key: cord-0853429-jeck91nr
authors: Heinrich, Fabian; Nentwich, Michael F; Bibiza-Freiwald, Eric; Nörz, Dominik; Roedl, Kevin; Christner, Martin; Hoffmann, Armin; Olearo, Flaminia; Kluge, Stefan; Aepfelbacher, Martin; Wichmann, Dominic; Lütgehetmann, Marc; Pfefferle, Susanne
title: SARS-CoV-2 blood RNA load predicts outcome in critically ill COVID-19 patients
date: 2021-10-06
journal: Open Forum Infect Dis
DOI: 10.1093/ofid/ofab509
sha: f0bb56a6e8980e4ca841ccb8db07ece6851038f1
doc_id: 853429
cord_uid: jeck91nr

BACKGROUND: SARS-CoV-2 RNA loads in patient specimens may act as a clinical outcome predictor in critically ill patients with COVID-19. METHODS: We evaluated the predictive value of viral RNA loads and courses in the blood compared to the upper and lower respiratory tract loads of critically ill COVID-19 patients. Daily specimen collection and viral RNA quantification by RT-qPCR was performed in all consecutive 170 COVID-19 patients between March 2020 and February 2021 during the entire ICU stay (4145 samples analyzed). Patients were grouped according to their 90-days outcome as survivors (n=100) or non-survivors (n=70). RESULTS: In non-survivors, blood SARS-CoV-2 RNA loads were significantly higher at the time of admission to the ICU (p=0.0009). Failure of blood RNA clearance was observed in 33/50 (66 %) of the non-survivors compared to 12/64 (19 %) of survivors (p<0.0001). As determined by multivariate analysis, taking sociodemographic and clinical parameters into account, blood SARS-CoV-2 RNA load represents a valid and independent predictor of outcome in critically ill COVID-19 patients (OR [log(10)]: 0.23 [0.12 – 0.42], p<0.0001) with a significantly higher effect for survival compared to the respiratory tract SARS-CoV-2 RNA loads (OR [log(10)]: 0.75 [0.66 – 0.85], p<0.0001). Blood RNA loads exceeding 2.51 x 10 (3) SARS-CoV-2 RNA copies/ml were found to indicate a 50% probability of death. Consistently, 29/33 (88%) of the non-survivors with failure of virus clearance exceeded this cut-off value constantly. CONCLUSION: Blood SARS-CoV-2 load is an important independent outcome predictor and should be further evaluated for treatment allocation and patient monitoring.

A c c e p t e d M a n u s c r i p t 4 Text (word count: 2924)

Risk assessment and stratification of Coronavirus disease 2019 (COVID-19) patients is challenging, notably in intensive care units (ICUs), as it is still unclear which factors correlate with severe courses or fatal outcomes. Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) RNA load in blood and respiratory tract specimens as detected by Reverse Transcription-Polymerase Chain Reaction (RT-qPCR) has been suggested to correlate with disease severity and mortality. However, previous studies on the impact of viremia on patient outcomes were mostly limited to single-point measurements and did not consider the level and course of viral loads 1-7 . By analyzing the course of viremia in a small cohort of critically ill patients with haemato-oncologic disorders, we recently reported that failure to clear SARS-CoV-2 RNA from the bloodstream is associated with a high risk of death 8, 9 , as confirmed in small patient cohorts 10,11 .

To investigate the prognostic value of viral load in a mixed patient population, we now conditions. Patients were grouped according to their 90-days outcome status as survivors (n=100) or non-survivors (n=70). Readmissions of patients were counted as one intensive care unit stay. Relevant co-variables evaluated were the age, sex, body mass index, preexisting medical conditions (i.e., prior myocardial infarction, congestive heart failure, peripheral vascular disease, rheumatologic disease, peptic ulcer disease, mild, moderate, or severe liver disease, diabetes mellitus, cerebrovascular (hemiplegia) event, moderate-tosevere renal disease, diabetes with chronic complications, cancer without metastases, leukaemia, lymphoma, metastatic solid tumour, acquired immune-deficiency as part of the Charlson comorbidity index; chronic lung diseases and arterial hypertension), immunosuppression due to pre-existing medical conditions, time from COVID-19 diagnosis until ICU admission, presence and degree of Acute Respiratory Distress Syndrome (ARDS) according to the Berlin definition, disease severity according to Simplified Acute Physiology Score II (SAPS II) and Sepsis-related organ failure assessment score (SOFA), need for mechanical ventilation (MV), need for extracorporeal membrane oxidation (ECMO), and need for COVID-19 related treatments (i.e., dexamethasone, remdesivir, monoclonal antibodies, therapeutic plasma exchange (TPE)).

The Ethics Committee of the Hamburg Chamber of Physicians was informed about the study. Due to the retrospective nature of the study, the need for informed consent was A c c e p t e d M a n u s c r i p t 6 waived (WF-094/21). Partial data of a subset of the cohort (30 out of 170) has been previously analyzed and published elsewhere 8,9 . Sampling, molecular diagnostic and epidemiology: c c e p t e d M a n u s c r i p t 7 1x10 3 copies/ml was set for quantification, RNA loads coming below this cut-off were excluded from the quantitative analysis. Of all patients, initial respiratory samples were analyzed in a multiplex typing PCR, identifying and distinguishing SARS-CoV-2 spike variants 16 .

Estimation of virus RNA clearance: Successful RNA clearance was defined as the absence of SARS CoV-2 RNA (in RT-qPCRs) from the respective compartment for at least three days.

Assuming non-parametric data distribution, the Wilcoxon-Mann-Whitney-U-Test was used to compare viral loads between two groups. Categorical variables were compared using the two-sided Fisher's exact test or two-sided Chi-squared test. The survival distribution of two groups was compared using the log-rank test. A generalized mixed model with logistic regression and Firth approximation was used to identify predictors of adverse outcomes in the URT, LRT, and blood. [<LoD -4.12 x 10 6 copies/ml]) (p<0.0001) (see figure 1b) .

During the course of the disease, maximum URT levels showed no significant differences between the two groups (median RNA load of < 1.00 x 10 3 SARS-CoV-2 RNA copies/ml, IQR: In the present study, we evaluated the prognostic value of SARS-CoV-2 RNA levels and kinetics in blood, upper and lower respiratory tract samples by daily molecular analyses of all three compartments in 170 critically ill patients. This in-depth look at the course of virological data contrasts with previous studies that analyzed single-point measurements 1,2,4,5,7 or only focused on individual compartments without quantifying viral loads 7, 17 . Two studies on blood SARS-CoV-2 RNA loads found increased mortality in viremic patients, yet findings are based on smaller cohorts and lower sampling frequencies 10,11 . A recent large study focused on respiratory specimens and highlighted the association of viral RNA load and infectivity in outpatients compared to inpatients, yet without addressing their predictive value for mortality and disease progression 18 .

Notably, and consistent with previous studies 6,10,11 , blood RNA loads on admission to the ICU were significantly elevated in patients with fatal outcomes (p=0.0009), while no significant difference in admission viral loads could be shown for the lower respiratory tract. Furthermore, the maximum blood and LRT viral RNA loads during the disease were significantly higher in non-survivors compared to survivors. Again, no such differences were observed for maximum URT viral RNA levels during the course of the disease.

Clearance of viral RNA from the bloodstream occurred more frequently (p<0.0001) and more rapidly (median time 9 days) in survivors than in non-survivors. Likewise, and similar to recently published data 8, 9, 11, 17 According to the model presented here, blood RNA levels exceeding 2.51 x 10 3 SARS-CoV-2 RNA copies/ml reflect a 50% probability of death. Considering this blood RNA load as a critical cut-off value, half of all patients in the survivor group compared to the majority in the non-survivor group exceeded this value at least once during the ICU stay. Notably, in 88% of the non-survivors, blood RNA loads remained constantly elevated above that cut-off value, while in all but 2 of the survivors, blood RNA loads declined below that threshold during the course of the disease.

The proportion of patients receiving COVID-19-related antiviral therapy (dexamethasone, remdesivir, or monoclonal antibodies) was comparable in survivors and non-survivors, though monoclonal antibody therapy was initiated in four of the non-survivors and none of the survivors. Altogether, the difference in successful viral blood clearance seems not to be attributable to specific therapeutic interventions.

However, the proportion of immunocompromised patients was higher among non-survivors in our study. Thus, our data prove evidence of an increased risk of SARS-CoV-2 viremia and associated mortality in this particular patient population [8] [9] [10] [11] . However, it is not yet possible A c c e p t e d M a n u s c r i p t 15 to conclude whether immunosuppression promotes viremia or whether, conversely, viremia exacerbates immunosuppression in critically ill patients.

Previous studies have shown that SARS-CoV-2 affects different organs besides the respiratory tract and that high viral loads in the affected organs correlate with increased mortality 19 .

Although our study does not identify viremia itself as the cause of death, our data indicate that patients with high levels of viremia and delayed virus clearance represent a vulnerable subgroup. This subgroup might particularly benefit from specific therapy such as monoclonal antibodies or direct antiviral substances. Moreover, monitoring of viremia could thus be useful for future patient management in the ICU.

However, it is currently difficult for diagnostic laboratories to offer reliable quantitative molecular SARS-CoV-2 diagnostics for specimens other than respiratory tract samples. FDAor CE approved molecular assays are missing for this purpose. Since the importance of virologic blood diagnostics is highlighted, indicating a high prognostic value for the patients' outcome in this study, such diagnostics might help clinicians in patient management assessing each patient's prognosis. Thus, there is an urgent need for the rapid evaluation and approval of blood RT-qPCRs for SARS-CoV-2 10,15 .

A c c e p t e d M a n u s c r i p t 16 We are aware that our study has limitations. The intermittent shortage of reagents and supplies lead to the use of a variety of assays for quantification. Blood samples were almost exclusively analyzed with the cobas 6800 system, but slight deviations in the quantification in URT and LRT cannot be completely ruled out given the multitude of assays. Also, the URT sampling frequency is lower than for LRT and blood because nasopharyngeal swabs were In summary, our data indicate that SARS-CoV-2 viremia is a better predictor of outcome than respiratory tract viral RNA load, and clearance of SARS-CoV-2 RNA from the bloodstream is strongly associated with survival. Thus, reliable quantification of SARS-CoV-2 RNA in the blood as part of clinical practice seems mandatory to assess patients' risk of fatal outcomes. Moreover, monitoring of viremia could be an important surrogate marker of the effectiveness of antiviral therapies. FDA approved assays are required for this purpose. to 1 x 10 1 copies/ml, samples < threshold of 1x10 3 copies/ml were set to 1 x 10 2 copies/ml to allow for logarithmic presentation.

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