key: cord-0732955-3guoti80
authors: Hagman, Karl; Hedenstierna, Magnus; Gille-Johnson, Patrik; Hammas, Berit; Grabbe, Malin; Dillner, Joakim; Ursing, Johan
title: SARS-CoV-2 RNA in serum as predictor of severe outcome in COVID-19: a retrospective cohort study
date: 2020-08-28
journal: Clin Infect Dis
DOI: 10.1093/cid/ciaa1285
sha: 7da7b8586a62ee2c0ee9dd6b29991d3a69cebe2f
doc_id: 732955
cord_uid: 3guoti80

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes Coronavirus disease 2019 (COVID-19). This study aimed to determine if SARS-CoV-2 RNA in serum at admission correlated with clinical outcome in COVID-19. METHODS: COVID-19 patients admitted to the Infectious Diseases department of a tertiary level Swedish hospital, and sampled for SARS-CoV-2 RNA in serum at admission, April 10 to June 30 2020 were included in a cohort. Primary outcomes were day 28 all-cause mortality and progress to critical disease. RESULTS: The cohort (N=167) consisted of 106 SARS-CoV-2 RNA serum negative and 61 positive patients. Median sampling time for initial SARS-CoV-2 in serum was 1 (IQR 1-2) day after admission corresponding to day 10 (IQR 8-12) after symptom onset. Median ages were 53 (IQR 44-67) and 63 (IQR 52-74) years for the PCR-negative and positive patients, respectively. In the serum PCR negative and positive groups 3/106 and 15/61 patients died, respectively. The hazard ratios for critical disease and all-cause mortality were 7.2 (95% CI 3.0-17) and 8.6 (95% CI 2.4-30), respectively for patients that were serum PCR positive compared to serum PCR negative. Conclusion: SARS-CoV-2 RNA in serum at hospital admission indicates a high-risk of progression to critical disease and death.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of the current Coronavirus disease 2019 (COVID-19) pandemic [1] . COVID-19 is characterised by fever, respiratory tract, and gastrointestinal symptoms with multiple organ involvement in severe disease [2] . A proportion of patients develop critical disease with Acute Respiratory Distress Syndrome and high mortality rates [2, 3] .

A "cytokine storm" has been proposed as the cause of severe COVID-19, based on the detection of high levels of cytokines [4] . Similar findings were described in critically ill patients with Severe Acute Respiratory Syndrome (SARS) [5] . In SARS, high serum viral load and detection of virus at multiple sites were predictive of adverse clinical outcome. These findings indicated that adverse outcome was associated with continued, uncontrolled viral replication [6] . Similarly, a high viral load in respiratory samples was associated with more severe disease in Middle East Respiratory Syndrome (MERS) and has also been described in COVID-19 [7] [8] [9] [10] . Oropharyngeal viral load also appears to increase with age in COVID- 19 [11] . In line with these findings, SARS-CoV-2 RNA in serum was detected in five of 17 critically ill patients of whom two died and in none of 31 patients with severe or moderate COVID-19 [12] . Moreover, in a very recent study, SARS-CoV-2 RNA was detected in serum in 43/58 (74%) of hospitalised COVID-19 patients with an ultrasensitive Polymerase Chain Reaction (PCR) method [13] . The presence and levels of SARS-CoV-2 RNA in serum were correlated to disease severity, and there was a tendency towards a higher risk of poor outcome in the serum positive group. An inability to control viral replication, and a disseminated hematogenous spread to multiple organs, is thus a possible cause of severe disease or possibly the driving force behind the inflammatory reaction that characterises severe COVID-19. Detection of viral-RNA in serum may thus be an early prognostic marker that is simpler to use compared to more complex risk scoring systems [14, 15] . However, the prognostic value of detecting serum SARS-CoV-2 RNA with a commonly available method has not been evaluated. The primary aim of this study was to determine if SARS-A c c e p t e d M a n u s c r i p t CoV-2 RNA in serum, detected using standard Reverse Transcriptase (RT) PCR methods, correlated with clinical outcome in COVID-19.

This was a retrospective cohort study of patients with confirmed COVID-19, consecutively admitted to the 38 bed Department of Infectious Diseases at Danderyd Hospital, between the 10 th of April and the 30 th of June 2020. Danderyd Hospital is a 500-bed tertiary care hospital in Stockholm, Sweden that serves a population of approximately 500.000 people.

The study was approved by the Swedish Ethical Review Authority (Registration number 2020-01770).

The study population consisted of patients with confirmed COVID-19 that were admitted to the Department of Infectious Diseases and had a SARS-CoV-2 RNA serum sample taken within three days of admission. All patients admitted to the hospital during the study period with symptoms suggestive of COVID-19 were screened for SARS-CoV-2 in airway (nasopharyngeal, throat, or sputum) samples by PCR assays. Patients with positive results were considered to have a confirmed diagnosis.

At the beginning of the study period the influx of patients was considerable and patients with confirmed COVID-19 were admitted to medical and surgical wards as well as the Department of Infectious Diseases. During this period there was no particular selection of any patient category to any particular ward.

A c c e p t e d M a n u s c r i p t A phase I/II safety and dose-finding study on convalescent plasma was conducted at the Department of Infectious Diseases during the study period. Febrile patients requiring supplemental oxygen therapy were initially eligible for entry into the phase I study. Subsequently, patients with SARS-CoV-2 RNA detected in serum were eligible for entry to the phase II study. No other interventional studies were ongoing in this population. No antivirals or chloroquine derivatives were in use for the treatment of COVID-19 during the study period. Corticosteroid treatment was introduced for patients with critical disease from the 18 th of June. 

The primary outcome was all-cause mortality at 28 days after first SARS-CoV-2 serum sampling.

The secondary outcome was progression to critical disease within 28 days of the initial SARS-CoV-2 serum sampling. Critical disease was defined as a composite of ICU-care and mortality. The hospital's ICU consisted of one ward for SARS-CoV-2 patients requiring mechanical ventilation, and two wards for patients requiring Non-Invasive Ventilation (NIV), High Flow Nasal Oxygen (HFNO) Therapy, or inotropic drugs. During the study period, the ICUs had an admission criterion of peripheral capillary A c c e p t e d M a n u s c r i p t oxygen saturation <90% despite 12 litres/min oxygen on a mask (PaO2/FiO2-ratio approximately 75).

At the Department of Infectious Diseases, oxygen was given using a mask but not using NIV or HFNO.

Patients that were SARS-CoV-2 serum PCR negative at study entry, in effect on admission, were classed as PCR negative. Patients that were SARS-CoV-2 serum PCR positive at entry were classed as PCR positive. Continuous variables are presented as medians (interquartile range -IQR) and were calculated using quantile regression with bootstrap (100 reps). Hazard Ratios (HR) were calculated with 95% confidence intervals (95% CI) using univariate and multivariate Cox regression. Variables that have been previously described as predictors for severe outcome (age, gender, body mass index (BMI), hypertension, diabetes, C-reactive protein, lymphocyte count, cardiovascular and pulmonary disease), were included in the univariate analyses [19] . Due to missing data and difficulties with standardized interpretations, lactate dehydrogenase and chest x-rays were not included in the statistical analyses. Variables with a p-value < 0.05 in the univariate analysis were included in the multivariate analysis. The proportional hazards assumption was tested with a log-log plot of survival.

We constructed Kaplan-Meier survival curves for all-cause mortality and progression to critical disease. The log-rank test was used to test for equality of survivor functions. All tests were two-sided and a p-value <0.05 was considered significant. Statistical analyses were done with STATA version 13.1 (StataCorp, TX, USA).

A total of 258 patients with confirmed COVID-19 were admitted to the Department of Infectious Diseases during the study period. Of these, 167 were tested for SARS-CoV-2 RNA in serum within three days of admission. SARS-CoV-2 RNA in serum was detected in 61/167 (37%) patients and not detected in 106/167 (63%) patients. A total of 91 patients were excluded from the study. Of these, A c c e p t e d M a n u s c r i p t 77 were not tested, 10 tested more than three days after admission, and four tested during/after ICU-care. The flow of patients through the study are shown in Figure 1 . Baseline characteristics are shown in Table 1 . Routine serum sampling of patients admitted to the Department of Infectious Diseases was implemented from the 10 th of May until the 30 th of June. In this subgroup, 69/106 (65%) patients were PCR negative and 37/106 (35%) were PCR positive.

The median time from admission to initial PCR testing in serum was 1 (IQR 1-2) day in both the PCR negative and positive group. The median durations of illness before testing were 10 days (IQR 8-12) and 9 days (IQR 8-11) in the PCR negative and positive groups, respectively. Median time to first outcome was 2 (IQR 1-2) days after sampling in the PCR positive group. 

Convalescent plasma was given to 28/61 serum PCR positive patients. Plasma from several donors at 7-fold varying doses were used. The proportion of patients that died amongst serum PCR positive patients that did and did not receive convalescent plasma were 6/28 and 9/33, respectively.

A c c e p t e d M a n u s c r i p t

The principal finding was that detection of SARS-CoV-2 RNA in serum, within three days of admission, identified a population with a seven-fold increased risk of progress to critical disease and an eight-fold increased risk of death, compared to patients in whom SARS-CoV-2 RNA was not detected in serum. Equally valuable, not detecting SARS-CoV-2 RNA in serum indicated a high chance of uncomplicated recovery. The data are consistent with a study which reported SARS-CoV-2 RNA in the serum of critically ill patients only (N=5) and a recent study that identified higher serum viral loads in more severely ill patients [12, 13] . Veyer et. al. also showed a trend towards worse outcome in the PCR positive group. The results are also in line with SARS, MERS, and COVID-19 data indicating that detection of virus in serum and viral load in respiratory samples correlated with outcome [6, 7, 9, 10] . However, detection of SARS-CoV-2 in serum was not significantly associated with disease severity in a cohort of 96 patients [8] . A possible explanation is that disease severity was based on clinical characteristics in that cohort, in which there was zero mortality. In the current analysis, the presence of SARS-CoV-2 RNA in serum was assessed as an early predictor of clinical outcome, essentially asking the question the other way around. Thus, these data do not necessarily oppose each other and detection of SARS-CoV-2 in the serum of hospitalized patients seems to be an early indicator of a population at a significantly increased risk of critical disease and death. However, the results need to be verified in other populations.

As shown previously, age was also an independent predictor of critical disease and death [11] . In addition, the proportion of serum PCR positive patients increased with age, especially in those over the age of 60 years. An age correlated increased nasopharyngeal viral load has been reported previously supporting this result [9] . These data suggest that there is an age correlated, decreased ability to control viral replication that may in turn be causally linked to severe disease. Furthermore, SARS-CoV-2 was found in serum of 78% (28/36) of patients admitted to Danderyd hospital ICU due to an anticipated need of invasive ventilation during a similar time period (unpublished data), which A c c e p t e d M a n u s c r i p t matches the 88% (23/26) PCR positive patients with critical disease in a study by Veyer et. al. [13] .

Taken together this suggests that most patients who developed critical disease had a continued, age correlated, uncontrolled viral replication as found in SARS [6] .

Dysregulation of the inflammatory response resulting in a "cytokine storm" has been proposed as an important factor in the pathogenesis of severe COVID-19 [4] . In line with this, high IL-6 levels commonly occur in critically ill patients [3, 12, 20, 21] . Moreover, detection of SARS-CoV-2 in serum correlated with extremely high levels of IL-6 [12] . Detection of SARS-CoV-2 in serum could thus be a result of leakage from tissues damaged by the inflammatory response. However, the presence of SARS-CoV-2 in serum prior to the development of severe disease suggests that the virus is, at least initially, driving the inflammatory response. Detecting SARS-CoV-2 in serum is in line with the multiorgan involvement seen in patients with critical disease and autopsy data reporting viral infection in several organs, indicating hematogenic spread of the virus [2, 22, 23] .

The serum PCR CT values in this study were high with low variance as reported previously, and unlike CT values from respiratory samples where virus is known to replicate [8] . The low variance and the dichotomous nature of PCR positivity suggests a total viral load reaching a detectable level, predictive of a severe course. In a study using an ultrasensitive PCR method, the viral load in serum differed significantly between patients of different disease severity and 74% of tested patients were PCR positive [13] . This difference is most likely due to a greater method sensitivity enabling detection of even lower RNA levels in serum, of potential clinical utility in the future.

Early identification of groups with a high or low risk of critical disease, using a commonly available PCR method, helps us make informed treatment decisions. Recent preliminary data indicate that remdesivir given early to moderately ill patients may be an effective COVID-19 treatment [24] .

Serum PCR positive patients are a group in which to primarily assess the efficacy of remdesivir while the supply is limited. Other potential antiviral therapies such as convalescent plasma may also be most effective if given to serum PCR positive patients before the development of critical disease. The patients receiving convalescent plasma, the incidence of serious adverse events was low [25] .

Thirdly, lactate dehydrogenase and radiological results have been described as predictors for severe outcome. Unfortunately, we were not able to include these variables in our statistical models due to missing data and difficulties with standardized interpretations.

SARS-CoV-2 RNA in serum at admission to hospital is associated with a seven-fold increased risk of progression to critical disease and an eight-fold increased risk of death. PCR positivity in serum at admission is thus a predictor for risk of progression to critical illness, and identifies a group of patients to whom treatment should be prioritized in a setting with limited drug availability. A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 

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