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1.
Viruses ; 14(8):1754, 2022.
Article in English | MDPI | ID: covidwho-1987986

ABSTRACT

SARS-CoV-2 outbreaks on 69 Dutch mink farms in 2020 were studied to identify risk factors for virus introduction and transmission and to improve surveillance and containment measures. Clinical signs, laboratory test results, and epidemiological aspects were investigated, such as the date and reason of suspicion, housing, farm size and distances, human contact structure, biosecurity measures, and presence of wildlife, pets, pests, and manure management. On seven farms, extensive random sampling was performed, and age, coat color, sex, and clinical signs were recorded. Mild to severe respiratory signs and general diseases such as apathy, reduced feed intake, and increased mortality were detected on 62/69 farms. Throat swabs were more likely to result in virus detection than rectal swabs. Clinical signs differed between virus clusters and were more severe for dark-colored mink, males, and animals infected later during the year. Geographical clustering was found for one virus cluster. Shared personnel could explain some cases, but other transmission routes explaining farm-to-farm spread were not elucidated. An early warning surveillance system, strict biosecurity measures, and a (temporary) ban on mink farming and vaccinating animals and humans can contribute to reducing the risks of the virus spreading and acquisition of potential mutations relevant to human and animal health.

2.
Sci Immunol ; : eabq4450, 2022 Jun 23.
Article in English | MEDLINE | ID: covidwho-1901912

ABSTRACT

The emergence and rapid spread of SARS-CoV-2 variants may impact vaccine efficacy significantly. The Omicron variant termed BA.2, which differs substantially from BA.1 based on genetic sequence, is currently replacing BA.1 in several countries, but its antigenic characteristics have not yet been assessed. Here, we used antigenic cartography to quantify and visualize antigenic differences between early SARS-CoV-2 variants (614G, Alpha, Beta, Gamma, Zeta, Delta and Mu) using hamster antisera obtained after primary infection. We first verified that the choice of the cell line for the neutralization assay did not affect the topology of the map substantially. Antigenic maps generated using pseudotyped SARS-CoV-2 on the widely used VeroE6 cell line and the human airway cell line Calu-3 generated similar maps. Maps made using authentic SARS-CoV-2 on Calu-3 cells also closely resembled those generated with pseudotyped viruses. The antigenic maps revealed a central cluster of SARS-CoV-2 variants, which grouped based on mutual spike mutations. Whereas these early variants are antigenically similar, clustering relatively close to each other in antigenic space, Omicron BA.1 and BA.2 have evolved as two distinct antigenic outliers. Our data show that BA.1 and BA.2 both escape vaccine-induced antibody responses as a result of different antigenic characteristics. Thus, antigenic cartography could be used to assess antigenic properties of future SARS-CoV-2 variants of concern that emerge and to decide on the composition of novel spike-based (booster) vaccines.

4.
J Virol Methods ; 300: 114397, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1540816

ABSTRACT

Here we describe a SARS-CoV-2 variant with diminished amplification of the ORF ORF1ab target in the Cobas® dual-target SARS-CoV-2 assay resulting in a discrepancy of Ct-values (Ct-value 20.7 for the E-gene and Ct-value 30.2 for ORF1ab). Five unique nucleotide mutations were identified in ORF1ab: C11450A (nsp10) C14178T (RdRp), G15006T (RdRp), G18394T (Hel), and G20995T (Hel). This case highlights the importance of surveillance of genomic regions used in molecular diagnostics and the importance of the public release of target regions used to update commercial and in-house developed SARS-CoV-2 PCR tests. This work underpins the importance of using dual-targets in molecular diagnostic assays to limit the change of false-negative results due to primer and/or probe mismatches.


Subject(s)
COVID-19 , SARS-CoV-2 , Diagnostic Tests, Routine , Humans , RNA, Viral , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity
5.
Nat Commun ; 12(1): 6802, 2021 11 23.
Article in English | MEDLINE | ID: covidwho-1532052

ABSTRACT

In the first wave of the COVID-19 pandemic (April 2020), SARS-CoV-2 was detected in farmed minks and genomic sequencing was performed on mink farms and farm personnel. Here, we describe the outbreak and use sequence data with Bayesian phylodynamic methods to explore SARS-CoV-2 transmission in minks and humans on farms. High number of farm infections (68/126) in minks and farm workers (>50% of farms) were detected, with limited community spread. Three of five initial introductions of SARS-CoV-2 led to subsequent spread between mink farms until November 2020. Viruses belonging to the largest cluster acquired an amino acid substitution in the receptor binding domain of the Spike protein (position 486), evolved faster and spread longer and more widely. Movement of people and distance between farms were statistically significant predictors of virus dispersal between farms. Our study provides novel insights into SARS-CoV-2 transmission between mink farms and highlights the importance of combining genetic information with epidemiological information when investigating outbreaks at the animal-human interface.


Subject(s)
COVID-19/epidemiology , COVID-19/transmission , COVID-19/virology , Evolution, Molecular , Farms , Mink/virology , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Amino Acid Sequence , Animal Diseases/epidemiology , Animal Diseases/transmission , Animal Diseases/virology , Animals , Bayes Theorem , Disease Outbreaks , Humans , Netherlands/epidemiology , Phylogeny , SARS-CoV-2/isolation & purification , Sequence Analysis, Protein , Spike Glycoprotein, Coronavirus/classification , Spike Glycoprotein, Coronavirus/genetics
9.
Age Ageing ; 50(5): 1454-1463, 2021 09 11.
Article in English | MEDLINE | ID: covidwho-1406457

ABSTRACT

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.


Subject(s)
COVID-19 , SARS-CoV-2 , Disease Outbreaks , Humans , Metadata , Nursing Homes
10.
Nat Med ; 27(9): 1518-1524, 2021 09.
Article in English | MEDLINE | ID: covidwho-1402106

ABSTRACT

The current coronavirus disease 2019 (COVID-19) pandemic is the first to apply whole-genome sequencing near to real time, with over 2 million severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome sequences generated and shared through the GISAID platform. This genomic resource informed public health decision-making throughout the pandemic; it also allowed detection of mutations that might affect virulence, pathogenesis, host range or immune escape as well as the effectiveness of SARS-CoV-2 diagnostics and therapeutics. However, genotype-to-phenotype predictions cannot be performed at the rapid pace of genomic sequencing. To prepare for the next phase of the pandemic, a systematic approach is needed to link global genomic surveillance and timely assessment of the phenotypic characteristics of novel variants, which will support the development and updating of diagnostics, vaccines, therapeutics and nonpharmaceutical interventions. This Review summarizes the current knowledge on key viral mutations and variants and looks to the next phase of surveillance of the evolving pandemic.


Subject(s)
COVID-19/epidemiology , Epidemiological Monitoring , Genome, Viral/genetics , Molecular Epidemiology/methods , SARS-CoV-2/genetics , Base Sequence/genetics , Clinical Decision-Making , Databases, Genetic , Humans , Public Health , Whole Genome Sequencing
11.
Occup Environ Med ; 78(12): 893-899, 2021 12.
Article in English | MEDLINE | ID: covidwho-1388534

ABSTRACT

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.


Subject(s)
Dust/analysis , Environmental Exposure , Farms , Mink/virology , Occupational Exposure , RNA, Viral/isolation & purification , SARS-CoV-2/isolation & purification , Animals , Humans , Netherlands/epidemiology
12.
Transbound Emerg Dis ; 2021 Jun 03.
Article in English | MEDLINE | ID: covidwho-1388408

ABSTRACT

Animals like mink, cats and dogs are susceptible to SARS-CoV-2 infection. In the Netherlands, 69 out of 127 mink farms were infected with SARS-CoV-2 between April and November 2020 and all mink on infected farms were culled after SARS-CoV-2 infection to prevent further spread of the virus. On some farms, (feral) cats and dogs were present. This study provides insight into the prevalence of SARS-CoV-2-positive cats and dogs in 10 infected mink farms and their possible role in transmission of the virus. Throat and rectal swabs of 101 cats (12 domestic and 89 feral cats) and 13 dogs of 10 farms were tested for SARS-CoV-2 using PCR. Serological assays were performed on serum samples from 62 adult cats and all 13 dogs. Whole Genome Sequencing was performed on one cat sample. Cat-to-mink transmission parameters were estimated using data from all 10 farms. This study shows evidence of SARS-CoV-2 infection in 12 feral cats and 2 dogs. Eleven cats (18%) and two dogs (15%) tested serologically positive. Three feral cats (3%) and one dog (8%) tested PCR-positive. The sequence generated from the cat throat swab clustered with mink sequences from the same farm. The calculated rate of mink-to-cat transmission showed that cats on average had a chance of 12% (95%CI 10%-18%) of becoming infected by mink, assuming no cat-to-cat transmission. As only feral cats were infected it is most likely that infections in cats were initiated by mink, not by humans. Whether both dogs were infected by mink or humans remains inconclusive. This study presents one of the first reports of interspecies transmission of SARS-CoV-2 that does not involve humans, namely mink-to-cat transmission, which should also be considered as a potential risk for spread of SARS-CoV-2.

14.
Clin Infect Dis ; 73(Suppl 2): S163-S169, 2021 07 30.
Article in English | MEDLINE | ID: covidwho-1373635

ABSTRACT

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.


Subject(s)
COVID-19 , SARS-CoV-2 , Disease Outbreaks , Humans , Netherlands , Nursing Homes
15.
Nature ; 595(7869): 713-717, 2021 07.
Article in English | MEDLINE | ID: covidwho-1287812

ABSTRACT

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.


Subject(s)
COVID-19/transmission , COVID-19/virology , SARS-CoV-2/isolation & purification , COVID-19/epidemiology , COVID-19/prevention & control , Europe/epidemiology , Genome, Viral/genetics , Humans , Incidence , Locomotion , Phylogeny , Phylogeography , SARS-CoV-2/classification , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Time Factors , Travel/statistics & numerical data
16.
Int J Infect Dis ; 109: 24-32, 2021 Aug.
Article in English | MEDLINE | ID: covidwho-1272475

ABSTRACT

OBJECTIVES: To obtain insight into SARS-CoV-2 clustering and transmission routes during outbreaks in the predominantly migrant workforce of the fruit and vegetable packaging industry of South Holland, the Netherlands, May to July 2020. DESIGN: This mixed-methods study applied direct observation and interviews, epidemiologic investigation, source and contact data analysis and whole-genome sequencing. RESULTS: We detected 46 SARS-CoV-2 cases and 4 outbreaks with a proportional representation of labour migrant and native workers in 6 unrelated facilities. Complete viral genome sequences revealed at least 3 clusters of native workers and labour migrants, 2 within and 1 between facilities. On-site inspections found adequate implementation of preventative measures to which both native workers and labour migrants showed suboptimal adherence. Being a labour migrant was associated with living in shared housing, but not with more contacts or different sources. CONCLUSIONS: The fruit and vegetable packaging industry gave the impression of sufficient preparedness and control. Suboptimal adherence to the facilities' preventative guidelines could have facilitated work floor transmission. Community and household transmission are likely to have contributed to outbreaks. We encourage further research into risk factors for transmission in labour migrants and application of these insights into targeted public health policy.


Subject(s)
COVID-19 , Transients and Migrants , Cluster Analysis , Disease Outbreaks , Fruit , Humans , Netherlands/epidemiology , SARS-CoV-2 , Vegetables
17.
Sci Immunol ; 6(59)2021 05 25.
Article in English | MEDLINE | ID: covidwho-1243688

ABSTRACT

The emergence of SARS-CoV-2 variants harboring mutations in the spike (S) protein has raised concern about potential immune escape. Here, we studied humoral and cellular immune responses to wild type SARS-CoV-2 and the B.1.1.7 and B.1.351 variants of concern in a cohort of 121 BNT162b2 mRNA-vaccinated health care workers (HCW). Twenty-three HCW recovered from mild COVID-19 disease and exhibited a recall response with high levels of SARS-CoV-2-specific functional antibodies and virus-specific T cells after a single vaccination. Specific immune responses were also detected in seronegative HCW after one vaccination, but a second dose was required to reach high levels of functional antibodies and cellular immune responses in all individuals. Vaccination-induced antibodies cross-neutralized the variants B.1.1.7 and B.1.351, but the neutralizing capacity and Fc-mediated functionality against B.1.351 was consistently 2- to 4-fold lower than to the homologous virus. In addition, peripheral blood mononuclear cells were stimulated with peptide pools spanning the mutated S regions of B.1.1.7 and B.1.351 to detect cross-reactivity of SARS-CoV-2-specific T cells with variants. Importantly, we observed no differences in CD4+ T-cell activation in response to variant antigens, indicating that the B.1.1.7 and B.1.351 S proteins do not escape T-cell-mediated immunity elicited by the wild type S protein. In conclusion, this study shows that some variants can partially escape humoral immunity induced by SARS-CoV-2 infection or BNT162b2 vaccination, but S-specific CD4+ T-cell activation is not affected by the mutations in the B.1.1.7 and B.1.351 variants.


Subject(s)
Antibodies, Viral/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , COVID-19 Vaccines/immunology , Cell Line , Cross Reactions/immunology , Humans , Immunologic Memory/immunology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Vaccination
18.
BMC Infect Dis ; 21(1): 418, 2021 May 04.
Article in English | MEDLINE | ID: covidwho-1216884

ABSTRACT

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.


Subject(s)
COVID-19/epidemiology , Long-Term Care/statistics & numerical data , Nursing Homes/statistics & numerical data , SARS-CoV-2/genetics , Aged , Aged, 80 and over , COVID-19/etiology , COVID-19/virology , Cross-Sectional Studies , Disease Outbreaks , Female , Germany , Health Personnel , Humans , Infection Control , Male , Middle Aged , Netherlands/epidemiology , Real-Time Polymerase Chain Reaction , Whole Genome Sequencing
19.
Science ; 371(6525): 172-177, 2021 01 08.
Article in English | MEDLINE | ID: covidwho-1066803

ABSTRACT

Animal experiments have shown that nonhuman primates, cats, ferrets, hamsters, rabbits, and bats can be infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition, SARS-CoV-2 RNA has been detected in felids, mink, and dogs in the field. Here, we describe an in-depth investigation using whole-genome sequencing of outbreaks on 16 mink farms and the humans living or working on these farms. We conclude that the virus was initially introduced by humans and has since evolved, most likely reflecting widespread circulation among mink in the beginning of the infection period, several weeks before detection. Despite enhanced biosecurity, early warning surveillance, and immediate culling of animals in affected farms, transmission occurred between mink farms in three large transmission clusters with unknown modes of transmission. Of the tested mink farm residents, employees, and/or individuals with whom they had been in contact, 68% had evidence of SARS-CoV-2 infection. Individuals for which whole genomes were available were shown to have been infected with strains with an animal sequence signature, providing evidence of animal-to-human transmission of SARS-CoV-2 within mink farms.


Subject(s)
COVID-19/transmission , COVID-19/virology , Mink , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Zoonoses , Animals , COVID-19/epidemiology , COVID-19/veterinary , Disease Outbreaks , Farms , Humans , Likelihood Functions , Mutation , Netherlands/epidemiology , Phylogeny , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/classification , SARS-CoV-2/physiology , Whole Genome Sequencing , Zoonoses/transmission , Zoonoses/virology
20.
J Infect Public Health ; 13(12): 1878-1884, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-894048

ABSTRACT

BACKGROUND: SARS-CoV-2 has spread globally. Currently, literature of SARS-CoV-2 in neonates is scarce. We present a case of a neonate with a high viral load and prolonged virus shedding. METHODS: Epidemiology, clinical characteristics, treatment, laboratory data and follow-up information and the treatment of a neonate with COVID-19 were recorded. RESULTS: A 7-day-old boy was admitted to the hospital with fever, lethargy and apnoea. He was found SARS-CoV-2 RNA positive with an exceptionally high viral load in nasopharyngeal swab and stool. The father and two maternity nurses at home had detectable SARS-CoV-2 RNA as well. Sequencing showed all strains belonged to the same cluster. The father was asymptomatic and the maternity nurses developed symptoms after visiting. In the mother, no SARS-CoV-2 RNA could be found. Six days after admission, the neonate was discharged after clinical improvement with oral antibiotics because of a possible pyelonephritis. Monitoring the course of this infection showed that SARS-CoV-2 RNA was detectable in the nasopharynx until day 19 and in stool until day 42 after symptom onset. CONCLUSIONS: This case shows that neonates can have a high viral load of SARS-CoV-2 and can shed the virus for over one month in stool. Despite the high viral load in the neonate, the mother and a sibling did not get infected.


Subject(s)
COVID-19/diagnosis , SARS-CoV-2 , COVID-19/complications , COVID-19/virology , Diagnosis, Differential , Fever/etiology , Humans , Infant, Newborn , Male , Viral Load , Virus Shedding
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