Multiple pathogen contamination of water, hands, and fomites in rural Nepal and the effect of WaSH interventions.

Water, Sanitation, and Hygiene (WaSH) interventions are the most effective in reducing diarrheal disease severity and prevalence. However, very few studies have investigated the effectiveness of WaSH intervention in reducing pathogen presence and concentration. In this study, we employed a microfluidic PCR approach to quantify twenty bacterial pathogens in water (n = 360), hands (n = 180), and fomite (n = 540) samples collected in rural households of Nepal to assess the pathogen exposures and the effect of WaSH intervention on contamination and exposure rates. The pathogen load and the exposure pathways for each pathogen in intervention and control villages were compared to understand the effects of WaSH intervention. Pathogens were detected in higher frequency and concentration from fomites samples, toilet handle (21.42%; 5.4,0 95%CI: mean log10 of 4.69, 5.96), utensils (23.5%; 5.47, 95%CI: mean log10 of 4.77, 6.77), and water vessels (22.42%; 5.53, 95%CI: mean log10 of 4.79, 6.60) as compared to cleaning water (14.36%; 5.05, 95%CI: mean log10 of 4.36, 5.89), drinking water (14.26%; 4.37, 85%CI: mean log10 of 4.37, 5.87), and hand rinse samples (16.92%; 5.49, 95%CI: mean log10 of 4.77, 6.39). There was no clear evidence that WaSH intervention reduced overall pathogen contamination in any tested pathway. However, we observed a significant reduction (p < 0.05) in the prevalence, but not concentration, of some target pathogens, including Enterococcus spp. in the intervention village compared to the control village for water and hands rinse samples. Conversely, no significant reduction in target pathogen concentration was observed for water and hand rinse samples. In swab samples, there was a reduction mostly in pathogen concentration rather than pathogen prevalence, highlighting that a reduction in pathogen prevalence was not always accompanied by a reduction in pathogen concentration. This study provides an understanding of WaSH intervention on microbe concentrations. Such data could help with better planning of intervention activities in the future.

Assessment of pathogens in flood waters in coastal rural regions: Case study after Hurricane Michael and Florence.

The severity of hurricanes, and thus the associated impacts, is changing over time. One of the understudied threats from damage caused by hurricanes is the potential for cross-contamination of water bodies with pathogens in coastal agricultural regions. Using microbiological data collected after hurricanes Florence and Michael, this study shows a dichotomy in the presence of pathogens in coastal North Carolina and Florida. Salmonella typhimurium was abundant in water samples collected in the regions dominated by swine farms. A drastic decrease in Enterococcus spp. in Carolinas is indicative of pathogen removal with flooding waters. Except for the abundance presence of Salmonella typhimurium, no significant changes in pathogens were observed after Hurricane Michael in the Florida panhandle. We argue that a comprehensive assessment of pathogens must be included in decision-making activities in the immediate aftermath of hurricanes to build resilience against risks of pathogenic exposure in rural agricultural and human populations in vulnerable locations.

Microbial assessment of water, sanitation, and hygiene (WaSH) in temporary and permanent settlements two years after Nepal 2015 earthquake.

Disaster-induced displacement often causes people to live in temporary settlements that have limited infrastructure and access to water, sanitation, and hygiene (WaSH). Reducing the risk of diarrheal diseases in such situations requires knowing how housing influences the presence of pathogens in water and the interaction between human settlements and exposure to pathogens. A cross-sectional study was conducted in May 2017 in two communities hard-hit by the Nepal 2015 earthquake: one recovered with newly reconstructed houses, and one recovered with residents still living in sheet metal temporary shelters constructed after the earthquake. We collected 60 water (30 drinking water and 30 cleaning water), 30 hand rinse, and 90 environmental swab samples (30 toilet handles, 30 utensils, and 30 water vessels) from selected households in each location and quantified 22 bacterial pathogens using microfluidic quantitative polymerase chain reaction (mfqPCR). A total of 59 samples were randomly selected for amplicon-based sequencing of the 16S rRNA, and it identified bacterial community profiles between these two settlements and their association with target genes of pathogenic bacteria. Target genes like uidA of Escherichia coli and the mip gene of Legionella pnuemophila showed significantly high frequency in specific sample types in temporary settlements than in permanent settlements. A significantly high concentration was observed in temporary settlements for Enterococcus spp. and S. typhimurium, specifically in swab samples. There was a sharp distinction of microbial community profiles between water and hand rinse samples with environmental swab samples, with a large abundance of potentially pathogenic bacteria in swab samples in both settlements. This observation highlighted that fomite could be an important transmission route for pathogens in rural settings and designing key interventions to target different stages of transmission pathways is essential. Overall findings from this study suggest that the recovered settlement with higher quality housing may be less impacted by fecal contamination than recovering settlements and that interventions should be designed to disrupt multiple transmission pathways to reduce pathogen exposure.

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.

Distribution and Antibiotic Resistance Profiles of Salmonella enterica in Rural Areas of North Carolina After Hurricane Florence in 2018.

In this study, water samples were analyzed from a rural area of North Carolina after Hurricane Florence in 2018 and the distribution of the ttrC virulence gene of Salmonella enterica were investigated. We also examined the distribution of culturable S. enterica and determined their antibiotic resistance profiles. Antibiotic resistance genes (ARGs) in the classes of aminoglycoside, beta-lactam, and macrolide-lincosamide-streptogramin B (MLSB) were targeted in this study. The ttrC gene was detected in 23 out of 25 locations. There was a wider and higher range of the ttrC gene in flooded water versus unflooded water samples (0-2.12 × 105 copies/L vs. 0-4.86 × 104 copies/L). Culturable S. enterica was isolated from 10 of 25 sampling locations, which was less prevalent than the distribution of the ttrC gene. The antibiotic resistance profiles were not distinct among the S. enterica isolates. The aminoglycoside resistance gene aac(6')-Iy had the highest relative abundance (around 0.05 copies/16S rRNA gene copy in all isolates) among all ARGs. These findings suggested that the 2018 flooding event led to higher copy numbers of the ttrC genes of S. enterica in some flooded water bodies compared to those in unflooded water bodies. The high ARG level and similar ARG profiles were observed in all S. enterica isolates from both flooded and unflooded samples, suggesting that the antibiotic resistance was prevalent in S. enterica within this region, regardless of flooding.

Quantification and Comparison of Risks Associated with Wastewater Use in Spray Irrigation.

In the U.S., spray irrigation is the most common method used in agriculture and supplementing with animal wastewater has the potential to reduce water demands. However, this could expose individuals to respiratory pathogens such as Legionella pneumophila and nontuberculosis Mycobacteria (NTM). Disinfection with methods like anaerobic digestion is an option but can increase concentrations of cytotoxic ammonia (personal communication). Our study aimed to model the annual risks of infection from these bacterial pathogens and the air concentrations of ammonia and determine if anaerobically digesting this wastewater is a safe option. Air dispersion modeling, conducted in AERMOD, generated air concentrations of water during the irrigation season (May-September) for the years 2013-2018. These values fed into the quantitative microbial risk assessments for the bacteria and allowed calculation of ammonia air concentrations. The outputs of these models were compared to the safety thresholds of 10-4 infections/year and 0.5 mg/m3 , respectively, to determine their potential for negative health outcomes. It was determined that infection from NTM was not a concern for individuals near active spray irrigators, but that infection with L. pneumophila could be a concern, with a maximum predicted annual risk of infection of 3.5 × 10-3 infections/year and 25.2% of parameter combinations exceeding the established threshold. Ammonia posed a minor risk, with 1.5% of parameter combinations surpassing the risk threshold of 0.5 mg/m3 . These findings suggest that animal wastewater should be anaerobically digested prior to use in irrigation to remove harmful pathogens.

Assessment of microbial risks by characterization of Escherichia coli presence to analyze the public health risks from poor water quality in Nepal.

The combination of natural disasters and inadequate infrastructure introduce unique challenges in providing safe drinking water in low-income countries. For example, Nepal faces several challenges in managing sporadic diarrheal outbreaks across the country, given its complex geographic terrain, and its susceptibility to extreme natural events like earthquakes, floods, and landslides. To assess the risks of diarrheal diseases caused by fecal contamination in several water sources in different geographical regions of Nepal, we conducted a two months cross-sectional study throughout 37 out of 75 districts in Nepal, including the ones affected by Nepal 2015 earthquake. Quantitative Microbial Risk Assessment (QMRA) was applied to estimate the human health risk based on Escherichia coli (E. coli) count for 2,822 water samples collected at source and households. Disease burden calculations suggested that Hilly and Terai (low-land) regions are at the highest risk with 0.27 and 0.16 DALYs per person per year (DALYpppy), respectively, whereas mountain region disease burden was 0.02 DALYpppy. The risk comparison among exposure pathways indicated that the water used in households, including drinking water and water for washing, posed higher risks than from source water, reservoir water or tap water, suggesting deteriorated sanitation conditions in households. These findings suggest that the combination of QMRA and spatial analysis can help to prioritize intervention activities after a major natural disaster.

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.

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.

The Effect of the 2015 Earthquake on the Bacterial Community Compositions in Water in Nepal.

We conducted a study to examine the effect of seasonal variations and the disruptive effects of the 2015 Nepal earthquake on microbial communities associated with drinking water sources. We first characterized the microbial communities of water samples in two Nepali regions (Kathmandu and Jhapa) to understand the stability of microbial communities in water samples collected in 2014. We analyzed additional water samples from the same sources collected from May to August 2015, allowing the comparison of samples from dry-to-dry season and from dry-to-monsoon seasons. Emphasis was placed on microbes responsible for maintaining the geobiochemical characteristics of water (e.g., ammonia-oxidizing and nitrite-oxidizing bacteria and archaea and sulfate-reducing bacteria) and opportunistic pathogens often found in water (Acinetobacter). When examining samples from Jhapa, we identified that most geobiochemical microbe populations remained similar. When examining samples from Kathmandu, the abundance of microbial genera responsible for maintaining the geobiochemical characteristics of water increased immediately after the earthquake and decreased 8 months later (December 2015). In addition, microbial source tracking was used to monitor human fecal contamination and revealed deteriorated water quality in some specific sampling sites in Kathmandu post-earthquake. This study highlights a disruption of the environmental microbiome after an earthquake and the restoration of these microbial communities as a function of time and sanitation practices.

Quantification of multiple waterborne pathogens in drinking water, drainage channels, and surface water in Kampala, Uganda, during seasonal variation.

Longitudinal water quality monitoring is important for understanding seasonal variations in water quality, waterborne disease transmission, and future implications for climate change and public health. In this study, microfluidic quantitative polymerase chain reaction (MFQPCR) was used to quantify genes from pathogens commonly associated with human intestinal infections in water collected from protected springs, a public tap, drainage channels, and surface water in Kampala, Uganda, from November 2014 to May 2015. The differences in relative abundance of genes during the wet and dry seasons were also assessed. All water sources tested contained multiple genes from pathogenic microorganisms, with drainage channels and surface waters containing a higher abundance of genes as compared to protected spring and the public tap water. Genes detected represented the presence of enterohemorrhagic Escherichia coli, Shigella spp., Salmonella spp., Vibrio cholerae, and enterovirus. There was an increased presence of pathogenic genes in drainage channels during the wet season when compared to the dry season. In contrast, surface water and drinking water sources contained little seasonal variation in the quantity of microbes assayed. These results suggest that individual water source types respond uniquely to seasonal variability and that human interaction with contaminated drainage waters, rather than direct ingestion of contaminated water, may be a more important contributor to waterborne disease transmission. Furthermore, future work in monitoring seasonal variations in water quality should focus on understanding the baseline influences of any one particular water source given their unique complexities.