Untangling introductions and persistence in COVID-19 resurgence in Europe.

After the first wave of SARS-CoV-2 infections in spring 2020, Europe experienced a resurgence of the virus starting in late summer 2020 that was deadlier and more difficult to contain1. Relaxed intervention measures and summer travel have been implicated as drivers of the second wave2. Here we build a phylogeographical model to evaluate how newly introduced lineages, as opposed to the rekindling of persistent lineages, contributed to the resurgence of COVID-19 in Europe. We inform this model using genomic, mobility and epidemiological data from 10 European countries and estimate that in many countries more than half of the lineages circulating in late summer resulted from new introductions since 15 June 2020. The success in onward transmission of newly introduced lineages was negatively associated with the local incidence of COVID-19 during this period. The pervasive spread of variants in summer 2020 highlights the threat of viral dissemination when restrictions are lifted, and this needs to be carefully considered in strategies to control the current spread of variants that are more transmissible and/or evade immunity. Our findings indicate that more effective and coordinated measures are required to contain the spread through cross-border travel even as vaccination is reducing disease burden.

West Nile virus spread in Europe: Phylogeographic pattern analysis and key drivers.

BACKGROUND: West Nile virus (WNV) outbreaks in birds, humans, and livestock have occurred in multiple areas in Europe and have had a significant impact on animal and human health. The patterns of emergence and spread of WNV in Europe are very different from those in the US and understanding these are important for guiding preparedness activities. METHODS: We mapped the evolution and spread history of WNV in Europe by incorporating viral genome sequences and epidemiological data into phylodynamic models. Spatially explicit phylogeographic models were developed to explore the possible contribution of different drivers to viral dispersal direction and velocity. A "skygrid-GLM" approach was used to identify how changes in environments would predict viral genetic diversity variations over time. FINDINGS: Among the six lineages found in Europe, WNV-2a (a sub-lineage of WNV-2) has been predominant (accounting for 73% of all sequences obtained in Europe that have been shared in the public domain) and has spread to at least 14 countries. In the past two decades, WNV-2a has evolved into two major co-circulating clusters, both originating from Central Europe, but with distinct dynamic history and transmission patterns. WNV-2a spreads at a high dispersal velocity (88km/yr-215 km/yr) which is correlated to bird movements. Notably, amongst multiple drivers that could affect the spread of WNV, factors related to land use were found to strongly influence the spread of WNV. Specifically, the intensity of agricultural activities (defined by factors related to crops and livestock production, such as coverage of cropland, pasture, cultivated and managed vegetation, livestock density) were positively associated with both spread direction and velocity. In addition, WNV spread direction was associated with high coverage of wetlands and migratory bird flyways. CONCLUSION: Our results suggest that-in addition to ecological conditions favouring bird- and mosquito- presence-agricultural land use may be a significant driver of WNV emergence and spread. Our study also identified significant gaps in data and the need to strengthen virological surveillance in countries of Central Europe from where WNV outbreaks are likely seeded. Enhanced monitoring for early detection of further dispersal could be targeted to areas with high agricultural activities and habitats of migratory birds.

Clinical and Virological Outcome of Monoclonal Antibody Therapies Across SARS-CoV-2 Variants in 245 Immunocompromised Patients: A Multicenter Prospective Cohort Study.

BACKGROUND: Immunocompromised patients (ICPs) have an increased risk for a severe and prolonged COVID-19. SARS-CoV-2 monoclonal antibodies (mAbs) were extensively used in these patients, but data from randomized trials that focus on ICPs are lacking. We evaluated the clinical and virological outcome of COVID-19 in ICPs treated with mAbs across SARS-CoV-2 variants. METHODS: In this multicenter prospective cohort study, we enrolled B-cell- and/or T-cell-deficient patients treated with casirivimab/imdevimab, sotrovimab, or tixagevimab/cilgavimab. SARS-CoV-2 RNA was quantified and sequenced weekly, and time to viral clearance, viral genome mutations, hospitalization, and death rates were registered. RESULTS: Two hundred and forty five patients infected with the Delta (50%) or Omicron BA.1, 2, or 5 (50%) variant were enrolled. Sixty-seven percent were vaccinated; 78 treated as outpatients, of whom 2 required hospital admission, but both survived. Of the 159 patients hospitalized at time of treatment, 43 (27%) required mechanical ventilation or died. The median time to viral clearance was 14 days (interquartile range, 7-22); however, it took >30 days in 15%. Resistance-associated spike mutations emerged in 9 patients in whom the median time to viral clearance was 63 days (95% confidence interval, 57-69; P < .001). Spike mutations were observed in 1 of 42 (2.4%) patients after treatment with 2 active mAbs, in 5 of 34 (14.7%) treated with actual monotherapy (sotrovimab), and 3 of 20 (12%) treated with functional monotherapy (ie, tixagevimab/cilgavimab against tixagevimab-resistant variant). CONCLUSIONS: Despite treatment with mAbs, morbidity and mortality of COVID-19 in ICPs remained substantial. Combination antiviral therapy should be further explored and may be preferred in severely ICPs.

Circulation, viral diversity and genomic rearrangement in mpox virus in the Netherlands during the 2022 outbreak and beyond.

Mpox is an emerging zoonotic disease which has now spread to over 113 countries as of August 2023, with over 89,500 confirmed human cases. The Netherlands had one of the highest incidence rates in Europe during the peak of the outbreak. In this study, we generated 158 near-complete mpox virus (MPXV) genomes (12.4% of nationwide cases) that were collected throughout the Netherlands from the start of the outbreak in May 2022 to August 2023 to track viral evolution and investigate outbreak dynamics. We detected 14 different viral lineages, suggesting multiple introductions followed by rapid initial spread within the country. The estimated evolutionary rate was relatively high compared to previously described in orthopoxvirus literature, with an estimated 11.58 mutations per year. Genomic rearrangement events occurred at a rate of 0.63% and featured a large deletion event. In addition, based on phylogenetics, we identified multiple potential transmission clusters which could be supported by direct source- and contact tracing data. This led to the identification of at least two main transmission locations at the beginning of the outbreak. We conclude that whole genome sequencing of MPXV is essential to enhance our understanding of outbreak dynamics and evolution of a relatively understudied and emerging zoonotic pathogen.

Ongoing mpox outbreak in Kamituga, South Kivu province, associated with monkeypox virus of a novel Clade I sub-lineage, Democratic Republic of the Congo, 2024.

Since the beginning of 2023, the number of people with suspected monkeypox virus (MPXV) infection have sharply increased in the Democratic Republic of the Congo (DRC). We report near-to-complete MPXV genome sequences derived from six cases from the South Kivu province. Phylogenetic analyses reveal that the MPXV affecting the cases belongs to a novel Clade I sub-lineage. The outbreak strain genome lacks the target sequence of the probe and primers of a commonly used Clade I-specific real-time PCR.

Sotrovimab Resistance and Viral Persistence After Treatment of Immunocompromised Patients Infected With the Severe Acute Respiratory Syndrome Coronavirus 2 Omicron Variant.

Viral evolution was evaluated in 47 immunocompromised patients treated with sotrovimab. Sequencing of SARS-CoV-2 following therapy was successful in 16. Mutations associated with sotrovimab resistance were documented in 6; viral replication continued after 30 days in 5. Combination antibody therapy may be required to avoid acquired resistance in immunocompromised patients.

High-titer convalescent plasma plus nirmatrelvir/ritonavir treatment for non-resolving COVID-19 in six immunocompromised patients.

OBJECTIVES: Immunocompromised patients have an increased risk of severe or prolonged COVID-19. Currently available drugs are registered to treat COVID-19 during the first 5 to 7 days after symptom onset. Data on the effectivity in immunocompromised patients with chronic non-resolving COVID-19 are urgently needed. Here, we report the outcome of patients treated with nirmatrelvir/ritonavir together with high-titer convalescent plasma (CP) in six immunocompromised patients with non-resolving COVID-19. METHODS: Immunocompromised patients with persisting COVID-19 (positive PCR with Ct values <30 for ≥20 days) received off-label therapy with nirmatrelvir/ritonavir. It was combined with CP containing BA.5 neutralizing titers of ≥1/640 whenever available. Follow-up was done by PCR and sequencing on nasopharyngeal swabs on a weekly basis until viral genome was undetectable consecutively. RESULTS: Five immunocompromised patients were treated with high-titer CP and 5 days of nirmatrelvir/ritonavir. One patient received nirmatrelvir/ritonavir monotherapy. Median duration of SARS-CoV-2 PCR positivity was 70 (range 20-231) days before nirmatrelvir/ritonavir treatment. In four patients receiving combination therapy, no viral genome of SARS-CoV-2 was detected on day 7 and 14 after treatment while the patient receiving nirmatrelvir/ritonavir monotherapy, the day 7 Ct value increased to 34 and viral genome was undetectable thereafter. Treatment was unsuccessful in one patient. In this patient, sequencing after nirmatrelvir/ritonavir treatment did not show protease gene mutations. CONCLUSIONS: In immunocompromised patients with non-resolving COVID-19, the combination of nirmatrelvir/ritonavir and CP may be an effective treatment. Larger prospective studies are needed to confirm these preliminary results and should compare different treatment durations.

SARS-CoV-2 in lions, gorillas and zookeepers in the Rotterdam Zoo, the Netherlands, a One Health investigation, November 2021.

In November 2021, seven western lowland gorillas and four Asiatic lions were diagnosed with COVID-19 at Rotterdam Zoo. An outbreak investigation was undertaken to determine the source and extent of the outbreak and to identify possible transmission routes. Interviews were conducted with staff to identify human and animal contacts and cases, compliance with personal protective equipment (PPE) and potential transmission routes. Human and animal contacts and other animal species suspected to be susceptible to SARS-CoV-2 were tested for SARS-CoV-2 RNA. Positive samples were subjected to sequencing. All the gorillas and lions that could be tested (3/7 and 2/4, respectively) were RT-PCR positive between 12 November and 10 December 2021. No other animal species were SARS-CoV-2 RNA positive. Forty direct and indirect human contacts were identified. Two direct contacts tested RT-PCR positive 10 days after the first COVID-19 symptoms in animals. The zookeepers' viral genome sequences clustered with those of gorillas and lions. Personal protective equipment compliance was suboptimal at instances. Findings confirm transmission of SARS-CoV-2 among animals and between humans and animals but source and directionality could not be established. Zookeepers were the most likely source and should have periodic PPE training. Sick animals should promptly be tested and isolated/quarantined.

Cryptic SARS-CoV-2 lineage identified on two mink farms as a possible result of long-term undetected circulation in an unknown animal reservoir, Poland, November 2022 to January 2023.

In late 2022 and early 2023, SARS-CoV-2 infections were detected on three mink farms in Poland situated within a few km from each other. Whole-genome sequencing of the viruses on two of the farms showed that they were related to a virus identified in humans in the same region 2 years before (B.1.1.307 lineage). Many mutations were found, including in the S protein typical of adaptations to the mink host. The origin of the virus remains to be determined.

Mpox outbreak among men who have sex with men in Amsterdam and Rotterdam, the Netherlands: no evidence for undetected transmission prior to May 2022, a retrospective study.

Since May 2022, over 21,000 mpox cases have been reported from 29 EU/EEA countries, predominantly among men who have sex with men (MSM). The Netherlands was the fourth most affected country in Europe, with more than 1,200 cases and a crude notification rate of 70.7 per million population. The first national case was reported on 10 May, yet potential prior transmission remains unknown. Insight into prolonged undetected transmission can help to understand the current outbreak dynamics and aid future public health interventions. We performed a retrospective study and phylogenetic analysis to elucidate whether undetected transmission of human mpox virus (hMPXV) occurred before the first reported cases in Amsterdam and Rotterdam. In 401 anorectal and ulcer samples from visitors to centres for sexual health in Amsterdam or Rotterdam dating back to 14 February 2022, we identified two new cases, the earliest from 6 May. This coincides with the first cases reported in the United Kingdom, Spain and Portugal. We found no evidence of widespread hMPXV transmission in Dutch sexual networks of MSM before May 2022. Likely, the mpox outbreak expanded across Europe within a short period in the spring of 2022 through an international highly intertwined network of sexually active MSM.

Clinical and In Vitro Evidence Favoring Immunoglobulin Treatment of a Chronic Norovirus Infection in a Patient With Common Variable Immunodeficiency.

BACKGROUND: Immunocompromised individuals can become chronically infected with norovirus, but effective antiviral therapies are not yet available. METHODS: Treatments with nitazoxanide, ribavirin, interferon alpha-2a, and nasoduodenally administered immunoglobulins were evaluated sequentially in an immunocompromised patient chronically infected with norovirus. In support, these components were also applied to measure norovirus inhibition in intestinal enteroid cultures in vitro. Viral RNA levels were determined in fecal and plasma samples during each treatment and viral genomes were sequenced. RESULTS: None of the antivirals resulted in a reduction of viral RNA levels in feces or plasma. However, during ribavirin treatment, there was an increased accumulation of virus genome mutations. In vitro, an effect of interferon alpha-2a on virus replication was observed and a genetically related strain was neutralized effectively in vitro using immunoglobulins and post-norovirus-infection antiserum. In agreement, after administration of immunoglobulins, the patient cleared the infection. CONCLUSIONS: Intestinal enteroid cultures provide a relevant system to evaluate antivirals and the neutralizing potential of immunoglobulins. We successfully treated a chronically infected patient with immunoglobulins, despite varying results reported by others. This case study provides in-depth, multifaceted exploration of norovirus treatment that can be used as a guidance for further research towards norovirus treatments.

Towards reliable whole genome sequencing for outbreak preparedness and response.

BACKGROUND: To understand the dynamics of infectious diseases, genomic epidemiology is increasingly advocated, with a need for rapid generation of genetic sequences during outbreaks for public health decision making. Here, we explore the use of metagenomic sequencing compared to specific amplicon- and capture-based sequencing, both on the Nanopore and the Illumina platform for generation of whole genomes of Usutu virus, Zika virus, West Nile virus, and Yellow Fever virus. RESULTS: We show that amplicon-based Nanopore sequencing can be used to rapidly obtain whole genome sequences in samples with a viral load up to Ct 33 and capture-based Illumina is the most sensitive method for initial virus determination. CONCLUSIONS: The choice of sequencing approach and platform is important for laboratories wishing to start whole genome sequencing. Depending on the purpose of genome sequencing the best choice can differ. The insights presented in this work and the shown differences in data characteristics can guide labs to make a well informed choice.

SARS-CoV-2 outbreaks in secondary school settings in the Netherlands during fall 2020; silent circulation.

BACKGROUND: In fall 2020 when schools in the Netherlands operated under a limited set of COVID-19 measures, we conducted outbreaks studies in four secondary schools to gain insight in the level of school transmission and the role of SARS-CoV-2 transmission via air and surfaces. METHODS: Outbreak studies were performed between 11 November and 15 December 2020 when the wild-type variant of SARS-CoV-2 was dominant. Clusters of SARS-CoV-2 infections within schools were identified through a prospective school surveillance study. All school contacts of cluster cases, irrespective of symptoms, were invited for PCR testing twice within 48 h and 4-7 days later. Combined NTS and saliva samples were collected at each time point along with data on recent exposure and symptoms. Surface and active air samples were collected in the school environment. All samples were PCR-tested and sequenced when possible. RESULTS: Out of 263 sampled school contacts, 24 tested SARS-CoV-2 positive (secondary attack rate 9.1%), of which 62% remained asymptomatic and 42% had a weakly positive test result. Phylogenetic analysis on 12 subjects from 2 schools indicated a cluster of 8 and 2 secondary cases, respectively, but also other distinct strains within outbreaks. Of 51 collected air and 53 surface samples, none were SARS-CoV-2 positive. CONCLUSION: Our study confirmed within school SARS-CoV-2 transmission and substantial silent circulation, but also multiple introductions in some cases. Absence of air or surface contamination suggests environmental contamination is not widespread during school outbreaks.

Unraveling the Modes of Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) During a Nursing Home Outbreak: Looking Beyond the Church Superspreading Event.

BACKGROUND: An outbreak of coronavirus disease 2019 (COVID-19) in a nursing home in the Netherlands, following an on-site church service held on 8 March 2020, triggered an investigation to unravel sources and chain(s) of transmission. METHODS: Epidemiological data were collected from registries and through a questionnaire among church attendees. Symptomatic residents and healthcare workers (HCWs) were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by reverse-transcription polymerase chain reaction and subjected to whole genome sequencing (WGS). Sequences from a selection of people from the same area were included as community reference. RESULTS: After the church service, 30 of 39 attendees (77%) developed symptoms; 14 (11 residents and 3 nonresidents) were tested and were positive for COVID-19. In the following 5 weeks, 62 of 300 residents (21%) and 30 of 640 HCWs (5%) tested positive for COVID-19; 21 of 62 residents (34%) died. The outbreak was controlled through a cascade of measures. WGS of samples from residents and HCWs identified a diversity of sequence types, grouped into 8 clusters. Seven resident church attendees all were infected with distinct viruses, 4 of which belonged to 2 larger clusters in the nursing home. CONCLUSIONS: Although initial investigation suggested the church service as the source of the outbreak, detailed analysis showed a more complex picture, most consistent with widespread regional circulation of the virus in the weeks before the outbreak, and multiple introductions into the nursing home before the visitor ban. The findings underscore the importance of careful outbreak investigations to understand SARS-CoV-2 transmission to develop evidence-based mitigation measures.

COVID-19: first long-term care facility outbreak in the Netherlands following cross-border introduction from Germany, March 2020.

BACKGROUND: The Dutch province of Limburg borders the German district of Heinsberg, which had a large cluster of COVID-19 cases linked to local carnival activities before any cases were reported in the Netherlands. However, Heinsberg was not included as an area reporting local or community transmission per the national case definition at the time. In early March, two residents from a long-term care facility (LTCF) in Sittard, a Dutch town located in close vicinity to the district of Heinsberg, tested positive for COVID-19. In this study we aimed to determine whether cross-border introduction of the virus took place by analysing the LTCF outbreak in Sittard, both epidemiologically and microbiologically. METHODS: Surveys and semi-structured oral interviews were conducted with all present LTCF residents by health care workers during regular points of care for information on new or unusual signs and symptoms of disease. Both throat and nasopharyngeal swabs were taken from residents suspect of COVID-19, based on regional criteria, for the detection of SARS-CoV-2 by Real-time Polymerase Chain Reaction. Additionally, whole genome sequencing was performed using a SARS-CoV-2 specific amplicon-based Nanopore sequencing approach. Moreover, twelve random residents were sampled for possible asymptomatic infections. RESULTS: Out of 99 residents, 46 got tested for COVID-19. Out of the 46 tested residents, nineteen (41%) tested positive for COVID-19, including 3 asymptomatic residents. CT-values for asymptomatic residents seemed higher compared to symptomatic residents. Eleven samples were sequenced, along with three random samples from COVID-19 patients hospitalized in the regional hospital at the time of the LTCF outbreak. All samples were linked to COVID-19 cases from the cross-border region of Heinsberg, Germany. CONCLUSIONS: Sequencing combined with epidemiological data was able to virtually prove cross-border transmission at the start of the Dutch COVID-19 epidemic. Our results highlight the need for cross-border collaboration and adjustment of national policy to emerging region-specific needs along borders in order to establish coordinated implementation of infection control measures to limit the spread of COVID-19.

Occupational and environmental exposure to SARS-CoV-2 in and around infected mink farms.

OBJECTIVE: Unprecedented SARS-CoV-2 infections in farmed minks raised immediate concerns regarding transmission to humans and initiated intensive environmental investigations to assess occupational and environmental exposure. METHODS: Air sampling was performed at infected Dutch mink farms, at farm premises and at nearby residential sites. A range of other environmental samples were collected from minks' housing units, including bedding materials. SARS-CoV-2 RNA was analysed in all samples by quantitative PCR. RESULTS: Inside the farms, considerable levels of SARS-CoV-2 RNA were found in airborne dust, especially in personal inhalable dust samples (approximately 1000-10 000 copies/m3). Most of the settling dust samples tested positive for SARS-CoV-2 RNA (82%, 75 of 92). SARS-CoV-2 RNA was not detected in outdoor air samples, except for those collected near the entrance of the most recently infected farm. Many samples of minks' housing units and surfaces contained SARS-CoV-2 RNA. CONCLUSIONS: Infected mink farms can be highly contaminated with SARS-CoV-2 RNA. This warns of occupational exposure, which was substantiated by considerable SARS-CoV-2 RNA concentrations in personal air samples. Dispersion of SARS-CoV-2 to outdoor air was found to be limited and SARS-CoV-2 RNA was not detected in air samples collected beyond farm premises, implying a negligible risk of environmental exposure to nearby communities. Our occupational and environmental risk assessment is in line with whole genome sequencing analyses showing mink-to-human transmission among farm workers, but no indications of direct zoonotic transmission events to nearby communities.

Are presymptomatic SARS-CoV-2 infections in nursing home residents unrecognised symptomatic infections? Sequence and metadata from weekly testing in an extensive nursing home outbreak.

BACKGROUND: Sars-CoV-2 outbreaks resulted in a high case fatality rate in nursing homes (NH) worldwide. It is unknown to which extent presymptomatic residents and staff contribute to the spread of the virus. AIMS: To assess the contribution of asymptomatic and presymptomatic residents and staff in SARS-CoV-2 transmission during a large outbreak in a Dutch NH. METHODS: Observational study in a 185-bed NH with two consecutive testing strategies: testing of symptomatic cases only, followed by weekly facility-wide testing of staff and residents regardless of symptoms. Nasopharyngeal and oropharyngeal testing with RT-PCR for SARs-CoV-2, including sequencing of positive samples, was conducted with a standardised symptom assessment. RESULTS: 185 residents and 244 staff participated. Sequencing identified one cluster. In the symptom-based test strategy period, 3/39 residents were presymptomatic versus 38/74 residents in the period of weekly facility-wide testing (P-value < 0.001). In total, 51/59 (91.1%) of SARS-CoV-2 positive staff was symptomatic, with no difference between both testing strategies (P-value 0.763). Loss of smell and taste, sore throat, headache or myalga was hardly reported in residents compared to staff (P-value <0.001). Median Ct-value of presymptomatic residents was 21.3, which did not differ from symptomatic (20.8) or asymptomatic (20.5) residents (P-value 0.624). CONCLUSIONS: Symptoms in residents and staff are insufficiently recognised, reported or attributed to a possible SARS-CoV-2 infection. However, residents without (recognised) symptoms showed the same potential for viral shedding as residents with symptoms. Weekly testing was an effective strategy for early identification of SARS-Cov-2 cases, resulting in fast mitigation of the outbreak.

Economic evaluation of whole genome sequencing for pathogen identification and surveillance - results of case studies in Europe and the Americas 2016 to 2019.

BackgroundWhole genome sequencing (WGS) is increasingly used for pathogen identification and surveillance.AimWe evaluated costs and benefits of routine WGS through case studies at eight reference laboratories in Europe and the Americas which conduct pathogen surveillance for avian influenza (two laboratories), human influenza (one laboratory) and food-borne pathogens (five laboratories).MethodsThe evaluation focused on the institutional perspective, i.e. the 'investment case' for implementing WGS compared with conventional methods, based on costs and benefits during a defined reference period, mostly covering at least part of 2017. A break-even analysis estimated the number of cases of illness (for the example of Salmonella surveillance) that would need to be avoided through WGS in order to 'break even' on costs.ResultsOn a per-sample basis, WGS was between 1.2 and 4.3 times more expensive than routine conventional methods. However, WGS brought major benefits for pathogen identification and surveillance, substantially changing laboratory workflows, analytical processes and outbreaks detection and control. Between 0.2% and 1.1% (on average 0.7%) of reported salmonellosis cases would need to be prevented to break even with respect to the additional costs of WGS.ConclusionsEven at cost levels documented here, WGS provides a level of additional information that more than balances the additional costs if used effectively. The substantial cost differences for WGS between reference laboratories were due to economies of scale, degree of automation, sequencing technology used and institutional discounts for equipment and consumables, as well as the extent to which sequencers are used at full capacity.

Detection of West Nile virus in a common whitethroat (Curruca communis) and Culex mosquitoes in the Netherlands, 2020.

On 22 August, a common whitethroat in the Netherlands tested positive for West Nile virus lineage 2. The same bird had tested negative in spring. Subsequent testing of Culex mosquitoes collected in August and early September in the same location generated two of 44 positive mosquito pools, providing first evidence for enzootic transmission in the Netherlands. Sequences generated from the positive mosquito pools clustered with sequences that originate from Germany, Austria and the Czech Republic.