Application of wastewater-based epidemiology for monitoring COVID-19 in hospital and housing wastewaters.

An alternative and complementary diagnostic method of surveillance is provided by wastewater-based surveillance (WBS), particularly in low-income nations like Nepal with scant wastewater treatment facilities and clinical testing infrastructure. In this study, a total of 146 water samples collected from two hospitals (n = 63) and three housing wastewaters (n = 83) from the Kathmandu Valley over the period of March 2021-Febraury 2022 were investigated for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using quantitative reverse transcription TaqMan PCR assays targeting the N and E genes. Of the total, 67 % (98/146) samples were positive for SARS-CoV-2 RNA either by using N- or E-gene assay, with concentrations ranging from 3.6 to 9.1 log10 copies/L. There was a significant difference found between positive ratio (Chi-square test, p < 0.05) and concentration (t-test, p = 0.009) of SARS-CoV-2 RNA detected from hospital wastewater and housing waters. Wastewater data are correlated with COVID-19 active cases, indicating significance in specific areas like the Hospital (APFH) (p < 0.05). According to the application of a bivariate linear regression model (p < 0.05), the concentrations of N gene may be used to predict the COVID-19 cases in the APFH. Remarkably, SARS-CoV-2 RNA was detected prior to, during, and following clinical case surges, implying that wastewater surveillance could serve as an early warning system for public health decisions. The significance of WBS in tracking and managing pandemics is emphasized by this study, especially in resource-constrained settings.

Quantification of multiple respiratory viruses in wastewater in the Kathmandu Valley, Nepal: Potential implications of wastewater-based epidemiology for community disease surveillance in developing countries.

Despite being the major cause of death, clinical surveillance of respiratory viruses at the community level is very passive, especially in developing countries. This study focused on the surveillance of three respiratory viruses [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IFV-A), and respiratory syncytial virus (RSV)] in the Kathmandu Valley, Nepal, by implication of wastewater-based epidemiology (WBE). Fifty-one untreated wastewater samples were from two wastewater treatment plants (WWTPs) between April and October 2022. Among eight combinations of the pre-evaluated methods, the combination of concentration by simple centrifugation, pretreatment by DNA/RNA Shield (Zymo Research), and extraction by the QIAamp Viral RNA Mini Kit (QIAGEN) showed the best performance for detecting respiratory viruses. Using this method with a one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR), SARS-CoV-2 RNA was successfully detected from both WWTPs (positive ratio, 100 % and 81 %) at concentrations of 5.6 ± 0.6 log10 copies/L from each WWTP. Forty-six SARS-CoV-2 RNA-positive samples were further tested for three mutation site-specific one-step RT-qPCR (L452R, T478K, and E484A/G339D), where G339D/E484A mutations were frequently detected in both WWTPs (96 %). IFV-A RNA was more frequently detected in WWTP A (84 %) compared to WWTP B (38 %). RSV RNA was also detected in both WWTPs (28 % and 8 %, respectively). This is the first study on detecting IFV-A and RSV in wastewater in Nepal, showing the applicability and importance of WBE for respiratory viruses in developing countries where clinical data are lacking.

Evaluating acute gastroenteritis-causing pathogen reduction in wastewater and the applicability of river water for wastewater-based epidemiology in the Kathmandu Valley, Nepal.

Rapid urbanization and population growth without the implementation of proper waste management are capable of contaminating water sources, which can lead to acute gastroenteritis. This study examined the detection and reduction of five gastroenteritis-causing enteropathogens, Salmonella, Campylobacter coli, Campylobacter jejuni, Clostridium perfringens, and genogroup IV norovirus, and one respiratory pathogen, influenza A virus, in two municipal wastewater treatment plants (WWTP) using an oxidation ditch system (WWTP A; n = 20) and a stabilization pond system (WWTP B; n = 18) in the Kathmandu Valley, Nepal, collected between August 2017 and August 2019. All enteropathogens were detected in wastewater via quantitative PCR. The concentrations of the pathogens ranged from 5.7 to 7.9 log10 copies/L in WWTP A and from 4.9 to 8.1 log10 copies/L in WWTP B. The log10 reduction values of the pathogens ranged from 0.3 to 1.0 in WWTP A and from -0.1 to 0.2 in WWTP B. The association between the pathogen concentrations and the number of clinical cases in the corresponding week could not be evaluated; however, the consistent detection of pathogens in the wastewater despite low number of case reports suggested the use of wastewater-based epidemiology (WBE) for early warning of acute gastroenteritis (AGE) in the Kathmandu Valley. The pathogens were also detected in river water at approximately 7.0 log10 copies/L and exhibited no significant difference in concentration compared to wastewater, suggesting the applicability of river water for WBE of AGE. Insufficient treatment of all pathogens in the wastewater was observed, suggesting the need for full rehabilitation of the treatment plants. However, the influent may be utilized for early detection of AGE-causing pathogens in the city, whereas the river water may serve as an alternative in areas without connection to the WWTPs.

Detection of SARS-CoV-2 RNA in wastewater, river water, and hospital wastewater of Nepal.

The applicability of wastewater-based epidemiology (WBE) has been extensively studied throughout the world with remarkable findings. This study reports the presence and reduction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at two wastewater treatment plants (WWTPs) of Nepal, along with river water, hospital wastewater (HWW), and wastewater from sewer lines collected between July 2020 and February 2021. SARS-CoV-2 RNA was detected in 50%, 54%, 100%, and 100% of water samples from WWTPs, river hospitals, and sewer lines, respectively, by at least one of four quantitative PCR assays tested (CDC-N1, CDC-N2, NIID_2019-nCOV_N, and N_Sarbeco). The CDC-N2 assay detected SARS-CoV-2 RNA in the highest number of raw influent samples of both WWTPs. The highest concentration was observed for an influent sample of WWTP A (5.5 ± 1.0 log10 genome copies/L) by the N_Sarbeco assay. SARS-CoV-2 was detected in 47% (16/34) of the total treated effluents of WWTPs, indicating that biological treatments installed at the tested WWTPs are not enough to eliminate SARS-CoV-2 RNA. One influent sample was positive for N501Y mutation using the mutation-specific qPCR, highlighting a need for further typing of water samples to detect Variants of Concern. Furthermore, crAssphage-normalized SARS-CoV-2 RNA concentrations in raw wastewater did not show any significant association with the number of new coronavirus disease 2019 (COVID-19) cases in the whole district where the WWTPs were located, suggesting a need for further studies focusing on suitability of viral as well as biochemical markers as a population normalizing factor. Detection of SARS-CoV-2 RNA before, after, and during the peaking in number of COVID-19 cases suggests that WBE is a useful tool for COVID-19 case estimation in developing countries.

Persistence and occurrence of SARS-CoV-2 in water and wastewater environments: a review of the current literature.

As the world continues to cope with the COVID-19 pandemic, emerging evidence indicates that respiratory transmission may not the only pathway in which the virus can be spread. This review paper aims to summarize current knowledge surrounding possible fecal-oral transmission of SARS-CoV-2. It covers recent evidence of proliferation of SARS-CoV-2 in the gastrointestinal tract, as well as presence and persistence of SARS-CoV-2 in water, and suggested future directions. Research indicates that SARS-CoV-2 can actively replicate in the human gastrointestinal system and can subsequently be shed via feces. Several countries have reported SARS-CoV-2 RNA fractions in wastewater systems, and various factors such as temperature and presence of solids have been shown to affect the survival of the virus in water. The detection of RNA does not guarantee infectivity, as current methods such as RT-qPCR are not yet able to distinguish between infectious and non-infectious particles. More research is needed to determine survival time and potential infectivity, as well as to develop more accurate methods for detection and surveillance.

Occurrence of SARS-CoV-2 RNA in Six Municipal Wastewater Treatment Plants at the Early Stage of COVID-19 Pandemic in The United States.

In this study, we investigated the occurrence of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) RNA in primary influent (n = 42), secondary effluent (n = 24) and tertiary treated effluent (n = 34) collected from six wastewater treatment plants (WWTPs A-F) in Virginia (WWTP A), Florida (WWTPs B, C, and D), and Georgia (WWTPs E and F) in the United States during April-July 2020. Of the 100 wastewater samples analyzed, eight (19%) untreated wastewater samples collected from the primary influents contained SARS-CoV-2 RNA as measured by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays. SARS-CoV-2 RNA were detected in influent wastewater samples collected from WWTP A (Virginia), WWTPs E and F (Georgia) and WWTP D (Florida). Secondary and tertiary effluent samples were not positive for SARS-CoV-2 RNA indicating the treatment processes in these WWTPs potentially removed SARS-CoV-2 RNA during the secondary and tertiary treatment processes. However, further studies are needed to understand the log removal values (LRVs) and transmission risks of SARS-CoV-2 RNA through analyzing wastewater samples from a wider range of WWTPs.

Surveillance of SARS-CoV-2 RNA in wastewater: Methods optimisation and quality control are crucial for generating reliable public health information.

Monitoring for SARS-CoV-2 RNA in wastewater through the process of wastewater-based epidemiology (WBE) provides an additional surveillance tool, contributing to community-based screening and prevention efforts as these measurements have preceded disease cases in some instances. Numerous detections of SARS-CoV-2 RNA have been reported globally using various methods, demonstrating the technical feasibility of routine monitoring. However, in order to reliably interpret data produced from these efforts for informing public health interventions, additional quality control information and standardization in sampling design, sample processing, and data interpretation and reporting is needed. This review summarizes published studies of SARS-CoV-2 RNA detection in wastewater as well as available information regarding concentration, extraction, and detection methods. The review highlights areas for potential standardization including considerations related to sampling timing and frequency relative to peak fecal loading times; inclusion of appropriate information on sample volume collected; sample collection points; transport and storage conditions; sample concentration and processing; RNA extraction process and performance; effective volumes; PCR inhibition; process controls throughout sample collection and processing; PCR standard curve performance; and recovery efficiency testing. Researchers are recommended to follow the Minimum Information for Publication of Quantitative Real-Time PCR (MIQE) guidelines. Adhering to these recommendations will enable robust interpretation of wastewater monitoring results and improved inferences regarding the relationship between monitoring results and disease cases.

First detection of SARS-CoV-2 RNA in wastewater in North America: A study in Louisiana, USA.

We investigated the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater samples in southern Louisiana, USA. Untreated and treated wastewater samples were collected on five occasions over a four-month period from January to April 2020. The wastewater samples were concentrated via ultrafiltration (Method A), and an adsorption-elution method using electronegative membranes (Method B). SARS-CoV-2 RNA was detected in 2 out of 15 wastewater samples using two reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays (CDC N1 and N2). None of the secondary treated and final effluent samples tested positive for SARS-CoV-2 RNA. To our knowledge, this is the first study reporting the detection of SARS-CoV-2 RNA in wastewater in North America, including the USA. However, concentration methods and RT-qPCR assays need to be refined and validated to increase the sensitivity of SARS-CoV-2 RNA detection in wastewater.

Reduction of Pathogenic and Indicator Viruses at a Drinking Water Treatment Plant in Southern Louisiana, USA.

Monthly sampling was conducted at a drinking water treatment plant (DWTP) in Southern Louisiana, USA from March 2017 to February 2018 to determine the prevalence and reduction efficiency of pathogenic and indicator viruses. Water samples were collected from the DWTP at three different treatment stages (raw, secondary-treated, and chlorinated drinking water) and subjected to quantification of seven pathogenic viruses and three indicator viruses [pepper mild mottle virus (PMMoV), tobacco mosaic virus (TMV), and crAssphage] based on quantitative polymerase chain reaction. Among the seven pathogenic viruses tested, only Aichi virus 1 (AiV-1) (7/12, 58%) and noroviruses of genogroup II (NoVs-GII) (2/12, 17%) were detected in the raw water samples. CrAssphage had the highest positive ratio at 78% (28/36), and its concentrations were significantly higher than those of the other indicator viruses for all three water types (P < 0.05). The reduction ratios of AiV-1 (0.7 ± 0.5 log10; n = 7) during the whole treatment process were the lowest among the tested viruses, followed by crAssphage (1.1 ± 1.9 log10; n = 9), TMV (1.3 ± 0.9 log10; n = 8), PMMoV (1.7 ± 0.8 log10; n = 12), and NoVs-GII (3.1 ± 0.1 log10; n = 2). Considering the high abundance and relatively low reduction, crAssphage was judged to be an appropriate process indicator during drinking water treatment. To the best of our knowledge, this is the first study to assess the reduction of crAssphage and TMV during drinking water treatment.

Reduction of Human Enteric and Indicator Viruses at a Wastewater Treatment Plant in Southern Louisiana, USA.

This study assessed wastewater quality through the quantification of four human enteric viruses and the applicability of pepper mild mottle virus (PMMoV) and tobacco mosaic virus (TMV) as indicators of viral reduction during wastewater treatment. Thirty-three samples were collected from three steps of a wastewater treatment plant in Southern Louisiana, USA for a year between March 2017 and February 2018. Noroviruses of genogroup I were the most prevalent human enteric viruses in influent samples. The concentrations of PMMoV in influent samples (5.9 ± 0.7 log10 copies/L) and biologically treated effluent samples (5.9 ± 0.5 log10 copies/L) were significantly higher than those of TMV (P < 0.05), and the reduction ratio of PMMoV (1.0 ± 0.8 log10) was found comparable to those of TMV and Aichi virus 1. Because of the high prevalence, high correlations with human enteric viruses, and lower reduction ratios, PMMoV was deemed an appropriate indicator of human enteric viral reduction during wastewater treatment process.

Arsenic disturbs the gut microbiome of individuals in a disadvantaged community in Nepal.

Arsenic is ubiquitous in nature, highly toxic, and is particularly abundant in Southern Asia. While many studies have focused on areas like Bangladesh and West Bengal, India, disadvantaged regions within Nepal have also suffered from arsenic contamination levels, with wells and other water sources possessing arsenic contamination over the recommended WHO and EPA limit of 10 µg/L, some wells reporting levels as high as 500 µg/L. Despite the region's pronounced arsenic concentrations within community water sources, few investigations have been conducted to understand the impact of arsenic contamination on host gut microbiota health. This study aims to examine differential arsenic exposure on the gut microbiome structure within two disadvantaged communities in southern Nepal. Fecal samples (n = 42) were collected from members of the Mahuawa (n = 20) and Ghanashyampur (n = 22) communities in southern Nepal. The 16S rRNA gene was amplified from fecal samples using Illumina-tag PCR and subject to high-throughput sequencing to generate the bacterial community structure of each sample. Bioinformatics analysis and multivariate statistics were conducted to identify if specific fecal bacterial assemblages and predicted functions were correlated with urine arsenic concentration. Our results revealed unique assemblages of arsenic volatilizing and pathogenic bacteria positively correlated with increased arsenic concentration in individuals within the two respective communities. Additionally, we observed that commensal gut bacteria negatively correlated with increased arsenic concentration in the two respective communities. Our study has revealed that arsenic poses a broader human health risk than was previously known. It is influential in shaping the gut microbiome through its enrichment of arsenic volatilizing and pathogenic bacteria and subsequent depletion of gut commensals. This aspect of arsenic has the potential to debilitate healthy humans by contributing to disorders like heart and liver cancers and diabetes, and it has already been shown to contribute to serious diseases and disorders, including skin lesions, gangrene and several types of skin, renal, lung, and liver cancers in disadvantaged areas of the world like Nepal.

Applicability of crAssphage, pepper mild mottle virus, and tobacco mosaic virus as indicators of reduction of enteric viruses during wastewater treatment.

This study was conducted to evaluate the applicability of crAssphage, pepper mild mottle virus (PMMoV), and tobacco mosaic virus (TMV) as indicators of the reduction of human enteric viruses during wastewater treatment. Thirty-nine samples were collected from three steps at a wastewater treatment plant (raw sewage, secondary-treated sewage, and final effluent) monthly for a 13-month period. In addition to the three indicator viruses, eight human enteric viruses [human adenoviruses, JC and BK polyomaviruses, Aichi virus 1 (AiV-1), enteroviruses, and noroviruses of genogroups I, II, and IV] were tested by quantitative PCR. Indicator viruses were consistently detected in the tested samples, except for a few final effluents for crAssphage and TMV. The mean concentrations of crAssphage were significantly higher than those of most tested viruses. The concentrations of crAssphage in raw sewage were positively correlated with the concentrations of all tested human enteric viruses (p <0.05), suggesting the applicability of crAssphage as a suitable indicator to estimate the concentrations of human enteric viruses in raw sewage. The reduction ratios of AiV-1 (1.8 ± 0.7 log10) were the lowest among the tested viruses, followed by TMV (2.0 ± 0.3 log10) and PMMoV (2.0 ± 0.4 log10). Our findings suggested that the use of not only AiV-1 and PMMoV but also TMV as indicators of reductions in viral levels can be applicable during wastewater treatment.

Detection of Pathogenic Viruses, Pathogen Indicators, and Fecal-Source Markers within Tanker Water and Their Sources in the Kathmandu Valley, Nepal.

Tanker water is used extensively for drinking as well as domestic purposes in the Kathmandu Valley of Nepal. This study aimed to investigate water quality in terms of microbial contamination and determine sources of fecal pollution within these waters. Thirty-one samples from 17 tanker filling stations (TFSs) and 30 water tanker (WT) samples were collected during the dry and wet seasons of 2016. Escherichia coli was detected in 52% of the 31 TFS samples and even more frequently in WT samples. Of the six pathogenic viruses tested, enteroviruses, noroviruses of genogroup II (NoVs-GII), human adenoviruses (HAdVs), and group A rotaviruses were detected using quantitative PCR (qPCR) at 10, five, four, and two TFSs, respectively, whereas Aichi virus 1 and NoVs-GI were not detected at any sites. Index viruses, such as pepper mild mottle virus and tobacco mosaic virus, were detected using qPCR in 77% and 95% out of 22 samples, respectively, all of which were positive for at least one of the tested pathogenic viruses. At least one of the four human-associated markers tested (i.e., BacHum, HAdVs, and JC and BK polyomaviruses) was detected using qPCR in 39% of TFS samples. Ruminant-associated markers were detected at three stations, and pig- and chicken-associated markers were found at one station each of the suburbs. These findings indicate that water supplied by TFSs is generally of poor quality and should be improved, and proper management of WTs should be implemented.

Performance Evaluation of Human-Specific Viral Markers and Application of Pepper Mild Mottle Virus and CrAssphage to Environmental Water Samples as Fecal Pollution Markers in the Kathmandu Valley, Nepal.

Monitoring of environmental water is crucial to protecting humans and animals from possible health risks. Although numerous human-specific viral markers have been designed to track the presence of human fecal contamination in water, they lack adequate sensitivity and specificity in different geographical regions. We evaluated the performances of six human-specific viral markers [Aichi virus 1 (AiV-1), human adenoviruses (HAdVs), BK and JC polyomaviruses (BKPyVs and JCPyVs), pepper mild mottle virus (PMMoV), and crAssphage] using 122 fecal-source samples collected from humans and five animal hosts in the Kathmandu Valley, Nepal. PMMoV and crAssphage showed high sensitivity (90-100%) with concentrations of 4.5-9.1 and 6.2-7.0 log10 copies/g wet feces (n = 10), respectively, whereas BKPyVs, JCPyVs, HAdVs, and AiV-1 showed poor performances with sensitivities of 30-40%. PMMoV and crAssphage were detected in 40-100% and 8-90%, respectively, of all types of animal fecal sources and showed no significantly different concentrations among most of the fecal sources (Kruskal-Wallis test, P > 0.05), suggesting their applicability as general fecal pollution markers. Furthermore, a total of 115 environmental water samples were tested for PMMoV and crAssphage to identify fecal pollution. PMMoV and crAssphage were successfully detected in 62% (71/115) and 73% (84/115) of water samples, respectively. The greater abundance and higher mean concentration of crAssphage (4.1 ± 0.9 log10 copies/L) compared with PMMoV (3.3 ± 1.4 log10 copies/L) indicated greater chance of detection of crAssphage in water samples, suggesting that crAssphage could be preferred to PMMoV as a marker of fecal pollution.

Co-Infection by Waterborne Enteric Viruses in Children with Gastroenteritis in Nepal.

Enteric viruses are highly contagious and a major cause of waterborne gastroenteritis in children younger than five years of age in developing world. This study examined the prevalence of enteric virus infection in children with gastroenteritis to identify risk factors for co-infections. In total, 107 stool samples were collected from patients with acute gastroenteritis along with samples of their household drinking water and other possible contamination sources, such as food and hand. The presence of major gastroenteritis-causing enteric virus species (group A rotaviruses, enteroviruses, adenoviruses, and noroviruses of genogroup I) in stool and water samples was examined using quantitative polymerase chain reaction. Among the 107 stool samples tested, 103 (96%) samples contained at least one of the four tested enteric viruses, and the combination of group A rotaviruses and enteroviruses was the most common co-infection (52%, n = 54/103). At least one viral agent was detected in 16 (16%) of 103 drinking water samples. Identical enteric viruses were detected in both the stool and water samples taken from the same patients in 13% of cases (n = 13/103). Group A rotaviruses were most frequently found in children suffering from acute diarrhea. No socio-demographic and clinical factors were associated with the risk of co-infection compared with mono-infection. These less commonly diagnosed viral etiological agents in hospitals are highly prevalent in patients with acute gastroenteritis.

Hospital based surveillance and molecular characterization of rotavirus in children less than 5 years of age with acute gastroenteritis in Nepal.

BACKGROUND: Rotavirus remains a significant causative agent of childhood acute gastroenteritis, particularly among children less than 5 years of age. Although precise data on childhood mortality associated with diarrheal disease in Nepal is not available, it is estimated that22% of all rotavirus deaths globally occurs in neighboring country of India. In spite of the substantial burden of rotavirus gastroenteritis in the Indian subcontinent, rotavirus vaccine has not been introduced in Nepal. Continuous surveillance for monitoring rotavirus disease burden and molecular characterization is needed prior to rotavirus vaccine introduction in Nepal. METHODS: A total of 3310 stool samples (2849 hospitalized cases and 461 non-hospitalized cases), were collected from patients <5 years of age from January 2013 to December 2016 and tested for rotavirus antigen by ELISA (ProSpecT, USA). A subset of ELISA positive stool samples was genotyped. Demographic data were collected. RESULTS: During the four-year surveillance period, the overall burden of rotavirus infection was 24% among hospitalized children which was much higher than among non-hospitalized children (12%). The majority of children hospitalized with rotavirus gastroenteritis were less than 2 years of age (86%). Rotavirus-associated gastroenteritis hospitalizations occur year-round in Nepal, but a distinct peak in winter (up to 40% among hospitalized) was observed. Of 735 ELISA positive samples, 492 were genotyped by RT-PCR. The most prevalent genotype was G12P[6] (45.3%), followed byG2P[4](12.2%), G1P[8] (9.6%), G9P[4](7.3%), and G9P[8](4.5%). Mixed infection accounted for 4.4% of cases, 6.2% were partially typed and 10.5% of the samples were G and P untypable. CONCLUSIONS: A high burden of rotavirus gastroenteritis and a diversity of circulating rotavirus strains in Nepal were observed. Recommendation to introduce a rotavirus vaccine with known vaccine effectiveness would help in reducing the severity of Rotavirus diarrheal disease in children less than 5 years of age.

Gut microbiome transition across a lifestyle gradient in Himalaya.

The composition of the gut microbiome in industrialized populations differs from those living traditional lifestyles. However, it has been difficult to separate the contributions of human genetic and geographic factors from lifestyle. Whether shifts away from the foraging lifestyle that characterize much of humanity's past influence the gut microbiome, and to what degree, remains unclear. Here, we characterize the stool bacterial composition of four Himalayan populations to investigate how the gut community changes in response to shifts in traditional human lifestyles. These groups led seminomadic hunting-gathering lifestyles until transitioning to varying levels of agricultural dependence upon farming. The Tharu began farming 250-300 years ago, the Raute and Raji transitioned 30-40 years ago, and the Chepang retain many aspects of a foraging lifestyle. We assess the contributions of dietary and environmental factors on their gut-associated microbes and find that differences in the lifestyles of Himalayan foragers and farmers are strongly correlated with microbial community variation. Furthermore, the gut microbiomes of all four traditional Himalayan populations are distinct from that of the Americans, indicating that industrialization may further exacerbate differences in the gut community. The Chepang foragers harbor an elevated abundance of taxa associated with foragers around the world. Conversely, the gut microbiomes of the populations that have transitioned to farming are more similar to those of Americans, with agricultural dependence and several associated lifestyle and environmental factors correlating with the extent of microbiome divergence from the foraging population. The gut microbiomes of Raute and Raji reveal an intermediate state between the Chepang and Tharu, indicating that divergence from a stereotypical foraging microbiome can occur within a single generation. Our results also show that environmental factors such as drinking water source and solid cooking fuel are significantly associated with the gut microbiome. Despite the pronounced differences in gut bacterial composition across populations, we found little differences in alpha diversity across lifestyles. These findings in genetically similar populations living in the same geographical region establish the key role of lifestyle in determining human gut microbiome composition and point to the next challenging steps of determining how large-scale gut microbiome reconfiguration impacts human biology.

Development of a Quantitative PCR Assay for Arcobacter spp. and its Application to Environmental Water Samples.

Arcobacter spp. are emerging pathogens associated with gastroenteritis in humans. The objective of this study was to develop a highly sensitive and broadly reactive quantitative PCR (qPCR) assay for Arcobacter spp. and to apply the developed assay to different water sources in the Kathmandu Valley, Nepal. Fifteen samples to be analyzed by next-generation sequencing were collected from 13 shallow dug wells, a deep tube well, and a river in the Kathmandu Valley in August 2015. Among the 86 potential pathogenic bacterial genera identified, Acinetobacter, Pseudomonas, Flavobacterium, and Arcobacter were detected with relatively high abundance in 15, 14, 12, and 8 samples, respectively. A primer pair was designed with maximal nucleotide homologies among Arcobacter spp. by comparing the sequences of 16S rRNA genes. These primers were highly specific to most of the known species of Arcobacter and quantified between 1.0×101 and 6.4×106 copies reaction-1 and sometimes detected as few as 3 copies reaction-1. The qPCR assay was used to quantify Arcobacter spp. in bacterial DNA in not only the above 15 water samples, but also in 33 other samples collected from 15 shallow dug wells, 6 shallow tube wells, 5 stone spouts, 4 deep tube wells, and 3 springs. Thirteen (27%) out of 48 samples tested were positive for Arcobacter spp., with concentrations of 5.3-9.1 log copies 100 mL-1. This qPCR assay represents a powerful new tool to assess the prevalence of Arcobacter spp. in environmental water samples.

Presence of Human Enteric Viruses, Protozoa, and Indicators of Pathogens in the Bagmati River, Nepal.

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 < 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 < 0.05), suggesting that these IPP can be used to estimate the presence of enteric viruses in the Bagmati River water.

Prevalence and associated risk factors of Giardia duodenalis infection among school-going children in Nepal.

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.