Clinical and virological characteristics of hospitalised COVID-19 patients in a German tertiary care centre during the first wave of the SARS-CoV-2 pandemic: a prospective observational study

Purpose Adequate patient allocation is pivotal for optimal resource management in strained healthcare systems, and requires detailed knowledge of clinical and virological disease trajectories. The purpose of this work was to identify risk factors associated with need for invasive mechanical ventilation (IMV), to analyse viral kinetics in patients with and without IMV and to provide a comprehensive description of clinical course. Methods A cohort of 168 hospitalised adult COVID-19 patients enrolled in a prospective observational study at a large European tertiary care centre was analysed. Results Forty-four per cent (71/161) of patients required invasive mechanical ventilation (IMV). Shorter duration of symptoms before admission (aOR 1.22 per day less, 95% CI 1.10–1.37, p < 0.01) and history of hypertension (aOR 5.55, 95% CI 2.00–16.82, p < 0.01) were associated with need for IMV. Patients on IMV had higher maximal concentrations, slower decline rates, and longer shedding of SARS-CoV-2 than non-IMV patients (33 days, IQR 26–46.75, vs 18 days, IQR 16–46.75, respectively, p < 0.01). Median duration of hospitalisation was 9 days (IQR 6–15.5) for non-IMV and 49.5 days (IQR 36.8–82.5) for IMV patients. Conclusions Our results indicate a short duration of symptoms before admission as a risk factor for severe disease that merits further investigation and different viral load kinetics in severely affected patients. Median duration of hospitalisation of IMV patients was longer than described for acute respiratory distress syndrome unrelated to COVID-19. Supplementary Information The online version contains supplementary material available at 10.1007/s15010-021-01594-w.


Introduction
The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic places an unprecedented burden on healthcare systems worldwide. Host factors predictive of severe clinical course and adverse outcome in patients with coronavirus disease 2019 (COVID-19) include older age, male gender, and pre-existing chronic comorbidities [1][2][3][4][5][6]. Several risk scores containing clinical characteristics, laboratory assessments, and biomarkers have been proposed for improved patient management and resource allocation [5,7]. The members of the Pa-COVID Study Group are mentioned in "Acknowledgements".
The data reflect the situation in a tertiary care referral centre for the treatment of patients with acute respiratory distress syndrome (ARDS), including veno-venous extracorporeal membrane oxygenation (vvECMO) therapy, and an associated certified weaning centre during the first months of the COVID-19 pandemic, before treatment with dexamethasone became standard of care [18]. Specific aims of this work were to identify risk factors associated with need for IMV, to analyse viral kinetics in patients with and without IMV, and to provide a comprehensive description of clinical course and outcome.

Study cohort and data collection
Data collection was performed within the Pa-COVID-19 study, a prospective observational cohort study conducted at Charité-Universitätsmedizin Berlin, as described [13]. Adult patients admitted between March 1st and June 30th, 2020, with PCR-confirmed SARS-CoV-2 infection were included if patients or their legal representatives gave informed consent. We recorded epidemiological and demographic data, medical history, history of present illness, symptoms, clinical course, treatment, and outcomes upon enrolment and longitudinally during hospitalisation. The study was approved by the ethics committee of Charité-Universitätsmedizin Berlin (EA2/066/20), conducted according to the Declaration of Helsinki and Good Clinical Practice principles (ICH 1996) and is registered in the German and WHO international clinical trials registry (DRKS00021688).
The primary objective of this first analysis of the Pa-COVID-19 cohort was to identify risk factors and virological and laboratory parameters associated with need for IMV. Comorbidities were classified using the Charlson comorbidity Index (CCI) [19]. ARDS was defined according to the Berlin definition of ARDS [20]. Sequential Organ Failure Assessment (SOFA) score [21] was calculated from data recorded in the ICU data management system. The following predefined events were assessed in all patients: (1) sepsis (defined according to sepsis-3 criteria [22], (2) venous thromboembolic events (VTE; pulmonary embolism or deep vein thrombosis), (3) neurologic events (haemorrhagic/ischaemic stroke, delirium, intensive care unitacquired weakness, ICUAW), epileptic seizure, meningitis and encephalitis). Treatment was unaffected by participation in the study. Patient allocation was performed according to structured regional processes [23] and management of critically ill patients following current guidelines as described [24,25]. Duration of symptoms was only analysed for patients with reliable information on symptom onset in the patient chart given by patients themselves or relatives.
All laboratory assessments were carried out in accredited laboratories at Charité-Universitätsmedizin Berlin. SARS-CoV-2 viral concentration was measured in respiratory samples (naso-or oropharyngeal swabs) by real-time RT-PCR [26]. Viral concentration is given as log 10 genome copies per swab or initial 1 mL sampling buffer.

Statistical analyses
Distribution of continuous variables was summarised by median and interquartile range (IQR) values or mean and standard deviation (SD), as appropriate. The differences of continuous variables between groups were examined by Welch's t test or, in absence of normal distribution, by Mann-Whitney U test. Categorical variables were compared using Chi-square tests. For all analyses, complete cases were used for the respective evaluation.
We conducted a multiple logistic regression with "need for invasive mechanical ventilation" as a binary dependent variable and age, BMI, hypertension, diabetes, and time between symptom onset and admission to hospital as independent variables. The covariates were chosen taking into account current evidence, results from univariate testing, and sample size. We performed univariate tests regarding all available patient factors and association with organ support treatment, complications, and outcome. Continuous variables were treated as follows: age was categorized in accordance with other reports on patients with COVID-19 [1], BMI and CCI were dichotomized at the median. Patients with therapy limitations (Do Not Intubate (DNI) or Do Not Resuscitate (DNR) orders) at the respective time point were excluded for comparison between non-IMV and IMV patients, and for analyses of course-of-organ support and mortality. Non-survivors were excluded from comparison between short-(< 15 days) or long-term (≥ 15 days) IMV. For analysis of SOFA score, scores were extracted from electronic patient charts and the highest score per day was included for calculation of means. The neurological component of the SOFA score was not taken into consideration due to patient sedation and incomplete documentation during daily discontinuation of sedation. Patients who died or were transferred to other centres were excluded from calculation of length of hospital stay. For analyses of routine laboratory parameters within 72 h of first admission, first-available parameters were included.
We compared viral concentration between non-IMV and IMV patients, and regressed viral concentration on the duration from symptom onset to admission using both the first positive RT-PCR result and the RT-PCR with the highest viral concentration. For the calculation of viral concentration decline, we estimated the slope parameter from a linear regression of at least four viral concentration measurements over time for each patient. If available, the first of at least two final negative RT-PCR results was included, in which case the viral concentration of the negative RT-PCR was set to 2.0 in accordance with the RT-PCR limit of detection and sample dilution factor of ~ 20 [26,27]. We calculated shedding duration as the time from symptom onset to the date of the first of at least two final negative RT-PCR results.
Analyses were conducted with R (version 3.6.1), JMP (version 14.2.0), and statsmodels (version 0.12.0) in Python 3.7.9. A p value < 0.05 indicates statistical significance, although all results have to be considered as nonconfirmatory. For this reason, no adjustment for multiple testing was done.

Baseline characteristics
Between March 1st and June 30th 2020, a total of 347 adult patients with COVID-19 were hospitalised at Charité-Universitätsmedizin Berlin. This analysis includes 168 adult patients who consented to participation in the prospective observational study (Fig. 1). Sixty-five per cent (110/168) were directly admitted to our centre, whereas 29.8% (50/168) were referred due to ARDS or other conditions requiring tertiary care. Four per cent (7/168) were hospitalised for other reasons and coincidentally diagnosed with SARS-CoV-2 infection during routine screening, and one patient (0.6%) was admitted due to a late complication of COVID-19.
Baseline characteristics are shown in Table 1

ARDS and organ support treatment
Fifty-two per cent (88/168) of patients developed ARDS. One of them was already dependent on long-term Fig. 1 Study cohort flowchart. A total of 347 adult patients were hospitalised with COVID-19 during the study period from March 1st until June 30th at Charité-Universitätsmedizin Berlin. Of these, 168 patients could be enrolled in the prospective observational study, whereas 179 denied. Among the included patients, 88 had acute res-piratory distress syndrome (ARDS). One patient with ARDS was already invasively ventilated and six of them had DNI/DNR (do not intubate/do not resuscitate) orders in place, resulting in 81 patients requiring respiratory support. Of those, 71 patients were intubated and ten required only high-flow nasal cannula oxygen therapy Patients with therapy limitations were excluded from comparison and statistical analysis of IMV versus non-IMV patients. Asymptomatic patients were excluded from analyses of symptom characterisation. Independent variables incorporated into the multiple logistic regression model: Age, BMI, hypertension, diabetes, and length between symptom onset and admission IMV invasive mechanical ventilation, OR odds ratio, CI confidence interval, IQR interquartile range, BMI body mass index, CCI Charlson comorbidity index, ARB angiotensin II receptor blockers, ACE-i angiotensin-converting enzyme inhibitor, ARDS acute respiratory distress syndrome a Inverse adjusted OR and confidence interval As no evidence of efficacy of antiviral and anti-inflammatory treatments was available at the time, these were not used systematically and only in a small subgroup of patients of this cohort. Seventeen per cent (29/168) of all patients received corticosteroids in ≥ 40 mg prednisolone equivalent for ≥ 1 day. For details see Supplementary Table 3.

Virological and routine laboratory data
Viral concentration data were available for 166/168 patients. On average, each patient had seven RT-PCR tests (SD: 5.3, min = 1, max = 29, including positive and negative result) from the day of symptom onset (or 10 days from first admission, if the date of symptom onset was not available) to the end of hospitalisation, with tests performed every 8.4 days on average (SD: 8.8). Eighty-six patients had two final negative RT-PCR tests at the end of the disease course. Median first-measured viral concentration differed by 0.68 log 10 viral copies between IMV and non-IMV patients (5.9, IQR 4.68-7.28 vs 5.22 log 10 viral copies, IQR 4.49-7.28, respectively; p = 0.12, Fig. 2a), and median highest viral concentration by 1.19 log 10 viral copies (6.7, IQR 5.35-7.62 vs 5.51 log 10 viral copies, IQR 4.7-7.62, respectively; p = 0.02, Fig. 2b). Decline of viral concentration ( Supplementary Fig. 1) was significantly slower in IMV versus non-IMV patients (-0.13, IQR -0.19 to -0.08 vs -0.22 log 10 viral concentration decrease / day, IQR -0.3 to -0.08, respectively; p < 0.01) (Fig. 2c, Supplementary  Fig. 2a-d). The duration of shedding was significantly longer in IMV patients than in non-IMV patients (median 33, IQR 26-46.75 vs 18 days, IQR 16-46.75, p < 0.01) (Fig. 2d). We found no association between viral concentration and the duration from symptom onset to admission and no difference in first or in highest viral concentrations in patients requiring long versus short IMV ( Supplementary Fig. 3a-d, Supplementary Table 1).
A statistically significant difference between non-IMV and IMV patients was observed in the levels of C-reactive protein (CRP), procalcitonin (PCT), Interleukin-6 (IL-6), lactate dehydrogenase, ferritin, leukocyte count, lymphocyte count, neutrophil-to-lymphocyte ratio, creatinine, urea, aspartate aminotransferase, creatine kinase, N-terminal prohormone of brain natriuretic peptide, and troponin (Supplementary Table 4). The course of 12 routine laboratory parameters over time in non-IMV and IMV patients is shown in Fig. 3.

Complications during treatment
Eighteen per cent (31/168) of patients developed sepsis. Occurrence of sepsis was associated with organ replacement therapies, but not with patient-related risk factors (Supplementary Table 5). The first sepsis episode occurred after a median of 16 days (IQR 8-21) from hospital admission and 11.5 days (IQR 3-18.3 days) from intubation. In 15/31 (48.4%) of patients with sepsis, ECMO was initiated during or soon after occurrence of sepsis (median time from occurrence of sepsis and initiation of ECMO, 0 days, IQR 0-6.25 days).

Discussion
We analysed a detailed clinical and virological dataset from a prospective observational cohort of COVID-19 patients hospitalised in a tertiary, ECMO/ARDS referral and weaning centre in Germany during the first wave of the SARS-CoV-2 pandemic, before dexamethasone became standard of care.
We report a short duration of symptoms before clinical deterioration as a risk factor for severe COVID-19. Second, our results indicate that severely ill patients have higher maximal viral concentrations and a slower decline of viral concentration compared to mildly affected patients. Third, we describe a comparatively long duration of inpatient treatment for patients with IMV, largely exceeding that described for non-COVID-19 ARDS patients.
Rapid clinical deterioration-as reflected by short duration of symptoms before hospital admission-is a highly relevant risk factor for both need of IMV in a multivariable risk model and death in univariate analyses in our cohort. A shorter duration of symptoms was not associated with higher initial viral concentration. There was also no difference in initial viral concentration in more severely ill patients, i.e., those requiring IMV, compared to mildly ill patients. Yet, we found a significant increase of inflammatory markers such as CRP, PCT, and IL-6 at presentation and over time in IMV compared to non-IMV patients. Moreover, IMV patients had higher maximal concentrations, a slower decline as well as longer duration of shedding of SARS-CoV-2 compared to non-IMV patients. Higher maximal viral concentrations were also found in patients who died, compared to survivors.
These findings underline that the early inflammatory host response to SARS-CoV-2 determines the course of disease more than pathogen factors such as initial SARS-CoV-2 RNA concentration [2,28,29]. A short duration of symptoms before admission possibly reflects a rapid increase of the level of inflammation and might serve as an easy to assess prognostic factor for clinical deterioration, a finding that merits further exploration [30]. During the further course of COVID-19, severe disease is characterised by the inability to rapidly reduce viral particles. A possible explanation for this phenotype-rapid deterioration after symptom onset, high inflammatory markers, and lack of efficient clearing of viral particles-might be the various kinds of immune dysregulation, as reported by our group and others [15,31]. Conflicting results have been published regarding the association between clinical severity and viral concentration for SARS-CoV-2 so far [32][33][34][35][36]. This could possibly be explained by a rather unbalanced proportion of mildly and severely affected patients in the respective studies as compared to our cohort [35]. However, it is undetermined whether higher maximal viral concentration and longer duration of shedding are a possible cause or an indicator of more severe organ damage and disease.
Although a remarkable proportion (29.8%) of patients was transferred to our center due to severe ARDS, and overall 44.1% needed IMV, we report a comparatively low inhospital mortality of 13 [37] in cohorts from Germany with 17.2% [1] and 32.9% [37] ventilated patients, respectively. Of note, our data show a high median length of hospital stay of 49.5 days for patients requiring IMV. By comparison, the median length of hospital stay for non-COVID ARDS patients was 17 days in a recent global multi-centre prospective study [38]. There is growing evidence that the length of IMV-, ICU-, and inpatient treatment of patients with COVID-19 ARDS exceeds that of patients with ARDS unrelated to COVID-19 [30,37]. Despite the long median duration of hospital stay, a considerable percentage of patients could not successfully be weaned off the ventilator (18%), and 6% required ongoing oxygen therapy following discharge. The mere number of deceased patients therefore depicts the burden of disease of COVID-19 only very incompletely, particularly with respect to long-term morbidity. The prospective approach of our study will allow us to evaluate long-term complications in the aftermath of COVID-19.
Prospective observational studies are often hampered by selection of patients with relatively mild disease courses due to need for informed consent. The high proportion of severely affected patients in this study cohort indicates that this specific selection bias does not apply to our data. On the contrary, it is rather the selection of severely ill patients referred for ARDS management that might have led to a bias. On the other hand, one-fifth of patients was only mildly affected and did not require oxygen therapy, representing a sub-cohort admitted for clinical observation or due to lacking the possibility of self-isolation. Other major limitations of our work are its monocentricity and small sample size.
Our data give a comprehensive description of the clinical course, virological characteristics, organ support treatment, complications, and outcome of a representative cohort of patients in an unrestricted tertiary care healthcare setting with comparatively low mortality. The reported findings will be of value for comparison of clinical courses in the dexamethasone era and for sample size calculation for interventional studies in similar settings.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.