Pilot study on wastewater surveillance of dengue virus RNA: Lessons, challenges, and implications for future research

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.Peer reviewe

Maculosin, a non-toxic antioxidant compound isolated from Streptomyces sp. KTM18

Context Streptomyces species are prolific sources of bioactive secondary metabolites known especially for their antimicrobial and anticancer activities. Objective This study sought to isolate and characterize antioxidant molecules biosynthesized by Streptomyces sp. KTM18. The antioxidant potential of an isolated compound and its toxicity were accessed. Materials and methods The compound was purified using bioassay-guided chromatography techniques. Nuclear magnetic resonance (NMR) experiments were carried out for structure elucidation. The antioxidant potential of the isolated compound was determined using DPPH free radical scavenging assay. The toxicity of the isolated compound was measured using a brine shrimp lethality (BSL) assay. Results Ethyl acetate extract of Streptomyces sp. KTM18 showed more than 90% inhibition of DPPH free radical at 50 mu g/mL of the test concentration. These data were the strongest among 13 Streptomyces isolates (KTM12-KTM24). The active molecule was isolated and characterized as maculosin (molecular formula, C14H16N2O3 as determined by the [M + H](+) peak at 261.1259). The DPPH free radical scavenging activity of pure maculosin was higher (IC50, 2.16 +/- 0.05 mu g/mL) than that of commercial butylated hydroxyanisole (BHA) (IC50, 4.8 +/- 0.05 mu g/mL). No toxicity was observed for maculosin (LD50, <128 mu g/mL) in brine shrimp lethality assay (BSLA) up to the compound's antioxidant activity (IC50) concentration range. The commercial standard, berberine chloride, showed toxicity in BSLA with an LD50 value of 8.63 +/- 0.15 mu g/mL. Conclusions Maculosin may be a leading drug candidate in various cosmetic and therapeutic applications owing to its strong antioxidant and non-toxic properties

Monkeypox Outbreak: Wastewater and Environmental Surveillance Perspective

Monkeypox disease (MPXD), a viral disease caused by monkeypox virus (MPXV), is an emerging zoonotic disease endemic in some countries of Central and Western Africa but seldom reported outside the affected region. Since May 2022, MPXD has been reported at least in 74 countries globally, prompting the World Health Organization to declare the MPXD outbreak a Public Health Emergency of International Concern. As of July 24, 2022, 92% (68/74) of the countries with reported MPXD cases had no historical MPXD case reports. From the One Health perspective, the spread of MPXV in the environment poses a risk not only to humans but also to small mammals and may, ultimately, spread to potent novel host populations. Wastewater-based surveillance (WBS), has been extensively utilized for monitoring communicable diseases, particularly during the ongoing coronavirus disease, the COVID-19 pandemic It helped to monitor infectious disease caseloads as well as specific viral variants circulating in communities. The detection of MPXV DNA in various body fluids, including respiratory and nasal secretions, saliva, urine, feces, and semen of infected individuals, supports the possibility of using WBS as an early proxy for the detection of MPXV infections. WBS of MPXV DNA can be used to monitor MPXV activity/trends in sewerage network areas even before detecting laboratory-confirmed clinical cases within a community. However, several factors affect the detection of MPXV in wastewater including, but not limited to, routes and duration time of virus shedding by infected individuals, infection rates in the relevant affected population, environmental persistence, the processes and analytical sensitivity of the used methods. Further research is needed to identify the key factors that impact the detection of MPXV biomarkers in wastewater and improve the utility of WBS of MPXV as an early warning and monitoring tool for safeguarding human health. In this review, we shortly summarize aspects of MPXV outbreak relevant to wastewater monitoring and discuss the challenges associated with WBS.Peer reviewe

Monkeypox outbreak : Wastewater and environmental surveillance perspective

Monkeypox disease (MPXD), a viral disease caused by the monkeypox virus (MPXV), is an emerging zoonotic disease endemic in some countries of Central and Western Africa but seldom reported outside the affected region. Since May 2022, MPXD has been reported at least in 74 countries globally, prompting the World Health Organization to declare the MPXD outbreak a Public Health Emergency of International Concern. As of July 24, 2022; 92 % (68/74) of the countries with reported MPXD cases had no historical MPXD case reports. From the One Health perspective, the spread of MPXV in the environment poses a risk not only to humans but also to small mammals and may, ultimately, spread to potent novel host populations. Wastewater-based surveillance (WBS) has been extensively utilized to monitor communicable diseases, particularly during the ongoing COVID-19 pandemic. It helped in monitoring infectious disease caseloads as well as specific viral variants circulating in communities. The detection of MPXV DNA in lesion materials (e.g. skin, vesicle fluid, crusts), skin rashes, and various body fluids, including respiratory and nasal secretions, saliva, urine, feces, and semen of infected individuals, supports the possibility of using WBS as an early proxy for the detection of MPXV infections. WBS of MPXV DNA can be used to monitor MPXV activity/trends in sewerage network areas even before detecting laboratory-confirmed clinical cases within a community. However, several factors affect the detection of MPXV in wastewater including, but not limited to, routes and duration time of virus shedding by infected individuals, infection rates in the relevant affected population, environmental persistence, the processes and analytical sensitivity of the used methods. Further research is needed to identify the key factors that impact the detection of MPXV biomarkers in wastewater and improve the utility of WBS of MPXV as an early warning and monitoring tool for safeguarding human health. In this review, we shortly summarize aspects of the MPXV outbreak relevant to wastewater monitoring and discuss the challenges associated with WBS.publishedVersionPeer reviewe

First environmental surveillance for the presence of SARS-CoV-2 RNA in wastewater and river water in Japan

Wastewater-based epidemiology is a powerful tool to understand the actual incidence of coronavirus disease 2019 (COVID-19) in a community because severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, can be shed in the feces of infected individuals regardless of their symptoms. The present study aimed to assess the presence of SARS-CoV-2 RNA in wastewater and river water in Yamanashi Prefecture, Japan, using four quantitative and two nested PCR assays. Influent and secondary-treated (before chlorination) wastewater samples and river water samples were collected five times from a wastewater treatment plant and three times from a river, respectively, between March 17 and May 7, 2020. The wastewater and river water samples (200-5000 mL) were processed by using two different methods: the electronegative membrane-vortex (EMV) method and the membrane adsorption-direct RNA extraction method. Based on the observed concentrations of indigenous pepper mild mottle virus RNA, the EMV method was found superior to the membrane adsorption-direct RNA extraction method. SARS-CoV-2 RNA was successfully detected in one of five secondary-treated wastewater samples with a concentration of 2.4 x 10(3) copies/L by N_Sarbeco qPCR assay following the EMV method, with sequence confirmation of the qPCR product, whereas all the influent samples were tested negative for SARS-CoV-2 RNA. This result could be attributed to higher limit of detection for influent (4.0 x 10(3)-82 x 10(4) copies L) with a lower filtration volume (200 mL) compared to that for secondary-treated wastewater (1.4 x 10(2)-2.5 x 10(3) copies/L) with a higher filtration volume of 5000 mL. None of the river water samples tested positive for SARS-CoV-2 RNA Comparison with the reported COVID-19 cases in Yamanashi Prefecture showed that SARS-CoV-2 RNA was detected in the secondary-treated wastewater sample when the cases peaked in the community. This is the first study reporting the detection of SARS-CoV-2 RNA in wastewater in Japan. (C) 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

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

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 &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