One of the distinguishing features of this research is its long-term scope, spanning both the acute pandemic phase and the post-pandemic transition period. Most wastewater surveillance studies published to date have focused on relatively short windows, often limited to pandemic waves of SARS-CoV-2, which restricts the ability to draw conclusions about how viral circulation behaves under more ordinary epidemiological conditions. By continuing collection through the period when public health interventions were lifted and society returned to pre-pandemic patterns of contact, the researchers were able to observe the re-establishment of seasonal respiratory virus dynamics that had been dramatically suppressed during 2020 and much of 2021. This before-and-after contrast is scientifically precious because it documents, in a single location with consistent methodology, how the near-total interruption of transmission for viruses such as influenza and respiratory syncytial virus was followed by unusual out-of-season resurgences and subsequently by a gradual return to typical winter seasonality.

The concept of a middle-size city is relevant to the broader applicability of wastewater surveillance. Much of the foundational work in this field has been conducted in large metropolitan areas, where sewersheds serve millions of people and dilution effects are substantial but signal magnitude is high. Smaller cities present a different set of conditions: the contributing population is smaller, which can make signals more sensitive to localized outbreaks but also more variable, and the sewer network characteristics, industrial discharges, and demographic composition differ from those of megacities. Demonstrating that a standardized analytical pipeline can produce interpretable, reproducible data in a middle-size urban setting strengthens the case for deploying such systems across heterogeneous municipalities, which is precisely what many national and regional surveillance programs in Europe now aim to do under frameworks supported by the European Commission and coordinated through initiatives involving public health institutes across member states.

Methodologically, long-term wastewater studies of respiratory viruses must contend with several persistent analytical challenges. Viral RNA in sewage degrades over time depending on temperature, pH, and the presence of nucleases, so normalization strategies are needed to distinguish true changes in viral shedding from artifacts introduced by variable wastewater flow, rainfall dilution, or sample processing efficiency. Common approaches include normalizing to fecal indicators such as human adenovirus or pepper mild mottle virus, or to physicochemical parameters like ammonium concentration and flow volume. Recovery controls, typically spiked surrogate viruses, allow laboratories to estimate extraction efficiency for each sample. The choice of concentration method, whether electronegative membrane filtration, ultrafiltration, or polyethylene glycol precipitation, influences sensitivity for different viruses. Studies that maintain the same protocol over years, as this one did, gain an important advantage: temporal comparisons become more reliable because methodological noise is held constant, allowing genuine epidemiological trends to stand out more clearly.

The multipathogen panel typical of such studies generally includes SARS-CoV-2, influenza A and B viruses, respiratory syncytial virus, and often additional targets such as human metapneumovirus, parainfluenza viruses, seasonal coronaviruses, rhinoviruses, and adenoviruses. Quantitative reverse transcription PCR remains the workhorse detection technology because it provides absolute or relative quantification with well-characterized performance. Multiplexing several assays in a single reaction conserves sample volume and reduces cost, which matters when hundreds of samples are processed over multi-year campaigns. The resulting time series can be analyzed for peak timing, peak height, epidemic onset, and the lead time between wastewater signal and clinical indicators such as hospital admissions or sentinel physician reports. Across many studies, wastewater signals for influenza and RSV have tended to lead or coincide with clinical peaks by roughly one to two weeks, a window that can be operationally meaningful for hospital preparedness, staffing decisions, and the timing of public health communications.

The pandemic-to-post-pandemic transition also offers a natural experiment in viral interference and immune landscape dynamics. During the period of intense SARS-CoV-2 circulation and non-pharmaceutical interventions, the near-disappearance of influenza and RSV created a substantial immunity debt, particularly among children born during those years who had never encountered RSV. When restrictions eased, many countries in the Northern Hemisphere, including Spain, experienced an out-of-season RSV wave in the summer of 2021 and an unusually early and intense influenza and RSV season in late 2022. A wastewater time series that spans these events provides an independent record of how quickly viral circulation rebounded and how the relative timing of different pathogens shifted, information that is difficult to reconstruct from clinical data alone because testing practices for non-COVID respiratory viruses were themselves disrupted during the pandemic.

Another dimension of long-term wastewater data is its potential to capture the emergence and replacement of SARS-CoV-2 variants. Variant-specific assays or sequencing of wastewater samples can reveal the rise of Alpha, Delta, Omicron, and subsequent lineages weeks before genomic surveillance of clinical samples detects the same shifts, simply because wastewater aggregates infections across the whole community without the sampling biases introduced by who gets tested. Even when the primary focus of a study is quantitative viral load rather than lineage tracking, the overall SARS-CoV-2 signal reflects the cumulative effect of variant-driven changes in transmissibility, immune evasion, and shedding kinetics. The post-pandemic period, characterized by the evolution of Omicron sublineages and the transition of COVID-19 toward an endemic, wave-like pattern, is particularly interesting in this respect, as wastewater data can help define whether SARS-CoV-2 settles into winter seasonality similar to influenza or retains a distinct periodicity.

From a public health operations standpoint, the value of a multi-year dataset lies in establishing baselines. A single season of data cannot tell decision-makers whether a given viral load measurement represents a normal winter peak or an anomalous surge. After several years of consistent monitoring, thresholds can be defined empirically, for example as multiples of the median off-season concentration, and these thresholds can trigger predefined responses such as enhanced clinical testing, hospital surge planning, or targeted vaccination campaigns. The European Union’s recommendation in 2023 that member states extend wastewater surveillance beyond SARS-CoV-2 to include other pathogens of concern reflects exactly this logic: sustained, standardized monitoring is what converts raw measurements into actionable intelligence. Studies conducted in individual cities, with fully documented protocols and openly reported concentrations, provide the calibration points that such larger programs depend upon.

It is also worth noting the complementary relationship between wastewater surveillance and clinical sentinel systems. Clinical data provide information that wastewater cannot: which individuals are infected, their age distribution, vaccination status, symptom severity, and the identification of specific strains through patient sampling. Wastewater data, conversely, provide population-level coverage without dependence on healthcare-seeking behavior or testing policy, and they are available even when clinical laboratories scale back routine respiratory panels during off-seasons. Integrating the two streams, for instance by correlating wastewater concentrations with hospitalization rates or by using wastewater to trigger more intensive clinical sampling, generally yields better situational awareness than either source alone. The Spanish middle-size city dataset, by covering both pandemic and post-pandemic phases, illustrates how this integration can be evaluated across very different epidemiological regimes, from emergency-driven mass testing to routine seasonal monitoring.

Finally, the scientific community’s interest in studies of this kind reflects a broader shift in how infectious disease surveillance is conceptualized. Rather than reacting to outbreaks after they become clinically visible, public health authorities increasingly seek leading indicators drawn from environmental monitoring, genomic sequencing, and digital data sources. Wastewater surveillance occupies a central place in this vision because it is relatively inexpensive per capita, technologically accessible to regional laboratories, and demonstrably effective across a growing list of pathogens, including not only respiratory viruses but also enteroviruses, hepatitis A, mpox, and antimicrobial resistance genes. Long-term, single-site studies with consistent methodology, such as the one conducted in this Spanish city, serve as the empirical backbone for this transition, demonstrating that the signals are stable, interpretable, and reproducible over years rather than weeks, and that the infrastructure built during the COVID-19 emergency can be repurposed into durable, routine surveillance capacity capable of informing responses to future epidemic threats.

Subject of Research: Pandemic and post-pandemic dynamics of respiratory viruses in a Spanish middle-size city using a long-term wastewater surveillance

Article Title: Pandemic and post-pandemic dynamics of respiratory viruses in a Spanish middle-size city using a long-term wastewater surveillance

Article References: Casado-Martín, L., Hernández, M., Pérez-Alonso, D., Yeramian, N., Alves-Elois, M., Dorighello-Cadamuro, R., Fongaro, G., Eiros, J. M., & Rodríguez-Lázaro, D. (2026). Pandemic and post-pandemic dynamics of respiratory viruses in a Spanish middle-size city using a long-term wastewater surveillance. npj Viruses. https://doi.org/10.1038/s44298-026-00232-2

Image Credits: AI Generated

DOI: 10.1038/s44298-026-00232-2

Keywords: Pandemic, post-pandemic, dynamics, respiratory, viruses, Spanish, middle-size, city, long-term, wastewater, surveillance, scientific research

The research, led by Selwin G. Samuel, Hsu-Wen Tseng, Bastien Rival, and colleagues including senior author Kylie A. Alexander, builds on a growing body of evidence that inflammation is a central engine of NHO development. Clinicians have long observed retrospective associations between NHO and conditions that inflame the body—smoking, pressure ulcers, pneumonia, urinary tract infections, and polytrauma. Even more intriguingly, heterotopic ossification has been documented in patients with severe COVID-19 who had no central nervous system injury at all, hinting that a sufficiently intense systemic infection might be capable of driving ectopic bone formation on its own. The new work provides the mechanistic backbone for these clinical observations, showing in precise molecular detail how the molecular signatures of pathogens can fan the flames of abnormal bone growth.

The team’s experimental platform is a mouse model in which the spinal cord is surgically transected between the eleventh and thirteenth thoracic vertebrae, followed by a controlled injury to the hamstring muscle induced by cardiotoxin purified from snake venom. In this setting, the injured muscle, deprived of normal neural regulation and flooded with inflammatory signals, becomes fertile ground for ectopic ossification. Previous work by the same group had established a critical piece of the puzzle: lipopolysaccharide, or LPS, a molecular component of the outer membrane of gram-negative bacteria such as E. coli, worsened NHO in mice in a dose-dependent manner. That effect depended on Toll-like receptor 4, or TLR4, and its signaling adaptor TRIF, revealing for the first time that a single bacterial molecule could amplify pathological bone formation through a defined immune receptor pathway.

But LPS is just one molecular signature among many. Pathogens carry an arsenal of conserved molecular structures, collectively called pathogen-associated molecular patterns, or PAMPs, which are detected by pattern recognition receptors, or PRRs, displayed on the surfaces of and within immune and non-immune cells. When a PRR binds its ligand, it triggers downstream inflammatory cascades, notably through the NF-κB and interferon signaling pathways, releasing cytokines that orchestrate the immune response. The research team asked a deceptively simple question: if one bacterial PAMP can worsen NHO, what about the rest of the pathogen world?

The first step was to map which pattern recognition receptors are actually present in the cells that matter. Using flow cytometry to sort individual cell populations from mouse muscle—satellite cells, which regenerate muscle fibers; fibro-adipogenic progenitors, or FAPs, which are the mesenchymal cells that turn into bone in NHO; endothelial cells; and monocytes/macrophages—the researchers quantified messenger RNA for a broad panel of PRRs. The results were revealing. Macrophages expressed essentially the full complement of these sensors. More surprisingly, several receptors, including TLR3, RIG-I, STING, MDA-5, PKR, NOD1, and NOD2, were detectable in every cell type isolated from muscle. In human samples, FAPs isolated from muscles surrounding surgically excised NHO biopsies at Raymond Poincaré Hospital in France expressed TLR4, TLR6, NOD1, and several viral RNA sensors, while CD14-positive blood monocytes carried a complementary set including TLR7, TLR8, TLR9, and the fungal-sensing lectins Dectin-1, Dectin-2, and Mincle. Muscle tissue, in other words, is thoroughly wired to detect infection.

With that receptor map in hand, the team conducted a systematic in vivo screen, injecting purified PAMPs representing bacterial, viral, and fungal signatures into mice undergoing the SCI-plus-muscle-injury procedure, then measuring ectopic bone volumes by micro-computed tomography at seven and twenty-one days after surgery. The outcomes split into two camps. Agonists that activate TLR2-containing receptor complexes—the synthetic lipopeptides Pam3CSK4 and Pam2CSK4, which mimic bacterial lipoproteins, and lipoteichoic acid from gram-positive bacteria—all significantly exacerbated NHO in a dose-dependent fashion, with effects visible as early as day seven. CpG oligodeoxynucleotide ODN1668, which mimics bacterial DNA by activating TLR9, also increased NHO volumes, though its effect emerged more slowly, becoming apparent by day twenty-one.

The exceptions proved equally informative. Flagellin, the protein that forms bacterial tails and activates TLR5 as well as cytosolic NLRC4 and NAIP5 receptors, had no effect on NHO even at the highest dose mice could tolerate. The study’s title captures this nuance precisely: many, but not all, pathogen-associated molecular patterns aggravate neurogenic heterotopic ossification. This selectivity suggests that the pro-ossification signal is not simply generic inflammation, but a specific receptor-driven program that certain microbial signatures engage and others do not.

To understand the cellular machinery connecting infection to bone, the researchers turned to in vitro systems. When PAMPs were applied directly to purified FAPs, most had little effect on calcium mineralization, the laboratory readout of bone formation. But when macrophages were stimulated with PAMPs and their conditioned medium was transferred to FAP cultures, mineralization surged. The message was clear: macrophages act as intermediaries, translating the molecular language of pathogens into signals that directly reprogram muscle progenitor cells toward an osteoblastic fate. The team pinpointed two key messengers in that translation: the inflammatory cytokines interleukin-1 and oncostatin M. When human FAPs were treated with conditioned medium from PAMP-stimulated human CD14-positive monocytes, blocking the interleukin-1 receptor with an antagonist, or neutralizing oncostatin M with a specific monoclonal antibody, markedly reduced the calcium mineralization and expression of RUNX2, the master transcription factor of osteogenic differentiation. Both IL-1 and OSM had previously been implicated in NHO in the group’s earlier work; the new study cements them as convergent, druggable endpoints of PAMP-driven signaling.

The clinical implications are substantial. SCI patients are disproportionately vulnerable to infections—pneumonia and urinary tract infections are among the most common complications of paralysis, and the study notes that the animal model itself requires prophylactic antibiotics and manual bladder expression to prevent urosepsis. The findings suggest that a bacterial lung infection or a fungal urinary colonization during the critical early weeks after injury could biochemically amplify the deposition of ectopic bone in the very muscles surrounding immobilized joints. They may also explain why retrospective cohorts consistently report higher NHO prevalence in patients with infections, pressure ulcers, and tracheostomies, and why heterotopic bone appeared in intubated, severely inflamed COVID-19 patients without any central nervous system injury.

Beyond explaining risk, the work opens potential therapeutic avenues. If TLR2 signaling is a major driver, then TLR2 inhibitors—such as the compound C29 used in the mouse experiments—might blunt NHO exacerbation during infections. More immediately, the confirmation that IL-1 and oncostatin M sit at the convergence point of multiple PAMP pathways strengthens the case for targeting these cytokines. Interleukin-1 receptor antagonists are already approved drugs for autoinflammatory diseases, and neutralizing antibodies against oncostatin M are in clinical development for other inflammatory conditions, meaning repurposing trials for NHO prevention are at least conceivable. Because the only reliable preventive measure currently available is cyclooxygenase inhibition, based on retrospective evidence alone, the field is clearly in need of mechanism-based interventions.

The study also carries a caveat worth noting: the mouse experiments used female animals, in part because manual bladder expression is easier and safer in females, and infection-associated NHO risk in humans will need direct clinical validation. Still, by systematically surveying the pathogen landscape rather than a single molecule, the researchers have transformed NHO from an inexplicable complication into a process with identifiable immune triggers, intermediate cell types, and downstream cytokine effectors. For the hundreds of thousands of people worldwide living with spinal cord injury, that molecular clarity is the first step toward keeping their joints—and their independence—from being locked away by bone that should never have formed.