Assessing infectivity of emerging enveloped viruses in wastewater and sewage sludge: Relevance and procedures.

The emergence of SARS-CoV-2 has heightened the need to evaluate the detection of enveloped viruses in the environment, particularly in wastewater, within the context of wastewater-based epidemiology. The studies published over the past 80 years focused primarily on non-enveloped viruses due to their ability to survive longer in environmental matrices such as wastewater or sludge compared to enveloped viruses. However, different enveloped viruses survive in the environment for different lengths of time. Therefore, it is crucial to be prepared to assess the potential infectious risk that may arise from future emerging enveloped viruses. This will require appropriate tools, notably suitable viral concentration methods that do not compromise virus infectivity. This review has a dual purpose: first, to gather all the available literature on the survival of infectious enveloped viruses, specifically at different pH and temperature conditions, and in contact with detergents; second, to select suitable concentration methods for evaluating the infectivity of these viruses in wastewater and sludge. The methodology used in this data collection review followed the systematic approach outlined in the PRISMA (Preferred Reporting Items for Systematic Review and Meta-Analysis) guidelines. Concentration methods cited in the data gathered are more tailored towards detecting the enveloped viruses' genome. There is a lack of suitable methods for detecting infectious enveloped viruses in wastewater and sludge. Ultrafiltration, ultracentrifugation, and polyethylene glycol precipitation methods, under specific/defined conditions, appear to be relevant approaches. Further studies are necessary to validate reliable concentration methods for detecting infectious enveloped viruses. The choice of culture system is also crucial for detection sensitivity. The data also show that the survival of infectious enveloped viruses, though lower than that of non-enveloped ones, may enable environmental transmission. Experimental data on a wide range of enveloped viruses is required due to the variability in virus persistence in the environment.

BA.2.86 variant emergence and spread dynamics through wastewater monitoring in Paris, France.

Numerous SARS-CoV-2 variants are emerging as the epidemic continues, inducing new waves of contamination. In July 2023, a new variant named BA.2.86 was identified, raising concerns about its potential for viral escape, even in an immune population. The reduction in patient-centered testing and the identification of variants by sequencing means that we are now blind to the spread of this new variant. The aim of this study was to track the signature of this variant in wastewater in Paris, France. This variant showed a very rapid spread, highly correlated with national flash studies involving sequencing of clinical samples, but with a moderate impact on virus circulation. This easy-to-implement approach enabled us to monitor the emergence and spread of this new variant in real time at low cost.

Implementation of a Sensitive Method to Assess High Virus Retention Performance of Low-Pressure Reverse Osmosis Process.

Human enteric viruses are important etiological agents of waterborne diseases. Environmental waters are usually contaminated with low virus concentration requiring large concentration factors for effective detection by (RT)-qPCR. Low-pressure reverse osmosis is often used to remove water contaminants, but very few studies focused on the effective virus removal of reverse osmosis treatment with feed concentrations as close as possible to environmental concentrations and principally relied on theoretical virus removal. The very low viral concentrations usually reported in the permeates (i.e. at least 5 log of removal rate) mean that very large volumes of water need to be analysed to have sufficient sensitivity and assess the process efficiency. This study evaluates two methods for the concentration of adenoviruses, enteroviruses and MS2 bacteriophages at different viral concentrations in large (< 200 L) and very large (> 200 L) volumes. The first method is composed of two ultrafiltration membranes with low-molecular weight cut-offs while the second method primarily relies on adsorption and elution phases using electropositive-charged filters. The recovery rates were assessed for both methods. For the ultrafiltration-based protocol, recovery rates were similar for each virus studied: 80% on average at high virus concentrations (106-107 viruses L-1) and 50% at low virus concentrations (103-104 viruses L-1). For the electropositive-charged filter-based method, the average recoveries obtained were about 36% for ADV 41, 57% for CV-B5 and 1.6% for MS2. The ultrafiltration-based method was then used to evaluate the performance of a low-pressure reverse osmosis lab-scale pilot plant. The retentions by reverse osmosis were similar for all studied viruses and the validated recovery rates applied to the system confirmed the reliability of the concentration method. This method was effective in concentrating all three viruses over a wide range of viral concentrations. Moreover, the second concentration method using electropositive-charged filters was studied, allowing the filtration of larger volumes of permeate from a semi-industrial low-pressure reverse osmosis pilot plant. This reference method was used because of the inability of the UF method to filter volumes on the order of one cubic metre.

Reduction in SARS-CoV-2 Virus Infectivity in Human and Hamster Feces.

OBJECTIVE: There is extensive evidence that SARS-CoV-2 replicates in the gastrointestinal tract. However, the infectivity of virions in feces is poorly documented. Although the primary mode of transmission is airborne, the risk of transmission from contaminated feces remains to be assessed. DESIGN: The persistence of SARS-CoV-2 (infectivity and RNA) in human and animal feces was evaluated by virus isolation on cell culture and RT-qPCR, respectively. The exposure of golden Syrian hamsters to experimentally contaminated feces through intranasal inoculation has also been tested to assess the fecal-oral transmission route. RESULTS: For periods that are compatible with average intestinal transit, the SARS-CoV-2 genome was noticeably stable in human and animal feces, contrary to the virus infectivity that was reduced in a time- and temperature-dependent manner. In human stools, this reduction was variable depending on the donors. Viral RNA was excreted in the feces of infected hamsters, but exposure of naïve hamsters to feces of infected animals did not lead to any productive infection. Conversely, hamsters could be experimentally infected following exposure to spiked fresh feces. CONCLUSION: Infection following exposure to naturally contaminated feces has been suspected but has not been established so far. The present work demonstrates that SARS-CoV-2 rapidly lost infectivity in spiked or naturally infected feces. Although the possibility of persistent viral particles in human or animal feces cannot be fully ruled out, SARS-CoV-2 transmission after exposure to contaminated feces is unlikely.

From Alpha to Omicron BA.2: New digital RT-PCR approach and challenges for SARS-CoV-2 VOC monitoring and normalization of variant dynamics in wastewater.

Throughout the COVID-19 pandemic, new variants have continuously emerged and spread in populations. Among these, variants of concern (VOC) have been the main culprits of successive epidemic waves, due to their transmissibility, pathogenicity or ability to escape the immune response. Quantification of the SARS-CoV-2 genomes in raw wastewater is a reliable approach well-described and widely deployed worldwide to monitor the spread of SARS-CoV-2 in human populations connected to sewage systems. Discrimination of VOCs in wastewater is also a major issue and can be achieved by genome sequencing or by detection of specific mutations suggesting the presence of VOCs. This study aimed to date the emergence of these VOCs (from Alpha to Omicron BA.2) by monitoring wastewater from the greater Paris area, France, but also to model the propagation dynamics of these VOCs and to characterize the replacement kinetics of the prevalent populations. These dynamics were compared to various individual-centered public health data, such as regional incidence and the proportions of VOCs identified by sequencing of strains isolated from patient. The viral dynamics in wastewater highlighted the impact of the vaccination strategy on the viral circulation within human populations but also suggested its potential effect on the selection of variants most likely to be propagated in immunized populations. Normalization of concentrations to capture population movements appeared statistically more reliable using variations in local drinking water consumption rather than using PMMoV concentrations because PMMoV fecal shedding was subject to variability and was not sufficiently relevant in this study. The dynamics of viral spread was observed earlier (about 13 days on the wave related to Omicron VOC) in raw wastewater than the regional incidence alerting to a possible risk of decorrelation between incidence and actual virus circulation probably resulting from a lower severity of infection in vaccinated populations.

SARS-CoV-2 Whole-Genome Sequencing Using Oxford Nanopore Technology for Variant Monitoring in Wastewaters.

Since the beginning of the Coronavirus Disease-19 (COVID-19) pandemic, multiple Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) mutations have been reported and led to the emergence of variants of concern (VOC) with increased transmissibility, virulence or immune escape. In parallel, the observation of viral fecal shedding led to the quantification of SARS-CoV-2 genomes in wastewater, providing information about the dynamics of SARS-CoV-2 infections within a population including symptomatic and asymptomatic individuals. Here, we aimed to adapt a sequencing technique initially designed for clinical samples to apply it to the challenging and mixed wastewater matrix, and hence identify the circulation of VOC at the community level. Composite raw sewage sampled over 24 h in two wastewater-treatment plants (WWTPs) from a city in western France were collected weekly and SARS-CoV-2 quantified by RT-PCR. Samples collected between October 2020 and May 2021 were submitted to whole-genome sequencing (WGS) using the primers and protocol published by the ARTIC Network and a MinION Mk1C sequencer (Oxford Nanopore Technologies, Oxford, United Kingdom). The protocol was adapted to allow near-full genome coverage from sewage samples, starting from ∼5% to reach ∼90% at depth 30. This enabled us to detect multiple single-nucleotide variant (SNV) and assess the circulation of the SARS-CoV-2 VOC Alpha, Beta, Gamma, and Delta. Retrospective analysis of sewage samples shed light on the emergence of the Alpha VOC with detection of first co-occurring signature mutations in mid-November 2020 to reach predominance of this variant in early February 2021. In parallel, a mutation-specific qRT-PCR assay confirmed the spread of the Alpha VOC but detected it later than WGS. Altogether, these data show that SARS-CoV-2 sequencing in sewage can be used for early detection of an emerging VOC in a population and confirm its ability to track shifts in variant predominance.

Monitoring of Leptospira species diversity in freshwater bathing area and in rats in Paris, France.

Leptospirosis is a neglected zoonotic disease with a worldwide distribution caused by bacterial pathogenic Leptospira. Rodents are considered as the main reservoir of Leptospira and transmission usually occurs through exposure to urine-contaminated environment. However, interactions between environment, rodent reservoir and human leptospirosis remain poorly studied. Here, we evaluated the concentration of Leptospira in surface water and captured rats in the city of Paris (France) from 2018 to 2020 using an integrity qPCR (Quantitative Polymerase Chain Reaction). All environmental samples (n = 1031) were positive for saprophytic Leptospira but pathogenic Leptospira P1 group were only found in 40% (n = 363; 2018) to 0% (n = 264; 2020) of samples. In the same time, analysis of 200 brown rat corpses trapped in the city, showed about 15% of positivity for Leptospira but the different method used for rats conservation (based on presence or absence of conservative agent) showed important variations in the Leptospira prevalence. Metagenomic analysis, based on rrs gene sequencing, was also carried out to evaluate the distribution of Leptospira in samples. Results could indicate that some species of Leptospira are found in surface waters as well as rats, but further study is needed to accurately describe the nature of the link between these two reservoirs. Quantification of Leptospira and pathogenic species description circulating inside animal reservoir living in the vicinity of freshwater in urban areas, will be helpful to understand the eco-epidemiology of leptospirosis and to establish prevention and intervention strategies, especially in the context of organization of recreative activity events in these urban areas.

A nationwide indicator to smooth and normalize heterogeneous SARS-CoV-2 RNA data in wastewater.

Since many infected people experience no or few symptoms, the SARS-CoV-2 epidemic is frequently monitored through massive virus testing of the population, an approach that may be biased and may be difficult to sustain in low-income countries. Since SARS-CoV-2 RNA can be detected in stool samples, quantifying SARS-CoV-2 genome by RT-qPCR in wastewater treatment plants (WWTPs) has been carried out as a complementary tool to monitor virus circulation among human populations. However, measuring SARS-CoV-2 viral load in WWTPs can be affected by many experimental and environmental factors. To circumvent these limits, we propose here a novel indicator, the wastewater indicator (WWI), that partly reduces and corrects the noise associated with the SARS-CoV-2 genome quantification in wastewater (average noise reduction of 19%). All data processing results in an average correlation gain of 18% with the incidence rate. The WWI can take into account the censorship linked to the limit of quantification (LOQ), allows the automatic detection of outliers to be integrated into the smoothing algorithm, estimates the average measurement error committed on the samples and proposes a solution for inter-laboratory normalization in the absence of inter-laboratory assays (ILA). This method has been successfully applied in the context of Obépine, a French national network that has been quantifying SARS-CoV-2 genome in a representative sample of French WWTPs since March 5th 2020. By August 26th, 2021, 168 WWTPs were monitored in the French metropolitan and overseas territories of France. We detail the process of elaboration of this indicator, show that it is strongly correlated to the incidence rate and that the optimal time lag between these two signals is only a few days, making our indicator an efficient complement to the incidence rate. This alternative approach may be especially important to evaluate SARS-CoV-2 dynamics in human populations when the testing rate is low.

First evidence of SARS-CoV-2 genome detection in zebra mussel (Dreissena polymorpha).

The uses of bivalve molluscs in environmental biomonitoring have recently gained momentum due to their ability to indicate and concentrate human pathogenic microorganisms. In the context of the health crisis caused by the COVID-19 epidemic, the objective of this study was to determine if the SARS-CoV-2 ribonucleic acid genome can be detected in zebra mussels (Dreissena polymorpha) exposed to raw and treated urban wastewaters from two separate plants to support its interest as bioindicator of the SARS-CoV-2 genome contamination in water. The zebra mussels were exposed to treated wastewater through caging at the outlet of two plants located in France, as well as to raw wastewater in controlled conditions. Within their digestive tissues, our results showed that SARS-CoV-2 genome was detected in zebra mussels, whether in raw and treated wastewaters. Moreover, the detection of the SARS-CoV-2 genome in such bivalve molluscans appeared even with low concentrations in raw wastewaters. This is the first detection of the SARS-CoV-2 genome in the tissues of a sentinel species exposed to raw and treated urban wastewaters. Despite the need for development for quantitative approaches, these results support the importance of such invertebrate organisms, especially zebra mussel, for the active surveillance of pathogenic microorganisms and their indicators in environmental waters.

Effect of disinfection agents and quantification of potentially viable Leptospira in fresh water samples using a highly sensitive integrity-qPCR assay.

Leptospirosis is an emerging worldwide zoonotic disease, but the general biology of the causative agents is still poorly understood. Humans are an occasional host. The main risk factors are water-associated exposure during professional or recreational activities or during outbreaks in endemic areas. Detecting the presence of pathogenic bacteria in aquatic environments and their capacity to resist various inactivation processes are research fields that need to be further developed. In addition, the methods used for detecting and enumerating Leptospira still need to be improved. We aimed to describe a new quantitative polymerase chain reaction coupled to propidium monoazide treatment (PMAqPCR) that targets not only total Leptospira but also discriminates pathogenic from non-pathogenic Leptospira while also addressing PCR inhibitors, a frequently encountered problem when studying environmental water. In a second step, the killing efficiency of Leptospira to different treatments was tested and PMAqPCR compared to culture-based enumeration. This provided information about the effects of temperature, as well as ultraviolet and chlorine disinfection, that are both related to water treatment processes, in particular for the production of drinking water, on the persistence of both saprophytic and pathogenic Leptospira. Finally, PMAqPCR was used for the detection of Leptospira in freshwater samples for a proof-of-concept. In conclusion, our method could be used for routine freshwater monitoring and allows better evaluation of the presence of Leptospira, allowing evaluation of the bacterial dynamics in a designated area or assessment of the efficacy of water disinfection processes.

Effects of Dysbiosis and Dietary Manipulation on the Digestive Microbiota of a Detritivorous Arthropod.

The crucial role of microbes in the evolution, development, health, and ecological interactions of multicellular organisms is now widely recognized in the holobiont concept. However, the structure and stability of microbiota are highly dependent on abiotic and biotic factors, especially in the gut, which can be colonized by transient bacteria depending on the host's diet. We studied these impacts by manipulating the digestive microbiota of the detritivore Armadillidium vulgare and analyzing the consequences on its structure and function. Hosts were exposed to initial starvation and then were fed diets that varied the different components of lignocellulose. A total of 72 digestive microbiota were analyzed according to the type of the diet (standard or enriched in cellulose, lignin, or hemicellulose) and the period following dysbiosis. The results showed that microbiota from the hepatopancreas were very stable and resilient, while the most diverse and labile over time were found in the hindgut. Dysbiosis and selective diets may have affected the host fitness by altering the structure of the microbiota and its predicted functions. Overall, these modifications can therefore have effects not only on the holobiont, but also on the "eco-holobiont" conceptualization of macroorganisms.

Spatio-temporal survey of opportunistic premise plumbing pathogens in the Paris drinking water distribution system.

Opportunistic premise plumbing pathogens present in drinking water are linked to a significant number of infections for health compromised patients. However, their monitoring is not required in current water potability standards and they have been poorly studied in a full-scale network. In this study, we quantified, by qPCR, three opportunistic pathogens, Mycobacterium spp., Legionella pneumophila, Pseudomonas aeruginosa throughout the Paris drinking water network over a one-year sampling campaign. While Mycobacteria spp. seemed ubiquitous whatever the distribution system and the time of the year, the occurrence of L. pneumophila and P. aeruginosa showed seasonal variations. Unlike L. pneumophila and P. aeruginosa, the concentration (copies number/L) of Mycobacterium spp. varied between sampling sites. The variation in microbial numbers did not demonstrate any correlations with temperature, pH, chlorine, conductivity, orthophosphate or nitrate levels. In conclusion, Mycobacterium spp. are common inhabitants of the Paris network while L. pneumophila and P. aeruginosa presence fluctuate over space and time. Such qPCR approach would help to better understand the behaviour of opportunistic premise plumbing pathogens.

Targeted Metagenomics of Microbial Diversity in Free-Living Amoebae and Water Samples.

The presence of Legionella spp. in natural and man-made water systems is a great public health concern and heavily depends on the presence of free-living amoebae. Taking advantage of the development and affordability of next-generation sequencing technology, we present here a method to characterize the whole bacterial community directly from water samples, as well as from isolated free-living amoebae.

Microbiome of drinking water: A full-scale spatio-temporal study to monitor water quality in the Paris distribution system.

The microbiological water quality of drinking water distribution system (DWDS) is of primary importance for human health. High-throughput sequencing has gained more and more attention in the last decade to describe this microbial diversity in water networks. However, there are few studies describing this approach on large drinking water distribution systems and for extended periods of time. To fill this gap and observe the potential subtle variation in microbiota of a water network through time and space, we aimed to apply high-throughput sequencing of the 16S rRNA gene approach to characterize bacterial communities of the Paris' DWDS over a one-year period. In this study, the Paris network, composed of four different DWDSs, was sampled at 31 sites, each month for one year. The sampling campaign was one of the largest described so far (n = 368) and the importance of key spatio-temporal and physico-chemical parameters was investigated. Overall, 1321 taxa were identified within the Paris network, although only fifteen of them were found in high relative abundance (>1%) in all samples. Two genera, Phreatobacter and Hyphomicrobium were dominant. The whole bacterial diversity was not significantly affected between the four DWDSs (spatial parameter) and by physico-chemical parameters. However, the bacterial diversity was slightly modified over the one-year period (temporal parameter) as we were able to observe DWDS microbiome perturbations, presumably linked to a preceding flood event. Comparison of high-throughput sequencing of the 16S rRNA gene amplicons vs. cultivation-based techniques showed that only 1.8% of bacterial diversity was recovered through cultivation. High throughput sequencing has made it possible to monitor DWDS more accurately than conventional methods by describing the whole diversity and detecting slight fluctuations in bacterial communities. This method would be further used to supervise drinking water networks, to follow any perturbations due to internals events (such as treatments) or external events (such as flooding).

Importance of Local and Regional Scales in Shaping Mycobacterial Abundance in Freshwater Lakes.

Biogeographical studies considering the entire bacterial community may underestimate mechanisms of bacterial assemblages at lower taxonomic levels. In this context, the study aimed to identify factors affecting the spatial and temporal dynamic of the Mycobacterium, a genus widespread in aquatic ecosystems. Nontuberculous mycobacteria (NTM) density variations were quantified in the water column of freshwater lakes at the regional scale (annual monitoring of 49 lakes in the Paris area) and at the local scale (2-year monthly monitoring in Créteil Lake) by real-time quantitative PCR targeting the atpE gene. At the regional scale, mycobacteria densities in water samples ranged from 6.7 × 103 to 1.9 × 108 genome units per liter. Density variations were primarily explained by water pH, labile iron, and dispersal processes through the connection of the lakes to a river. In Créteil Lake, no spatial variation of mycobacterial densities was noticed over the 2-year monthly survey, except after large rainfall events. Indeed, storm sewer effluents locally and temporarily increased NTM densities in the water column. The temporal dynamic of the NTM densities in Créteil Lake was associated with suspended solid concentrations. No clear seasonal variation was noticed despite a shift in NTM densities observed over the 2012-2013 winter. Temporal NTM densities fluctuations were well predicted by the neutral community model, suggesting a random balance between loss and gain of mycobacterial taxa within Créteil Lake. This study highlights the importance of considering multiple spatial scales for understanding the spatio-temporal dynamic of bacterial populations in natural environments.