, a cluster of cases of pneumonia of unknown etiology were reported linked to a market in Wuhan, China 1. The causative agent was identified as the species Severe acute respiratory syndrome-related coronavirus and was named SARS-CoV-2 (ref. 2). By 16 April the virus had spread to 185 different countries, infected over 2,000,000 people and resulted in over 130,000 deaths 3. In the Netherlands, the first case of SARS-CoV-2 was notified on 27 February. The outbreak started with several different introductory events from Italy, Austria, Germany and France followed by local amplification in, and later also outside, the south of the Netherlands. The combination of near to real-time whole-genome sequence analysis and epidemiology resulted in reliable assessments of the extent of SARS-CoV-2 transmission in the community, facilitating early decision-making to control local transmission of SARS-CoV-2 in the Netherlands. We demonstrate how these data were generated and analyzed, and how SARS-CoV-2 whole-genome sequencing, in combination with epidemiological data, was used to inform public health decision-making in the Netherlands. Whole-genome sequencing (WGS) is a powerful tool to understand the transmission dynamics of outbreaks and inform outbreak control decisions 4-7. Evidence of this was seen during the 2014-2016 West African Ebola outbreak when real-time WGS was used to help public health decision-making, a strategy dubbed 'precision public health pathogen genomics' 8,9. Immediate sharing and analysis of data during outbreaks is now recommended as an integral part of outbreak response 10-12. Feasibility of real-time WGS requires access to sequence platforms that provide reliable sequences, access to metadata for interpretation, and data analysis at high speed and low cost. Therefore, WGS for outbreak support is an active area of research. Nanopore sequencing has been employed in recent outbreaks of Usutu, Ebola, Zika and yellow fever virus owing to the ease of use and relatively low start-up cost 4-7. The robustness of this method has recently been validated using Usutu virus 13,14. In the Netherlands, the first COVID-19 case was confirmed on 27 February and WGS was performed in near to real-time using an amplicon-based sequencing approach. From 22 January, symptomatic travelers from countries where SARS-CoV-2 was known to circulate were routinely tested. The first case of SARS-CoV-2 infection in the Netherlands was identified on 27 February in a person with recent travel history to Italy and an additional case was identified one day later, also in a person with recent travel history to Italy. The genomes of these first two positive samples were generated and analyzed by 29 February. These two viruses clustered differently in the phylogenetic tree, confirming separate introductions (Fig. 1a). The advice to test hospitalized patients with serious respiratory infections was issued on 24 February and subsequent attempts to identify possible local transmission chains triggered testing for SARS-CoV-2 on a large scale in h...

The objective of the present study was the development of a diagnostic reverse transcription (RT)-PCR for the specific detection of enterovirus (EV) RNA in clinical specimens controlled by an internal control (IC) RNA. The IC RNA contains the same primer binding sites as EV RNA but has a different probe region. The IC RNA was packaged into an MS2 phage core particle (armored) and was added to the clinical sample to allow monitoring of both extraction efficiency and RT-PCR efficiency. Serial dilutions of the IC RNA were made, and the detection limit of the RT-PCR was tested in a background of EV RNA-negative cerebrospinal fluid. The sensitivity and specificity of the RT-PCR assay were tested by using all 64 known EV serotypes, several non-EV serotypes, and two Quality Control for Molecular Diagnostics ( The human enteroviruses (EVs) are members of the family Picornaviridae, are ubiquitous, and are mainly enterically transmitted. EVs have traditionally been identified by serotype-specific antisera in a virus-neutralizing test, and 66 EV types are known to infect humans (19). The 66 EV serotypes were initially recognized and divided into five major groups: polioviruses (PV; types 1 to 3), coxsackieviruses A (CVAs; types 1 to 22 and 24), coxsackieviruses B (CVBs; types 1 to 6), echoviruses (types 1 to 7, 9, 11 to 27, and 29 to 33), and EV types 68 to 71 (17). Recent molecular analyses have proved that echovirus types 22 and 23 are genetically distinct from the members of the genus Enterovirus and have been reclassified in a separate genus, Parechovirus, in the family Picornaviridae (13,20,23).Infections with EVs cause a wide range of clinical outcomes, such as asymptomatic infections, aseptic meningitis (meningeal inflammation in the absence of a bacterial pathogen), encephalitis, paralytic poliomyelitis, and myocarditis. Although the majority of EV infections do not cause significant disease, infection can cause serious illness, especially in infants and immune-compromised patients. EV infections are the most common cause of aseptic meningitis and account for 80 to 90% of all cases of central nervous system infections for which a possible causative agent is identified (24). In the neonate, aseptic meningitis-induced complications and poor outcomes of EV infections generally occur within the first 2 days of life (1, 2). Aseptic meningitis in immune-competent adults is characterized by sudden onset of fever, but neurological abnormalities are rare, and both short-term and long-term outcomes are generally good. Encephalitis caused by EV infections is a less common but a more severe disease than aseptic meningitis (18,29,30). Immune-compromised children and adults who are infected with EV may develop chronic meningitis and encephalitis, which may last for years before becoming fatal (16).The early clinical symptoms of meningitis caused by viruses, bacteria, and fungi are quite similar and are difficult to distinguish, but the diagnosis, therapy, and outcome of disease caused by these pathogens vary considerably. A reli...

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Screening of antibodies to hepatitis C virus (HCV) is widely used for monitoring the prevalence of HCV infections and to assess HCV infectivity. Among HCV-infected individuals in the general population, the interval between the detection of HCV RNA and the development of HCV antibodies is usually 5 to 6 weeks, but in rare cases, seroconversion may be prolonged up to 6 to 9 months. In this study, we tested for the presence of HCV RNA during the antibody-undetectable period of 19 drug-injecting HCV seroconverters to gain insight into the antibody-negative carrier status in this population. HCV seroconversion status was determined by testing the first and last serum samples obtained from each subject, using third-generation antibody screening and confirmation assays. Serial samples were tested for HCV-specific antibodies to establish the moment of seroconversion and HCV RNA by single reverse transcriptase-polymerase chain reaction (RT-PCR) and branched DNA assay (bDNA) in serum. Plasma and peripheral blood mononuclear cells (PBMCs) were independently collected and tested for HCV RNA. HCV RNA-positivity was confirmed by Southern blot hybridization and sequencing of serial samples. The 19 HCV seroconverters had a mean follow-up of 5 years (range, 1 to 8 years). Of the 19, 4 were human immunodeficiency virus (HIV)-infected before HCV seroconversion. HCV RNA was detected in serum before seroconversion in 12 (63.2%) of the 19 HCV seroconverters, independent of HIV status. In 7 of these 12, the antibody-undetectable period was relatively short (2 to 10 months). The other 5, who were all HIV-negative before HCV seroconversion, had intermittent low levels of HCV RNA before seroconversion for a period of more than 12 months, with a mean of 40.8 months (range, 13 to 94 months). In all 5 individuals, independent repeats of the experiments confirmed the presence of HCV RNA in serum, and in 3 of these individuals, HCV-positivity was confirmed in independently collected plasma and PBMC samples. Low levels of HCV RNA may be present during prolonged antibody-undetectable periods before seroconversion in a number of injecting drug users. Independent of HIV status, their immune system appears to be unable to respond to these low HCV RNA levels and was sometimes only activated after reinfections with distinct HCV genotypes. These results indicate that primary HCV infection may not always elicit the rapid emergence of HCV antibodies and suggests that persistent low levels of HCV RNA (regardless of the genotype) may not elicit at all or delay antibody responses for prolonged periods of time.