Routine monitoring and wastewater-based bio-surveillance represents a strong supporting tool for following and understanding SARS-CoV-2 endemicity or epidemic dynamics in society. The main objective of the research was a two-year qualitative and quantitative monitoring program of SARS-CoV-2 in sewage in a model urban agglomeration (the capital city), including the development of an algorithm for action in this area and a joint evaluation of procedures with a water and sewage company (public administration sector). To resolve problems, we continuously investigated inflows and outflows of four wastewater treatment plants and raw sewage of the whole catchment (including healthcare units, airport and military areas) within the Warsaw capital city for SARS-CoV-2, during two waves of COVID-19 in 2023 and 2024. The screening was performed by RT-qPCR (N1, N2, E probes) and NGS metagenomics analysis. The average cumulative load of the virus in sewage for the whole tested period was 3.5 × 10⁵ genomic copies per litre. The highest load was found for wastewater inflows and the lowest one was found for housing estates and wastewater outflows. Higher viral concentrations in the wastewater than expected in medical records suggested a higher number of undiagnosed COVID-19 infections within the community. The obtained genotypes segregated within 11 clades (21I to 24 F), making '23 and '24 waves distinguishable. We consider our results and methodological approaches hold significant potential for public health, with particular emphasis on the needs of the Directive (EU) 2024/3019 of the European Parliament and of the Council.

Despite the vast amount of water on Earth, only a small percent is suitable for consumption, and these resources are diminishing. Moreover, water resources are unevenly distributed, leading to significant disparities in access to drinking water between countries and populations. Increasing consumption and the expanding human population necessitate the development of novel wastewater treatment technologies and the use of water treatment byproducts in other areas, such as fertilisers. However, water treatment sludge often cannot be used to enhance crop production due to the presence of parasite eggs, particularly from roundworms (Ascaridae family), which are resistant to environmental factors and can pose a threat for several years. Legislation prohibits the use of sludge containing parasite eggs as fertiliser. In some cases, water may not contain parasite eggs but larvae, which require different detection methods. Additionally, the presence of eggs does not necessarily indicate danger since they may lose infectivity due to prolonged storage or exposure to chemical compounds in the sewage. This paper reviews European Union regulations on wastewater treatment, the selected parasitic diseases related to the presence of parasites in wastewater, the spectrum of detection methods, and highlights differences in viability and invasiveness, which is intended to draw attention to the need to determine both biological properties of parasites.

Objectives: Wastewater-based bio-surveillance is essential for monitoring pathogens in communities. This study examines whether supernatant and the sediment left after centrifuging sewage should be tested separately or together. The goal is to assess how useful sediment information is for making accurate public health decisions. Materials and methods: Total nucleic acids were isolated from wastewater inflows and outflows in Warsaw, including contributions from healthcare facilities, the airport, and residential areas, to detect SARS-CoV-2, ESKAPE pathogens, re-emerging pathogens, alarming pathogens, drug-resistance patterns, and pathotypes during two consecutive waves of COVID-19. Total DNA was extracted from both sewage water and solid phases using vacuum pressure and semi-automated methods. This DNA was then amplified using RT-qPCR and qPCR in in vitro diagnostic (IVD) tests. The analysis focused on the presence of E, N1, and N2 markers for SARS-CoV-2, sequences indicating pathotypes (such as stx , eae , ipaH , elt , and est ), and genome determinants of antibiotic resistance genes in bacteria (including van , bla , mec , SA442/CoA, SCCmec-orfX, and mcr ). Results: A two-year study involving 80 locations in an urban catchment revealed both qualitative and quantitative differences in the phases following centrifugation of nearly 1,000 sewage samples. In 350 grams of sediment and over 60 micrograms of various TNA fractions, we observed frequent positive results in the sediments, while these results were absent in the supernatant (19:7). Conclusion: The careful consideration of post-centrifugation phases simplifies probe selection methods, enhances the accuracy of epidemiological inferences, and enables effective responses to public health challenges, whether during normal operations or times of crisis. Additionally, quickly and accurately determining the approximate number of infected individuals can help prevent the manipulation of epidemiological data.

In the review, the collection of population genetics papers from 1973 to 2025 comprises 400 publications, 81 of which were significant and consulted with representatives from water and sewage companies. Reviewed Proteobacteria (mean H_S = 0.42), Firmicutes (mean H_S = 0.43), Actinobacteria (mean H_S = 0.33), and Spirochaetes (mean H_S = 0.54) represent the 60 species under investigation through the lens of “h” coefficients related to gene diversity and expected heterozygosity. The research also included ESKAPE, emerging pathogens, bacterial indicators of wastewater treatment efficiency, environmental sanitary surveillance and public health. The restoration of the expected heterozygosity for haploids “h” was proposed in wastewater-based epidemiology as an innovative tool for public health. The unique “h” coefficient allows for the comparison of genetic variability in various organisms, regardless of their ploidy, using multiple markers and traits. The parameter represents a noble character for both the variability of phenotypes (proteins) and genotypes (nucleic acids). Leveraging the genetic diversity highlighted by the “h” coefficient can support wastewater-based epidemiology, offering the ability to predict the stages and trajectories of disease outbreaks.

Ticks from the Hyalomma genus have recently garnered public attention in countries in Northern and Central Europe, as they are transported by migratory birds and might have established stable populations due to climate warming. The main threat associated with Hyalomma ticks is their ability to transmit Crimean–Congo hemorrhagic fever virus (CCHFV), which can be fatal in up to 40% of cases. Here, we collected Hyalomma ticks from migratory birds during annual ringing actions in the spring seasons of 2023 and 2024. Four ticks were found on birds from the Acrocephalus genus and two on Hippolais icterina. The ticks were examined for the presence of Rickettsia spp., Babesia microti, Babesia divergens, Coxiella burnetii, Borreliella burgdorferi (s. l.), Anaplasma phagocytophilum, West Nile virus, and CCHF virus (CCHFV). The collected H. rufipes specimens were negative for tested pathogens, except for two ticks collected in 2024, which were positive for Rickettsia aeschlimannii. The data show that Hyalomma ticks are efficiently transported on birds preferring reedbeds or deciduous trees. The possibility of the occurrence of CCHF or rickettsiosis (induced by R. aeschlimannii) is currently assessed as low. Nevertheless, we have shown the transfer of Hyalomma ticks to Poland and indicated the need for careful future epidemiological monitoring of the presence of Hyalomma ticks.

Municipal wastewater catchments are significant sources of symbiotic, opportunistic, and drug-resistant bacteria that can acquire or enhance their resistance. This “re-emergence of threats” presents a serious public health issue, especially during crisis conditions (COVID-19). The aim of this study was to propose a sampling algorithm, identify strategically important sites within the wastewater catchment, expand the range of bacterial indicators, and rapidly confirm potential threats using in vitro diagnostics in both raw and treated sewage. A parallel objective was to evaluate the effectiveness of sequencing methods in pre-epidemic studies. The research was conducted as part of a long-term surveillance program at transportation hubs, healthcare facilities, residential complexes, and wastewater treatment plants in Warsaw, Poland. Total nucleic acid (TNA) was isolated from samples, amplified using qPCR kits, and selected samples underwent next-generation sequencing. In raw sewage, the most frequently detected regions included sequences complementary to the bacterial vanB gene, the Integron Verona-encoded metallo-β-lactamase (VIM) gene, and markers for Mycobacterium tuberculosis. Treated environmental effluents primarily contained DNA fragments complementary to the bacterial vanB sequence, along with genes related to resistance against oxacillin and imipenem, as well as the VIM gene and Legionella sp. 16S rRNA sequencing of the sewage samples revealed the presence of 12 promising bacterial indicators relevant to public health and environmental hygiene. The proposed algorithm aligns with the provisions of EU Directive 2024/3019 and in vitro diagnostic tests demonstrated the potential and utility of rapid screening analyses of wastewater during disruptions or crises, as well as in situations involving equipment shortages and logistical challenges in healthcare. The proposed expanded range of bioindicators could expedite decision-making regarding preventive measures and medical support for various counties, hospitals, and transportation hubs in large urban areas. The sequencing results further emphasized the need to broaden the scope of the bacterial indicators for environmental surveillance.