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Dengue virus (DENV) is an enveloped, single-stranded RNA virus that causes approximately 390 million infections, leading to 40,000 deaths annually. Due to the increasing trend of urbanization, water supply scarcity, and climate change, dengue is regarded as the “disease of the future,” requiring robust surveillance for the early detection of DENV infection. Since the virus is shed in urine and saliva and persists in wastewater at different temperatures, our study conducted wastewater surveillance as a novel approach to monitor dengue outbreaks in the Kathmandu Valley, Nepal. The viral concentrates (n = 34), which were previously collected and concentrated from municipal and hospital wastewater, and river water using the electronegative membrane-vortex method, were tested for DENV using quantitative reverse transcription polymerase chain reaction (RT-qPCR) and digital PCR (RT-dPCR). Pepper mild mottle virus, a process control and endogenous biomarker, was detected in all the samples with concentrations ranging from 8.0 to 10.0 log10 copies/L, whereas DENV was not detected in any sample using RT-dPCR and RT-qPCR. The undetected DENV in this study could be attributed to the collection of grab wastewater samples during a low relative prevalence of dengue infection in the region, insufficient sample volume processed, probable viral nucleic acid degradation due to storage of viral concentrate at -25 °C for a long period of time, or inefficiency of the primary concentration method used. This study highlights critical gaps in knowledge and provides recommendations for future implementation of wastewater surveillance of dengue outbreaks, especially in regions where dengue was recently introduced, clinical surveillance is limited, and wastewater surveillance for polio has been adopted.

In resource-limited settings, fecal indicator bacterial analysis is the only microbiological water quality test performed, and emerging contaminants, such as antibiotic resistance genes (ARGs), are often neglected. To address this knowledge gap, water samples were collected from shallow wells (n = 24), deep wells (n = 16), stone spouts (n = 14), springs (n = 8), tanker filling stations (TFS; n = 12), water tankers (n = 12), and drinking water treatment plants (DWTPs; n = 6 each of raw and treated water) of the Kathmandu Valley, Nepal, between December 2015 and August 2016 to investigate the presence of five ARGs (blaNDM-1, blaCTX−M, tetB, qnrS, and sul1) and class one integrase gene (intI1). All ARGs were detected in water samples from shallow wells and stone spouts, whereas blaNDM-1 and blaCTX−M were not detected in deep wells and springs. tetB and intI1 were detected at a greater number of water tanker samples than TFS, indicating that water tankers were not disinfected regularly. Only sul1 and intI1 were detected in treated water of DWTPs. Persistence of intI1 throughout the water treatment process and significantly strong correlation (ρ > 0.5, p < 0.05) with the majority of ARGs included in our study suggested the suitability of intI1 to assess the contamination and fate of ARGs in drinking water of the valley. Low free chlorine levels in treated water before pipeline distribution urge water quality managers to evaluate tap water for pathogens and ARGs. Further studies are recommended on emerging contaminants, such as antibiotics, in drinking water sources that are not routinely assessed but can provide selective pressure for the spread of antibiotic resistance in the environment.

Continuous discharge of enteric viruses through treated wastewater is a potential threat to the environment and humans. This study aimed to investigate the occurrence and reduction of enteric viruses at two wastewater treatment plants (WWTPs) in the Kathmandu Valley, Nepal. Twenty-two samples (from the influent samples and effluent samples, n = 11 each) were collected at two WWTPs (WWTP A and WWTP B, installing oxidation ditch and non-aerated lagoon systems, respectively) between April and August 2018. Human adenoviruses, JC and BK polyomaviruses, Aichi virus 1, enteroviruses, noroviruses of genogroup I (NoVs-GI), group A rotaviruses, and human cosavirus, along with plant viruses (pepper mild mottle and tobacco mosaic viruses), were detected using quantitative PCR. The most prevalent enteric virus was NoVs-GI (100%), followed by human adenoviruses (95%), and enteroviruses and JC polyomaviruses (91%). Log10 reduction values of all tested viruses were found to be lower than those of fecal indicator bacteria. Both WWTPs were less efficient for the reduction of pathogens (p