This prospective observational study was approved by the National Ethical Review Agency (EPM; No. 2020 − 01623). Informed consent was obtained from the patient, or next of kin if the patient was unable to give consent. The Declaration of Helsinki and its subsequent revisions were followed. The protocol of the study was registered a priori (ClinicalTrials ID: NCT04316884). STROBE guidelines were followed for reporting.
Data collection and patient cohort
The study was performed at the General intensive care unit (ICU) at Uppsala University Hospital, a tertiary care hospital in Sweden, at the time of the study only admitting COVID-19 patients. All adult patients with COVID-19 admitted to the ICU, during March to June 2020, were screened for eligibility and asked for consent. COVID-19 was diagnosed with positive polymerase chain reaction (PCR) for SARS-Cov-2 on nasopharyngeal swabs [12, 13].
Apart from demographical data, clinical data were recorded prospectively including medical history, medications, physiological data, level of organ support and date of death. Simplified acute physiology score 3 (SAPS3), Sequential Organ Failure Assessment (SOFA) score, and organ support data were collected as reported in the results. Blood samples were collected on admission to the ICU and daily during the ICU stay. Full blood count (FBC), plasma C-reactive protein (CRP), procalcitonin, IL-6, fibrin d-dimer, troponin and pro-brain natriuretic peptide-NT (pro-BNP-NT); kidney function tests: plasma creatinine and cystatin C; liver function tests: plasma bilirubin, alanine aminotransferase, aspartate aminotransferase (AST), alkaline phosphatase (ALP) were performed in the hospital central laboratory. FBC was analyzed on a Sysmex XN™ instrument (Sysmex, Kobe, Japan) while plasma CRP, ferritin, troponin I, kidney and liver markers were analyzed on an Architect ci16200 (Abbott Laboratories, Abbott Park, IL, US). Acute kidney injury (AKI) was defined according to the KDIGO AKI definitions. IL-6 was measured by a commercial sandwich ELISA kit, (D6050, R&D Systems, Minneapolis, MN).
Sample collection and virus detection
Peripheral blood was collected from patients with COVID-19 into EDTA-containing tubes and plasma was separated using centrifugation at 3000 g for 10 min. After separation, all plasma samples were stored at -80 °C. Total nucleic acid was extracted from 200 µl plasma samples using eMAG (Biomerieux) according to manufacturer’s instruction with an elution volume of 60 µl and stored at 4 °C.
Viral RNA in plasma was determined by real-time RT-PCR recognizing the SARS-CoV-2 N-gene with the 2019-nCoV N1 reagent set from the previously described protocol from Center for Disease Control (CDC) of the United States . For reverse transcription and real-time PCR we used the Taqman Fast Virus 1-step Master Mix (ThermoFischer Scientific) according to the manufacturer’s instructions. The reactions were performed with a sample volume of 10 µl in a total volume of 25 µl. Primer and probe concentrations were as follows: Forward primer, 400 nM ; Reverse primer, 800 nM; and Probe, 200 nM. The probe was labeled with Yakima Yellow as flurophore with internal ZEN and terminal 3IABkFQ as quenchers. The real-time PCR analysis was performed on a RotorGene Q instrument (Qiagen) with the software v2.3.1. The thermal cycling steps were: 50 °C for 15 min, 95 °C for 2 min, and 45 cycles of 95 °C for 15 s and 60 °C for 30 s.
For qualitative analysis, a Ct value of < 32 was defined as a positive result, and a Ct value of ≥ 32 was defined as a negative result. It should be noted that the threshold value is platform-dependent and as comparison, we use a four Ct steps higher value for the same reaction on QuantStudio 6 Pro (Applied Biosystems).
For quantitative analysis, we used imported standard curves based on previously determined PCR efficiency and adjustment against a reference point analysed in the same run. As external calibrator, we used the ISO 13485 certified molecular standard Quantitative Synthetic SARS-CoV-2 RNA: ORF, E, N (VR-3276SD, ATCC,). The reaction showed linearity over 6 orders of magnitudes with 109 copies/ml and 300 copies/ml as the upper and lower limits of quantitative detection, respectively. Since the quantification has limited precision close to the detection limit, we defined clinically significant RNAemia as > 1000 RNA copies/mL plasma.
The number of patients was defined by including all patients consenting to the study. Assuming that at least 20% of the patients would have RNAemia, we would have analyze samples from at least 78 patients to find a 10% difference with a power of 0.8.
Data are presented as median (IQR) or as number of observations (percent of total number of observations). To compare groups Mann–Whitney U test was used. Spearman Rank correlation test was used for assessing the association between continuous variables. Logistic regression was performed with organ failure, organ support and 30-day mortality as dependent variables with RNA copies and age as sole predictors to calculate odds ratios. As the number of observations were limited, we used age as a surrogate for the pre COVID-19 risk of death. Therefore, we also assessed if the RNA copies after adjusting for age was an independent predictor of these outcomes. We performed a sensitivity analysis defining RNAemia as RNA copies above the Limit of detection (> 300 RNA copies/mL plasma) when relevant. The proportion of missing data was low, < 10%, and was therefore not imputed. For calculations and figures, STATISTICA™ software, version 13.5 (TIBCO Software Inc, Tulsa, OK) was used. p < 0.05 was considered significant where relevant.