Target enrichment metaviromics enables comprehensive surveillance of coronaviruses in environmental and animal samples.

The COVID-19 pandemic has underscored the importance of understanding the role of animals in the transmission of coronaviruses (CoVs) and their impact on human health. A One Health approach, integrating human, animal, and environmental health, is essential for effective CoVs control. Next-generation sequencing has played a pivotal role in identifying and monitoring the evolution of novel CoVs strains, like SARS-CoV-2. However, viral occurrence and diversity studies in environmental and animal samples are challenging because of the complexity of viral communities and low abundance of viruses in these samples. Target enrichment sequencing (TES) has emerged as a valuable tool for investigating viral families in challenging samples. This approach involves the specific capture and enrichment of viral genomes using sequence-specific probes, thereby enhancing the efficiency of detection and characterization. In this study, we aimed to develop and validate a TES panel to study CoVs in various complex environmental and animal derived samples. The results demonstrated the panel's effectiveness in capturing and sequencing a wide diversity of CoVs providing valuable insights into their abundance and host diversity in urban wastewater, farm animal corpses lixiviates and bat guano samples. In sewage samples, CoVs were detected solely when TES was employed while in guano samples, sequencing of CoVs species was achieved in 2 out of 4 samples showing an almost three-logarithmic increase in the number of reads obtained in comparison with the untargeted approach. For animal lixiviates, only the TES application enabled the acquisition of CoVs reads. The information obtained can significantly contribute to early detection, surveillance, and control measures for CoVs, including viral discovery and potential spillover events. Additionally, this sequencing panel shows potential for studying other significant viral families and monitoring viral diversity in different animal populations.

Exploring viral contamination in urban groundwater and runoff.

The reliance of the global population on urban aquifers is steadily increasing, and urban aquifers are susceptible to pathogenic contamination through sources such as sewer leakage or urban runoff. However, there is insufficient monitoring of groundwater quality in urban areas. In this study, quantitative polymerase chain reaction (qPCR) was employed to evaluate the presence of human fecal viral indicators and viral pathogens in urban wastewater (n = 13) and groundwater (n = 12) samples from four locations in Barcelona with different degrees of urbanization, as well as in runoff samples (n = 2). Additionally, a target enrichment sequencing (TES) approach was utilized to explore the viral diversity within groundwater and runoff samples, offering insights into viral contamination and potential virus transmission routes in urban areas. Human adenovirus (HAdV) was identified in all wastewater samples, 67 % (8/12) of groundwater samples, and one runoff sample by qPCR indicating human viral fecal contamination. The viral pathogen Norovirus genogroup GI (NoV GI) was detected in wastewater and two winter groundwater samples from highly and medium urbanized areas. NoV genogroup GII (NoV GII), Enterovirus (EV) and SARS-CoV-2 were exclusively detected in wastewater. Human and other vertebrate viruses were detected in groundwater and runoff samples using TES. This study gives insights about the virome present in urban water sources, emphasizing the need for thorough monitoring and deeper understanding to address emerging public health concerns.

Cognitive profile in multiple sclerosis and post-COVID condition: a comparative study using a unified taxonomy.

Post-COVID condition (PCC) and multiple sclerosis (MS) share some clinical and demographic features, including cognitive symptoms and fatigue. Some pathophysiological mechanisms well-known in MS, such as autoimmunity, neuroinflammation and myelin damage, have also been implicated in PCC. In this study, we aimed to compare the cognitive phenotypes of two large cohorts of patients with PCC and MS, and to evaluate the relationship between fatigue and cognitive performance. Cross-sectional study including 218 patients with PCC and 218 with MS matched by age, sex, and years of education. Patients were evaluated with a comprehensive neuropsychological protocol and were categorized according to the International Classification of Cognitive Disorders system. Fatigue and depression were also assessed. Cognitive profiles of PCC and MS largely overlapped, with a greater impairment in episodic memory in MS, but with small effect sizes. The most salient deficits in both disorders were in attention and processing speed. The severity of fatigue was greater in patients with PCC. Still, the correlations between fatigue severity and neuropsychological tests were more prominent in the case of MS. There were no differences in the severity of depression among groups. Our study found similar cognitive profiles in PCC and MS. Fatigue was more severe in PCC, but was more associated with cognitive performance in MS. Further comparative studies addressing the mechanisms related to cognitive dysfunction and fatigue may be of interest to advance the knowledge of these disorders and develop new therapies.

Assessing environmental exposure to viruses in wastewater treatment plant and swine farm scenarios with next-generation sequencing and occupational risk approaches.

Occupational exposure to pathogens can pose health risks. This study investigates the viral exposure of workers in a wastewater treatment plant (WWTP) and a swine farm by analyzing aerosol and surfaces samples. Viral contamination was evaluated using quantitative polymerase chain reaction (qPCR) assays, and target enrichment sequencing (TES) was performed to identify the vertebrate viruses to which workers might be exposed. Additionally, Quantitative Microbial Risk Assessment (QMRA) was conducted to estimate the occupational risk associated with viral exposure for WWTP workers, choosing Human Adenovirus (HAdV) as the reference pathogen. In the swine farm, QMRA was performed as an extrapolation, considering a hypothetical zoonotic virus with characteristics similar to Porcine Adenovirus (PAdV). The modelled exposure routes included aerosol inhalation and oral ingestion through contaminated surfaces and hand-to-mouth contact. HAdV and PAdV were widespread viruses in the WWTP and the swine farm, respectively, by qPCR assays. TES identified human and other vertebrate viruses WWTP samples, including viruses from families such as Adenoviridae, Circoviridae, Orthoherpesviridae, Papillomaviridae, and Parvoviridae. In the swine farm, most of the identified vertebrate viruses were porcine viruses belonging to Adenoviridae, Astroviridae, Circoviridae, Herpesviridae, Papillomaviridae, Parvoviridae, Picornaviridae, and Retroviridae. QMRA analysis revealed noteworthy risks of viral infections for WWTP workers if safety measures are not taken. The probability of illness due to HAdV inhalation was higher in summer compared to winter, while the greatest risk from oral ingestion was observed in workspaces during winter. Swine farm QMRA simulation suggested a potential occupational risk in the case of exposure to a hypothetical zoonotic virus. This study provides valuable insights into WWTP and swine farm worker's occupational exposure to human and other vertebrate viruses. QMRA and NGS analyses conducted in this study will assist managers in making evidence-based decisions, facilitating the implementation of protection measures, and risk mitigation practices for workers.

Wastewater-based epidemiology applied at the building-level reveals distinct virome profiles based on the age of the contributing individuals.

BACKGROUND: Human viruses released into the environment can be detected and characterized in wastewater. The study of wastewater virome offers a consolidated perspective on the circulation of viruses within a population. Because the occurrence and severity of viral infections can vary across a person's lifetime, studying the virome in wastewater samples contributed by various demographic segments can provide valuable insights into the prevalence of viral infections within these segments. In our study, targeted enrichment sequencing was employed to characterize the human virome in wastewater at a building-level scale. This was accomplished through passive sampling of wastewater in schools, university settings, and nursing homes in two cities in Catalonia. Additionally, sewage from a large urban wastewater treatment plant was analysed to serve as a reference for examining the collective excreted human virome. RESULTS: The virome obtained from influent wastewater treatment plant samples showcased the combined viral presence from individuals of varying ages, with astroviruses and human bocaviruses being the most prevalent, followed by human adenoviruses, polyomaviruses, and papillomaviruses. Significant variations in the viral profiles were observed among the different types of buildings studied. Mamastrovirus 1 was predominant in school samples, salivirus and human polyomaviruses JC and BK in the university settings while nursing homes showed a more balanced distribution of viral families presenting papillomavirus and picornaviruses and, interestingly, some viruses linked to immunosuppression. CONCLUSIONS: This study shows the utility of building-level wastewater-based epidemiology as an effective tool for monitoring the presence of viruses circulating within specific age groups. It provides valuable insights for public health monitoring and epidemiological studies.

Unraveling brain fog in post-COVID syndrome: Relationship between subjective cognitive complaints and cognitive function, fatigue, and neuropsychiatric symptoms.

BACKGROUND AND PURPOSE: "Brain fog" is a frequent and disabling symptom that can occur after SARS-CoV-2 infection. However, its clinical characteristics and the relationships among brain fog and objective cognitive function, fatigue, and neuropsychiatric symptoms (depression, anxiety) are still unclear. In this study, we aimed to examine the characteristics of brain fog and to understand how fatigue, cognitive performance, and neuropsychiatric symptoms and the mutual relationships among these variables influence subjective cognitive complaints. METHODS: A total of 170 patients with cognitive complaints in the context of post-COVID syndrome were evaluated using a comprehensive neuropsychological protocol. The FLEI scale was used to characterize subjective cognitive complaints. Correlation analysis, regression machine-learning algorithms, and mediation analysis were calculated. RESULTS: Cognitive complaints were mainly attention and episodic memory symptoms, while executive functions (planning) issues were less often reported. The FLEI scale, a mental ability questionnaire, showed high correlations with a fatigue scale and moderate correlations with the Stroop test, and anxiety and depressive symptoms. Random forest algorithms showed an R2 value of 0.409 for the prediction of FLEI score, with several cognitive tests, fatigue and depression being the best variables used in the prediction. Mediation analysis showed that fatigue was the main mediator between objective and subjective cognition, while the effect of depression was indirect and mediated through fatigue. CONCLUSIONS: Brain fog associated with COVID-19 is mainly characterized by attention and episodic memory, and fatigue, which is the main mediator between objective and subjective cognition. Our findings contribute to understanding the pathophysiology of brain fog and emphasize the need to unravel the main mechanisms underlying brain fog, considering several aspects.

Hippocampal subfield abnormalities and biomarkers of pathologic brain changes: from SARS-CoV-2 acute infection to post-COVID syndrome.

BACKGROUND: Cognitive deficits are among the main disabling symptoms in COVID-19 patients and post-COVID syndrome (PCS). Within brain regions, the hippocampus, a key region for cognition, has shown vulnerability to SARS-CoV-2 infection. Therefore, in vivo detailed evaluation of hippocampal changes in PCS patients, validated on post-mortem samples of COVID-19 patients at the acute phase, would shed light into the relationship between COVID-19 and cognition. METHODS: Hippocampal subfields volume, microstructure, and perfusion were evaluated in 84 PCS patients and compared to 33 controls. Associations with blood biomarkers, including glial fibrillary acidic protein (GFAP), myelin oligodendrocyte glycoprotein (MOG), eotaxin-1 (CCL11) and neurofilament light chain (NfL) were evaluated. Besides, biomarker immunodetection in seven hippocampal necropsies of patients at the acute phase were contrasted against eight controls. FINDINGS: In vivo analyses revealed that hippocampal grey matter atrophy is accompanied by altered microstructural integrity, hypoperfusion, and functional connectivity changes in PCS patients. Hippocampal structural and functional alterations were related to cognitive dysfunction, particularly attention and memory. GFAP, MOG, CCL11 and NfL biomarkers revealed alterations in PCS, and showed associations with hippocampal volume changes, in selective hippocampal subfields. Moreover, post mortem histology showed the presence of increased GFAP and CCL11 and reduced MOG concentrations in the hippocampus in post-mortem samples at the acute phase. INTERPRETATION: The current results evidenced that PCS patients with cognitive sequalae present brain alterations related to cognitive dysfunction, accompanied by a cascade of pathological alterations in blood biomarkers, indicating axonal damage, astrocyte alterations, neuronal injury, and myelin changes that are already present from the acute phase. FUNDING: Nominative Grant FIBHCSC 2020 COVID-19. Department of Health, Community of Madrid. Instituto de Salud Carlos III through the project INT20/00079, co-funded by European Regional Development Fund "A way to make Europe" (JAMG). Instituto de Salud Carlos III (ISCIII) through Sara Borrell postdoctoral fellowship Grant No. CD22/00043) and co-funded by the European Union (MDC). Instituto de Salud Carlos III through a predoctoral contract (FI20/000145) (co-funded by European Regional Development Fund "A way to make Europe") (MVS). Fundación para el Conocimiento Madri+d through the project G63-HEALTHSTARPLUS-HSP4 (JAMG, SOM).

Transcranial direct current stimulation for post-COVID fatigue: a randomized, double-blind, controlled pilot study.

Fatigue is one of the most frequent and disabling symptoms of the post-COVID syndrome. In this study, we aimed to assess the effects of transcranial direct current stimulation on fatigue severity in a group of patients with post-COVID syndrome and chronic fatigue. We conducted a double-blind, parallel-group, sham-controlled study to evaluate the short-term effects of anodal transcranial direct current stimulation (2 mA, 20 min/day) on the left dorsolateral prefrontal cortex. The modified fatigue impact scale score was used as the primary endpoint. Secondary endpoints included cognition (Stroop test), depressive symptoms (Beck depression inventory) and quality of life (EuroQol-5D). Patients received eight sessions of transcranial direct current stimulation and were evaluated at baseline, immediately after the last session, and one month later. Forty-seven patients were enrolled (23 in the active treatment group and 24 in the sham treatment group); the mean age was 45.66 ± 9.49 years, and 37 (78.72%) were women. The mean progression time since the acute infection was 20.68 ± 6.34 months. Active transcranial direct current stimulation was associated with a statistically significant improvement in physical fatigue at the end of treatment and 1 month as compared with sham stimulation. No significant effect was detected for cognitive fatigue. In terms of secondary outcomes, active transcranial direct current stimulation was associated with an improvement in depressive symptoms at the end of treatment. The treatment had no effects on the quality of life. All the adverse events reported were mild and transient, with no differences between the active stimulation and sham stimulation groups. In conclusion, our results suggest that transcranial direct current stimulation on the dorsolateral prefrontal cortex may improve physical fatigue. Further studies are needed to confirm these findings and optimize stimulation protocols.

Surveillance of SARS-CoV-2 in sewage from buildings housing residents with different vulnerability levels.

During the last three years, various restrictions have been set up to limit the transmission of the Coronavirus Disease (COVID-19). While these rules apply at a large scale (e.g., country-wide level) human-to-human transmission of the virus that causes COVID-19, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), occurs at a small scale. Different preventive policies and testing protocols were implemented in buildings where COVID-19 poses a threat (e.g., elderly residences) or constitutes a disruptive force (e.g., schools). In this study, we sampled sewage from different buildings (a school, a university campus, a university residence, and an elderly residence) that host residents of different levels of vulnerability. Our main goal was to assess the agreement between the SARS-CoV-2 concentration in wastewater and the policies applied in these buildings. All buildings were sampled using passive samplers while 24 h composite samples were also collected from the elderly residence. Results showed that passive samplers performed comparably well to composite samples while being cost-effective to keep track of COVID-19 prevalence. In the elderly residence, the comparison of sampling protocols (passive vs. active) combined with the strict clinical testing allowed us to compare the sensitivities of the two methods. Active sampling was more sensitive than passive sampling, as the former was able to detect a COVID-19 prevalence of 0.4 %, compared to a prevalence of 2.2 % for passive sampling. The number of COVID-19-positive individuals was tracked clinically in all the monitored buildings. More frequent detection of SARS-CoV-2 in wastewater was observed in residential buildings than in non-residential buildings using passive samplers. In all buildings, sewage surveillance can be used to complement COVID-19 clinical testing regimes, as the detection of SARS-CoV-2 in wastewater remained positive even when no COVID-19-positive individuals were reported. Passive sampling is useful for building managers to adapt their COVID-19 mitigation policies.

The Catalan Surveillance Network of SARS-CoV-2 in Sewage: design, implementation, and performance.

Wastewater-based epidemiology has shown to be an efficient tool to track the circulation of SARS-CoV-2 in communities assisted by wastewater treatment plants (WWTPs). The challenge comes when this approach is employed to help Health authorities in their decision-making. Here, we describe the roadmap for the design and deployment of SARSAIGUA, the Catalan Surveillance Network of SARS-CoV-2 in Sewage. The network monitors, weekly or biweekly, 56 WWTPs evenly distributed across the territory and serving 6 M inhabitants (80% of the Catalan population). Each week, samples from 45 WWTPs are collected, analyzed, results reported to Health authorities, and finally published within less than 72 h in an online dashboard ( https://sarsaigua.icra.cat ). After 20 months of monitoring (July 20-March 22), the standardized viral load (gene copies/day) in all the WWTPs monitored fairly matched the cumulative number of COVID-19 cases along the successive pandemic waves, showing a good fit with the diagnosed cases in the served municipalities (Spearman Rho = 0.69). Here we describe the roadmap of the design and deployment of SARSAIGUA while providing several open-access tools for the management and visualization of the surveillance data.

Viral metagenomics reveals persistent as well as dietary acquired viruses in Antarctic fur seals.

Viruses linked to animals inhabiting Antarctic latitudes remain poorly studied. Remote environments hosting large pinniped populations may be prone to exposure of immunologically naïve animals to new infectious agents due to increasing human presence or introduction of new animal species. Antarctic fur seals (Arctocephalus gazella) inhabiting the Western Antarctic Peninsula and the South Shetland Islands are challenged because of climate change and increased anthropogenic activity. In the present study, the fecal and serum virome of A. gazella was characterized by applying target enrichment next generation sequencing. The resulting viromes were dominated by CRESS-DNA sequences. Viruses known to infect vertebrate and invertebrate hosts were also observed in fecal samples. Fur seal picornavirus was present in all the fecal pools studied suggesting it is a prevalent virus in these species. Six different viruses presenting similarities with previously described A. gazella viruses or other otariids and mammal viruses were identified as potential new A. gazella viruses. Also, diet-derived viruses such as crustacean viruses were present in fecal content. Penguin viruses, but not fish viruses, were also detected. Obtained results contribute to a better understanding of the viral community present in these species, which is relevant for its conservation.

Fatigue and Cognitive Dysfunction Are Associated with Occupational Status in Post-COVID Syndrome.

Post-COVID syndrome (PCS) is a medical condition characterized by the persistence of a wide range of symptoms after acute infection by SARS-CoV-2. The work capacity consequences of this disorder have scarcely been studied. We aimed to analyze the factors associated with occupational status in patients with PCS. This cross-sectional study involved 77 patients with PCS on active work before SARS-CoV-2 infection. Patients were evaluated 20.71 ± 6.50 months after clinical onset. We conducted a survey on occupational activity and cognitive and clinical symptoms. The association between occupational activity and fatigue, depression, anxiety, sleep quality, and cognitive testing was analyzed. Thirty-eight (49.4%) patients were working, and thirty-nine (50.6%) patients were not. Of those not working at the moment of the assessment, 36 (92.3%) patients were on sick leave. In 63 patients (81.8% of the sample), sick leave was needed at some point due to PCS. The mean duration of sick leave was 12.07 ± 8.07 months. According to the patient's perspective, the most disabling symptoms were cognitive complaints (46.8%) and fatigue (31.2%). Not working at the moment of the assessment was associated with higher levels of fatigue and lower cognitive performance in the Stroop test. No association was found between occupational status with depression and anxiety questionnaires. Our study found an influence of PCS on work capacity. Fatigue and cognitive issues were the most frequent symptoms associated with loss of work capacity.

Neuropsychological Predictors of Fatigue in Post-COVID Syndrome.

Fatigue is one of the most disabling symptoms in several neurological disorders and has an important cognitive component. However, the relationship between self-reported cognitive fatigue and objective cognitive assessment results remains elusive. Patients with post-COVID syndrome often report fatigue and cognitive issues several months after the acute infection. We aimed to develop predictive models of fatigue using neuropsychological assessments to evaluate the relationship between cognitive fatigue and objective neuropsychological assessment results. We conducted a cross-sectional study of 113 patients with post-COVID syndrome, assessing them with the Modified Fatigue Impact Scale (MFIS) and a comprehensive neuropsychological battery including standardized and computerized cognitive tests. Several machine learning algorithms were developed to predict MFIS scores (total score and cognitive fatigue score) based on neuropsychological test scores. MFIS showed moderate correlations only with the Stroop Color-Word Interference Test. Classification models obtained modest F1-scores for classification between fatigue and non-fatigued or between 3 or 4 degrees of fatigue severity. Regression models to estimate the MFIS score did not achieve adequate R2 metrics. Our study did not find reliable neuropsychological predictors of cognitive fatigue in the post-COVID syndrome. This has important implications for the interpretation of fatigue and cognitive assessment. Specifically, MFIS cognitive domain could not properly capture actual cognitive fatigue. In addition, our findings suggest different pathophysiological mechanisms of fatigue and cognitive dysfunction in post-COVID syndrome.

Evaluation of a virus concentration method based on ultrafiltration and wet foam elution for studying viruses from large-volume water samples.

Assessing the presence of viruses in large-volume samples involves cumbersome methods that require specialized training and laboratory equipment. In this study, a large volume concentration (LVC) method, based on dead-end ultrafiltration (DEUF) and Wet Foam Elution™ technology, was evaluated in different type of waters and different microorganisms. Its recovery efficiency was evaluated through different techniques (infectivity assays and molecular detection) by spiking different viral surrogates (bacteriophages PhiX174 and MS2 and Coxsackie virus B5 (CVB5) and Escherichia coli (E. coli). Furthermore, the application of a secondary concentration step was evaluated and compared with skimmed milk flocculation. Viruses present in river water, seawater and groundwater samples were concentrated by applying LVC method and a centrifugal ultrafiltration device (CeUF), as a secondary concentration step and quantified with specific qPCR Human adenoviruses (HAdV) and noroviruses (NoVs). MS2 was used as process control, obtaining a mean viral recovery of 22.0 ± 12.47%. The presence of other viruses was also characterized by applying two different next-generation sequencing approaches. LVC coupled to a secondary concentration step based on CeUF allowed to detect naturally occurring viruses such as HAdV and NoVs in different water matrices. Using HAdV as a human fecal indicator, the highest viral pollution was found in river water samples (100% of positive samples), followed by seawater (83.33%) and groundwater samples (66.67%). The LVC method has also proven to be useful as a virus concentration method in the filed since HAdV and NoVs were detected in the river water and groundwater samples concentrated in the field. All in all, LVC method presents high concentration factor and a low limit of detection and provides viral concentrates useful for subsequent molecular analysis such as PCR and massive sequencing.

Quantitative GC-TCD Measurements of Major Flatus Components: A Preliminary Analysis of the Diet Effect.

The impact of diet and digestive disorders in flatus composition remains largely unexplored. This is partially due to the lack of standardized sampling collection methods, and the easy atmospheric contamination. This paper describes a method to quantitatively determine the major gases in flatus and their application in a nutritional intervention. We describe how to direct sample flatus into Tedlar bags, and simultaneous analysis by gas chromatography-thermal conductivity detection (GC-TCD). Results are analyzed by univariate hypothesis testing and by multilevel principal component analysis. The reported methodology allows simultaneous determination of the five major gases with root mean measurement errors of 0.8% for oxygen (O2), 0.9% for nitrogen (N2), 0.14% for carbon dioxide (CO2), 0.11% for methane (CH4), and 0.26% for hydrogen (H2). The atmospheric contamination was limited to 0.86 (95% CI: [0.7-1.0])% for oxygen and 3.4 (95% CI: [1.4-5.3])% for nitrogen. As an illustration, the method has been successfully applied to measure the response to a nutritional intervention in a reduced crossover study in healthy subjects.

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance.

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.

Looking for a needle in a haystack. SARS-CoV-2 variant characterization in sewage.

SARS-CoV-2 variants are emerging worldwide, and monitoring them is key in providing early warnings. Here, we summarize the different analytical approaches currently used to study the dissemination of SARS-CoV-2 variants in wastewater and discuss their advantages and disadvantages. We also provide preliminary results of two sensitive and cost-effective approaches: variant-specific reverse transcription-nested PCR assays and a nonvariant-specific amplicon deep sequencing strategy that targets three key regions of the viral spike protein. Next-generation sequencing approaches enable the simultaneous detection of signature mutations of different variants of concern in a single assay and may be the best option to explore the real picture at a particular time. Targeted PCR approaches focused on specific signature mutations will need continuous updating but are sensitive and cost-effective.

Exploring the diversity of coronavirus in sewage during COVID-19 pandemic: Don't miss the forest for the trees.

In the wake of the COVID-19 pandemic, the use of next generation sequencing (NGS) has proved to be an important tool for the genetic characterization of SARS-CoV-2 from clinical samples. The use of different available NGS tools applied to wastewater samples could be the key for an in-depth study of the excreted virome, not only focusing on SARS-CoV-2 circulation and typing, but also to detect other potentially pandemic viruses within the same family. With this aim, 24-hours composite wastewater samples from March and July 2020 were sequenced by applying specific viral NGS as well as target enrichment NGS. The full virome of the analyzed samples was obtained, with human Coronaviridae members (CoV) present in one of those samples after applying the enrichment. One contig was identified as HCoV-OC43 and 8 contigs as SARS-CoV-2. CoVs from other animal hosts were also detected when applying this technique. These contigs were compared with those obtained from contemporary clinical specimens by applying the same target enrichment approach. The results showed that there is a co-circulation in urban areas of human and animal coronaviruses infecting domestic animals and rodents. NGS enrichment-based protocols might be crucial to describe the occurrence and genetic characteristics of SARS-CoV-2 and other Coronaviridae family members within the excreted virome present in wastewater.

NGS Techniques Reveal a High Diversity of RNA Viral Pathogens and Papillomaviruses in Fresh Produce and Irrigation Water.

Fresh fruits and vegetables are susceptible to microbial contamination at every stage of the food production chain, and as a potential source of pathogens, irrigation water quality is a critical factor. Next-generation sequencing (NGS) techniques have been flourishing and expanding to a wide variety of fields. However, their application in food safety remains insufficiently explored, and their sensitivity requires improvement. In this study, quantitative polymerase chain reaction (qPCR) assays showed low but frequent contamination of common circulating viral pathogens, which were found in 46.9% of samples of fresh produce: 6/12 lettuce samples, 4/12 strawberries samples, and 5/8 parsley samples. Furthermore, the application of two different NGS approaches, target enrichment sequencing (TES) for detecting viruses that infect vertebrates and amplicon deep sequencing (ADS), revealed a high diversity of viral pathogens, especially Norovirus (NoV) and Human Papillomavirus (HPV), in fresh produce and irrigation water. All NoV and HPV types found in fresh fruit and vegetable samples were also detected in irrigation water sources, indicating that these viruses are common circulating pathogens in the population and that irrigation water may be the most probable source of viral pathogens in food samples.

Certified Smoking Cessation Units in Spain: High Potential for Detection of Undiagnosed Chronic Obstructive Pulmonary Disease and Profile of Newly Diagnosed Cases.

PURPOSE: Smokers attending smoking cessation units (SCUs) may offer a unique opportunity for early recognition of undiagnosed chronic obstructive pulmonary disease (COPD). We aimed at assessing the impact of SCUs on the early diagnosis of COPD and describing the clinical and smoking profile of newly-diagnosed COPD cases at SCUs certified by the Spanish Society of Respiratory Diseases and Thoracic Surgery (SEPAR). PATIENTS AND METHODS: A multicenter cross-sectional observational cohort study (DIPREPOQ study) was performed in eight SEPAR-certified SCUs in Spain. Adult current smokers with no previously diagnosed respiratory disease and having one o more respiratory symptoms were included. Lung functional tests were performed and previously undiagnosed COPD cases were identified and characterized based on national guidelines. RESULTS: Out of 401 individuals newly attending the SCUs, 252 participants were included and 73 (28.9%) met the definition of previously undiagnosed COPD. A characterization of patients with COPD being newly recognized in SCUs showed: age (mean±SD) 61±9 years; men 59%; active work status 53.1%; functional class I/II dyspnea 82.8%, GOLD state mild/moderate/severe 57%/31%/12%; non-exacerbators 90%, CAT 14±4; emphysema in X-rays 40%. Most common co-morbidities were cardiovascular and psychiatric (anxiety and depression) ones. Usual smoking history included a lengthy smoking history (41±9 years) and a current consumption of 24±9 cigarettes/day. CONCLUSIONS AND IMPLICATIONS: Consistently certified SCUs can have a substantial contribution to early diagnosis of COPD. A typical profile of newly detected cases is reported, with most patients being men at their early sixties, with mild symptoms and with high and lengthy smoking history. Our study reports a high usefulness of lung functional tests to detect undiagnosed COPD in appropriately selected participants attending SCUs at a large national scale, using a standardized methodology. This is likely to be facilitated by the certification of SCUs using well-defined requirements by national scientific societies.