Wastewater-based surveillance for tracing the circulation of Dengue and Chikungunya viruses

Background Arboviral diseases, transmitted by infected arthropods, pose significant economic and societal threats. Their global distribution and prevalence have increased in recent years, driven by factors such as climate change, biodiversity loss and urbanization. These diseases are often underestimated due to uneven surveillance and asymptomatic cases. Current surveillance relies on monitoring vector occurrence and spatial distribution, as well as syndromic monitoring. In this work, we aimed to explore the utility of wastewater-based surveillance as an additional, added-value tool for vector-borne viruses tracking. Methods A retrospective wastewater-based surveillance survey was conducted at ten wastewater treatment plants covering a large part of Portugal mainland (North, Lisboa and Vale do Tejo and South regions). Using RT-qPCR, we quantified the RNA concentrations of Dengue (DENV) and Chikungunya (CHIKV) viruses for a period of 12 months ( n = 273 raw wastewater samples), ranging May 2022 - April 2023. Findings DENV was detected in 25% of the samples, with concentrations spanning from 3·5 to 6·8 log copies/L. CHIKV was detectable in 11% of the samples, with concentrations up to 6·3 log copies/L. Notably, the occurrence of DENV and CHIKV was rather similar between the three regions. The Lisboa and Vale do Tejo region exhibited in general the highest median concentration for DENV and CHIKV following normalization with crAssphage (1·5 × 10-4 and 1·1 × 10-3, respectively). Interpretation We demonstrate the efficacy of wastewater-based surveillance as a potent tool for gauging the epidemiological landscape of both DENV and CHIKV in mainland Portugal, where autochthonous cases have not been detected yet. Therefore, WBS should be integrated as a supplementary component to standard surveillance strategies. Evidence before this study Wastewater serves as a valuable resource for wastewater-based surveillance (WBS). This surveillance method involves analyzing biomarkers of human metabolism, activities and lifestyle in wastewater to gain insights into public health trends. Previously, WBS has been employed to track chemical substances like illicit drugs, including cocaine, as well as oseltamivir during the 2009 influenza pandemic. It has also played a crucial role in the global polio eradication program by contributing to assess poliovirus circulation in populations and evaluating immunization effectiveness. During the SARS-CoV-2 pandemic, there was a significant boost in wastewater-based surveillance. To the best of our knowledge, our study represents the first extensive application of this approach to comprehensively investigate and identify the presence of vector-borne viruses RNA such as chikungunya and dengue. In December 2022, we conducted a search on PubMed and Google Scholar for publications using the search terms “wastewater” and “dengue virus” or “chikungunya virus” or “West Nile virus” or “Zika virus”, encompassing manuscripts in all languages. Our search yielded only one study that focused on detecting arboviruses in wastewater, specifically Dengue. Surprisingly, the authors were unable to detect viral RNA despite the reported cases of Dengue between 2017 and 2019, when the wastewater samples were collected. This period included a major outbreak in 2019, with more than 7,000 confirmed cases. Remarkably, no other study has shed light on the presence and concentration of Dengue and Chikungunya viral RNA in wastewater. Added value of this study In response to the gap identified in the literature review, this study makes a significant contribution by reporting for the first time the presence and concentration of arboviral RNA, specifically focusing on the Dengue and Chikungunya viruses. Notably, it has demonstrated the circulation of these viruses, even when only a few travel-associated cases have been reported throughout the years and where no autochthonous have been reported within the community, although the vector ( Aedes albopictus ) has been identified in several occasions in mainland Portugal. This research underscores the vital role of wastewater-based surveillance in gaining insights into the circulation trends of underreported pathogens within communities at the sewershed level. Implications of the available information Arboviral infections are significantly underreported, especially in non-endemic countries, and they can lead from mild to severe health issues and even mortality. The symptoms are unspecific and resemble those of various other illnesses, such as Influenza and COVID-19. Consequently, many infected individuals may not seek medical assistance, and when they do, their infections might be misdiagnosed. This results in surveillance for arboviral infections being biased toward individuals presenting with unusual symptoms or severe illness, or returning from endemic regions, leading to delayed reporting and limited temporal and spatial accuracy. In contrast, wastewater surveillance provides results within 24 hours of sample collection and offers a representation of the population served by the sewer system. This includes asymptomatic individuals and those with mild symptoms who may not seek medical care. This approach increases the likelihood of detecting viral circulation and thus prompt public health intervention, namely through vector control, limiting the potential transmission to new hosts, breaking the cycle of transmission. Wastewater surveillance data can play a crucial role in informing public health and environmental decision-making. It can reveal the presence of unaccounted-for infections, indicating the need for control and mitigation strategies, both at the population and mosquito vector levels. Although the arboviral activity is assumed residual in Portugal, wastewater monitoring can be used in complement to the efforts that are already done under the National Vector Surveillance Network. Moreover, this data can be used to communicate with the general public, encouraging them to take actions that can help control the mosquito population, such as reducing stagnant water sources where mosquitoes lay their eggs. This is especially important for vulnerable populations, enabling them to make more informed decisions to mitigate the underlying risks effectively. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by the European Union through the Emergency Support Instrument [Support to the Member States to establish national systems, local collection points, and digital infrastructure for monitoring Covid19 and its variants in wastewater Portugal; Grant Agreement No. 060701/2021/864489/SUB/ENV.C2] and by Fundacao para a Ciencia e Tecnologia (FCT), through the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR) program, project Surveillance of Emerging Pathogens and Antibiotic Resistances in Aquatic Ecosystems (SARA), grant number Aquatic/0006/2020 ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors