Long-term wastewater monitoring of SARS-CoV-2 viral loads and variants at the major international passenger hub Amsterdam Schiphol Airport: A valuable addition to COVID-19 surveillance.

Wastewater-based epidemiological surveillance at municipal wastewater treatment plants has proven to play an important role in COVID-19 surveillance. Considering international passenger hubs contribute extensively to global transmission of viruses, wastewater surveillance at this type of location may be of added value as well. The aim of this study is to explore the potential of long-term wastewater surveillance at a large passenger hub as an additional tool for public health surveillance during different stages of a pandemic. Here, we present an analysis of SARS-CoV-2 viral loads in airport wastewater by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) from the beginning of the COVID-19 pandemic in Feb 2020, and an analysis of SARS-CoV-2 variants by whole-genome next-generation sequencing from Sep 2020, both until Sep 2022, in the Netherlands. Results are contextualized using (inter)national measures and data sources such as passenger numbers, clinical surveillance data and national wastewater surveillance data. Our findings show that wastewater surveillance was possible throughout the study period, irrespective of measures, as viral loads were detected and quantified in 98.6 % (273/277) of samples. Emergence of SARS-CoV-2 variants, identified in 91.0 % (161/177) of sequenced samples, coincided with increases in viral loads. Furthermore, trends in viral load and variant detection in airport wastewater closely followed, and in some cases preceded, trends in national daily average viral load in wastewater and variants detected in clinical surveillance. Wastewater-based epidemiology at a large international airport is a valuable addition to classical COVID-19 surveillance and the developed expertise can be applied in pandemic preparedness plans for other (emerging) pathogens in the future.

Inferring hospital admissions from SARS-CoV-2 virus loads in wastewater in The Netherlands, August 2020 - February 2022.

Wastewater-based surveillance enables tracking of SARS-CoV-2 circulation at a local scale in near-real time. Here we investigate the relation between virus loads and the number of hospital admissions in the Netherlands. Inferred virus loads from August 2020 until February 2022 in each of the 344 Dutch municipalities are analysed in a Bayesian multilevel Poisson regression to relate virus loads to daily age-stratified (in groups of 20 years) hospital admissions. Covariates include municipal vaccination coverages stratified by age and dose (first, second, and booster) and prevalence of the circulating coronavirus variants (wildtype, Alpha, Delta, and Omicron (BA.1 and BA.2)). Our model captures the relation between hospital admissions and virus loads well. Estimated hospitalisation rates per 1,000,000 persons per day at a virus load of 1013 particles range from 0.18 (95 % Prediction Interval (PI): 0.046-0.48) in children (0-19 years) to 20.1 (95 % PI: 9.46-36.8) in the oldest age group (80 years and older) in an unvaccinated population with only wildtype SARS-CoV-2 circulation. The analyses indicate a nearly twofold (1.92 (95 % PI: 1.78-2.05)) decrease in the expected number of hospitalisations at a given virus load between the Alpha and the Omicron variant. Our analyses show that virus load estimates in wastewater are closely related to the expected number of hospitalisations and provide an attractive tool to detect increased SARS-CoV-2 circulation at a local scale, even when there are few hospital admissions. Our analyses enable integration of data at the municipality level into meaningful conversion rates to translate virus loads at a local level into expected numbers of hospital admissions, which would allow for a better interpretation of virus loads detected in wastewater.

PCR standard curve quantification in an extensive wastewater surveillance program: results from the Dutch SARS-CoV-2 wastewater surveillance.

Since the start of the COVID-19 pandemic in 2020, wastewater surveillance programs were established, or upscaled, in many countries around the world and have proven to be a cost-effective way of monitoring infectious disease pathogens. Many of these programs use RT-qPCR, and quantify the viral concentrations in samples based on standard curves, by including preparations of a reference material with known nucleic acid or virus concentrations in the RT-qPCR analyses. In high-throughput monitoring programs it is possible to combine data from multiple previous runs, circumventing the need for duplication and resulting in decreased costs and prolonged periods during which the reference material is obtained from the same batch. However, over time, systematic shifts in standard curves are likely to occur. This would affect the reliability and usefulness of wastewater surveillance as a whole. We aim to find an optimal combination of standard curve data to compensate for run-to-run measurement variance while ensuring enough flexibility to capture systematic longitudinal shifts. Based on more than 4000 observations obtained with the CDC N1 and N2 assays, taken as a part of the National Sewage Surveillance program at the Dutch National Institute for Public Health and the Environment, we show that seasonal and long-term shifts in RT-qPCR efficiency and sensitivity occur. We find that in our setting, using five days of standard-curve data to quantify, results in the least error prone curve or best approximation. This results in differences up to 100% in quantified viral loads when averaged out over a nationwide program of >300 treatment plants. Results show that combining standard curves from a limited set of runs can be a valid approach to quantification without obscuring the trends in the viral load of interest.

Regional reemergence of a SARS-CoV-2 Delta lineage amid an Omicron wave detected by wastewater sequencing.

The implementation and integration of wastewater-based epidemiology constitutes a valuable addition to existing pathogen surveillance systems, such as clinical surveillance for SARS-CoV-2. In the Netherlands, SARS-CoV-2 variant circulation is monitored by performing whole-genome sequencing on wastewater samples. In this manuscript, we describe the detection of an AY.43 lineage (Delta variant) amid a period of BA.5 (Omicron variant) dominance in wastewater samples from two wastewater treatment plants (WWTPs) during the months of August and September of 2022. Our results describe a temporary emergence, which was absent in samples from other WWTPs, and which coincided with peaks in viral load. We show how these lineage estimates can be traced back to lineage-specific substitution patterns. The absence of this variant from reported clinical data, but high associated viral loads suggest cryptic transmission. Our findings highlight the additional value of wastewater surveillance for generating insights into circulating pathogens.

Thermal Inactivation of Hepatitis E Virus in Pork Products Estimated with a Semiquantitative Infectivity Assay.

Hepatitis E virus genotype 3 (HEV-3) is a food-borne pathogen causative of hepatitis E infections in humans. In Europe, HEV-3 is mainly transmitted through the consumption of raw or undercooked pork. In order to determine the effectiveness of control measures that can be taken in the industry or by the consumer, it is pivotal to determine the infectivity of HEV present in pork products after thermal food-processing steps. First, we implemented a method for the detection of infectious HEV-3c and HEV-3e in a cell culture medium and in extracts from inoculated pork products. Next, we investigated the effect of the thermal inactivation of HEV by mimicking food-processing steps specific for dried sausage and liver homogenate matrices. After four weeks, HEV-inoculated dried sausage subjected to 21 °C or lower temperatures was still infectious. For the liver homogenate, the highest HEV-3c/e inactivation of the conditions tested was observed at 71 °C for five min or longer. Finally, our method was able to successfully detect and estimate viral loads of infectious HEV in naturally infected pig livers. Our data provide a basis for the future use of the quantitative microbial risk assessment of infectious HEV in pork products that are subjected to thermal food processing steps.

Patterns of SARS-CoV-2 circulation revealed by a nationwide sewage surveillance programme, the Netherlands, August 2020 to February 2022.

BackgroundSurveillance of SARS-CoV-2 in wastewater offers a near real-time tool to track circulation of SARS-CoV-2 at a local scale. However, individual measurements of SARS-CoV-2 in sewage are noisy, inherently variable and can be left-censored.AimWe aimed to infer latent virus loads in a comprehensive sewage surveillance programme that includes all sewage treatment plants (STPs) in the Netherlands and covers 99.6% of the Dutch population.MethodsWe applied a multilevel Bayesian penalised spline model to estimate time- and STP-specific virus loads based on water flow-adjusted SARS-CoV-2 qRT-PCR data for one to four sewage samples per week for each of the more than 300 STPs.ResultsThe model captured the epidemic upsurges and downturns in the Netherlands, despite substantial day-to-day variation in the measurements. Estimated STP virus loads varied by more than two orders of magnitude, from ca 1012 virus particles per 100,000 persons per day in the epidemic trough in August 2020 to almost 1015 per 100,000 in many STPs in January 2022. The timing of epidemics at the local level was slightly shifted between STPs and municipalities, which resulted in less pronounced peaks and troughs at the national level.ConclusionAlthough substantial day-to-day variation is observed in virus load measurements, wastewater-based surveillance of SARS-CoV-2 that is performed at high sampling frequency can track long-term progression of an epidemic at a local scale in near real time.

Norovirus Outbreak Associated with Swimming in a Recreational Lake Not Influenced by External Human Fecal Sources in The Netherlands, August 2012.

Swimming in fecally contaminated recreational water may lead to gastrointestinal illness. A recreational water-associated outbreak of norovirus (NoV) infections affecting at least 100 people in The Netherlands occurred in August 2012. Questionnaire responses from patients indicated swimming in recreational lake Zeumeren as the most likely cause of illness. Most patients visited the lake during the weekend of 18⁻19 August, during which the weather was exceptionally warm (maximum temperatures 32⁻33 °C), and visitor numbers elevated. Patients, mostly children, became ill with gastroenteritis 1⁻6 days (median 2 days) after exposure. Four stool samples from patients were NoV GI positive. Subsurface sandy soil from one of the beaches where most patients swam was NoV GI positive; the water sample was negative. The epidemiological curve and the timeline of investigation based on reported symptoms demonstrate the difficulty in discovering the source in recreational water outbreaks. A NoV outbreak in a recreational lake that is not subjected to external fecal contamination sources shows the need for active communication about human shedding of viruses during and after diarrheal episodes and the advice to refrain from swimming, even a few weeks after the symptoms have resolved.

Aichi virus in sewage and surface water, the Netherlands.

Detection of Aichi virus in humans was initially reported in Japan in 1989. To establish a timeline for the prevalence of Aichi virus infection among humans in the Netherlands, we conducted molecular analysis of archival water samples from 1987-2000 and 2009-2012. Aichi virus RNA was detected in 100% (8/8) of sewage samples and 100% (7/7) of surface water samples collected during 1987-2000 and 100% (8/8) of sewage samples and 71% (5/7) of surface water samples collected during 2009-2012. Several genotype A and B Aichi virus lineages were observed over the 25-year period studied, but the time course of viral genetic diversity showed recent expansion of the genotype B population over genotype A. Our results show that Aichi virus has been circulating among the human population in the Netherlands since before its initial detection in humans was reported and that genotype B now predominates in this country.

Sources of hepatitis E virus genotype 3 in The Netherlands.

Non-travel-related hepatitis E virus (HEV) genotype 3 infections in persons in the Netherlands may have a zoonotic, foodborne, or water-borne origin. Possible reservoirs for HEV transmission by water, food, and animals were studied. HEV genotype 3/open reading frame 2 sequences were detected in 53% of pig farms, 4% of wild boar feces, and 17% of surface water samples. HEV sequences grouped within 4 genotype 3 clusters, of which 1 is so far unique to the Netherlands. The 2 largest clusters contained 35% and 43% of the animal and environmental sequences and 75% and 6%, respectively, of human HEV sequences obtained from a study on Dutch hepatitis E patients. This finding suggests that infection risk may be also dependent on transmission routes other than the ones currently studied. Besides the route of exposure, virus characteristics may be an important determinant for HEV disease in humans.

Increased hepatitis E virus prevalence on Dutch pig farms from 33 to 55% by using appropriate internal quality controls for RT-PCR.

Pigs have been suggested to be a potential reservoir for locally acquired human hepatitis E virus (HEV) infections in the Netherlands. To study possible trends in HEV prevalence in the Dutch pig population, 97 pig farms have been screened for the presence of HEV in stools. The prevalence rate of HEV was estimated at 55% (53/97) in 2005, indicating a significant increase as compared to the prevalence rate of 22% (25/115) as was reported in 1999. The current data suggest that this increase is due to the inclusion of appropriate quality assurance controls such as internal amplification controls for RT-PCR. The abundant presence of pigs excreting HEV raises concerns on potential zoonotic transmission of the virus, either by exposure through the environment or by consumption of contaminated pork products. Moreover, one of the detected strains belonged to a European cluster which was not detected in the Netherlands before, suggesting that HEV strains spread through European countries. These data demonstrate the need to include appropriate controls in diagnostic assays, especially in complex matrices such as feces which are known to contain PCR inhibitory substances.

Cryptosporidium and Giardia in commercial and non-commercial oysters (Crassostrea gigas) and water from the Oosterschelde, The Netherlands.

The intestinal parasites Cryptosporidium and Giardia cause gastro-enteritis in humans and can be transmitted via contaminated water. Oysters are filter feeders that have been demonstrated to accumulate pathogens such as Salmonella, Vibrio, norovirus and Cryptosporidium from contaminated water and cause foodborne infections. Oysters are economically important shellfish that are generally consumed raw. Commercial and non-commercial oysters (Crassostrea gigas) and oyster culture water from the Oosterschelde, The Netherlands, were examined for the presence of Cryptosporidium oocysts and Giardia cysts. Nine of 133 (6.7%) oysters from two non-commercial harvesting sites contained Cryptosporidium, Giardia or both. Six of 46 (13.0%) commercial oysters harboured Cryptosporidium or Giardia in their intestines. Data on the viability of (oo)cysts recovered from Oosterschelde oysters were not obtained, however viable (oo)cysts were detected in surface waters that enter the Oosterschelde oyster harvesting areas. The detection of Cryptosporidium and Giardia in oysters destined for human consumption has implications for public health only when human pathogenic (oo)cysts that have preserved infectivity during their stay in a marine environment are present. Our data suggest that consumption of raw oysters from the Oosterschelde may occasionally lead to cases of gastro-intestinal illness.

Real-time detection of noroviruses in surface water by use of a broadly reactive nucleic acid sequence-based amplification assay.

Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection assays for viruses in water are often repressed by inhibitory factors present in the environment, like humic acids and heavy metals. To study the effect of environmental inhibitors on the performance of nucleic acid sequence-based amplification (NASBA), we developed a real-time norovirus NASBA targeting part of the RNA-dependent RNA polymerase (RdRp) gene. Specificity of the assay was studied with 33 divergent clones that contained part of the targeted RdRp gene of noroviruses from 15 different genogroups. Viral RNA originated from commercial oysters, surface waters, and sewage treatment plants in The Netherlands. Ninety-seven percent of the clones derived from human noroviruses were detected by real-time NASBA. Two clones containing animal noroviruses were not detected by NASBA. We compared the norovirus detection by real-time NASBA with that by conventional reverse transcriptase PCR (RT-PCR) with large-volume river water samples and found that inhibitory factors of RT-PCR had little or no effect on the performance of the norovirus NASBA. This consequently resulted in a higher sensitivity of the NASBA assay than of the RT-PCR. We show that by combining an efficient RNA extraction method with real-time NASBA the sensitivity of norovirus detection in water samples increased at least 100 times, which consequently has implications for the outcome of the infectious risk assessment.

Isolation and detection of enterovirus RNA from large-volume water samples by using the NucliSens miniMAG system and real-time nucleic acid sequence-based amplification.

Concentration of water samples is a prerequisite for the detection of the low virus levels that are present in water and may present a public health hazard. The aim of this study was to develop a rapid, standardized molecular method for the detection of enteroviruses in large-volume surface water samples, using a concentration method suitable for the detection of infectious viruses as well as virus RNA. Concentration of water was achieved by a conventional filter adsorption-elution method and ultrafiltration, resulting in a 10,000-fold concentration of the sample. Isolation of virus RNA by a silica-based RNA extraction method was compared with the nonmagnetic and magnetic NucliSens RNA isolation methods. By using the silica-based RNA extraction method in two out of five samples, enterovirus RNA was detected, whereas four out of five samples were positive following RNA isolation with magnetic silica beads. Moreover, estimated RNA levels increased at least 100 to 500 times. Furthermore, we compared enterovirus detection by an in-house reverse transcription (RT)-PCR with a novel commercially available real-time nucleic acid sequence-based amplification (NASBA) assay. We found that the rapid real-time NASBA assay was slightly less sensitive than our in-house RT-PCR. The advantages, however, of a commercial real-time NASBA assay, like the presence of an internal control RNA, standardization, and enormous decrease in turnaround time, makes it an attractive alternative to RT-PCR.