ABSTRACT
This year-long research analysed emerging risks in influent, effluent wastewaters and biosolids from six wastewater treatment plants in Spain's Valencian Region. Specifically, it focused on human enteric and respiratory viruses, bacterial and viral faecal contamination indicators, extended spectrum beta-lactamases-producing Escherichia coli and antibiotic resistance genes. Additionally, particles and microplastics in biosolid and wastewater samples were assessed. Human enteric viruses were prevalent in influent wastewater, with limited post-treatment reduction. Wastewater treatment effectively eliminated respiratory viruses, except for low levels of SARS-CoV-2 in effluent and biosolid samples, suggesting minimal public health risk. Antibiotic resistance genes and microplastics were persistently found in effluent and biosolids, thus indicating treatment inefficiencies and potential environmental dissemination. This multifaced research sheds light on diverse contaminants present after water reclamation, emphasizing the interconnectedness of human, animal, and environmental health in wastewater management. It underscores the need for a One Health approach to address the United Nations Sustainable Development Goals.
ABSTRACT
The COVID-19 pandemic has posed a significant global threat, leading to several initiatives for its control and management. One such initiative involves wastewater-based epidemiology, which has gained attention for its potential to provide early warning of virus outbreaks and real-time information on its spread. In this study, water samples from two wastewater treatment plants (WWTPs) located at the south east of Spain (Region of Murcia) namely Murcia, and Cartagena, were analyzed by RT-qPCR, Phylogenetic Analysis, and Machine Learning Approach. The aim was to determine whether SARS-CoV-2 detection in the WWTPs of these two cities could serve as a proxy for the virus's spread in the population. The results confirmed that the levels of SARS-CoV-2 in these wastewater samples changed concerning the number of SARS-CoV-2 cases detected in the population and variant occurrences were in line with clinical reported data. Additionally, the phylogenetic analysis showed that samples obtained in close sampling times exhibited a higher similarity than those obtained more distantly in time. A second analysis using a machine learning approach based on the mutations found in the SARS-CoV-2 spike protein was also conducted. Hierarchical Clustering (HC) was used as an efficient unsupervised approach for data analysis. Results indicated that samples obtained in October 2022 in Murcia and Cartagena were significantly different, which corresponded well with the different virus variants circulating in the two locations. The proposed methods in this study are adequate for comparing the Accumulated Natural Vector (ANV) of the SARS-CoV-2 sequences as a preliminary evaluation of potential changes in the variants that are circulating in a given population at a specific time point.
Subject(s)
COVID-19 , Cluster Headache , Crohn DiseaseABSTRACT
Besides nasopharyngeal swabs, monkeypox virus (MPXV) DNA has been detected in a variety of samples such as saliva, semen, urine and fecal samples. Using the environmental surveillance network previously developed in Spain for the routine wastewater surveillance of SARS-CoV-2 (VATar COVID-19), we have analyzed the presence of MPXV DNA in wastewater from different areas of Spain. Samples (n=312) from 24 different wastewater treatment plants were obtained between May 9 (week 22_19) and August 4 (week 22_31), 2022. Following concentration of viral particles by flocculation, a qPCR procedure allowed us to detect MPXV DNA in 63 wastewater samples collected from May 16 to August 4, 2022, with values ranging between 2.2 per 103 to 8.7 per 104 genome copies (gc)/L. This study shows that MPXV DNA can be reproducibly detected by qPCR in longitudinal samples collected from different Spanish wastewater treatment plants. According to data from the National Epidemiological Surveillance Network (RENAVE) in Spain a total of 6,119 cases have been confirmed as of August 19, 2022. However, and based on the wastewater data, the reported clinical cases seem to be underestimated and asymptomatic infections may be more frequent than expected.
Subject(s)
COVID-19ABSTRACT
Background: Since its first identification in the United Kingdom in late 2020, the highly transmissible B.1.1.7 variant of SARS-CoV-2, become dominant in several European countries raising great concern. Aim: The aim of this study was to develop a duplex real-time RT-qPCR assay to detect, discriminate and quantitate SARS-CoV-2 variants containing one of its mutation signatures, the HV69/70 deletion, to trace the community circulation of the B.1.1.7 variant in Spain through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19). Results: B.1.1.7 variant was first detected in sewage from the Southern city of Malaga (Andalucia) in week 20_52, and multiple introductions during Christmas holidays were inferred in different parts of the country, earlier than clinical epidemiological reporting by the local authorities. Wastewater-based B.1.1.7 tracking showed a good correlation with clinical data and provided information at the local level. Data from WWTPs which reached B.1.1.7 prevalences higher than 90% for at least 2 consecutive weeks showed that 8.1+/-1.8 weeks were required for B.1.1.7 to become dominant. Conclusion: The study highlights the applicability of RT-qPCR-based strategies to track specific mutations of variants of concern (VOCs) as soon as they are identified by clinical sequencing, and its integration into existing wastewater surveillance programs, as a cost-effective approach to complement clinical testing during the COVID-19 pandemic.
Subject(s)
COVID-19ABSTRACT
BackgroundDuring the COVID-19 pandemic in 2020, breastfeeding in women positive for SARS-CoV-2 was compromised due to contradictory data regarding potential viral transmission. However, growing evidence confirms the relevant role of breast milk in providing passive immunity by generating and transmitting specific antibodies against the virus. Thus, our study aimed to develop and validate a specific protocol to detect SARS-CoV-2 in breast milk matrix as well as to determine the impact of maternal SARS-CoV-2 infection on presence, concentration, and persistence of specific SARS-CoV-2 antibodies. Study design/MethodsA prospective multicenter longitudinal study in Spain was carried out from April to December 2020. A total of 60 mothers with SARS-CoV-2 infection and/or recovered from COVID-19 were included (n=52 PCR-diagnosed and n=8 seropositive). Data from maternal-infant clinical records and symptomatology were collected. A specific protocol was validated to detect SARS-CoV-2 RNA in breast milk, targeting the N1 region of the nucleocapsid gene and the envelope (E) gene. Presence and levels of SARS-CoV-2 specific immunoglobulins (Igs) -IgA, IgG, and IgM-in breast milk samples from COVID-19 patients and from 13 women before the pandemic were also evaluated. ResultsAll breast milk samples showed negative results for SARS-CoV-2 RNA presence. We observed high intra- and inter-individual variability in the antibody response to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein for each of the three isotypes IgA, IgM and IgG. Protease domain (MPro) antibodies were also detected in milk. In general, 82.9 % of the milk samples were positive for at least one of the three antibody isotypes, being 52.86 % of those positive for all three Igs. Positivity rate for IgA was relatively stable over time (65.2 - 87.5 %), whereas it raised continuously for IgG (47.8 % the first ten days to 87.5 % from day 41 up to day 206 post-PCR confirmation). ConclusionsConsidering the lack of evidence for SARS-CoV-2 transmission through breast milk, our study confirms the safety of breastfeeding practices and highlights the relevance of virus-specific SARS-CoV-2 antibody transfer, that would provide passive immunity to breastfed infants and protect them against COVID-19 disease. This study provides crucial data to support official breastfeeding recommendations based on scientific evidence.
Subject(s)
COVID-19ABSTRACT
Background: Isolation, contact tracing and restrictions on social movement are being globally implemented to prevent and control onward spread of SARS-CoV-2, even though the infection risk modelled on RNA detection by RT-qPCR remains biased as viral shedding and infectivity are not discerned. Thus, we aimed to develop a rapid viability RT-qPCR procedure to infer SARS-CoV-2 infectivity in clinical specimens and environmental samples. Methods: We screened monoazide dyes and platinum compounds as viability molecular markers on five SARS-CoV-2 RNA targets. A platinum chloride-based viability RT-qPCR was then optimized using genomic RNA, and inactivated SARS-CoV-2 particles inoculated in buffer, stool, and urine. Our results were finally validated in nasopharyngeal swabs from persons who tested positive for COVID-19 and in wastewater samples positive for SARS-CoV-2 RNA. Findings: We established a rapid viability RT-qPCR that selectively detects potentially infectious SARS-CoV-2 particles in complex matrices. In particular, the confirmed positivity of nasopharyngeal swabs following the viability procedure suggests their potential infectivity, while the complete prevention of amplification in wastewater indicated either non-infectious particles or free RNA. Interpretation: The viability RT-qPCR approach provides a more accurate ascertainment of the infectious viruses detection and it may complement analyses to foster risk-based investigations for the prevention and control of new or re-occurring outbreaks with a broad application spectrum. Fundings: This work was supported by Spanish Scientific Research Council (CSIC), Generalitat Valenciana, and MICINN co-founded by AEI/FEDER, UE.
Subject(s)
COVID-19 , Severe Acute Respiratory SyndromeABSTRACT
The use of SARS-CoV-2 metagenomics in wastewater can allow the detection of variants circulating at community level. After comparing with clinical databases, we identified three novel variants in the spike gene, and six new variants in the spike detected for the first time in Spain. We finally support the hypothesis that this approach allows the identification of unknown SARS-CoV-2 variants or detected at only low frequencies in clinical genomes.
ABSTRACT
ObjectivesNear-source tracking of SARS-CoV-2 RNA in the sewage drains serving particular buildings may allow rapid identification of SARS-CoV-2 infection cases or local outbreaks. In this pilot study, we investigated whether this was the case for nursing homes (NH). MethodsThe study involved five NH (from A to E) affiliated to the Clinico-Malvarrosa Health Department, Valencia (Spain). These were nursing or mixed nursing/care homes of different sizes, altogether providing care for 472 residents attended by a staff of 309. Near-source sewage samples were screened for presence of SARS-CoV-2 RNA by RT-qPCR at least 5 days per week during the study period. SARS-CoV-2 RNA testing in nasopharyngeal swabs from residents and staff was performed with the TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific, Massachusetts, USA). ResultsSARS-CoV-2 RNA was detected in wastewater samples from four of the five NH. SARS-CoV-2 infection cases were documented in three of these four NH. Of the two NH without SARS-CoV-2 infection cases, no SARS-CoV-2 RNA was detected in sewer samples from one facility, while it was repeatedly detected in samples from the other. Presence of SARS-CoV-2 RNA in sewage preceded identification of isolated cases among residents or staff or outbreak declaration in two NH, with lag times ranging from 5 to 19 days. ConclusionOur study demonstrated that intermittent or persistent detection of SARS-CoV-2 RNA in NH sewers can provide an early warning of subsequent individual cases or outbreaks in these facilities.
Subject(s)
COVID-19ABSTRACT
Background: The COVID-19 disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a rapidly emerging pandemic which has enforced extreme containment measures worldwide. In the absence of a vaccine or efficient treatment, cost-effective epidemiological surveillance strategies are urgently needed. Methods: Here, we have used RT-qPCR for SARS-CoV-2 detection in a series of longitudinal metropolitan wastewaters samples collected during the earliest stages of the epidemic in the Region of Valencia, Spain. Results: We were able to consistently detect SARS-CoV-2 RNA in samples taken when communicated cases in that region were only incipient. We also find that the wastewater viral RNA context increased rapidly and anticipated the subsequent ascent in the number of declared cases. Interpretation: Our results strongly suggest that the virus was undergoing community transmission earlier than previously believed, and show that wastewater analysis is a sensitive and cost-effective strategy for COVID-19 epidemiological surveillance. Routine implementation of this surveillance tool would significantly improve our preparedness against new or re-occurring viral outbreaks.
Subject(s)
COVID-19 , Crohn DiseaseABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 200,000 reported COVID-19 cases in Spain resulting in more than 20,800 deaths as of April 21, 2020. Faecal shedding of SARS-CoV-2 RNA from COVID-19 patients has extensively been reported. Therefore, we investigated the occurrence of SARS-CoV-2 RNA in six wastewater treatments plants (WWTPs) serving the major municipalities within the Region of Murcia (Spain), the area with the lowest COVID-19 prevalence within Iberian Peninsula. Firstly, an aluminum hydroxide adsorption-precipitation concentration method was tested using a porcine coronavirus (Porcine Epidemic Diarrhea Virus, PEDV) and mengovirus (MgV). The procedure resulted in average recoveries of 10.90 and 10.85% in influent water and 3.29 and 6.19 in effluent water samples for PEDV and MgV, respectively. Then, the method was used to monitor the occurrence of SARS-CoV-2 from March 12 to April 14, 2020 in influent, secondary and tertiary effluent water samples. By using the real-time RT-PCR (RT-qPCR) Diagnostic Panel validated by US CDC that targets three regions of the virus nucleocapsid (N) gene, we estimated quantification of SARS-CoV-2 RNA titers in untreated wastewater waters of 5.38 log genomic copies/L on average. Two secondary water samples resulted positive (2 out of 18) and all tertiary water samples tested as negative (0 out 12). This environmental surveillance data were compared to declared COVID-19 cases at municipality level, revealing that SARS-CoV-2 was circulating among the population even before the first cases were reported by local or national authorities in many of the cities where wastewaters have been sampled. The detection of SARS-CoV-2 in wastewater in early stages of the spread of COVID-19 highlights the relevance of this strategy as an early indicator of the infection within a specific population. At this point, this environmental surveillance could be implemented by municipalities right away as a tool, designed to help authorities to coordinate the exit strategy to gradually lift its coronavirus lockdown.