key: cord-0739207-xklgzm52
authors: Soria, M. E.; Corton, M.; Martinez-Gonzalez, B.; Lobo-Vega, R.; Vazquez-Sirvent, L.; Lopez-Rodriguez, R.; Almoguera, B.; Mahillo, I.; Minguez, P.; Herrero, A.; Taracido, J. C.; Macias-Valcayo, A.; Esteban, J.; Fernandez, R.; Gadea, I.; Ruiz-Hornillos, J.; Ayuso, C.; Perales, C.
title: High SARS-CoV-2 viral load is associated with a worse clinical outcome of COVID-19 disease
date: 2020-11-16
journal: nan
DOI: 10.1101/2020.11.13.20229666
sha: 3459af4e03cb72253bcdb284aff960f7dd254afe
doc_id: 739207
cord_uid: xklgzm52

COVID-19 severity and progression are determined by several host and virological factors that may influence the final outcome of SARS-CoV-2-infected patients. The objective of this work is to determine a possible association between the viral load, obtained from nasopharyngeal swabs, and the severity of the infection in a cohort of 448 SARS-CoV-2-infected patients from a hospital in Madrid during the first outbreak of the pandemic in Spain. To perform this, we have clinically classified patients as mild, moderate and severe COVID-19 according to a number of clinical parameters such as hospitalization requirement, need of oxygen therapy, admission to intensive care units and/or exitus. Here we report a statistically significant correlation between viral load and disease severity, being high viral load associated with worse clinical prognosis, independently of several previously identified risk factors such as age, sex, hypertension, cardiovascular disease, diabetes, obesity, and lung disease (asthma and chronic obstructive pulmonary disease). The data presented here reinforce the viral load as a potential biomarker for predicting disease severity in SARS-CoV-2-infected patients. It is also an important parameter in viral evolution since it relates to the numbers and types of variant genomes present in a viral population, a potential determinant of disease progression.

Coronavirus SARS-CoV-2 emerged in the human population in 2019 is the causal agent of the new pandemic disease COVID-19 (1) . The virus has spread rapidly worldwide, and at the time of this writing there are 46,403,652 confirmed cases, and 1, 198 ,569 deaths in 219 countries worldwide, according to the WHO (https://covid19.who.int/); these numbers are increasing daily. Evolution of a virus in a specific host is defined by a number of closely related parameters such as viral load, replication rate, genetic heterogeneity and viral fitness that may influence virus adaptability, viral pathogenesis and disease progression (2, 3) . The replicative capacity of a virus is clinically relevant because it largely determines the viral load in infected individuals, and viral load influences disease manifestations (4) . In the case of SARS-CoV-2, some studies have correlated viral load with disease severity whereas in others this correlation was not clear. A positive correlation was reported in a cohort of SARS-CoV-2-infected patients from China, showing that the viral load detected in the respiratory tract was positively linked to lung disease severity (5) . In a related study, the . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 16, 2020. ; https://doi.org/10.1101/2020.11.13.20229666 doi: medRxiv preprint analysis of the viral RNA level in upper respiratory tract samples from 76 patients with COVID-19 revealed significantly lower Ct values (cycle threshold which is inversely correlated with viral RNA level), and longer virus-shedding periods in those patients classified as severe, as compared with those that exhibited mild disease (6) .

Additionally, a prospective study in a large hospitalized cohort of 1,145 infected patients documented a significant lower probability of survival in patients with high viral load than in those with low viral load (7) . In contrast, in other studies, it was observed that the diagnostic viral load level was lower in hospitalized than in nonhospitalized patients, resulting in a lack of correlation of viral load with admission to intensive care unit (ICU), length of oxygen support, and overall patient survival (8) .

Thus, the dynamics of viral load and its connection with different clinical parameters remain poorly characterized, and large studies with additional cohorts worldwide are needed to define the possible association and the predictive value of the viral load regarding disease progression and mortality. Viral RNA load calculated from nasopharyngeal swabs might be added to other predictive parameters to complete an early risk stratification of COVID-19 patients (9).

Given the disparate results trying to correlate viral load with COVID-19 disease is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 16, 2020. ; https://doi.org/10.1101/2020.11.13.20229666 doi: medRxiv preprint a measurement of viral load) in SARS-CoV-2-infected patients correlates positively with the disease progression and poor prognosis.

Regarding host factors, it has been established that age greater than 65 is a risk factor to develop acute respiratory distress syndrome (ARDS), a major complication of COVID-19 pneumonia, and that the risk of death increases with advanced age (10) . As a second factor, disease severity and mortality for males is significantly higher than for females, being older men the population most at risk (11, 12) . Additionally, several comorbidities have been potentially associated with poor outcome including hypertension (high blood pressure), cardiovascular disease, diabetes, obesity, and lung disease (asthma, EPOC) (13) . We have included these risk factor data in our patient cohort to assess their alignment with our disease severity-viral RNA load correlation.

Interestingly, a significant association between viral load and infection severity was still observed after adjusting for age, sex, hypertension, cardiovascular disease, diabetes, obesity, asthma and chronic obstructive pulmonary disease (COPD) (see ANCOVA tests in Table 1 ). The significant difference in viral load between the three groups was not attributable to the percentage of hospitalization, the percentage of ICU admission or the percentage of the different types of oxigenotherapy (see ANCOVA tests in Table 1 ).

Thus, our results with a SARS-CoV-2 population from a cohort which is different from those in previous studies on viral load influence on disease, suggest a positive correlation between viral load and COVID-19 disease severity. This conclusion is also in agreement with two studies showing a longer persistence of high viral load viruses in respiratory samples of patients with severe disease than those with mild disease, suggesting that the viral load may be a prognostic parameter (14, 15) .

To monitor the average number of days between the symptom onset and the sample extraction in our cohort, we collected these data for 102 patients. On average NP swabs were obtained at 7.84 ± 6.40 days after symptoms onset, which is within the time interval of active infection. It has been described that the highest SARS-CoV-2 viral load in throat swabs ─ and consequently the highest transmissibility peaks─ is around 5-6 days after the symptom onset (16) , but this is an average that depends on each patient; the transmissibility window extends from a few days before symptom onset up to 30 days in patients with severe disease (14, 17) .

A correlation between high viral load and disease severity has been also found in other viral infections. Among children naturally infected with respiratory syncytial virus (RSV), increased viral load was associated with clinical severity of disease defined as . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 16, 2020. an increased risk for intensive care, prolonged hospitalization, or the development of respiratory failure (18) . Examination of hepatitis A virus (HAV) RNA from sera by real-time PCR resulted in higher initial viral load in patients with severe outcomes such as fulminant hepatitis and severe acute hepatitis than in patients with less severe infection (19) . Viral load in patients infected with the pandemic type A influenza virus H1N1 (2009) who suffered pneumonia was higher than in patients with milder disease (those with bronchitis or upper respiratory tract infection), suggesting that the viral load is also an important predictive value in influenza infection (20) . Other instances of a connection of viral load with disease progression have been reviewed (2) .

In terms of viral quasispecies dynamics, a key question regarding clinical implications is that a population with a large viral load will contain a broader mutant repertoire than a population with low viral load. Even if each individual mutant might be present at the same frequency in both populations (as it corresponds to mutant frequency being an "intrinsic" property), the mutant repertoire is an "extrinsic" property of the population, a key distinction that has been numerically studied (21) . Thus, viral load is a potential source of variants that enhance the probability of infection to alternative cell types in the course of infection which is increasingly viewed as a stepwise adaptation process (2) (3) (4) 21) .

Data collected included patient demographics, risk factors for SARS-CoV-2 disease and clinical information related to the time of SARS-CoV-2 diagnosis (Table 1) .

Patients were classified according to the following COVID-19-associated parameters:

(1) need of hospital admission, (2) need for mechanical ventilation, (3) admission to the ICU, and (4) exitus attributed to COVID-19. Patients were classified as mild, moderate and severe cases according to the requirement and the type of hospitalization: (1) mild symptoms (neither hospital admission nor ICU) (n=110), (2) moderate symptoms (hospitalization without ICU) (n=236), and (3) severe symptoms (hospitalization with admission to the ICU, and/or exitus) (n=102). Exceptions to these criteria are detailed in Table 1 . The clinical relevance was defined before the data analysis was performed.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 16, 2020. ; https://doi.org/10.1101/2020.11.13.20229666 doi: medRxiv preprint

Nasopharyngeal swabs were collected at FJD hospital by trained medical personnel from all patients included in the study due to suspected COVID-19 infection.

After collection, the NP samples were transferred to viral transport media and transported to the Microbiology Department for molecular testing. RT-PCR to obtain diagnostic SARS-CoV-2 viral load was performed using the kit VIASURE Real Time PCR Detection Kits by CerTest BIOTEC following the manufacturer instructions. The Ct values were calculated using SARS-CoV-2-specific oligonucleotides directed to the ORF1ab.

The statistically significance of differences among viral load values according to the infection severity was calculated with the ANOVA test and t-test with Bonferroni correction using GraphPad Prism 7.00. The association between viral load and disease severity adjusted by risk factors was calculated with ANCOVA test using software R version 4.0.2.

All samples were collected according to WHO guidelines. This study was approved by 

We acknowledge all people in the Clinical Microbiology Department of the FJD for helping with the sample and data collection. We thank all health-care professionals who attended COVID-19 patients and collected the clinical samples that were included in this study in a difficult moment of the COVID-19 epidemic in Spain. We thank José . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 16, 2020. ; https://doi.org/10.1101/2020.11.13.20229666 doi: medRxiv preprint María Aguado and Octavio Carretero for their support in the whole project. We are indebted to E. Domingo for encouragement and critical reading of the manuscript, and to Nuria Verdaguer and Enrique Marcos for valuable discussions about SARS-CoV-2 scientific findings. This work was supported by Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (COVID-19 Research Call COV20/00181)co financed by European Development Regional Fund "A way to achieve Europe".

The work was also supported by grants CSIC-COV19-014 from Consejo Superior de . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 16, 2020. ; https://doi.org/10.1101/2020.11.13.20229666 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 16, 2020. ; https://doi.org/10.1101/2020.11.13.20229666 doi: medRxiv preprint

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Viral quasispecies evolution

Viral quasispecies

Virus as Populations

Clinical and biochemical indexes from 2019-nCoV infected patients linked to viral loads and lung injury

Viral dynamics in mild and severe cases of COVID-19

SARS-CoV-2 viral load predicts COVID-19 mortality. The Lancet Respiratory medicine

Association of initial viral load in SARS-CoV-2 patients with outcome and symtoms

IL-6-based mortality risk model for hospitalized patients with COVID-19

Clinical, molecular, and epidemiological characterization of the SARS-CoV-2 virus and the Coronavirus Disease 2019 (COVID-19), a comprehensive literature review. Diagnostic Microbiology and Infectious Disease

New Insights of Emerging SARS-CoV-2: Epidemiology, Etiology, Clinical Features, Clinical Treatment, and Prevention

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Clinical course and predictors of 60-day mortality in 239 critically ill patients with COVID-19: a multicenter retrospective study from Wuhan

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

Longitudinal analyses reveal immunological misfiring in severe COVID-19

Temporal dynamics in viral shedding and transmissibility of COVID-19

Viral load of SARS-CoV-2 in clinical samples

Respiratory syncytial virus load, viral dynamics, and disease severity in previously healthy naturally infected children

Hepatitis A viral load in relation to severity of the infection

Correlation of pandemic (H1N1) 2009 viral load with disease severity and prolonged viral shedding in children

From quasispecies theory to viral quasispecies: how complexity has permeated virology