Urban wastewater is a direct by-product of human activities in urban environments, with its composition reflecting the occurrence and levels of microbiological, chemical, and physical pollutants in the population. In recent years, growing evidence supports the use of WWS as an indicator for the presence and detection of circulating pathogens within the community [2, 32]. Furthermore, urban wastewater serves as a reservoir and source for AMR, making monitoring AMR in wastewater a crucial tool to comprehend the burden, transmission, and persistence of AMR in society [33].
The dual purpose of the two surveys was to assess the current state of WWS practices mainly in Europe and to pinpoint gaps and limitations in existing methodologies and practices. Additionally, the surveys aimed to evaluate the specific needs of laboratories to address these gaps, and to anticipate future challenges within the WWS community.
Notably, the primary focus of WWS in Europe revolves around SARS-CoV-2 monitoring, with nearly all participating laboratories engaged in this task. This underscores the concerted efforts directed towards efficient SARS-CoV-2 surveillance to mitigate the negative effects of the pandemic.
During the COVID-19 pandemic, the use of WWS for SARS-CoV-2 and its variants has emerged as a powerful independent and complementary tool to clinical epidemiology, providing valuable insights into the virus's presence and circulation within a certain community. Several laboratories have shown that the detection of new SARS-CoV-2 variants in wastewater samples precedes its identification at the clinical level [17, 34]. This emphasises the role of WWS as a proactive and early warning system, offering valuable insights for both the ongoing monitoring of SARS-CoV-2 and the timely identification of new viral variants within the population [14].
Numerous assays have been published in the scientific literature for the detection of SARS-CoV-2 in wastewater, and a number of papers evaluate the efficacy of different procedures applied in the analytical workflow [31, 35–40]. Additionally, the evolving nature of the virus poses a challenge as certain variants may elude detection by current assays or acquired viral genomic mutations influence assay performance [14]. Therefore, performance of the assays needs to be continuously verified and new assays eventually developed.
The surveys reveal that most of the participants have implemented an extensive arrays of quality control measures to guarantee the efficient execution of their workflows. This aligns well with the fact that many of the replies were received by national laboratories involved in SARS-CoV-2 surveillance programs. Most of these laboratories hold ISO accreditation (e.g. ISO/IEC 17025, ISO 15189), indicating the integration of general procedures into their workflows to maintain high-quality standards in their operations and services. This commitment to quality control ensures the reliability and accuracy of laboratory processes, contributing to the overall integrity of the generated results. Such commitment is further evidenced by participants' declaration on method validation specific to their context.
However, monitoring SARS-CoV-2 in wastewater systems poses significant challenges [41] [42]. Despite extensive research efforts, a lack of standardised methods for viral RNA concentration, extraction, and quantification in WWS of SARS-CoV-2 complicates the comparison of data among laboratories. The absence of a uniform approach has led to variations in results, as observed in inter-laboratory comparisons conducted by various groups [41] [42], with no single explanation accounting for such discrepancies. This lack of standardisation limits the effectiveness and reliability of WWS for SARS-CoV-2 monitoring [43]. The results of the surveys confirm this variability at large scale, considering not only academia and industry approaches but also the different methodologies used by national laboratories. Enhancing coordination, communication and fostering collaboration between (cross-border) laboratories may facilitate the exchange of experiences and identification of best practices.
The lack of standardised protocols and harmonised quality assurance and quality control (QA/QC) procedures, specifically underscores the necessity for certified reference materials in this domain. The development and utilization of these certified reference materials is an important requirement towards establishing essential common benchmarks for ensuring precision, reliability, and consistency of results across various laboratories and studies.
While laboratories currently employ control materials, their use tends to be fragmented, heterogeneous, and limited to specific steps within the analytical workflow. Indeed, the survey results highlight the absence of a current consensus on the optimal WWS normalization parameters e.g. to control the RNA extraction step and/or foecal content needed to establish even more robust correlations between SARS-CoV-2 WWS data and COVID-19 clinical case numbers. The use of surrogate viruses comes also with its weaknesses, as these viruses do not always resemble the physical and chemical characteristics of SARS-CoV-2 [44]. This reveals a critical gap and an urgent need for improvement, particularly in the development of reference materials certified for copy numbers concentration and sequence identity, ideally closely related to the virus target. The introduction of such materials, as spikes at the initial stages of the process, could significantly enhance the accuracy of pathogen recovery rate calculation. The use of faecal controls would also ensures accurate interpretation and comparison of data, accounting for variations in the composition of the matrix and providing more reliable insights into viral presence and trends.
It was also observed (with few exception) that participating laboratories did not engage in PT exercises for SARS-CoV-2 surveillance. The limited participation in PT exercises for SARS-CoV-2 surveillance may be attributed to the lack of organised PT initiatives specifically tailored for laboratories involved in WWS. This highlights the necessity of organising PTs to assess laboratory performance, identify potential gaps, uncover best practices, and foster the continual improvement of laboratory capabilities.
The utility of WWS extends beyond COVID-19, as it has successfully aided in the monitoring and management of various infectious diseases, including Hepatitis A, Hepatitis E, and Polio [2]. In addition, wastewater serves as an important tool for the analysis of AMR patterns, providing valuable insights into the prevalence and evolution of antimicrobial-resistant microorganisms within communities [26, 30, 45].
Looking ahead, prioritising AMR monitoring becomes imperative, aligning with the WHO Global Action Plan on AMR. This includes monitoring drug resistance in fungi, an aspect not yet explored according to survey results. This initiative is closely intertwined with the overarching goal of reducing or optimising the use of antimicrobial medicines in both human and animal health. In line with these global efforts, the Council Recommendation, on stepping up EU actions to combat AMR, adopted in June 2023, sets ambitious targets for 2030 [29]. These targets include a 20% reduction in the total consumption of antibiotics in humans, illustrating the EU's intensified efforts and commitment to combating AMR.
Recognised as one of the top 10 global public health threats by WHO [25, 46], AMR emerges prominently in the survey responses as a major concern for the future. However, the survey results reveal that AMR analysis in the wastewater community is still in its initial stages, involving less than 40% of the laboratories in this study and focus mainly on beta-lactam resistant gram-negative bacteria, which are categorised as priority by WHO [45]. In agreement with recent reports [30, 33, 47], the results of our surveys confirm knowledge gaps in AMR surveillance, as lack of standardised protocols and lack of standard monitoring targets and reference materials. Despite the advantages of wastewater-based AMR surveillance to offer a community-wide perspective, there is a clear need for improvement [33]. Addressing these deficiencies requires specific actions across various domains through a 'One Health' approach—a collaborative, trans-disciplinary, and multi-sectoral strategy promoting cooperation across human health, animal health, agrifood systems, and the environment as recently stated in the United Nations Environment Programme [27] and recommended by the European Council [28]. Researchers and public health professionals should work together to identify the microorganisms requiring control. This may involve the selection of common AMR markers and the establishment of acceptable concentration limits. These concerted efforts should provide clear guidance for effective intervention measures, ultimately mitigating the risks posed by specific pathogens to the community.
As observed for SARS-CoV-2, participants in our surveys express a critical need to enhance the accuracy of their results, with 80% acknowledging this imperative. A potential solution, identified by participants, is the use of reference materials, and the survey results indicate that whole organism reference materials or nucleic acid reference materials certified for their sequence and concentrations are the preferred choices among laboratories. Harmonized results among laboratories analysing wastewater samples are crucial to align temporal trends, ensuring quantitative data expressed in a uniform unit. This standardization is pivotal for establishing a comprehensive EU overview, especially in the face of emerging viruses, enabling effective monitoring and response across diverse laboratories and countries.
In conclusion, the insights gained from these surveys are expected to guide future directions in WWS, highlighting the importance of continued innovation and standardisation in the field. Moving forward in a post-COVID era, the lessons learned and the knowledge acquired will undoubtedly play a crucial role in shaping the development of resilient and robust public health surveillance systems, capable of detecting a wide range of pathogens and AMR in wastewater. High quality, comprehensive and real-time surveillance data are needed to support the use of WWS for regulatory purposes. This, in turn, will enhance our ability to respond to future public health challenges with greater speed and precision.