

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

DOI： 10.1016/j.scitotenv.2022.159166

Other Link： https://doi.org/10.1016/j.scitotenv.2022.159166

Language：English   Publishing type：Research paper (scientific journal)  

Wastewater-based epidemiology (WBE) has been considered as a promising approach for population-wide surveillance of coronavirus disease 2019 (COVID-19). Many studies have successfully quantified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentration in wastewater (CRNA). However, the correlation between the CRNA and the COVID-19 clinically confirmed cases in the corresponding wastewater catchments varies and the impacts of environmental and other factors remain unclear. A systematic review and meta-analysis were conducted to identify the correlation between CRNA and various types of clinically confirmed case numbers, including prevalence and incidence rates. The impacts of environmental factors, WBE sampling design, and epidemiological conditions on the correlation were assessed for the same datasets. The systematic review identified 133 correlation coefficients, ranging from -0.38 to 0.99. The correlation between CRNA and new cases (either daily new, weekly new, or future cases) was stronger than that of active cases and cumulative cases. These correlation coefficients were potentially affected by environmental and epidemiological conditions and WBE sampling design. Larger variations of air temperature and clinical testing coverage, and the increase of catchment size showed strong negative impacts on the correlation between CRNA and COVID-19 case numbers. Interestingly, the sampling technique had negligible impact although increasing the sampling frequency improved the correlation. These findings highlight the importance of viral shedding dynamics, in-sewer decay, WBE sampling design and clinical testing on the accurate back-estimation of COVID-19 case numbers through the WBE approach.

DOI： 10.1016/j.jhazmat.2022.129848

Scopus

PubMed

Other Link： https://doi.org/10.1016/j.jhazmat.2022.129848

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are continuously emerging, highlighting the importance of regular surveillance of SARS-CoV-2 and other epidemiologically significant pathogenic viruses in the current context. Reverse transcription-quantitative PCR (RT-qPCR) is expensive, time-consuming, labor-intensive, requires a large reagent volume, and only tests a few targets in a single run. High-throughput qPCR (HT-qPCR) utilizing the Biomark HD system (Fluidigm) can be used as an alternative. This study applied an HT-qPCR to simultaneously detect SARS-CoV-2, SARS-CoV-2 nucleotide substituted RNA, and other pathogenic viruses in wastewater. Wastewater samples were collected from the coronavirus disease 2019 (COVID-19) quarantine facility between October 2020 and February 2021 (n = 4) and from the combined and separated sewer lines of a wastewater treatment plant (WWTP) in Yokkaichi, Mie Prefecture, Japan, between March and August 2021 (n = 23 each). The samples were analyzed by HT-qPCR using five SARS-CoV-2, nine SARS-CoV-2 spike gene nucleotide substitution-specific, five pathogenic viruses, and three process control assays. All samples from the quarantine facility tested positive for SARS-CoV-2 and the nucleotide substitutions N501Y and S69-70 del (Alpha variant) were detected in the December 2020 sample, coinciding with the first clinical case in Japan. Only three WWTP samples were positive when tested with a single SARS-CoV-2 assay, whereas more than eight samples were positive when tested with all assays, indicating that using multiple assays increases the likelihood of detection. The nucleotide substitution L452R (Delta variant) was detected in the WWTP samples of Mie Prefecture in April 2021, but the detection of Delta variant from patients had not been reported until May 2021. Aichi virus 1 and norovirus GII were prevalent in WWTP samples. This study demonstrated that HT-qPCR may be the most time- and cost-efficient method for tracking COVID-19 and broadly monitoring community health.

DOI： 10.1016/j.scitotenv.2022.158659

PubMed

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.envc.2022.100614

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.scitotenv.2022.157869

Other Link： https://doi.org/10.1016/j.scitotenv.2022.157613

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.scitotenv.2022.157613

Other Link： https://doi.org/10.1016/j.scitotenv.2022.157613

Authorship：Lead author,　Last author,　Corresponding author   Language：Japanese   Publishing type：Research paper (research society, symposium materials, etc.)  

Authorship：Last author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：春恒社  

Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本工業出版  

Other Link： https://www.nikko-pb.co.jp/products/detail.php?product_id=5359

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

Antibiotic-resistant bacteria-associated infections are responsible for more than 1.2 million annual deaths worldwide. In low- and middle-income countries (LMICs), the consumption of antibiotics for human and veterinary uses is not regulated effectively. Overused and misused antibiotics can end up in aquatic environments, which may act as a conduit for antibiotic resistance dissemination. However, data on the prevalence of antibiotic resistance determinants in aquatic environments are still limited for LMICs. In this study, we evaluated the prevalence and concentration of antibiotic resistance genes (ARGs) in different drinking and environmental water sources collected from the Kathmandu Valley, Nepal, using droplet digital polymerase chain reaction to understand the current situation of ARG contamination. River water and shallow dug well water sources were the most contaminated with ARGs. Almost all samples contained sul1 (94%), and intI1 and tet(A) were detected in 83 and 60% of the samples, respectively. Maximum ARG concentration varied between 4.2 log₁₀ copies/100 ml for mecA and 9.3 log₁₀ copies/100 ml for sul1. Significant positive correlations were found between ARGs (r > 0.5, p < 0.01), except for mecA, qnrS, and vanA. As sul1 and intI1 were detected in almost all samples, the presence of these genes in a given sample may need to be considered as background antibiotic resistance in LMICs. Therefore, monitoring of ARGs, such as β-lactam ARGs, quinolone resistance genes, and vancomycin resistance genes, may provide a better picture of the antibiotic resistance determinants in aquatic environments of LMICs.

DOI： 10.3389/fmicb.2022.894014

Other Link： https://www.frontiersin.org/articles/10.3389/fmicb.2022.894014/abstract

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

Viral indicators of human-fecal contamination in wastewaters and environmental waters have been getting much attention in the past decade. Cross-assembly phage (crAssphage) is the most abundant DNA virus in human feces. Recently, the usefulness of crAssphage as a microbial source tracking and water quality monitoring tool for human-fecal contamination has been highlighted. Here, we conducted a comprehensive review on crAssphage in water, focusing on detection methodology, concentration range in various waters and wastewaters, specificity to human-fecal contamination, and reduction in wastewater treatment systems. This review highlights that crAssphage is globally distributed in wastewaters and various fecal-contaminated water bodies at high concentrations without seasonal fluctuations. CrAssphage is highly specific to human-fecal contamination and is rarely found in animal feces. It also has a good potential as a performance indicator to ensure virus reduction in wastewater treatment systems. Accordingly, crAssphage could be an effective tool for monitoring of human-fecal contamination and potential presence of fecal pathogenic microbes in environmental waters. Bridging the research gaps highlighted in this review would make crAssphage a powerful tool to support the control of water-related health risks.

DOI： 10.1016/j.watres.2022.118827

Scopus

PubMed

Other Link： https://doi.org/10.1016/j.watres.2022.118827

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

DOI： 10.3390/w14142224

Other Link： https://doi.org/10.3390/w14142224

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Language：English   Publishing type：Research paper (international conference proceedings)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

Language：English   Publishing type：Research paper (scientific journal)  

As a cost-effective and objective population-wide surveillance tool, wastewater-based epidemiology (WBE) has been widely implemented worldwide to monitor the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentration in wastewater. However, viral concentrations or loads in wastewater often correlate poorly with clinical case numbers. To date, there is no reliable method to back-estimate the coronavirus disease 2019 (COVID-19) case numbers from SARS-CoV-2 concentrations in wastewater. This greatly limits WBE in achieving its full potential in monitoring the unfolding pandemic. The exponentially growing SARS-CoV-2 WBE dataset, on the other hand, offers an opportunity to develop data-driven models for the estimation of COVID-19 case numbers (both incidence and prevalence) and transmission dynamics (effective reproduction rate). This study developed artificial neural network (ANN) models by innovatively expanding a conventional WBE dataset to include catchment, weather, clinical testing coverage and vaccination rate. The ANN models were trained and evaluated with a comprehensive state-wide wastewater monitoring dataset from Utah, USA during May 2020 to December 2021. In diverse sewer catchments, ANN models were found to accurately estimate the COVID-19 prevalence and incidence rates, with excellent precision for prevalence rates. Also, an ANN model was developed to estimate the effective reproduction number from both wastewater data and other pertinent factors affecting viral transmission and pandemic dynamics. The established ANN model was successfully validated for its transferability to other states or countries using the WBE dataset from Wisconsin, USA.

DOI： 10.1016/j.watres.2022.118451

Scopus

PubMed

Other Link： https://doi.org/10.1016/j.watres.2022.118451

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.scitotenv.2022.153816

Other Link： https://www.sciencedirect.com/science/article/pii/S0048969722009081

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.envc.2022.100527

Other Link： https://doi.org/10.1016/j.envc.2022.100527

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

DOI： 10.3390/w14081266

Other Link： https://www.mdpi.com/2073-4441/14/8/1266

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

DOI： 10.1016/j.scitotenv.2021.150722

PubMed

Other Link： https://doi.org/10.1016/j.scitotenv.2021.150722

Language：English   Publishing type：Research paper (scientific journal)  

Other Link： https://www.jswe.or.jp/aboutus/pdf/Manual%20for%20Detection%20of%20SARS-CoV-2%20RNA%20in%20Wastewater.pdf

Authorship：Lead author,　Last author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：環境新聞社  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

DOI： 10.3390/w13233412

Other Link： https://www.mdpi.com/2073-4441/13/23/3412

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.envc.2021.100281

Other Link： https://www.sciencedirect.com/science/article/pii/S2667010021002602

Authorship：Lead author,　Last author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：JSWE  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

DOI： 10.3390/w13192733

Other Link： https://www.mdpi.com/2073-4441/13/19/2733

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

We investigated the presence of enteric viruses and bacterial genetic markers to determine fecal contamination in groundwater samples from deep wells used for the public drinking water supply and private wells in Japan. Groundwater is often subjected to chlorination; malfunctions in chlorine treatment result in waterborne disease outbreaks.

DOI： 10.1128/AEM.00710-21

Other Link： https://journals.asm.org/doi/10.1128/AEM.00710-21

Authorship：Lead author,　Last author,　Corresponding author   Language：Japanese   Publishing type：Research paper (other science council materials etc.)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

Clostridium perfringens is classified into types A to G, and all types produce alpha-toxins; however, C. perfringens type F that is negative for phospholipase C (PLC) activity of alpha-toxin has been isolated from the environment and cases of humans afflicted by food poisoning. This study aimed to elucidate the distribution of PLC-negative C. perfringens type F in sewage influents and effluents. Influents and effluents of two wastewater treatment plants were collected monthly between July 2016 and January 2020 and between August 2018 and January 2020, respectively. Isolation rates of PLC-negative C. perfringens type F from sewage influents and effluents were 38% (33/86) and 22% (8/36), and the numbers of isolates were 43 and 13, respectively. The locus of the enterotoxin gene of all isolates was determined to be in a plasmid with an IS1151 sequence, and multilocus sequence typing revealed that all 17 representative isolates were assigned as sequence type 186. Sequencing of the plc gene of these representative isolates showed that nonsense mutation (p.W98*) causing alpha-toxin deficiency should be responsible for a loss of PLC enzymatic activity. These results suggest that alpha toxin-deficient C. perfringens type F is distributed in living and water environments since sewage influents contain community wastewater, and effluents contaminate the environment. Detection of C. perfringens type F, independent of PLC activity, should be carried out on human and environmental samples. IMPORTANCE Understanding the diversity of biochemical characteristics that may affect the identification of bacteria is essential. C. perfringens is a ubiquitous bacterium found in the environment, humans, and animals and is responsible for infectious disease in the intestine. Although the alpha-toxin of C. perfringens may be used for its detection, variants of the alpha-toxin lacking its activity have been isolated from soil and humans experiencing symptoms of diarrhea. It is valuable to disclose the prevalence of the alpha-toxin variant in the sewage of wastewater treatment plants, as it may reflect the hygienic condition of the community, as it would be a pollution source for the environment. This study shows the persistent existence and genetic characteristics of the alpha-toxin variant in sewage and reveals a lacking mechanism of the alpha-toxin activity and proposes the detection method of C. perfringens, independent of the alpha-toxin activity.

DOI： 10.1128/Spectrum.00214-21

PubMed

Other Link： https://journals.asm.org/doi/10.1128/Spectrum.00214-21

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

DOI： 10.1016/C2020-0-02703-9

Other Link： https://www.elsevier.com/books/environmental-resilience-and-transformation-in-times-of-covid-19/ramanathan/978-0-323-85512-9

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Microbial Ecology  

The abundance and diversity of anaerobic ammonium oxidation (anammox) bacteria were assessed in 152 groundwater samples in the Kathmandu Valley, Nepal. Anammox bacterial 16S rRNA genes were detected in 54% (37/68) of samples collected in the dry season at 1.6×105-8.8×106 copies L-1, and in 60% (50/84) of samples collected in the wet season at 4.3×104-1.2×107 copies L-1. The 16S rRNA genes of "Candidatus Brocadia", "Candidatus Anammoxoglobus", and five new deduced anammox bacterial phylotypes were detected in the shallow groundwater samples. Diverse anammox bacteria were broadly distributed in the shallow groundwater aquifer of the Kathmandu Valley.

DOI： 10.1264/jsme2.ME20143

PubMed

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Water Association  

<title>Abstract</title>
This mini review describes the current status and challenges regarding institutionalisation of wastewater surveillance systems against COVID-19. Monitoring SARS-CoV-2 in wastewater has been proposed to be a potential tool to understand the actual prevalence of COVID-19 in the community, and it could be an effective approach to monitor the trend during the COVID-19 pandemic. However, challenges to institutionalise wastewater surveillance systems are still abundant and unfolding at a rapid rate given that the international understanding regarding the scientific knowledge and socio-political impacts of COVID-19 are in the developing stages. To better understand the existing challenges and bottlenecks, a comparative study between Japan, Viet Nam, and Indonesia was carried out in the present study. Through gaining a better understanding of common issues as well as issues specific to each country, we hope to contribute to building a robust multistakeholder system to monitor SARS-CoV-2 in wastewater as an effective disease surveillance system for COVID-19.

DOI： 10.2166/wst.2020.558

Authorship：Last author   Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

DOI： 10.1016/j.envres.2020.110092

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s11270-020-04920-4

Authorship：Lead author,　Last author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：産業用水調査会  

Authorship：Last author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本分析化学会  

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本オゾン協会  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.scitotenv.2020.139960

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.scitotenv.2020.139076

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.scitotenv.2020.140405

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.coesh.2020.09.003

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s12560-020-09436-y

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s12560-020-09433-1

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

Clostridium perfringens is an important pathogen that is responsible for gastroenteritis; the causative agent for the symptoms is C. perfringens enterotoxin (CPE), which is mainly produced by type F C. perfringens. Since shellfishes may gather C. perfringens in the water environment, this study estimated the potential circulation of type F C. perfringens among humans, sewage, and Ruditapes philippinarum (asari clams) as a result of sewage pollution. A comparison of the characteristics among the isolates from 86 sewage influents, 36 effluents, 76 asari clams, and 37 humans was conducted. Serotyping, cpe genotyping, and toxin genotyping showed that C. perfringens with a plasmid IS1151 sequence downstream of cpe was predominant among sewage influents, effluents, humans, and asari clams. Multilocus sequence typing suggested that some isolates from a human, sewage influents, effluents, and asari clams were linked to each other. These results demonstrated that asari clams are the necessary infection sources of C. perfringens responsible for carriers and foodborne diseases, and that these pathogens from humans infected by asari clams can pollute the water environment. It is useful to assess bacteria such as C. perfringens isolates from sewage to estimate the trend of those from the community.

DOI： 10.3390/pathogens9080669

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.2166/wh.2020.223

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本水道新聞社  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.coesh.2020.02.006

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.coesh.2020.02.009

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3390/w12061729

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acs.est.0c02388

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s11270-020-04648-1

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本水道新聞社  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Nature  

DOI： 10.1038/s41598-020-60547-9

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3390/w12020450

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

Salmonella enterica is a major cause of gastroenteritis usually caused by animal-based contaminated foods. Since the current passive surveillance is not sufficient to detect all infections and infection sources, we determined the prevalence of Salmonella isolated from sewage influent of wastewater treatment plants (WWTPs) and compared the characteristics of human and food isolates to identify the infection sources. Sewage influent samples were collected monthly from two WWTPs located in the Yamanashi Prefecture, Japan, for three years. Serotypes, antimicrobial resistances, isolation periods, isolated areas, and pulsed-field gel electrophoresis patterns of six isolates belonging to five serotypes were consistent with those of the isolates from patients. Real-time PCR for Salmonella indicated that sewage influents reflect cases of patients infected with Salmonella, including unreported cases. Serovars Schwarzengrund and Anatum were predominant in sewage, but not in humans, and their characteristics were closely related or identical to those isolated from poultry heart and liver, respectively. These results suggest that sewage influent contains Salmonella isolates from humans and that some originated from unreported human cases infected by poultry-associated products. Therefore, it is necessary to take countermeasures against Salmonella infection based on the unreported cases, which would be disclosed by analysis of sewage influent.

DOI： 10.3390/pathogens9010052

PubMed

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s12560-019-09398-w

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

DOI： 10.3390/pathogens8040175

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s12560-019-09389-x

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3390/pathogens8030110

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3390/w11071416

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3390/pathogens8020081

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.26717/BJSTR.2019.17.003060

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

DOI： 10.3390/healthcare7010009

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

DOI： 10.3390/w10121796

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society of Civil Engineers  

DOI： 10.2208/jscejhe.74.5_I_349

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pone.0207727

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Microbial Ecology  

DOI： 10.1264/jsme2.ME18052

Language：English   Publishing type：Research paper (scientific journal)  

Background: Groundwater is a common domestic water source in developing countries, but is persistently contaminated with enteropathogens. However, studies on determinants of diarrhoea have predominantly focused on piped water. This study examines the relationship between groundwater microbial quality and household diarrhoea occurrence (HDO). Methods: Considering it as a proxy of enteropathogens, this study analysed Escherichia coli concentrations in groundwater wells. Ordinary kriging, a geostatistical technique in geographic information systems, was used to interpolate the E. coli concentration to survey points that had secondary survey data (n=942). The relationship between E. coli and HDO using simple and multivariate statistical analyses in SPSS was analysed. Results: A total of 77% of households used groundwater. One-third of households were without piped-water access (PWA), and these households were significantly more likely to use groundwater than those with PWA. Of the 87 households that reported HDO, 77% were groundwater users. Of the groundwater users, the households with HDO consumed groundwater with significantly higher E. coli concentrations than the households without HDO. Of the households without PWA, the increase in the E. coli concentration increased the odds of HDO (adjusted odds ratio=3.15; 95% CI=1.07-9.22). Conclusion: It is suggested that the groundwater microbial quality is a risk factor for HDO and illustrates this by an application of an interpolation technique relevant for developing countries.

DOI： 10.1093/inthealth/ihy037

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

Human astroviruses are associated with gastroenteritis and known to contaminate water environments. Three different genetic clades of astroviruses are known to infect humans and each clade consists of diverse strains. This study aimed to determine the occurrence and genetic diversity of astrovirus strains in water samples in different geographical locations, i.e., influent and effluent wastewater samples (n = 24 each) in Arizona, U.S., and groundwater (n = 37) and river water (n = 14) samples collected in the Kathmandu Valley, Nepal, using next-generation amplicon sequencing. Astrovirus strains including rare types (types 6 and 7 classical human astroviruses), emerging type (type 5 VA-astroviruses), and putative recombinants were identified. Feline astrovirus strains were collaterally identified and recombination between human and feline astroviruses was suggested. Classical- and VA-astroviruses seemed to be prevalent during cooler months, while MLB-astroviruses were identified only during warmer months. This study demonstrated the effectiveness of next-generation amplicon sequencing for identification and characterization of genetically diverse astrovirus strains in environmental water.

DOI： 10.1038/s41598-018-30217-y

PubMed

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing Inc.  

DOI： 10.1111/jam.13884

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.2521/jswtb.54.65

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society  

Increased demand for water reuse and reclamation accentuates the importance for optimal wastewater treatment to limit protozoa in effluents. Two wastewater treatment plants utilizing advanced Bardenpho were investigated over a 12-month period to determine the incidence and reduction of Cryptosporidium, Giardia, Cyclospora, and fecal indicators. Results were compared to facilities that previously operated in the same geographical area. Protozoa (oo)cysts were concentrated using an electronegative filter and subsequently detected by fluorescent microscopy and/or PCR methods. Cryptosporidium and Giardia were frequently detected in raw sewage, but Cyclospora was not detected in any wastewater samples. Facilities with Bardenpho treatment exhibited higher removals of (oo)cysts than facilities utilizing activated sludge or trickling filters. This was likely due to Bardenpho systems having increased solid wasting rates
however, this mechanism cannot be confirmed as sludge samples were not analyzed. Use of dissolved-air-flotation instead of sedimentation tanks did not result in more efficient removal of (oo)cysts. Concentrations of protozoa were compared with each other, Escherichia coli, somatic coliphage, and viruses (pepper mild mottle virus, Aichi virus 1, adenovirus, and polyomaviruses JC and BK). Although significant correlations were rare, somatic coliphage showed the highest potential as an indicator for the abundance of protozoa in wastewaters.

DOI： 10.1021/acs.est.7b05876

Scopus

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Ltd  

Waterborne human enteric viruses, such as noroviruses and adenoviruses, are excreted in the feces of infected individuals and transmitted via the fecal-oral route including contaminated food and water. Since viruses are normally present at low concentrations in aquatic environments, they should be concentrated into smaller volumes prior to downstream molecular biological applications, such as quantitative polymerase chain reaction (qPCR). This review describes recent progress made in the development of concentration and detection methods of human enteric viruses in water, and discusses their applications for providing a better understanding of the prevalence of the viruses in various types of water worldwide. Maximum concentrations of human enteric viruses in water that have been reported in previous studies are summarized to assess viral abundances in aquatic environments. Some descriptions are also available on recent applications of sequencing analyses used to determine the genetic diversity of viral genomes in water samples, including those of novel viruses. Furthermore, the importance and significance of utilizing appropriate process controls during viral analyses are discussed, and three types of process controls are considered: whole process controls, molecular process controls, and (reverse transcription (RT)-)qPCR controls. Although no standards have been established for acceptable values of virus recovery and/or extraction-(RT-)qPCR efficiency, use of at least one of these appropriate control types is highly recommended for more accurate interpretation of observed data.

DOI： 10.1016/j.watres.2018.02.004

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing Inc.  

Aims: To investigate the molecular epidemiology, especially temporal variations in genotype distribution, of sapoviruses and Aichi virus 1 (AiV-1) in Arizona, United States, by examining wastewater. Methods and Results: A total of 26 wastewater samples (13 influent and 13 effluent) were collected monthly from a wastewater treatment plant and viral strains were identified through nested reverse transcription-PCR followed by cloning and sequencing analysis. Identified sapovirus strains were classified into seven genotypes belonging to three genogroups (GI, GII, and GV): GI.1, GI.2, GI.3, GII.1, GII.2, GII.8 and GV.1, with a clear temporal shift. The majority of AiV-1 strains identified from the wastewater samples were classified into genotype B, and genotype A strains were identified in only two samples. Conclusions: We identified a number of sapovirus and AiV-1 strains belonging to multiple genotypes in wastewater samples collected over a 13-month period. Our results suggested a temporal shift in prevalent genotypes in the community. Significance and Impact of the Study: This is the first study elucidating the genotype distribution of human sapoviruses and AiV-1 in wastewater in the United States. Wastewater surveillance is especially useful for understanding molecular epidemiology of viruses that are less commonly tested in clinical diagnosis, including sapoviruses and AiV-1.

DOI： 10.1111/jam.13712

Scopus

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Quantification of waterborne pathogens in water sources is essential for alerting the community about health hazards. This study determined the presence of human enteric viruses and protozoa in the Bagmati River, Nepal, and detected fecal indicator bacteria (total coliforms, Escherichia coli, and Enterococcus spp.), human-fecal markers (human Bacteroidales and JC and BK polyomaviruses), and index viruses (tobacco mosaic virus and pepper mild mottle virus). During a one-year period between October 2015 and September 2016, a total of 18 surface water samples were collected periodically from three sites along the river. Using quantitative polymerase chain reaction, all eight types of human enteric viruses tested-including adenoviruses, noroviruses, and enteroviruses, were detected frequently at the midstream and downstream sites, with concentrations of 4.4-8.3 log copies/L. Enteroviruses and saliviruses were the most frequently detected enteric viruses, which were present in 72% (13/18) of the tested samples. Giardia spp. were detected by fluorescence microscopy in 78% (14/18) of the samples, with a lower detection ratio at the upstream site. Cryptosporidium spp. were detected only at the midstream and downstream sites, with a positive ratio of 39% (7/18). The high concentrations of enteric viruses suggest that the midstream and downstream regions are heavily contaminated with human feces and that there are alarming possibilities of waterborne diseases. The concentrations of enteric viruses were significantly higher in the dry season than the wet season (p &lt
0.05). There was a significant positive correlation between the concentrations of human enteric viruses and the tested indicators for the presence of pathogens (IPP) (p &lt
0.05), suggesting that these IPP can be used to estimate the presence of enteric viruses in the Bagmati River water.

DOI： 10.3390/pathogens7020038

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer New York LLC  

Irrigation water is a doorway for the pathogen contamination of fresh produce. We quantified pathogenic viruses [human adenoviruses, noroviruses of genogroups I and II, group A rotaviruses, Aichi virus 1 (AiV-1), enteroviruses (EnVs), and salivirus (SaliV)] and examined potential index viruses [JC and BK polyomaviruses (JCPyVs and BKPyVs), pepper mild mottle virus (PMMoV), and tobacco mosaic virus (TMV)] in irrigation water sources in the Kathmandu Valley, Nepal. River, sewage, wastewater treatment plant (WWTP) effluent, pond, canal, and groundwater samples were collected in September 2014, and in April and August 2015. Viruses were concentrated using an electronegative membrane-vortex method and quantified using TaqMan (MGB)-based quantitative PCR (qPCR) assays with murine norovirus as a molecular process control to determine extraction-reverse transcription-qPCR efficiency. Tested pathogenic viruses were prevalent with maximum concentrations of 5.5–8.8 log10 copies/L, and there was a greater abundance of EnVs, SaliV, and AiV-1. Virus concentrations in river water were equivalent to those in sewage. Canal, pond, and groundwater samples were found to be less contaminated than river, sewage, and WWTP effluent. Seasonal dependency was clearly evident for most of the viruses, with peak concentrations in the dry season. JCPyVs and BKPyVs had a poor detection ratio and correspondence with pathogenic viruses. Instead, the frequently proposed PMMoV and the newly proposed TMV were strongly predictive of the pathogen contamination level, particularly in the dry season. We recommend utilizing canal, pond, and groundwater for irrigation to minimize deleterious health effects and propose PMMoV and TMV as indexes to elucidate pathogenic virus levels in environmental samples.

DOI： 10.1007/s12560-017-9324-2

Scopus

Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Institute of Physics Publishing  

In this study, the prevalence of various waterborne pathogens in water samples collected in the Kathmandu Valley, Nepal, and the applicability of Escherichia coli as an indicator of pathogen contamination in groundwater were assessed. Fifty-three water samples, including shallow groundwater and river water, were analyzed to examine the presence of protozoan (oo)cysts via fluorescence microscopy and that of viral and bacterial genomes via quantitative PCR. At least one of the seven types of pathogens tested (i.e., Cryptosporidium, Giardia, human adenoviruses, noroviruses of genogroups I and II, group A rotaviruses, and Vibrio cholerae) was detected in 68% (15/22) of the shallow dug well water samples
groundwater in the shallow dug wells was more contaminated compared with that in shallow tube wells (8/15, 53%). River water and sewage samples were contaminated with extremely high concentrations of multiple pathogens, whereas a tap water sample supplied by a water tanker tested positive for human adenoviruses and V. cholerae. The detection of host-specific Bacteroidales genetic markers revealed the effects of human and animal feces on groundwater contamination. The tested pathogens were sometimes detected even in E. coli-negative groundwater samples, indicative of the limitations of using E. coli as an indicator for waterborne pathogens in groundwater.

DOI： 10.1088/1755-1315/120/1/012004

Scopus

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Verlag  

This study aimed to determine the prevalence of intestinal parasites and its associated risk factors among school-going children in Kathmandu, Nepal. Between August and September 2016, a total of 333 stool samples were collected from children at five public schools. The collected samples were subjected to formol-ether concentration, followed by conventional microscopic examination for intestinal parasites. The overall prevalence of intestinal parasites was 24.3% (81/333), with Giardia spp. showing the highest prevalence of 18.9% (63/333). Samples positive for Giardia spp. by microscopy were further subjected to quantitative polymerase chain reaction (qPCR) for G. duodenalis, resulting in a positive ratio of 100%. The positive ratio of Giardia spp. was considerably high among children consuming tanker water (27.3%), jar water (21.0%), and tap water (17.5%). Our results demonstrated that G. duodenalis remains predominant in school-going children in Nepal.

DOI： 10.1007/s00436-017-5706-5

Scopus

PubMed

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Bacteriological analysis of drinking water leads to detection of only conventional fecal indicator bacteria. This study aimed to explore and characterize bacterial diversity, to understand the extent of pathogenic bacterial contamination, and to examine the relationship between pathogenic bacteria and fecal indicator bacteria in different water sources in the Kathmandu Valley, Nepal. Sixteen water samples were collected from shallow dug wells (n = 12), a deep tube well (n= 1), a spring (n = 1), and rivers (n = 2) in September 2014 for 16S rRNA gene next-generation sequencing. A total of 525 generawere identified, ofwhich 81 genera were classified as possible pathogenic bacteria. Acinetobacter, Arcobacter, and Clostridium were detected with a relatively higher abundance (&gt;0.1% of total bacterial genes) in 16, 13, and 5 of the 16 samples, respectively, and the highest abundance ratio of Acinetobacter (85.14%) was obtained in the deep tube well sample. Furthermore, the blaOXA23-like genes of Acinetobacterwere detected using SYBR Green-based quantitative PCR in 13 (35%) of 37water samples, including the 16 samples that were analyzed for next-generation sequencing, with concentrations ranging 5.3-7.5 log copies/100mL. There was no sufficient correlation found between fecal indicator bacteria, such as Escherichia coli and total coliforms, and potential pathogenic bacteria, as well as the blaOXA(23-like) gene of Acinetobacter. These results suggest the limitation of using conventional fecal indicator bacteria in evaluating the pathogenic bacteria contamination of different water sources in the Kathmandu Valley. (C 2017 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.scitotenv.2017.05.105

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

AimsTo assess diarrhoeal risks from enteropathogenic Escherichia coli, Giardia and Cryptosporidium from consuming raw spinach, cabbage, carrots and tomatoes in Kathmandu Valley, Nepal.
Methods and ResultsThe annual infection risk was quantified using the probabilistic Quantitative Microbial Risk Assessment approach, which considered 12 vegetable washing combinations. A new model was used to estimate dose of pathogens per exposure comprising parameters such as pathogen concentration in vegetable wash water before selling and eating, vegetable consumption rate, remaining pathogen ratio after washing, remaining water on vegetables after washing and water treatment removal efficiency. When all washing combinations were considered, high infection risks above the acceptable level of -4log(10) infection per person per year were obtained, whereas the risk was reduced when other sources excluding river water were used. Assuming use of water treated with ceramic filters by all consumers, a 0-2log(10) reduction in the estimated risks was obtained, which was insufficient to achieve the required risk level.
ConclusionHigh risk of diarrhoea prevails among raw vegetable consumers in the valley.
Significance and Impact of the StudyIt is needed to protect vegetable washing water sources and establish advanced water treatment methods to achieve the required level of public health risk.

DOI： 10.1111/jam.13573

Web of Science

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Aichi virus 1 genomes were detected by quantitative PCR in groundwater from shallow dug (10/22) and tube wells (1/15), river water (14/14), and sewage (1/1), with the maximum concentration of 4.0 x 10(9) copies/l. Nucleotide sequencing analysis demonstrated the prevalence of genotype B in the virus positive samples.

DOI： 10.1007/s12560-017-9283-7

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The use of organic matter, rather than chemical fertilizers, is recommended for sustainable agriculture. The use of green manure is an environmentally friendly technique that is used to manage soil quality, and it has the potential to reduce the dependence on mineral fertilizers and to maintain soil organic matter content. A lysimeter facility, with 25-m(2) (5 m x 5 m) plots that were distributed randomly in blocks, was used in this study. Four experimental plots, chemical fertilizer, non-green manure, hairy vetch (Vicia villosa), and hairy vetch and a Crotalaria sp. (crop rotation), were set up in this lysimeter. The incorporation of hairy vetch as a green manure was conducted in April 2014, and we investigated its effects on fungal biomass, fungal diversity, the rate of phosphate-solubilizing fungi and soil phosphatase activity. Hairy vetch stimulated the activity of soil fungi by increasing fungal biomass and diversity, when compared with chemical fertilizer and non-green manure. Additionally, hairy vetch incorporation increased the abundance of a Cladosporium sp., as well as phosphatase activity in soil. To date, although studies of the incorporation of green manure have mainly focused on roles of bacteria, our results suggest that soil fungal activity could provide a reliable biological indicator, useful in assessing the effect of green manure fertilization.

DOI： 10.1016/j.apsoil.2017.04.015

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer  

We investigated the prevalence and genetic diversity of genogroup IV norovirus (GIV NoV) strains in wastewater in Arizona, United States, over a 13-month period. Among 50 wastewater samples tested, GIV NoVs were identified in 13 (26 %) of the samples. A total of 47 different GIV NoV strains were identified, which were classified into two genetically distinct clusters: the GIV.1 human cluster and a unique genetic cluster closely related to strains previously identified in Japanese wastewater. The results provide additional evidence of the considerable genetic diversity among GIV NoV strains through the analysis of wastewater containing virus strains shed from all populations.

DOI： 10.1007/s00705-016-3036-z

Web of Science

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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DOI： 10.2965/jwet.15-047

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Event date： 2024.1

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Event date： 2023.11 - 2023.12

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Venue：KDDI維新ホール   Country：Japan  

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Venue：香川大学   Country：Japan  

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Venue：大阪大学   Country：Japan  

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