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There is an ongoing debate on airborne transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) as a risk factor for infection. In this study, the level of SARS-CoV-2 in air and on surfaces of SARS-CoV-2 infected nursing home residents was assessed to gain insight in potential transmission routes.During outbreaks, air samples were collected using three different active and one passive air sampling technique in rooms of infected patients. Oropharyngeal swabs (OPS) of the residents and dry surface swabs were collected. Additionally, longitudinal passive air samples were collected during a period of 4 months in common areas of the wards. Presence of SARS-CoV-2 RNA was determined using RT-qPCR, targeting the RdRp- and E-genes. OPS, samples of two active air samplers and surface swabs with Ct value ≤35 were tested for the presence of infectious virus by cell culture. In total, 360 air and 319 surface samples from patient rooms and common areas were collected. In rooms of 10 residents with detected SARS-CoV-2 RNA in OPS, SARS-CoV-2 RNA was detected in 93 of 184 collected environmental samples (50.5%) (lowest Ct 29,5), substantially more than in the rooms of residents with negative OPS on the day of environmental sampling (n=2) (3.6%). SARS-CoV-2 RNA was most frequently present in the larger particle size fractions (>4 μm 60% (6/10); 1-4 μm 50% (5/10); in vitro was detected.In conclusion, SARS-CoV-2 is frequently detected in air and on surfaces in the immediate surroundings of room-isolated COVID-19 patients, providing evidence of environmental contamination. The environmental contamination of SARS-CoV-2 and infectious aerosols confirm the potential for transmission via air up to several meters.

IntroductionSelf-testing for COVID-19 infection with lateral flow assay SARS-CoV-2 rapid antigen detection tests (RDT), provides rapid results and could enable frequent and extensive testing in the community, thereby improving the control of SARS-CoV-2. The objective of this study is to evaluate the performance of self-testing using RDT without assistance.MethodsParticipants visiting a municipal SARS-CoV-2 testing centre, received self-testing kits containing either the BD Veritor System (BD RDT) or Roche SARS-CoV-2 antigen detection test (Roche RDT). Oro-nasopharyngeal swabs were collected from the participants for qRT-PCR testing. As a proxy for contagiousness, viral culture was performed on a selection of qRT-PCR positive samples to determine the Ct-value at which the chance of a positive culture was dropping below 0.5 (Ct-value cut-off). Sensitivity and specificity of self-testing were compared to qRT-PCR with a Ct-value below the Ct value cut-off. Determinants independently associated with a false-negative self-test result were determined.ResultsA total of 3,215 participants were included (BD RDT n=1604; Roche RDT n=1611). Sensitivity and specificity of self-testing compared to the qRT-PCR results with Ct-value below the Ct-value cut-off was 78.0% (95% CI:72.5-82.8) and 99.4% (95%CI: 99.0-99.6) respectively. Determinants independently associated with a false-negative self-testing results were: higher age, low viral load and finding self-testing difficult.DiscussionSelf-testing using currently available RDT’s has a high specificity and relatively high sensitivity to identify individuals with a high probability of contagiousness. The performance of two tests were comparable. This application has the potential for frequent and extensive testing which may be an aid to lift restrictions to society while controlling the spread of SARS-CoV-2.

BackgroundTo limit societal and economic costs of lockdown measures, public health strategies are needed that control the spread of SARS-CoV-2 and simultaneously allow lifting of disruptive measures. Regular universal random screening of large proportions of the population regardless of symptoms has been proposed as a possible control strategy.MethodsWe developed a mathematical model that includes test sensitivity depending on infectiousness for PCR-based and antigen-based tests, and different levels of onward transmission for testing and non-testing parts of the population. Only testing individuals participate in high-risk transmission events, allowing more transmission in case of unnoticed infection. We calculated the required testing interval and coverage to bring the effective reproduction number due to universal random testing (Rrt) below 1, for different scenarios of risk behavior of testing and non-testing individuals.FindingsWith R0 = 2.5, lifting all control measures for tested subjects with negative test results would require 100% of the population being tested every three days with a rapid test method with similar sensitivity as PCR-based tests. With remaining measures in place reflecting Re = 1.3, 80% of the population would need to be tested once a week to bring Rrt below 1. With lower proportions tested and with lower test sensitivity, testing frequency should increase further to bring Rrt below 1. With similar Re values for tested and non-tested subjects, and with tested subjects not allowed to engage in higher risk events, at least 80% of the populations needs to test every five days to bring Rrt below. The impact of the test-sensitivity on the reproduction number is far less than the frequency of testing.InterpretationRegular universal random screening followed by isolation of infectious individuals is not a viable strategy to reopen society after controlling a pandemic wave of SARS-CoV-2. More targeted screening approaches are needed to better use rapid testing such that it can effectively complement other control measures.FundingRECOVER (H2020-101003589) (MJMB), ZonMw project 10430022010001 (MK, HH), FCT project 131_596787873 (GR). ZonMw project 91216062 (MK)

BackgroundLimited information is available on whether blaKPC-containing plasmids from isolates in a hospital outbreak can be differentiated from epidemiologically unrelated blaKPC-containing plasmids based on sequence data.ObjectiveThis study aimed to evaluate the performance of three approaches to distinguish epidemiologically related from unrelated blaKPC-containing IncF plasmids.MethodEpidemiologically related isolates, were short- and long-read whole genome sequenced on an Illumina MiSeq and MinION sequencer. A hybrid assembly was performed and plasmid sequences were extracted from the assembly graph. Epidemiologically unrelated plasmid sequences were extracted from the GenBank. Pairwise comparisons were performed of epidemiologically related and unrelated plasmids based on SNP differences using snippy, phylogenetic distance using Roary and using a similarity index that penalizes size differences between plasmids (Stoesser-index). The percentage of pairwise comparisons misclassified as genetically related or as clonally unrelated was determined using different genetic thresholds for genetic relatedness for all three comparison methods.ResultsDespite the median number of SNP differences, Roary phylogenetic distance, and Stoesser-index differed between the epidemiologically related and unrelated plasmids, the range of differences overlapped between the two comparison groups for all three comparison methods. When using a genetic similarity threshold that classified 100% of epidemiologically related plasmid pairs as genetically related, the percentages of plasmids misclassified as epidemiologically related ranged from 6.7% (Roary) to 20.8% (Stoesser-index).DiscussionAlthough epidemiologically related plasmids can be distinguished from unrelated plasmids based on genetic similarity, epidemiologically related and unrelated blaKPC-containing IncF plasmids show a high degree of sequence similarity. The phylogenetic distance as determined using Roary showed the highest degree of discriminatory power between the epidemiologically related and unrelated plasmids.Impact statementAccurately distinguishing epidemiologically related from unrelated plasmids is essential to detect nosocomial plasmid transmission in outbreaks. However, limited information is available on whether blaKPC-containing plasmids from isolates in a hospital outbreak can be differentiated from epidemiologically unrelated blaKPC-containing plasmids based on sequence data. This study aimed to evaluate the performance of three approaches to distinguish epidemiologically related from unrelated blaKPC-containing IncF plasmids. Pairwise comparisons were performed of epidemiologically related and unrelated plasmids based on SNP differences using snippy, phylogenetic distance using Roary and using a similarity index that penalizes size differences between plasmids (Stoesser-index). Based on our results, epidemiologically related plasmids can be distinguished from unrelated plasmids based on genetic similarity. Despite this, epidemiologically related and unrelated blaKPC-containing IncF plasmids show a high degree of sequence similarity and judgements on the horizontal transfer of these plasmids during hospital outbreaks based on genetic identity should be made with caution. The phylogenetic distance determined using Roary showed the highest discriminatory power between the epidemiologically related and unrelated plasmids.Data summaryShort-and long-read sequence data of the epidemiologically related Enterobacteriaceae isolates included in this study are available from the publicly available European Nucleotide Archive of the European Bioinformatics Institute under study accession number: PRJEB41009. The authors confirm that all supporting data have been provided within the article and through the supplementary data files.

ObjectivesThis study was primarily conducted to evaluate clinical sensitivity and specificity of the SARS-CoV-2 rapid antigen test ‘BD Veritor System for Rapid Detection of SARS-CoV-2’ (VRD) compared to real time reverse transcriptase polymerase chain reaction (qRT-PCR). Furthermore, the VRD sensitivity for different Ct-value groups (Ct DesignProspective performance evaluation study.SettingMunicipal Health Service (GGD) COVID-19 test centres in West-Brabant, the NetherlandsParticipantsIn order to evaluate clinical specificity, 352 symptomatic adults (≥18 years) who presented at a participating GGD test centre for a COVID-19 test between September 28 and October 7 2020 were included. In order to evaluate clinical sensitivity, 123 symptomatic adults (≥18 years) who were tested positive with qRT-PCR in a participating GGD test centre between September 26 and October 6 were included.ResultsAn overall clinical specificity of 100% (95%CI: 98.9%-100%) and sensitivity of 80.7% (95% CI: 73,2%-86,9%) was found for the VRD compared to qRT-PCR. Sensitivity was the highest for low Ct-value categories and for specimen obtained within the first days after disease onset. For specimen obtained within 7 days after onset of symptoms, the overall sensitivity was 91.0% (95% CI: 82,4%-96,3%) and 98,6% (95% CI: 92,3%-100%) for samples with qRT-PCR Ct-value beneath 30.ConclusionThe VRD is a promising diagnostic test for COVID-19 community screening for symptomatic individuals within 7 days after symptom onset in function of disease control. The clinical sensitivity was highest when viral load was high, which correlated with the duration of symptoms. Further research on practical applicability and the optimal position of the test within the current testing landscape is needed.

It is well established that antibiotic treatment selects for resistance in pathogenic bacteria. However, the evolutionary responses of pathogen populations to antibiotic treatment during infections remain poorly resolved, especially in acute infections. Here we map the evolutionary responses to treatment in high definition through genomic and phenotypic characterization of >100 isolates from a patient with P. aeruginosa pneumonia. Antibiotic therapy (meropenem, colistin) caused a rapid crash of the P. aeruginosa population in the lung, but this decline was followed by the spread of meropenem resistance mutations that restrict antibiotic uptake (oprD) or modify LPS biosynthesis (wbpM). Low fitness strains with high-level meropenem resistance (oprD) were then replaced by high fitness strains with ‘anti-resistance’ mutations in the MexAB-OprM efflux pump, causing a rapid decline in resistance to both meropenem and a collateral loss of resistance to a broad spectrum of antibiotics. In contrast, we did not observe any evolutionary responses to antibiotic treatment in the intestinal population of P. aeruginosa. Carbapenem antibiotics are key to the treatment of infections caused by Gram negative pathogens, and our work highlights the ability of natural selection to drive both the rapid rise and fall of carbapenem resistance during acute infections.

BackgroundThere is a continuing need for in-depth and updated knowledge about the epidemiology of surgical site infections (SSIs) caused by Staphylococcus aureus to support the development of effective preventive interventions. The ASPIRE-SSI study aims primarily to determine the incidence of S. aureus SSIs and postoperative bloodstream infections (BSIs) in Europe and to assess their association with patient-related, pathogen-related, and contextual risk factors.MethodsASPIRE-SSI is a prospective, multicenter, observational cohort study primarily assessing the incidence of and risk factors for S. aureus SSI and postoperative BSI in Europe. Five thousand adult surgical patients (of which two-thirds will be S. aureus carriers and one-third non-carriers) undergoing several types of surgical procedures in sites located across Europe were enrolled in the study. Data and specimens were collected from these subjects who were followed for up to 90 days following surgery to assess study outcomes.Using advanced survival analyses and regression techniques (including competing risks models), we will determine event-specific and sub-distribution hazards to assess the independent associations of these study outcomes with risk factors. Additionally, a risk prediction model will be derived to quantify the risk of developing SSI or BSI due to S. aureus.DiscussionDespite the challenges, this study will provide important and contemporary information about the epidemiology of SSI and BSI (and other infections) caused by S. aureus in the current surgical population in Europe, thereby supporting the development of effective preventive interventions.Trial registrationclinicaltrial.gov number NCT02935244

Cefotaxime (CTX) is a commonly used third-generation cephalosporin (3GC) to treat infections caused by Escherichia coli. Two genetic mechanisms have been associated with 3GC resistance in E. coli. The first is the conjugative transfer of a plasmid harboring antibiotic resistance genes. The second is the introduction of mutations in the promoter region of the ampC β-lactamase gene that cause chromosomal-encoded β-lactamase hyperproduction. A wide variety of promoter mutations related to AmpC hyperproduction have been described. However, their link to a specific 3GC such as CTX resistance has not been reported. Here, we measured CTX MICs in 172 cefoxitin resistant E. coli isolates and performed genome-wide analysis of homoplastic mutations associated with CTX resistance by comparing Illumina whole-genome sequencing data of all isolates to a PacBio tailored-made reference chromosome. We mapped the mutations on the reference chromosome and determined their occurrence in the phylogeny, revealing extreme homoplasy at the −42 position of the ampC promoter. The 24 occurrences of a “T” at the −42 position rather than the wild type “C”, resulted from 18 independent C>T mutations in 5 phylogroups. The −42 C>T mutation was only observed in E. coli lacking a plasmid-encoded ampC gene. The association of the −42 C>T mutation with CTX resistance was confirmed to be significant (FDR < 0.05). To conclude, genome-wide analysis of homoplasy in combination with CTX resistance identifies the −42 C>T mutation of the ampC promotor as significantly associated with CTX resistance and underline the role of recurrent mutations in the spread of antibiotics resistance.Impact StatementIn the past decades, the worldwide spread of extended spectrum beta-lactamases (ESBLs) has led to a substantial increase in the prevalence of resistant common pathogens, thereby restricting available treatment options. Although acquired resistance genes, e.g. ESBLs, get most attention, chromosome-encoded resistance mechanisms may play an important role as well. In E. coli chromosome-encoded β-lactam resistance can be caused by alterations in the promoter region of the ampC gene. To improve our understanding of how frequently these alterations occur, a comprehensive interpretation of the evolution of these mutations is essential. This study is the first to apply genome-wide homoplasy analysis to better perceive adaptation of the E. coli genome to antibiotics. Thereby, this study grants insights into how chromosomal-encoded antibiotic resistance evolves and, by combining genome-wide association studies with homoplasy analyses, provides potential strategies for future association studies into the causes of antibiotics resistance.Data summaryAll data is available under BioProject: PRJNA592140. Raw Illumina sequencing data and metadata of all 171 E. coli isolates used in this study is available from the Sequence Read Archive database under accession no. SAMN15052485 to SAMN15052655. Full reference chromosome of ampC_0069 is available via GenBank accession no. CP046396.1 and NCBI Reference Sequence: NZ_CP046396.1.

BackgroundOn February 27, 2020, the first patient with COVID-19 was reported in the Netherlands. During the following weeks, nine healthcare workers (HCWs) were diagnosed with COVID-19 in two Dutch teaching hospitals, eight of whom had no history of travel to China or Northern-Italy. A low-threshold screening regimen was implemented to determine the prevalence and clinical presentation of COVID-19 among HCWs in these two hospitals.MethodsHCWs who suffered from fever or respiratory symptoms were voluntarily tested for SARS-CoV-2 by real-time reverse-transcriptase PCR on oropharyngeal samples. Structured interviews were conducted to document symptoms for all HCWs with confirmed COVID-19.FindingsThirteen-hundred fifty-three (14%) of 9,705 HCWs employed were tested, 86 (6%) of whom were infected with SARS-CoV-2. Most HCWs suffered from relatively mild disease and only 46 (53%) reported fever. Eighty (93%) HCWs met a case definition of fever and/or coughing and/or shortness of breath. None of the HCWs identified through the screening reported a travel history to China or Northern Italy, and 3 (3%) reported to have been exposed to an inpatient known with COVID-19 prior to the onset of symptoms.InterpretationWithin two weeks after the first Dutch case was detected, a substantial proportion of HCWs with fever or respiratory symptoms were infected with SARS-CoV-2, probably caused by acquisition of the virus in the community during the early phase of local spread. The high prevalence of mild clinical presentations, frequently not including fever, asks for less stringent use of the currently recommended case-definition for suspected COVID-19.RESEARCH IN PERSPECTIVEEvidence before this studyThis study was conducted in response to the global spread of SARS-CoV-2, and the detection of eight healthcare workers (HCWs) in two Dutch teaching hospitals within two weeks after the first patient with COVID-19 was detected in the Netherlands who had no history of travel to China or Northern-Italy, raising the question of whether undetected community circulation was occurring.Added value of this studyTo the best of our knowledge, this report is the first to describe the prevalence, the clinical presentation and early outcomes of COVID-19 in HCWs, which may be helpful for others seeking to identify HCWs suspected for COVID-19 in an outbreak situation.Implications of all the available evidenceWe describe that within two weeks after the first Dutch case was detected, a substantial proportion of HCWs with fever or (mild) respiratory symptoms were infected with SARS-CoV-2, probably caused by acquisition of the virus in the community during the early phase of local spread. The high prevalence of mild clinical presentations, frequently not including fever, asks for less stringent use of the currently recommended case-definition for suspected COVID-19.

The aim of this study was to determine the performance of both cefotaxime and ceftazidime containing agars on the specificity and sensitivity for chromosomal AmpC-hyperproducing and plasmid AmpC harboring Escherichia coli compared to ESBL-producing E. coli and E. coli without ESBL, pAmpC or cAmpC hyperproduction. Second, we evaluated the influence of adding cefoxitin to these agars for detection of both chromosomal AmpC-hyperproducing and plasmid AmpC harboring E. coli.Four different homemade screening agars with cefotaxime (1mg/L), ceftazidime (1mg/L), cefotaxime (1mg/L) with cefoxitin (8mg/L), and ceftazidime (1mg/L) with cefoxitin (8mg/L) were compared to each other for the identification of AmpC producing E. coli. A total of 40 isolates with plasmid encoded AmpC β-lactamases, 40 isolates with alterations in the promoter/attenuator region of the AmpC gene leading to hyperproduction of the β-lactamase, 40 isolates with ESBL genes and 39 isolates lacking both a AmpC and ESBL genotype were used to test the four agars.The sensitivity and specificity were 100% (95% confidence interval (95% CI) 96.1% to 100%) and 48.1% (95% CI 38.6%-60.2%), respectively, for the cefotaxime agar; 100% (95% CI 96.1% to 100%) and 49.41% (95% CI 39.8%-61.4%), respectively, for the ceftazidime agar; 96.3% (95% CI 89.1% to 99.2%) and 77.2% (95% CI 66.7%-85.2%) respectively, for the cefotaxime with cefoxitin agar; 98.8% (95% CI) 92.6% to 99.6%) and 81.0% (95% CI 70.9%-88.3%) respectively, for the ceftazidime agar with cefoxitin. The main reason for false-positive results were ESBL-harboring strains that grew on various agars; therefore, the specificity of each agar reported here was influenced mainly by the proportion of ESBL isolates tested. In conclusion addition of cefoxitin to cefotaxime and ceftazidime containing agars had little influence on sensitivity, but increased specificity for the detection of AmpC in E. coli.

ObjectivesCurrent guidelines for empirical antibiotic treatment poorly predict the presence of 3rd generation cephalosporin resistant Enterobacteriaceae (3GC-R EB) as a cause of infection, thereby increasing unnecessary carbapenem use. We aimed to develop diagnostic scoring systems to better predict the presence of 3GC-R EB as a cause of bacteraemia.MethodsA retrospective nested case-control study was performed that included patients ≥18 years in whom blood cultures were obtained and intravenous antibiotics were initiated. Each patient with 3GC-R EB bacteraemia was matched to four control infection episodes within the same hospital, based on blood culture date and onset location (community or hospital). Starting from 32 described clinical risk factors at infection onset, selection strategies were used to derive scoring systems for the probability of community- and hospital-onset 3GC-R EB bacteraemia.Results3GC-R EB bacteraemia occurred in 90 of 22,506 (0.4%) community-onset and in 82 of 8,110 (1.0%) hospital-onset infections, and these cases were matched to 360 community-onset and 328 hospital-onset control episodes, respectively. The derived community-onset and hospital-onset scoring system consisted of 6 and 9 predictors, respectively, with c-statistics of 0.807 (95% confidence interval 0.756-0.855) and 0.842 (0.794-0.887). With selected score cutoffs, the models identified 3GC-R EB bacteraemia with equal sensitivity as existing guidelines, but reduced the proportion of patients classified as at risk for 3GC-R EB bacteraemia (i.e. eligible for empiric carbapenem therapy) with 40% in patients with community-onset and 49% in patients with hospital-onset infection.ConclusionsThese prediction rules for 3GC-R EB bacteraemia may reduce unnecessary empiric carbapenem use.