Kei Nishida

Prevalence of protozoa, viruses, coliphages and indicator bacteria in groundwater and river water in the Kathmandu Valley, Nepal.

Limited information is available on the prevalence of waterborne pathogens in aquatic environments in developing countries. In this study, water samples were collected from nine shallow wells and a river in the Kathmandu Valley, Nepal, during the rainy season in 2009 and were subjected to detection of waterborne protozoa, viruses and coliphages using a recently developed method for simultaneous concentration of protozoa and viruses in water. Escherichia coli and total coliforms were also tested as indicator bacteria. At least one type of the five pathogens tested (Cryptosporidium, Giardia, human adenoviruses, and noroviruses of genogroups I and II) was detected in five groundwater samples (56%) (1000 ml each) from shallow wells. Compared with groundwater samples, the pathogens were more abundant in the river water sample (100ml); the concentrations of Cryptosporidium and Giardia were 140 oocysts/l and 8500 cysts/l, respectively, and the mean threshold cycle (Ct) values in real-time RT-PCR were 34.3, 36.8 and 34.0 for human adenoviruses and noroviruses of genogroups I and II, respectively. Genotyping of F-RNA coliphages by real-time RT-PCR was successfully used to differentiate human and animal faecal contamination in the samples. Moreover, for the groundwater samples, protozoa and viruses were detected only in E. coli-positive samples, suggesting that E. coli may be an appropriate indicator of pathogen contamination of valley groundwater.

Access to improved water and its relationship with diarrhoea in Kathmandu Valley, Nepal: a cross-sectional study

Objective To assess the associations between diarrhoea and types of water sources, total quantity of water consumed and the quantity of improved water consumed in rapidly growing, highly populated urban areas in developing countries. Design Cross-sectional analysis using population-representative secondary data obtained from an interview survey conducted by the Asian Development Bank for the 2009 Kathmandu Valley Water Distribution, Sewerage and Urban Development Project. Setting Kathmandu Valley, Nepal. Participants 2282 households. Methods A structured questionnaire was used to collect information from households on the quantity and sources of water consumed; health, socioeconomic and demographic status of households; drinking water treatment practices and toilet facilities. Results Family members of 179 households (7.8%) reported having developed diarrhoea during the previous month. For households in which family members consumed less than 100 L of water per capita per day (L/c/d), which is the minimum quantity recommended by WHO, the risk of contracting diarrhoea doubled (1.56-fold to 2.92-fold). In households that used alternative water sources (such as wells, stone spouts and springs) in addition to improved water (provided by a water management authority), the likelihood of contracting diarrhoea was 1.81-fold higher (95% CI 1.00 to 3.29) than in those that used only improved water. However, access to an improved water source was not associated with a lower risk of developing diarrhoea if optimal quantities of water were not consumed (ie, <100 L/c/d). These results were independent of socioeconomic and demographic variables, daily drinking water treatment practices, toilet facilities and residential areas. Conclusions Providing access to a sufficient quantity of water—regardless of the source—may be more important in preventing diarrhoea than supplying a limited quantity of improved water.

Characterization of microbial communities distributed in the groundwater pumped from deep tube wells in the Kathmandu Valley of Nepal

Although groundwater is a major water supply source in the Kathmandu Valley of Nepal, it is known that the groundwater has significant microbial contamination exceeding the drinking water quality standard recommended by the World Health Organization (WHO), and that this has been implicated in causing a variety of diseases among people living in the valley. However, little is known about the distribution of pathogenic microbes in the groundwater. Here, we analysed the microbial communities of the six water samples from deep tube wells by using the 16S rRNA gene sequences based culture-independent method. The analysis showed that the groundwater has been contaminated with various types of opportunistic microbes in addition to fecal microbes. Particularly, the clonal sequences related to the opportunistic microbes within the genus Acinetobacter were detected in all samples. As many strains of Acinetobacter are known as multi-drug resistant microbes that are currently spreading in the world, we conducted a molecular-based survey for detection of the gene encoding carbapenem-hydrolysing β-lactamase (bla(oxa-23-like) gene), which is a key enzyme responsible for multi-drug resistance, in the groundwater samples. Nested polymerase chain reaction (PCR) using two specific primer sets for amplifying bla(oxa-23-like) gene indicated that two of six groundwater samples contain multi-drug resistant Acinetobacter.

Characterization of the genes involved in nitrogen cycling in wastewater treatment plants using DNA microarray and most probable number-PCR

AbstractTo improve nitrogen removal performance of wastewater treatment plants (WWTPs), it is essential to understand the behavior of nitrogen cycling communities, which comprise various microorganisms. This study characterized the quantity and diversity of nitrogen cycling genes in various processes of municipal WWTPs by employing two molecular-based methods:most probable number-polymerase chain reaction (MPN-PCR) and DNA microarray. MPN-PCR analysis revealed that gene quantities were not statistically different among processes, suggesting that conventional activated sludge processes (CAS) are similar to nitrogen removal processes in their ability to retain an adequate population of nitrogen cycling microorganisms. Furthermore, most processes in the WWTPs that were researched shared a pattern:the nirS and the bacterial amoA genes were more abundant than the nirK and archaeal amoA genes, respectively. DNA microarray analysis revealed that several kinds of nitrification and denitrification genes were detected in both CAS and anaerobic-oxic processes (AO), whereas limited genes were detected in nitrogen removal processes. Results of this study suggest that CAS maintains a diverse community of nitrogen cycling microorganisms; moreover, the microbial communities in nitrogen removal processes may be specific.

Risk of diarrhoea from shallow groundwater contaminated with enteropathogens in the Kathmandu Valley, Nepal.

Shallow groundwater is the main water source among many alternatives in the Kathmandu Valley, Nepal, which has a rapidly growing population and intermittent piped water supply. Although human pathogens are detected in groundwater, its health effects are unclear. We estimated risk of diarrhoea from shallow groundwater use using quantitative microbial risk assessment. Escherichia coli, Giardia cyst and Cryptosporidium oocyst levels were analysed in dug and tube wells samples. E. coli concentrations were converted to those of enteropathogenic E. coli (EPEC). Risks from EPEC in dug wells and from Cryptosporidium and Giardia in both dug and tube wells were higher than the acceptable limit (<10⁻⁴ infections/person-year) for both drinking and bathing exposures. Risk from protozoan enteropathogens increased the total risk 10,000 times, indicating that ignoring protozoans could lead to serious risk underestimation. Bathing exposure considerably increased risk, indicating that it is an important pathway. Point-of-use (POU) water treatment decreased the risk six-fold and decreased risk overestimation. Because removal efficiency of POU water treatment has the largest impact on total risk, increasing the coverage and efficiency of POU water treatment could be a practical risk management strategy in the Kathmandu Valley and similar settings.

High-throughput DNA microarray detection of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley, Nepal.

Because of heavy dependence on groundwater for drinking water and other domestic use, microbial contamination of groundwater is a serious problem in the Kathmandu Valley, Nepal. This study investigated comprehensively the occurrence of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley by applying DNA microarray analysis targeting 941 pathogenic bacterial species/groups. Water quality measurements found significant coliform (fecal) contamination in 10 of the 11 investigated groundwater samples and significant nitrogen contamination in some samples. The results of DNA microarray analysis revealed the presence of 1–37 pathogen species/groups, including 1–27 biosafety level 2 ones, in 9 of the 11 groundwater samples. While the detected pathogens included several feces- and animal-related ones, those belonging to Legionella and Arthrobacter, which were considered not to be directly associated with feces, were detected prevalently. This study could provide a rough picture of overall pathogenic bacterial contamination in the Kathmandu Valley, and demonstrated the usefulness of DNA microarray analysis as a comprehensive screening tool of a wide variety of pathogenic bacteria.

Atmospheric nitrate leached from small forested watersheds during rainfall events: Processes and quantitative evaluation

To determine the availability of atmospheric NO3− deposition on forested ecosystems and to understand the interaction between the nitrogen cycle in a forest ecosystem and atmospheric nitrogen input/output, we quantitatively evaluated the atmospheric NO3− passing through forested watersheds by measuring δ18ONO3 leaching during rainfall events in two forest ecosystems (Su-A and Ab-S). Atmospheric NO3− leaching in rainfall events was clearly higher in Ab-S than in Su-A, even for a similar amount of rainfall, which demonstrated that atmospheric NO3− leaching differs among forested watersheds. Our observations suggest that a large part of the atmospheric NO3− leached from the watersheds was derived from surface soil, which was deposited before rainfall events occurred; however, direct atmospheric NO3− leaching via throughfall discharge also contributed, especially at the beginning of rainfall events. In Ab-S, 2.9–37.8% (average = 15.5%) of atmospheric NO3− deposition passed through the watershed, accounting for 3.1–49.8% (average, 26.4%) of the total NO3− leached during rainfall events. The NO3− input was not large, and the NO3− pool and net nitrification rate were small; therefore, nitrogen was not saturated in the soil at Ab-S. Nevertheless, some of the atmospheric NO3− deposition was not assimilated and was leached immediately. Moreover, our observations suggest that the hydrological characteristics of the watersheds, which control the ease of rainwater discharge, strongly influenced the rate of atmospheric NO3− leaching. This suggests that the hydrological characteristics of watersheds influence the availability of atmospheric NO3− deposition in forested ecosystems and the progression of nitrogen saturation.

Development of a whole community genome amplification-assisted DNA microarray method to detect functional genes involved in the nitrogen cycle

A novel DNA microarray analysis targeting key functional genes involved in most nitrogen cycling reactions was developed to comprehensively analyze microbial populations associated with the nitrogen cycle. The developed microarray contained 876 oligonucleotide probes based on the nucleotide sequences of the nif, amo, hao/hzo, nap, nar, nirK, nirS, nrf, cnor, qnor and nos genes. An analytical method combining detection by the designed microarray with whole community genome amplification was then applied to monitor the nitrogen cycling microorganisms in river water and wastewater treatment sludge samples. The developed method revealed that nitrogen cycling microorganisms in river water appeared to become less diverse in response to input of effluent from municipal wastewater treatment plants. Additionally, the nitrogen cycling community associated with anaerobic ammonium oxidation and partial nitrification reactors could be reasonably analyzed by the developed method. However, the results obtained for two activated sludge samples from municipal wastewater treatment plants with almost equivalent wastewater treatment performance differed greatly from each other. These results suggested that the developed method is useful for comprehensive analysis of nitrogen cycling microorganisms, although its applicability to complex samples with abundant untargeted populations should be further examined.

A novel water security index and well-being at micro level in urban areas of developing countries

A composite metric assessing water security’s physical dimension at the micro/ community level is lacking but is essential for setting priorities for program and policy implementations. We prepared an objective index (OI) of water security to measure the physical dimension using a model centered on household water-use behavior in developing countries’ urban areas. A cross-sectional household survey (n = 1500) with multi-stage cluster design was conducted from December 2015 to February 2016 in the Kathmandu Valley, which has faced long-term, severe water shortage. A structured questionnaire probed socio-demographic characteristics, water sources, frequency and quantity of water use, cost related to water, etc. A 15-item water insecurity scale was used to measure subjective and experiential dimension of water insecurity. The World Health Organization Quality of Life – BREF was used to measure quality of life (QoL). The QoL has been considered as proxy of well-being in this study. The OI measured differential water security within small cities, the utility’s service areas for instance, and identified area-specific key dimensions that need improvement. Overall, the OI and its key dimensions can be useful measures to design water-scarcity averting programs and policies, specific to a particular community’s needs. The increased OI values were significantly and positively associated with better physical and psychological health and better social relationship domains of QoL suggesting health implications of water security.

Groundwater use and diarrhoea in urban Nepal: novel application of a geostatistical interpolation technique linking environmental and epidemiologic survey data

Abstract 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.