Viral RNA load in plasma is associated with critical illness and a dysregulated host response in COVID-19

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Abstract

Background

COVID-19 can course with respiratory and extrapulmonary disease. SARS-CoV-2 RNA is detected in respiratory samples but also in blood, stool and urine. Severe COVID-19 is characterized by a dysregulated host response to this virus. We studied whether viral RNAemia or viral RNA load in plasma is associated with severe COVID-19 and also to this dysregulated response.

Methods

A total of 250 patients with COVID-19 were recruited (50 outpatients, 100 hospitalized ward patients and 100 critically ill). Viral RNA detection and quantification in plasma was performed using droplet digital PCR, targeting the N1 and N2 regions of the SARS-CoV-2 nucleoprotein gene. The association between SARS-CoV-2 RNAemia and viral RNA load in plasma with severity was evaluated by multivariate logistic regression. Correlations between viral RNA load and biomarkers evidencing dysregulation of host response were evaluated by calculating the Spearman correlation coefficients.

Results

The frequency of viral RNAemia was higher in the critically ill patients (78%) compared to ward patients (27%) and outpatients (2%) ( p < 0.001). Critical patients had higher viral RNA loads in plasma than non-critically ill patients, with non-survivors showing the highest values. When outpatients and ward patients were compared, viral RNAemia did not show significant associations in the multivariate analysis. In contrast, when ward patients were compared with ICU patients, both viral RNAemia and viral RNA load in plasma were associated with critical illness (OR [CI 95%], p): RNAemia (3.92 [1.183–12.968], 0.025), viral RNA load (N1) (1.962 [1.244–3.096], 0.004); viral RNA load (N2) (2.229 [1.382–3.595], 0.001). Viral RNA load in plasma correlated with higher levels of chemokines (CXCL10, CCL2), biomarkers indicative of a systemic inflammatory response (IL-6, CRP, ferritin), activation of NK cells (IL-15), endothelial dysfunction (VCAM-1, angiopoietin-2, ICAM-1), coagulation activation (D-Dimer and INR), tissue damage (LDH, GPT), neutrophil response (neutrophils counts, myeloperoxidase, GM-CSF) and immunodepression (PD-L1, IL-10, lymphopenia and monocytopenia).

Conclusions

SARS-CoV-2 RNAemia and viral RNA load in plasma are associated with critical illness in COVID-19. Viral RNA load in plasma correlates with key signatures of dysregulated host responses, suggesting a major role of uncontrolled viral replication in the pathogenesis of this disease.

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Most cited references 28

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Clinical Characteristics of Coronavirus Disease 2019 in China

Wei-jie Guan, Zheng-yi Ni, Yu Hu … (2020)

Abstract Background Since December 2019, when coronavirus disease 2019 (Covid-19) emerged in Wuhan city and rapidly spread throughout China, data have been needed on the clinical characteristics of the affected patients. Methods We extracted data regarding 1099 patients with laboratory-confirmed Covid-19 from 552 hospitals in 30 provinces, autonomous regions, and municipalities in mainland China through January 29, 2020. The primary composite end point was admission to an intensive care unit (ICU), the use of mechanical ventilation, or death. Results The median age of the patients was 47 years; 41.9% of the patients were female. The primary composite end point occurred in 67 patients (6.1%), including 5.0% who were admitted to the ICU, 2.3% who underwent invasive mechanical ventilation, and 1.4% who died. Only 1.9% of the patients had a history of direct contact with wildlife. Among nonresidents of Wuhan, 72.3% had contact with residents of Wuhan, including 31.3% who had visited the city. The most common symptoms were fever (43.8% on admission and 88.7% during hospitalization) and cough (67.8%). Diarrhea was uncommon (3.8%). The median incubation period was 4 days (interquartile range, 2 to 7). On admission, ground-glass opacity was the most common radiologic finding on chest computed tomography (CT) (56.4%). No radiographic or CT abnormality was found in 157 of 877 patients (17.9%) with nonsevere disease and in 5 of 173 patients (2.9%) with severe disease. Lymphocytopenia was present in 83.2% of the patients on admission. Conclusions During the first 2 months of the current outbreak, Covid-19 spread rapidly throughout China and caused varying degrees of illness. Patients often presented without fever, and many did not have abnormal radiologic findings. (Funded by the National Health Commission of China and others.)

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Endothelial cell infection and endotheliitis in COVID-19

Zsuzsanna Varga, Andreas J. Flammer, Peter Steiger … (2020)

Cardiovascular complications are rapidly emerging as a key threat in coronavirus disease 2019 (COVID-19) in addition to respiratory disease. The mechanisms underlying the disproportionate effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on patients with cardiovascular comorbidities, however, remain incompletely understood.1, 2 SARS-CoV-2 infects the host using the angiotensin converting enzyme 2 (ACE2) receptor, which is expressed in several organs, including the lung, heart, kidney, and intestine. ACE2 receptors are also expressed by endothelial cells. 3 Whether vascular derangements in COVID-19 are due to endothelial cell involvement by the virus is currently unknown. Intriguingly, SARS-CoV-2 can directly infect engineered human blood vessel organoids in vitro. 4 Here we demonstrate endothelial cell involvement across vascular beds of different organs in a series of patients with COVID-19 (further case details are provided in the appendix). Patient 1 was a male renal transplant recipient, aged 71 years, with coronary artery disease and arterial hypertension. The patient's condition deteriorated following COVID-19 diagnosis, and he required mechanical ventilation. Multisystem organ failure occurred, and the patient died on day 8. Post-mortem analysis of the transplanted kidney by electron microscopy revealed viral inclusion structures in endothelial cells (figure A, B ). In histological analyses, we found an accumulation of inflammatory cells associated with endothelium, as well as apoptotic bodies, in the heart, the small bowel (figure C) and lung (figure D). An accumulation of mononuclear cells was found in the lung, and most small lung vessels appeared congested. Figure Pathology of endothelial cell dysfunction in COVID-19 (A, B) Electron microscopy of kidney tissue shows viral inclusion bodies in a peritubular space and viral particles in endothelial cells of the glomerular capillary loops. Aggregates of viral particles (arrow) appear with dense circular surface and lucid centre. The asterisk in panel B marks peritubular space consistent with capillary containing viral particles. The inset in panel B shows the glomerular basement membrane with endothelial cell and a viral particle (arrow; about 150 nm in diameter). (C) Small bowel resection specimen of patient 3, stained with haematoxylin and eosin. Arrows point to dominant mononuclear cell infiltrates within the intima along the lumen of many vessels. The inset of panel C shows an immunohistochemical staining of caspase 3 in small bowel specimens from serial section of tissue described in panel D. Staining patterns were consistent with apoptosis of endothelial cells and mononuclear cells observed in the haematoxylin-eosin-stained sections, indicating that apoptosis is induced in a substantial proportion of these cells. (D) Post-mortem lung specimen stained with haematoxylin and eosin showed thickened lung septa, including a large arterial vessel with mononuclear and neutrophilic infiltration (arrow in upper inset). The lower inset shows an immunohistochemical staining of caspase 3 on the same lung specimen; these staining patterns were consistent with apoptosis of endothelial cells and mononuclear cells observed in the haematoxylin-eosin-stained sections. COVID-19=coronavirus disease 2019. Patient 2 was a woman, aged 58 years, with diabetes, arterial hypertension, and obesity. She developed progressive respiratory failure due to COVID-19 and subsequently developed multi-organ failure and needed renal replacement therapy. On day 16, mesenteric ischaemia prompted removal of necrotic small intestine. Circulatory failure occurred in the setting of right heart failure consequent to an ST-segment elevation myocardial infarction, and cardiac arrest resulted in death. Post-mortem histology revealed lymphocytic endotheliitis in lung, heart, kidney, and liver as well as liver cell necrosis. We found histological evidence of myocardial infarction but no sign of lymphocytic myocarditis. Histology of the small intestine showed endotheliitis (endothelialitis) of the submucosal vessels. Patient 3 was a man, aged 69 years, with hypertension who developed respiratory failure as a result of COVID-19 and required mechanical ventilation. Echocardiography showed reduced left ventricular ejection fraction. Circulatory collapse ensued with mesenteric ischaemia, and small intestine resection was performed, but the patient survived. Histology of the small intestine resection revealed prominent endotheliitis of the submucosal vessels and apoptotic bodies (figure C). We found evidence of direct viral infection of the endothelial cell and diffuse endothelial inflammation. Although the virus uses ACE2 receptor expressed by pneumocytes in the epithelial alveolar lining to infect the host, thereby causing lung injury, the ACE2 receptor is also widely expressed on endothelial cells, which traverse multiple organs. 3 Recruitment of immune cells, either by direct viral infection of the endothelium or immune-mediated, can result in widespread endothelial dysfunction associated with apoptosis (figure D). The vascular endothelium is an active paracrine, endocrine, and autocrine organ that is indispensable for the regulation of vascular tone and the maintenance of vascular homoeostasis. 5 Endothelial dysfunction is a principal determinant of microvascular dysfunction by shifting the vascular equilibrium towards more vasoconstriction with subsequent organ ischaemia, inflammation with associated tissue oedema, and a pro-coagulant state. 6 Our findings show the presence of viral elements within endothelial cells and an accumulation of inflammatory cells, with evidence of endothelial and inflammatory cell death. These findings suggest that SARS-CoV-2 infection facilitates the induction of endotheliitis in several organs as a direct consequence of viral involvement (as noted with presence of viral bodies) and of the host inflammatory response. In addition, induction of apoptosis and pyroptosis might have an important role in endothelial cell injury in patients with COVID-19. COVID-19-endotheliitis could explain the systemic impaired microcirculatory function in different vascular beds and their clinical sequelae in patients with COVID-19. This hypothesis provides a rationale for therapies to stabilise the endothelium while tackling viral replication, particularly with anti-inflammatory anti-cytokine drugs, ACE inhibitors, and statins.7, 8, 9, 10, 11 This strategy could be particularly relevant for vulnerable patients with pre-existing endothelial dysfunction, which is associated with male sex, smoking, hypertension, diabetes, obesity, and established cardiovascular disease, all of which are associated with adverse outcomes in COVID-19.

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Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes

Wei Zhang, Rong-Hui Du, Bei Li … (2020)

ABSTRACT In December 2019, a novel coronavirus (2019-nCoV) caused an outbreak in Wuhan, China, and soon spread to other parts of the world. It was believed that 2019-nCoV was transmitted through respiratory tract and then induced pneumonia, thus molecular diagnosis based on oral swabs was used for confirmation of this disease. Likewise, patient will be released upon two times of negative detection from oral swabs. However, many coronaviruses can also be transmitted through oral–fecal route by infecting intestines. Whether 2019-nCoV infected patients also carry virus in other organs like intestine need to be tested. We conducted investigation on patients in a local hospital who were infected with this virus. We found the presence of 2019-nCoV in anal swabs and blood as well, and more anal swab positives than oral swab positives in a later stage of infection, suggesting shedding and thereby transmitted through oral–fecal route. We also showed serology test can improve detection positive rate thus should be used in future epidemiology. Our report provides a cautionary warning that 2019-nCoV may be shed through multiple routes.

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Author and article information

Contributors

David J. Kelvin: dkelvin@jidc.org

Journal

Journal ID (nlm-ta): Crit Care

Title: Critical Care

Publisher: BioMed Central (London )

ISSN (Print): 1364-8535

ISSN (Electronic): 1466-609X

Publication date (Electronic): 14 December 2020

Publication date PMC-release: 14 December 2020

Publication date Collection: 2020

Volume: 24

Electronic Location Identifier: 691

Affiliations

[1 ]GRID grid.452531.4, Group for Biomedical Research in Sepsis (BioSepsis), , Instituto de Investigación Biomédica de Salamanca, (IBSAL), ; Paseo de San Vicente, 58-182, 37007 Salamanca, Spain

[2 ]GRID grid.411280.e, ISNI 0000 0001 1842 3755, Hospital Universitario Río Hortega, ; Calle Dulzaina, 2, 47012 Valladolid, Spain

[3 ]GRID grid.413448.e, ISNI 0000 0000 9314 1427, Centro de Investigación Biomédica en Red en Enfermedades Respiratorias (CIBERES), , Instituto de Salud Carlos III, ; Av. de Monforte de Lemos, 3-5, 28029 Madrid, Spain

[4 ]GRID grid.410526.4, ISNI 0000 0001 0277 7938, Department of Laboratory Medicine, , Hospital General Universitario Gregorio Marañón, ; Calle del Dr. Esquerdo, 46, 28007 Madrid, Spain

[5 ]GRID grid.4795.f, ISNI 0000 0001 2157 7667, Department of Medicine, Faculty of Medicine, , Universidad Complutense de Madrid, ; Plaza de Ramón y Cajal, s/n, 28040 Madrid, Spain

[6 ]GRID grid.410526.4, ISNI 0000 0001 0277 7938, Emergency Department, , Hospital General Universitario Gregorio Marañón, ; Calle del Dr. Esquerdo, 46, 28007 Madrid, Spain

[7 ]GRID grid.411280.e, ISNI 0000 0001 1842 3755, Microbiology Service, , Hospital Universitario Rio Hortega, ; Calle Dulzaina, 2, 47012 Valladolid, Spain

[8 ]GRID grid.413448.e, ISNI 0000 0000 9314 1427, Viral Infection and Immunity Unit, Centro Nacional de Microbiología, , Instituto de Salud Carlos III, ; Ctra. de Pozuelo, 28, 28222 Majadahonda, Spain

[9 ]GRID grid.414761.1, Hospital Universitario Infanta Leonor, ; Av. Gran Vía del Este, 80, 28031 Madrid, Spain

[10 ]GRID grid.411336.2, ISNI 0000 0004 1765 5855, Servicio de Microbiología Clínica, , Hospital Universitario Príncipe de Asturias, ; Carr. de Alcalá, s/n, 28805 Madrid, Spain

[11 ]GRID grid.411280.e, ISNI 0000 0001 1842 3755, Intensive Care Unit, , Hospital Universitario Rio Hortega, ; Calle Dulzaina, 2, 47012 Valladolid, Spain

[12 ]GRID grid.411280.e, ISNI 0000 0001 1842 3755, Emergency Department, , Hospital Universitario Rio Hortega, ; Calle Dulzaina, 2, 47012 Valladolid, Spain

[13 ]GRID grid.411057.6, ISNI 0000 0000 9274 367X, Intensive Care Unit, , Hospital Clínico Universitario de Valladolid, ; Av. Ramón y Cajal, 47003 Valladolid, Spain

[14 ]GRID grid.5239.d, ISNI 0000 0001 2286 5329, Department of Anesthesiology, , Facultad de Medicina de Valladolid, ; Av. Ramón y Cajal, 47005 Valladolid, Spain

[15 ]GRID grid.411280.e, ISNI 0000 0001 1842 3755, Anesthesiology and Reanimation Service, , Hospital Universitario Rio Hortega, ; Calle Dulzaina, 2, 47012 Valladolid, Spain

[16 ]GRID grid.410526.4, ISNI 0000 0001 0277 7938, Intensive Care Unit, , Hospital General Universitario Gregorio Marañón, ; Calle del Dr. Esquerdo, 46, 28007 Madrid, Spain

[17 ]GRID grid.411057.6, ISNI 0000 0000 9274 367X, Microbiology Service, , Hospital Clinico Universitario de Valladolid, ; Av. Ramón y Cajal, 47003 Valladolid, Spain

[18 ]GRID grid.84393.35, ISNI 0000 0001 0360 9602, Pulmonology Service, , Hospital Universitario y Politécnico de La Fe, ; Avinguda de Fernando Abril Martorell, 106, 46026 Valencia, Spain

[19 ]GRID grid.411969.2, ISNI 0000 0000 9516 4411, Clinical Microbiology Department, , Complejo Asistencial Universitario de León, ; Calle Altos de Nava, s/n, 24001 León, Spain

[20 ]GRID grid.459669.1, Microbiology Service, , Hospital Universitario de Burgos, ; Av. Islas Baleares, 3, 09006 Burgos, Spain

[21 ]GRID grid.411969.2, ISNI 0000 0000 9516 4411, Intensive Care Unit, , Complejo Asistencial Universitario de León, ; Calle Altos de nava, s/n, 24001 León, Spain

[22 ]GRID grid.5239.d, ISNI 0000 0001 2286 5329, Pneumology Service, Hospital Universitario Río Hortega/Biomedical Engineering Group, , Universidad de Valladolid, ; Calle Dulzaina, 2, 47012 Valladolid, Spain

[23 ]GRID grid.459669.1, Intensive Care Unit, , Hospital Universitario de Burgos, ; Av. Islas Baleares, 3, 09006 Burgos, Spain

[24 ]GRID grid.5239.d, ISNI 0000 0001 2286 5329, Clinical Analysis Service. Hospital, , Clínico Universitario de Valladolid, ; Av. Ramón y Cajal, 47003 Valladolid, Spain

[25 ]GRID grid.55602.34, ISNI 0000 0004 1936 8200, Department of Microbiology and Immunology, Faculty of Medicine, Canadian Center for Vaccinology CCfV, , Dalhousie University, ; Halifax, NS B3H 4R2 Canada

[26 ]GRID grid.411679.c, ISNI 0000 0004 0605 3373, Laboratory of Immunity, , Shantou University Medical College, ; 22 Xinling Rd., Jinping, Shantou, Guangdong China

[27 ]GRID grid.411057.6, ISNI 0000 0000 9274 367X, Department of Cardiovascular Surgery, , Hospital Clínico Universitario de Valladolid, ; Av. Ramón y Cajal, 47003 Valladolid, Spain

[28 ]GRID grid.411280.e, ISNI 0000 0001 1842 3755, Infectious Diseases Clinic, Internal Medicine Department, , Hospital Universitario Río Hortega, ; Calle Dulzaina, 2, 47012 Valladolid, Spain

[29 ]GRID grid.5841.8, ISNI 0000 0004 1937 0247, Department of Pulmonology, Hospital Clinic de Barcelona, Institut D Investigacions August Pi I Sunyer (IDIBAPS), , Universidad de Barcelona, ; Carrer del Rosselló, 149, 08036 Barcelona, Spain

[30 ]GRID grid.430994.3, ISNI 0000 0004 1763 0287, Intensive Care Department, Vall d’Hebron Hospital Universitari, SODIR Research Group, , Vall d’Hebron Institut de Recerca, ; Passeig de la Vall d’Hebron, 119, 08035 Barcelona, Spain

[31 ]Respiratory Department, Institut Ricerca Biomedica de Lleida, Av. Alcalde Rovira Roure, 80, 25198 Lleida, Spain

Article

Publisher ID: 3398

DOI: 10.1186/s13054-020-03398-0

PMC ID: 7734467

PubMed ID: 33317616

SO-VID: 8537e3a8-f765-4536-a0f0-bd45697b7e9a

License:

Open AccessThis 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://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

History

Date received : 3 September 2020

Date accepted : 18 November 2020

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100000024, Canadian Institutes of Health Research;

Award ID: CIHR OV2 – 170357

Award Recipient : Ali Toloue Ostadgavahi

Funded by: FundRef http://dx.doi.org/10.13039/501100004587, Instituto de Salud Carlos III;

Award ID: COV20/00110

Award ID: CD018/0123

Award Recipient : Ana P. Tedim

Funded by: Gerencia Regional de Salud de Castilla y León

Award ID: GRS COVID 53/A/20

Custom metadata

ScienceOpen disciplines: Emergency medicine & Trauma

Keywords: sars-cov-2,cytokine,sepsis,covid-19,plasma,rnaemia,viral rna load,icu

Data availability:

ScienceOpen disciplines: Emergency medicine & Trauma

Keywords: sars-cov-2, cytokine, sepsis, covid-19, plasma, rnaemia, viral rna load, icu

Viral RNA load in plasma is associated with critical illness and a dysregulated host response in COVID-19

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Abstract

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Results

Conclusions

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Novel Coronavirus Disease COVID-19

Most cited references 28

Clinical Characteristics of Coronavirus Disease 2019 in China

Endothelial cell infection and endotheliitis in COVID-19

Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes

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