The Catalan Surveillance Network of SARS-CoV-2 in Sewage: design, implementation, and performance.

Wastewater-based epidemiology has shown to be an efficient tool to track the circulation of SARS-CoV-2 in communities assisted by wastewater treatment plants (WWTPs). The challenge comes when this approach is employed to help Health authorities in their decision-making. Here, we describe the roadmap for the design and deployment of SARSAIGUA, the Catalan Surveillance Network of SARS-CoV-2 in Sewage. The network monitors, weekly or biweekly, 56 WWTPs evenly distributed across the territory and serving 6 M inhabitants (80% of the Catalan population). Each week, samples from 45 WWTPs are collected, analyzed, results reported to Health authorities, and finally published within less than 72 h in an online dashboard ( https://sarsaigua.icra.cat ). After 20 months of monitoring (July 20-March 22), the standardized viral load (gene copies/day) in all the WWTPs monitored fairly matched the cumulative number of COVID-19 cases along the successive pandemic waves, showing a good fit with the diagnosed cases in the served municipalities (Spearman Rho = 0.69). Here we describe the roadmap of the design and deployment of SARSAIGUA while providing several open-access tools for the management and visualization of the surveillance data.

Removal of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli, ST98, in Water for Human Consumption by Black Ceramic Water Filters in Low-Income Ecuadorian Highlands.

Fecal contamination in natural water sources is a common problem in low-income countries. Several health risks are associated with unprotected water sources, such as gastrointestinal infections caused by parasites, viruses, and bacteria. Moreover, antibiotic-resistant bacteria in water sources have become an increasing problem worldwide. This study aimed to evaluate the bacterial pathogens present in water within a rural context in Ecuador, along with the efficiency of black ceramic water filters (BCWFs) as a sustainable household water treatment. We monitored five natural water sources that were used for human consumption in the highlands of Ecuador and analyzed the total coliforms and E. coli before and after BCWF installation. The results indicated a variable bacterial contamination (29-300 colony-forming units/100mL) in all unfiltered samples, and they were considered as high risk for human consumption, but after filtration, no bacteria were present. Moreover, extended-spectrum beta-lactamase-producing E. coli with blaTEM, blaCTX-M9, and blaCTX-M1 genes, and two E. coli classified in the clonal complex ST10 (ST98) were detected in two of the locations sampled; these strains can severely impact public health. The clonal complex ST10, found in the E. coli isolates, possesses the potential to spread bacteria-resistant genes to humans and animals. The results of the use of BCWFs, however, argue for the filters' potential impact within those contexts, as the BCWFs completely removed even antibiotic-resistant contaminants from the water.

SARS-CoV-2 in river water: Implications in low sanitation countries.

Since the beginning of COVID-19 pandemic studies on viral shedding have reported that this virus is excreted in feces in most patients. High viral loads are found at the sewage pipeline or at the entrance of wastewater treatment plants from cities where the number of COVID-19 cases are significant. In Quito (Ecuador) as in many other cities worldwide, wastewater is directly discharged into natural waters. The aim of this study was to evaluate SARS-CoV-2 presence in urban streams from a low sanitation context. Three river locations along the urban rivers of Quito were sampled on the 5th of June during a peak of COVID-19 cases. River samples were evaluated for water quality parameters and afterwards, concentrated for viral analysis using skimmed milk flocculation method. The viral concentrates were quantified for SARS-CoV-2 (N1 and N2 target regions) and Human Adenovirus as a human viral indicator. The results showed that SARS-CoV-2 was detected for both target regions in all samples analyzed in a range of 2,91E+05 to 3,19E+06 GC/L for N1 and from 2,07E+05 to 2,22E+06 GC/L for N2. The high values detected in natural waters from a low sanitation region have several implications in health and ecology that should be further assessed.

Congenital Malaria in Newborns Delivered to Mothers with Malaria-Infected Placenta in Blue Nile State, Sudan.

Diagnosis of congenital malaria is complicated by the low density of the parasite circulating in the cord blood and/or the peripheral blood of the newborns. Molecular techniques are significantly more sensitive than blood smears in detecting low-level parasitemia. This study investigated the prevalence of congenital malaria by the use of the real-time polymerase chain reaction (real-time PCR) in 102 babies born to mothers with microscopically confirmed infected placenta from Blue Nile state, Sudan. At delivery time, placental, maternal peripheral and cord blood samples in addition to samples collected from the newborns' peripheral blood were examined for malaria infection using Giemsa-stained thick smear and parasite DNA detection by real-time PCR. The overall prevalence of congenital malaria includes the total babies with cord blood parasitaemia and peripheral blood parasitaemia was 18.6 and 56.8% using microscopy and real-time PCR, respectively. Even though all the neonates were aparasitaemic by microscopy, 19 (18.6%) of the babies had congenital malaria detected by real-time PCR, 15 (25.9%) of the babies with congenital malaria were born to mothers with both placental and peripheral blood malaria infections detected using the two techniques. Congenital malaria was significantly associated with cord blood malaria infections, maternal age and maternal haemoglobin level (p < 0.001). This first study investigating congenital malaria in Blue Nile state, Sudan shows that malaria-infected placenta resulted in infant and cord blood infections.

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples.

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent. At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes. The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Quito's virome: Metagenomic analysis of viral diversity in urban streams of Ecuador's capital city.

In Quito, the microbiological contamination of surface water represents a public health problem, mainly due to the lack of sewage treatment from urban wastewater. Contaminated water contributes to the transmission of many enteric pathogens through direct consumption, agricultural and recreational use. Among the different pathogens present in urban discharges, viruses play an important role on disease, being causes of gastroenteritis, hepatitis, meningitis, respiratory infections, among others. This study analyzes the presence of viruses in highly impacted surface waters of urban rivers using next-generation sequencing techniques. Three representative locations of urban rivers, receiving the main discharges from Quito sewerage system, were selected. Water samples of 500 mL were concentrated by skimmed-milk flocculation method and the viral nucleic acid was extracted and processed for high throughput sequencing using Illumina MiSeq. The results yielded very relevant data of circulating viruses in the capital of Ecuador. A total of 29 viral families were obtained, of which 26 species were associated with infections in humans. Among the 26 species identified, several were related to gastroenteritis: Human Mastadenovirus F, Bufavirus, Sapporovirus, Norwalk virus and Mamastrovirus 1. Also detected were: Gammapapillomavirus associated with skin infections, Polyomavirus 1 related to cases of kidney damage, Parechovirus A described as cause of neonatal sepsis with neurological affectations and Hepatovirus A, the etiologic agent of Hepatitis A. Other emergent viruses identified, of which its pathogenicity remains to be fully clarified, were: Bocavirus, Circovirus, Aichi Virus and Cosavirus. The wide diversity of species detected through metagenomics gives us key information about the public health risks present in the urban rivers of Quito. In addition, this study describes for the first time the presence of important infectious agents not previously reported in Ecuador and with very little reports in Latin America.

Atypical enteropathogenic Escherichia coli (aEPEC) in children under five years old with diarrhea in Quito (Ecuador)

Enteropathogenic Escherichia coli (EPEC) remain one the most important pathogens infecting children and they are one of the main causes of persistent diarrhea worldwide. In this study, we have isolated EPEC from 94 stool samples of children under five years old with diarrheal illness in the area of Quito (Ecuador), and we have determined the occurrence of the two subtypes of EPEC, typical EPEC (tEPEC) and atypical (aEPEC), by PCR amplification of the genes eae (attaching and effacing) and bfp (bundle- forming pilus). Typical EPEC is positive for eae and bfp genes while aEPEC is positive only for eae. Our results suggest that aEPEC is the most prevalent subtype in Quito (89.36 %), while subtype tEPEC is less prevalent (10.64 %) (AU)

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UV disinfection and flocculation-chlorination sachets to reduce hepatitis E virus in drinking water.

Hepatitis E Virus (HEV) is a major cause of waterborne outbreaks in areas with poor sanitation. As safe water supplies are the keystone for preventing HEV outbreaks, data on the efficacy of disinfection treatments are urgently needed. Here, we evaluated the ability of UV radiation and flocculation-chlorination sachets (FCSs) to reduce HEV in water matrices. The HEV-p6-kernow strain was replicated in the HepG2/C3A cell line, and we quantified genome number using qRT-PCR and infectivity using an immunofluorescence assay (IFA). UV irradiation tests using low-pressure radiation showed inactivation kinetics for HEV of 99.99% with a UV fluence of 232J/m(2) (IC 95%, 195,02-269,18). Moreover, the FCSs preparations significantly reduced viral concentrations in both water matrices, although the inactivation results were under the baseline of reduction (4.5 LRV) proposed by WHO guidelines.

Atypical enteropathogenic Escherichia coli (aEPEC) in children under five years old with diarrhea in Quito (Ecuador).

Enteropathogenic Escherichia coli (EPEC) remain one the most important pathogens infecting children and they are one of the main causes of persistent diarrhea worldwide. In this study, we have isolated EPEC from 94 stool samples of children under five years old with diarrheal illness in the area of Quito (Ecuador), and we have determined the occurrence of the two subtypes of EPEC, typical EPEC (tEPEC) and atypical (aEPEC), by PCR amplification of the genes eae (attaching and effacing) and bfp (bundle- forming pilus). Typical EPEC is positive for eae and bfp genes while aEPEC is positive only for eae. Our results suggest that aEPEC is the most prevalent subtype in Quito (89.36 %), while subtype tEPEC is less prevalent (10.64 %). [Int Microbiol 19(3):157-160 (2016)].

UVC Inactivation of dsDNA and ssRNA Viruses in Water: UV Fluences and a qPCR-Based Approach to Evaluate Decay on Viral Infectivity.

Disinfection by low-pressure monochromatic ultraviolet (UVC) radiation (253.7 nm) became an important technique to sanitize drinking water and also wastewater in tertiary treatments. In order to prevent the transmission of waterborne viral diseases, the analysis of the disinfection kinetics and the quantification of infectious viral pathogens and indicators are highly relevant and need to be addressed. The families Adenoviridae and Polyomaviridae comprise human and animal pathogenic viruses that have been also proposed as indicators of fecal contamination in water and as Microbial Source Tracking tools. While it has been previously suggested that dsDNA viruses may be highly resistant to UVC radiation compared to other viruses or bacteria, no information is available on the stability of polyomavirus toward UV irradiation. Here, the inactivation of dsDNA (HAdV2 and JCPyV) and ssRNA (MS2 bacteriophage) viruses was analyzed at increasing UVC fluences. A minor decay of 2-logs was achieved for both infectious JC polyomaviruses (JCPyV) and human adenoviruses 2 (HAdV2) exposed to a UVC fluence of 1,400 J/m(2), while a decay of 4-log was observed for MS2 bacteriophages (ssRNA). The present study reveals the high UVC resistance of dsDNA viruses, and the UV fluences needed to efficiently inactivate JCPyV and HAdV2 are predicted. Furthermore, we show that in conjunction with appropriate mathematical models, qPCR data may be used to accurately estimate virus infectivity.

Environmental Effectors on the Inactivation of Human Adenoviruses in Water.

Environmental factors are highly relevant to the global dissemination of viral pathogens. However, the specific contribution of major effectors such as temperature and sunlight on the inactivation of waterborne viruses is not well characterized. In this study, the effect of temperature (7, 20, and 37 °C), UVB and UVA radiation on viral inactivation was evaluated in phosphate buffered saline (PBS), mineral water, wastewater, 1,000-fold diluted wastewater and seawater. The stability of human adenoviruses infectivity, known as human pathogens and indicators of fecal contamination, was monitored during 24 h, both in the dark and exposed to UV radiation by immunofluorescence assays. In the dark, no Human adenovirus (HAdV) inactivation was observed in PBS and mineral water at any of the temperatures studied, whereas at 37 °C in reactors with higher microbial concentration (wastewater, diluted wastewater, and seawater), decays between 2.5 and 5 log were recorded. UVB radiation showed a dramatic effect on HAdV inactivation and 6-log were achieved in all reactors by the end of the experiments. The effect of UVA showed to be dependent on the water matrix analyzed. At 20 °C, HAdV showed a 2-log decay in all reactors radiation while at 37 °C, results in wastewater, diluted wastewater, and seawater reactors were equivalent to those observed in the dark. These results suggest UVB radiation as the major environmental factor challenging viral inactivation, followed by biotic activity indirectly associated to higher temperatures and finally, by UVA radiation.

Occurrence of water-borne enteric viruses in two settlements based in Eastern Chad: analysis of hepatitis E virus, hepatitis A virus and human adenovirus in water sources.

Hepatitis E virus (HEV) is a common cause of water-borne acute hepatitis in areas with poor sanitation. In 2004 an outbreak of HEV infection affected around 2,000 people in Eastern Chad (Dar Sila). This paper describes the decrease in the incidence of acute jaundice syndrome (AJS) from 2004 until 2009 when a mean incidence of 0.48 cases/1,000 people/year was recorded in the region. Outbreaks of AJS were identified in some of the camps in 2007 and 2008. Moreover, water samples from drinking water sources were screened for human adenoviruses considered as viral indicators and for hepatitis A virus and HEV. Screening of faecal samples from donkeys for HEV gave negative results. Some of the samples were also analysed for faecal coliforms showing values before disinfection treatment between 3 and >50 colony forming units per 100 mL. All water samples tested were negative for HEV and HAV; however, the presence of low levels of human adenoviruses in 4 out of 16 samples analysed indicates possible human faecal contamination of groundwater. Consequently, breakdowns in the treatment of drinking water and/or increased excretion of hepatitis viruses, which could be related to the arrival of a new population, could spread future outbreaks through drinking water.