key: cord-0921006-jpqljiib
authors: Boyapati, Anita; Wipperman, Matthew F; Ehmann, Peter J; Hamon, Sara; Lederer, David J; Waldron, Alpana; Flanagan, John J; Karayusuf, Elif; Bhore, Rafia; Nivens, Michael C; Hamilton, Jennifer D; Sumner, Giane; Sivapalasingam, Sumathi
title: Baseline SARS-CoV-2 Viral Load is Associated With COVID-19 Disease Severity and Clinical Outcomes: Post-Hoc Analyses of a Phase 2/3 Trial
date: 2021-09-08
journal: J Infect Dis
DOI: 10.1093/infdis/jiab445
sha: 51fcc04b0898090ab0f8ecb25156e8166369cb96
doc_id: 921006
cord_uid: jpqljiib

BACKGROUND: Elucidating the relationship between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load and clinical outcomes is critical for understanding COVID-19. METHODS: SARS-CoV-2 levels were analyzed by quantitative real-time polymerase chain reaction (RT-qPCR) of nasopharyngeal or oropharyngeal swab specimens collected at baseline and clinical outcomes were recorded over 60 days from 1362 COVID-19 hospitalized patients enrolled in a multicenter, randomized, placebo-controlled phase 2/3 trial of sarilumab for COVID-19 (NCT04315298). RESULTS: In post-hoc analyses, higher baseline viral load, measured by both RT-qPCR cycle threshold (Ct) and log(10) copies/mL, was associated with greater supplemental oxygenation requirements and disease severity at study entry. Higher baseline viral load was associated with higher mortality, lower likelihood of improvement in clinical status and supplemental oxygenation requirements, and lower rates of hospital discharge. Viral load was not impacted by sarilumab treatment over time versus placebo. CONCLUSIONS: These data support viral load as an important determinant of clinical outcomes in hospitalized patients with COVID-19 requiring supplemental oxygen or assisted ventilation.

Since the association of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with COVID-19 disease was established, studies have examined the kinetics and biological compartments of viral shedding across disease presentations [1] [2] [3] [4] . Data demonstrates the virus persists in the upper respiratory tract, particularly in the absence of an anti-viral therapeutic [5] . Viral shedding may have different clinical associations between outpatient and hospital settings. Furthermore, viral persistence is associated with seroconversion status; patients seronegative for endogenous anti-SARS-CoV-2 antibodies have higher viral loads in nasopharyngeal specimens than seropositive patients [1, 6] . In hospitalized patients, higher SARS-CoV-2 viral load was associated with intubation risk and mortality [7, 8] .

To explore the relationship between viral load and disease severity, baseline viral load, serology, supplemental oxygenation requirements, survival and recovery were evaluated.

In this adaptive, phase 2/3, randomized, double-blind, placebo-controlled trial, subjects aged ≥ 18 years, hospitalized with laboratory-confirmed SARS-CoV-2 infection (within 2 weeks of study) and COVID-19 pneumonia requiring supplemental oxygen and/or assisted ventilation were treated between March and July 2020 with intravenous (IV) sarilumab or placebo (NCT04315298). [9] Local institutional review boards or ethics committees at each center oversaw trial conduct and documentation. All patients provided written informed consent.

Specimen collection included nasopharyngeal (N = 1047) and oropharyngeal swabs (N = 315). Baseline refers to pre-dose collections post study randomization, required as part of the protocol but were missing in some randomized subjects. Subsequent testing was optional. Nucleic acid extraction and quantitative real-time polymerase chain reaction (RT-qPCR) was performed at Viracor-Eurofins laboratory (MO, USA). Details of the Emergency Use Authorization assay have been previously described [6] ; "Not Detected" results were transformed to 1, "Detected <714" results were transformed to half the lower limit of quantification of the assay, results greater than the upper limit of quantification (ULOQ) were transformed to the ULOQ prior to log 10 transformation for analysis.

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For all outcomes, patients who died were censored at day 60; patients who were alive were censored at day 60 or last follow-up date, whichever was earlier.

Time to all-cause mortality. Number of days to death (any cause) minus the first dose date +1 (assumed that patients were alive on first dose date and alive on date of death until death).

Time to clinical status improvement. Number of days to achieve ≥ 1-point increase in clinical status +1 using the seven-point ordinal scale [10]: 1, death; 2, hospitalized, requiring invasive mechanical ventilation (IMV) or extracorporeal membrane oxygenation; 3, hospitalized, requiring non-invasive ventilation or high-flow oxygen devices; 4, hospitalized, requiring supplemental oxygen; 5, hospitalized, not requiring supplemental oxygen but requiring ongoing medical care (any reason); 6, hospitalized, not requiring supplemental oxygen and no longer requiring ongoing medical care; and 7, not hospitalized.

Time to hospital discharge. Date of discharge minus the first dose date +1 day.

Time to improvement in oxygenation. Number of days from first dose to first improvement in oxygenation (SpO 2 /FiO 2 ratio ≥ nadir + 50), lasting ≥ 48 hours or until discharge, whichever was sooner.

Descriptive statistics grouped by disease severity are reported as mean (standard deviation [SD]) for normally distributed continuous variables, median (interquartile range [IQR]) for non-normally distributed continuous variables, and frequency (%) for categorical variables. Baseline viral load across disease strata were compared using Kruskal Wallis and post-hoc Dunn tests. Wilcoxon tests were used to compare baseline viral load between two groups and Spearman correlation was performed to assess relationships between baseline continuous demographic, clinical, laboratory, and virology variables. Survival analysis was performed for binary outcome variables with censored time-to-event information. Patients were grouped into viral load tertiles using baseline measurements. Hazard ratios (HR) for middle and high viral load groups, relative to low, were calculated. Since all-cause mortality is a competing risk with other outcomes, subdistribution HR (sHR) were calculated.

Covariates for all analyses included age, sex, race, ethnicity, baseline steroid use, duration of pneumonia pre-baseline, body mass index (BMI), diabetes, and hypertension. Treatment arm was included as a covariate in all analyses of longitudinal outcomes. A Type-I error rate of 0·05 was used as the threshold for statistical significance, with Bonferroni adjustment for multiple comparisons. Analyses were conducted using R version 3·6·1. M a n u s c r i p t

The phase 2/3 study included 1912 randomized (not all treated) patients from 62 sites with COVID-19 pneumonia who either required supplemental oxygen, were admitted in the intensive care unit (ICU), were immunocompromised, or had evidence of multisystem organ dysfunction (MSOD). The post-hoc analysis included 1362 patients (70% of randomized) with baseline virology measurements (after enrollment but prior to dosing) and disease strata at randomization. Disease strata included hospitalized patients who were receiving low flow oxygen (severe), critically ill patients who were receiving high flow oxygen (critical without IMV) or mechanical ventilation (critical with IMV), and patients with MSOD receiving extracorporeal life support, renal replacement therapy, or vasopressors (MSOD). The phase 3 immunocompromised stratum (n = 36) and patients enrolled in phase 3 cohorts receiving 800 mg sarilumab or placebo (N = 78) were excluded (Supplementary Figure 1 ).

Baseline demographics and clinical variables were similar to the overall patient population (Table 1) , except for BMI (higher in the MSOD stratum). A minority of patients (27%) were receiving concomitant corticosteroids at randomization.

The median duration of COVID-19 pneumonia symptom prior to baseline was 8 days (IQR 5-12), median time between positive diagnosis and enrollment was 3 days (IQR 2-6), and median duration of hospitalization was 4 days (IQR 3-7). Medical history variables were well matched except for admission to ICU, baseline fever, obesity, and vasopressor use (Table 1) . Within the critical stratum, IMV patients had more vasopressor use (27% vs 1%) and fever (61% vs 40%) at baseline versus those not on IMV. More patients with MSOD were on vasopressors at baseline versus other disease strata.

Information regarding baseline viral load, serology, oxygen device, clinical outcomes, and symptom duration is summarized in Table 2 .

Supplemental oxygen devices varied across disease strata as defined in the protocol-based randomization. Patients in the severe stratum had low oxygen requirements, and a simple oxygen face mask was the predominant device (75%). Patients in the critical stratum not on IMV received oxygen primarily by a non-rebreather face mask (41%) and high-flow nasal cannula (39%). Most patients with MSOD were on IMV (87%). Mortality was higher in critical patients on IMV and patients with MSOD, consistent with the primary study results. M a n u s c r i p t Differences in clinical outcomes and symptom duration were observed across disease severity (Table 2 ). Our study cohort overall had 29% mortality and 42% percent of patients on IMV at baseline, suggesting a critically ill population. Greater disease severity in critical patients on IMV and patients with MSOD was associated with higher rates of all-cause mortality and lower rates of clinical status improvement ≥1 point, improvement in oxygenation, and hospital discharge. Among survivors, greater disease severity was also associated with more days with symptoms, including fever, tachypnoea, hypoxemia, and requiring supplemental oxygen.

Of 1362 patients analyzed, 208 (15%) had undetectable viral levels at baseline. Baseline viral load was significantly higher in critical patients on IMV and patients with MSOD, versus severe patients or critical patients not on IMV ( Figure 1A ). Seropositivity rates (qualitative) and SI (quantitative) did not differ between disease strata ( Table 2) .

Baseline viral load was significantly higher in patients aged ≥ 60 years (4·42 log 10 copies/mL [IQR 3·04-6·04]) versus patients aged < 60 years (3·93 log 10 copies/mL [IQR 2·55-5·28]; P < ·001). Viral loads were similar across other variables, including sex, obesity, diabetes, and hypertension status. Table 1 for results on relationships between viral, clinical and laboratory measures and cytokine and inflammatory marker profiling.

To assess whether baseline viral load was predictive of longitudinal outcomes (all-cause mortality, clinical score improvement ≥1 point, improvement in oxygenation, and hospital discharge), we analyzed the contribution of treatment allocation and found that it did not result in significantly different rates of these outcomes. Therefore, we combined all subjects and grouped into tertiles to assess the prognostic value of baseline viral load. In addition, 384 patients were selected randomly from the low and high tertiles and had serostatus and SI evaluated. Seropositivity significantly varied between the low (98%) and high (85%) tertiles. SI in the low tertile (128 [IQR 54-193]) was significantly higher versus the high tertile (28 [IQR 3-89]) (Supplemental Figure 2 , P < ·001).

Survival analysis was also performed for outcome variables with censored time-to-event information. Cumulative incidence plots are shown in Figure 2 , and event rates and HR are presented in Table 3 . The results of the analysis described below were not substantially different if subjects with undetectable baseline viral load (N = 208) were excluded from the analysis.

All-cause mortality. 401 patients (29%) died by day 60. Patients who died had 15 times greater baseline viral load (5·04 log 10 copies/mL [IQR 3·57-6·40]) than patients who survived (3·87 log 10 copies/mL [IQR 2·55-5·19]; Supplementary Figure 3 ; P < ·001). Patients with high viral load experienced significantly greater mortality rates (HR 2·42 [95% CI 1·89-3·11]; Figure 2A ; P < ·001). By day 60, mortality in the low, middle, and high viral load groups were 20%, 25%, and 43%, respectively. A 10-fold (+1 log 10 copies/mL) greater viral load at baseline was associated with 22% increased odds of all-cause mortality. SI was not significantly different between patients who survived or died (P = ·57), nor among the high tertile patients who survived (median 23 [IQR: 3-6]) M a n u s c r i p t versus patients who died (median 39 ); P = ·52. Higher SI was consistently associated with worse clinical outcomes among patients with low viral load. In the low viral load tertile, the SI was higher in patients who died 150 (104-238) versus survivors 112 (47-172); P = ·01.

Clinical status improvement ≥1 point. Since prolonged hospitalization in patients with COVID-19 could be associated with numerous factors, we explored if baseline viral load impacted longitudinal supplemental oxygen requirements as reflected in an improvement in clinical status ( Figure 2B ). Patients who achieved ≥ 1-point clinical status improvement (3·80 log 10 copies/mL [IQR 2·55-5·11]) had approximately 15-fold lower baseline viral load versus patients who did not achieve clinical status improvement (4·97 log 10 copies/mL [IQR 3·49-6·20]). A 10-fold (+1 log 10 copies/mL) greater viral load at baseline was associated with 22% decreased odds of 1-point clinical status improvement. Supplementary Figure 3 ; P < .001). Additionally, we observed an association between baseline oxygenation status and ≥1-point clinical status improvement (Supplementary Figure 4) .

Patients with high viral load showed lower rates of oxygenation improvement versus low viral load patients (HR 0·64 [95% CI 0·54-0·75]; P < ·001). Patients in the middle tertile did not experience significantly lower rates (HR 0·90 [95% CI 0·77-1·05]; P= ·19; Figure 2C ).

Hospital discharge. 729 patients (54%) were discharged, but rates were much lower in IMV (31%) versus non-IMV patients (71%; Figure 2D ). Compared with the low viral load tertile, patients in both the middle (HR 0·76 [95% CI 0·64-0·89]; P = .002) and high tertiles (HR 0·41 [95% CI 0·34-0·50]; P < .001) were less likely to be discharged. By day 29 in the study, just 38% of patients in the high tertile had been discharged versus 58% in the middle and 68% in the low tertiles. A 10-fold (+1 log 10 copies/mL) greater viral load at baseline was associated with 23% decreased odds of hospital discharge.

Longitudinal SARS-CoV-2 virology data were available for a limited subset of subjects (Supplementary Figure 5, Supplementary Tables 2 and 3) . No significant differences in change in viral load from baseline were observed between treatment arms on days 4 and 7. These findings are consistent with sarilumab not having a direct anti-viral mechanism.

Absolute levels and changes in of viral load at days 4 and 7 were predictive of all clinical outcomes assessed (Supplemental Table 4 ).

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The relationship between SARS-CoV-2 viral load in hospitalized patients with COVID-19 and outcomes remains a key focus of clinical research. Early studies focused on the development of robust assays that could detect viral transcripts in clinical specimens to accurately diagnose infection. Additional studies characterized the time course of viral shedding in various biological specimens to understand persistence and to guide public health measures. Subsequent studies evaluated the relationship of seroconversion to symptoms and to viral persistence [1] . Viral persistence is still observed in individuals who generate anti-SARS-CoV-2 antibodies; however, serum antibody-positive individuals in the outpatient setting had lower viral load than serum antibody-negative patients [6] .

In hospitalized patients with COVID-19, significant efforts have been made to understand clinical and laboratory predictors of outcomes, such as requirements for ventilation and mortality. One of the first studies to demonstrate the association of viral load and risk of intubation and mortality was in a cohort of 678 patients at 2 centers in New York City [8] . Retrospective subgrouping by viral Ct values determined upon hospital admission were similar to our study and demonstrated that patients with Ct < 25 (high viral load) had 35% mortality versus patients with medium (17.6%) or low (6.2%) viral load. Subsequent analysis corroborated the association of higher viral load and in-hospital mortality in patients with and without cancer [7] . In contrast to these studies, a study of 205 subjects concluded that viral load was not associated with requirements for oxygen or overall survival [11] . Thus, there still exists a clear need to study these same questions in data collected in a multicenter study. Viral load in our study was determined at baseline, (~1-7 days after diagnosis and 4-12 days after pneumonia). A small subset had undetectable virus at trial initiation which could be due to levels below the lower limit of detection, false negatives, virus clearance before randomization, or virus persistence in different biological compartments that were not sampled. These patients still required hospitalization and supplemental oxygen. High baseline viral load was associated with greater disease severity at randomization and was highest in the patients on mechanical ventilation and those requiring extracorporeal life support. Patients with the highest viral loads were less likely to reduce oxygen support, less likely to be discharged, and more likely to die from COVID-19. Some of the aspects of our study include the variability in duration of illness and confirmation of SARS-CoV-2 prior to enrollment. In contrast to prior cohorts, our study cohort had 43%, 25%, and 20% in the high, middle, and low viral load subgroups, respectively. This suggests our cohort may have been sicker even though the timeframe of the study enrollments were similar.

Most of the patient subsets tested for serology were seropositive for anti-nucleocapsid antibodies at baseline. SI at baseline was positively correlated with duration of pneumonia prior to baseline, which ranged from 0-41 days (median 9 days). In patients enrolled closer to symptom onset, SI and seroconversion rates were significantly lower in patients with high viral load. This study did not evaluate neutralizing antibodies, which limits the interpretation of the serology status in this cohort, but the high viral load in hospitalized patients despite a seropositivity rate of > 80% suggests these antibodies are inadequate to control viral replication in this patient population. M a n u s c r i p t Limited longitudinal assessments were conducted to support conclusions about viral persistence and evaluate the contribution of anti-IL-6R blockade with sarilumab on viral load over time These data confirm that sarilumab did not have a direct anti-viral effect. Change in viral load at days 4 and 7 was predictive of clinical outcomes, with greater viral reductions observed in patients who survived and improved clinically.

There were several limitations in this study. Only 71% of randomized patients had available viral load for this analysis, however, baseline characteristics were similar between the overall study population and the subgroup included here. Serology testing was not available on all patients with virology, therefore, correlating serology status and viral load and clinical outcomes was challenging. However, most patients were serum antibody positive, despite having high viral loads and severe COVID-19, suggesting that serology testing may not be an ideal prognostic marker for disease progression. In addition, our study did not evaluate the viral variants with which patients were infected between March to July 2020 enrolled in this study. Despite this, our study encompasses centralized viral load measurements and standardized collection of clinical outcomes from a large multi-center trial providing a robust dataset to better understand the relationship between viral load and COVID-19 progression.

These analyses demonstrated that baseline viral load may be an important determinant of clinical outcomes in hospitalized patients with COVID-19. Recently, a phase 3 trial with REGEN-COV, a monoclonal antibody cocktail for the treatment of high-risk outpatients with COVID-19, demonstrated a significant reduction in viral load and COVID-19-related hospitalizations and death versus placebo, further supporting viral load in COVID-19 disease progression. M a n u s c r i p t Notes Acknowledgements. This study was supported by Sanofi and Regeneron Pharmaceuticals, Inc. The authors thank the patients, their families, and investigational site members. The authors wish to thank Georgia Bellingham and Lisa Boersma for operational support for virology testing. Manuscript support was provided by Prime Global. Contributors. AB, MFW, PJE, SCH contributed to the study design, analysis plan, implementation of the research. AB, MFW, PJE authored the manuscript. AB, EK, JF contributed to sample preparation and laboratory testing. AB, SS, DL, AW and RB contributed to the primary data acquisition and data cleaning. All authors participated in data analysis and interpretation as well as manuscript review and editing. PJE, AW and SCH had access to all data and verified the data and statistical analysis.

Data sharing. Qualified researchers may request access to study documents (including the clinical study report, study protocol with any amendments, blank case report form, statistical analysis plan) that support the methods and findings reported in this manuscript. Individual anonymized participant data will be considered for sharing once the indication has been approved by a regulatory body, if there is legal authority to share the data and there is not a reasonable likelihood of participant re-identification. Submit requests to https://vivli.org/. M a n u s c r i p t M a n u s c r i p t 

Virological assessment of hospitalized patients with COVID-2019

Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province, China

SARS-CoV-2 viral load is associated with increased disease severity and mortality

Association of SARS-CoV-2 Genomic Load with Outcomes in Patients with COVID-19

Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trial

REGN-COV2, a neutralizing antibody cocktail, in outpatients with Covid-19

SARS-CoV-2 Viral Load Predicts Mortality in Patients with and without Cancer Who Are Hospitalized with COVID-19

Impact of SARS-CoV-2 viral load on risk of intubation and mortality among hospitalized patients with coronavirus disease 2019

A Randomized Placebo-Controlled Trial of Sarilumab in Hospitalized Patients with Covid-19. medRxiv

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 Data are presented as n (%), mean (SD), or median (Q1-Q3).Demographic and medical history information from patients included in the analysis.Abbreviations: BMI, body-mass index; ICU, intensive care unit; IMV, invasive mechanical ventilation; MSOD,

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Hypoxemia (IQR) 6 (4-10) 11 (7- A c c e p t e d M a n u s c r i p t Data are presented as n (%), event rates (95% CI), or hazard ratios (95% CI).The sample was split into equal tertiles using baseline viral copies/mL. Survival analysis was performed for longitudinal outcomes in middle and high viral load groups relative to the low viral load group. Since all-cause mortality is a competing risk with the other three outcomes, sHRs were calculated. The variables in the covariateadjusted models include age, sex, race, ethnicity, steroid use, duration of pneumonia prior to baseline, bodymass index, diabetes, hypertension, and treatment arm.Abbreviation: sHR, subdistribution hazard ratio. A c c e p t e d M a n u s c r i p t Viral load tertiles were defined as follows: low (<3·32 log 10 copies/mL), middle (3·32-5·09 log 10 copies/mL), and high (>5·09 log 10 copies/mL). Tables of number of patients at risk at particular timepoints after baseline are shown below each plot.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