Culturing of SARS-CoV-2 from patient samples: Protocol for optimal virus recovery and assessment of infectious viral load.

Optimal sampling, preservation, and culturing of SARS-CoV-2 from COVID-19 patients are critical for successful recovery of virus isolates and to accurately estimate contagiousness of the patient. In this study, we investigated the influence of the type of sampling media, storage time, freezing conditions, sterile filtration, and combinations of these to determine the optimal pre-analytic conditions for virus recovery and estimation of infectious viral load in COVID-19 patients. Further, we investigated the viral shedding kinetics and mucosal antibody response in 38 COVID-19 hospitalized patients. We found Universal Transport Medium (Copan) to be the most optimal medium for preservation of SARS-CoV-2 infectivity. Our data showed that the probability of a positive viral culture was strongly correlated to Ct values, however some samples did not follow the general trend. We found a significant correlation between plaque forming units and levels of mucosal antibodies and found that high levels of mucosal antibodies correlated with reduced chance of isolating the virus. Our data reveals essential parameters to consider from specimen collection over storage to culturing technique for optimal chance of isolating SARS-CoV-2 and accurately estimating patient contagiousness.

Cross sectional study of multiresistant bacteria in Danish emergency departments: prevalence, patterns and risk factors for colonization (AB-RED project).

BACKGROUND: Multiresistant bacteria (MRB) is an increasing problem. Early identification of patients with MRB is mandatory to avoid transmission and to target the antibiotic treatment. The emergency department (ED) is a key player in the early identification of patients who are colonized with MRB. There is currently sparse knowledge of both prevalence and risk factors for colonization with MRSA, ESBL, VRE, CPE and CD in acutely admitted patients in Western European countries including Denmark. To develop evidence-based screening tools for identifying carriers of resistant bacteria among acutely admitted patients, systematic collection of information on risk factors and exposures is required. Since a geographical variation is suspected, it is desirable to include emergency departments across the country. The aim of this project is to provide a comprehensive overview of prevalence and risk factors for MRSA, ESBL, VRE, CPE and CD colonization in patients admitted to Danish ED's. The objectives are to describe the prevalence and demography of resistance, co-infections, to identify risk factors for carrier state and to develop and validate a screening tool for identification of carriers. METHODS: Multicenter descriptive and analytic cross-sectional survey from January-May 2018 of around 10.000 acutely admitted patients > 18 years in 8 EDs for carrier state and risk factors for antibiotic resistant bacteria. Information about the background and possible risk factors for carrier status together with swabs from the nose, throat and rectum is collected and analyzed for MRSA, ESBL, VRE, CPE and CD. The prevalence of the resistant bacteria are calculated at hospital level, regional level and national level and described with relation to residency, sex, age and risk factors. A screening model for identification of carrier stage of resistant bacteria is developed and validated. DISCUSSION: The study will provide the prevalence of colonized patients with resistant bacteria on arrival to the ED and variation in demographic patterns, and will develop a clinical tool to identify certain risk groups. This will enable the clinician to target antibiotic treatments and to reduce the in-hospital spreading of resistant bacteria. This knowledge is important for implementing and evaluating antimicrobial stewardships, screening and infection control strategies. TRIAL REGISTRATION: Clinicaltrials.gov : NCT03352167 (registration date: 20. November 2017).