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Prevalence of amyloid blood clots in COVID-19 plasma

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The rapid detection of COVID-19 uses genotypic testing for the presence of SARS-Cov-2 virus in nasopharyngeal swabs, but it can have a poor sensitivity. A rapid, host-based physiological test that indicated whether the individual was infected or not would be highly desirable. Coagulaopathies are a common accompaniment to COVID-19, especially micro-clots within the lungs. We show here that microclots can be detected in the native plasma of COVID-19 patient, and in particular that such clots are amyloid in nature as judged by a standard fluorogenic stain. This provides a rapid and convenient test (P<0.0001), and suggests that the early detection and prevention of such clotting could have an important role in therapy.
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1
Prevalence of amyloid blood clots in COVID-19 plasma
Etheresia Pretorius1* Chantelle Venter1, Gert Jacobus Laubscher2, Petrus Johannes
Lourens2 , Janami Steenkamp2, Douglas B Kell1,4,5*,
1* Department of Physiological Sciences, Faculty of Science, Stellenbosch University,
Stellenbosch, Private Bag X1 Matieland, 7602, South Africa
2 Stellenbosch MediClinic, Elsie du Toit street, Stellenbosch 7600, South Africa
3 PathCare Laboratories, PathCare Business Centre, Neels Bothma Street, N1 City,7460,
South Africa
4 Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and
Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Crown St,
Liverpool L69 7ZB, UK
5The Novo Nordisk Foundation Centre for Biosustainability, Building 220, Kemitorve
Technical University of Denmark, 2800 Kongens Lyngby, Denmark
*Corresponding authors:
*Etheresia Pretorius
Department of Physiological Sciences, Stellenbosch University, Private Bag X1 Matieland,
7602, SOUTH AFRICA
resiap@sun.ac.za
http://www.resiapretorius.net/
ORCID: 0000-0002-9108-2384
*Douglas B. Kell
Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and
Integrative Biology, University of Liverpool, Crown St, Liverpool L69 7ZB, UK
dbk@liv.ac.uk
http://dbkgroup.org/
ORCID: 0000-0001-5838-7963
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Prevalence of amyloid blood clots in COVID-19 plasma ................................................. 1
Keywords. COVID-19 – coagulopathies – amyloid – pathologies ............................... 2
Abstract ................................................................................................................................. 3
Introduction ........................................................................................................................... 3
Methods ................................................................................................................................ 4
Ethical considerations ......................................................................................................... 4
Patient sample ..................................................................................................................... 4
Results .................................................................................................................................. 5
Discussion .......................................................................................................................... 10
DECLARATIONS ............................................................................................................... 12
Funding ............................................................................................................................... 12
Competing interests .......................................................................................................... 12
Consent for publication ..................................................................................................... 12
References ......................................................................... Error! Bookmark not defined.
Keywords. COVID-19 coagulopathies amyloid pathologies
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3
Abstract
The rapid detection of COVID-19 uses genotypic testing for the presence of SARS-Cov-2 virus
in nasopharyngeal swabs, but it can have a poor sensitivity. A rapid, host-based physiological
test that indicated whether the individual was infected or not would be highly desirable.
Coagulaopathies are a common accompaniment to COVID-19, especially micro-clots within
the lungs. We show here that microclots can be detected in the native plasma of COVID-19
patient, and in particular that such clots are amyloid in nature as judged by a standard
fluorogenic stain. This provides a rapid and convenient test (P<0.0001), and suggests that the
early detection and prevention of such clotting could have an important role in therapy.
Introduction
The standard method for detecting infection with SARS-CoV-2 leading to COVID-19
disease involves a genotypic (PCR) test for the virus on nasopharyngeal swabs, but it
is not particularly pleasant and can have poor sensitivity (1-4). What would be
desirable is a rapid and phenotypic test on the host that indicates the presence, and if
possible the severity, of the consequences of infection. Presently, the standard
method for this is based on CT chest scans for pneumonia, which have high sensitivity
but lower specificity (see (5-7) and below), but this is neither cheap nor universally
available.
It is widely recognised (8-13) that extensive blood clotting has a major role in the
pathophysiology of COVID-19 disease severity and progression, yet so can excessive
bleeding (14, 15). The solution to this apparent paradox lies in the recognition (16) that
these phases are separated in time, with excessive clotting preceding the later
bleeding that is mediated by the clotting-induced depletion of fibrinogen and of von
Willebrand factor (VWF). This first phase is accompanied by partial fibrinolysis of the
formed clots, and an extent of D-dimer formation that is predictive of clinical outcomes
(17). These features, together with the accompanying decrease in platelets
(thrombocytopaenia), leads to the subsequent bleeding. Thus it is suggested that the
application of suitably monitored levels of anti-clotting agents in the earlier phase
provides for a much better outcome (10, 16).
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As well as the extent of clotting, including the life-threatening disseminated
intravascular coagulation (DIC) (12), a second issue pertains to its nature. Some years
ago, we discovered that in the presence of microbial cell wall components (18, 19),
and in a variety of chronic, inflammatory diseases (20-22) (including sepsis (23)),
blood fibrinogen can clot into an anomalous, amyloid form (24). These forms are easily
detected by a fluorogenic stain such as thioflavin T, or the so-called Amytracker stains
(25). In all cases, however, these experiments were performed in vitro using relevant
plasma, with clotting being induced by the addition of thrombin. This was also the case
for plasma from COVID-19 patients, but the signals were so massive that they were
essentially off the scale. However, as we report here, the plasma of COVID-19 patients
carries a massive load of preformed amyloid clots (with no thrombin being added), and
this therefore provides a rapid and convenient test for COVID-19.
Methods
Ethical considerations
Ethical approval for blood collection and analysis of the patients with COVID-19 and
healthy individuals, was given by the Health Research Ethics Committee (HREC) of
Stellenbosch University (reference number: 9521). This laboratory study was carried
out in strict adherence to the International Declaration of Helsinki, South African
Guidelines for Good Clinical Practice and the South African Medical Research Council
(SAMRC), Ethical Guidelines for research. Oral consent was obtained from all
participants prior to any sample collection.
Patient sample
Covid-19 patients
20 COVID-19-positive samples (11 males and 9 females) were obtained and blood
samples collected before treatment was embarked upon. Blood samples were
collected by JS. Platelet poor plasma (PPP) prepared and stored at -80°C, until
fluorescent microscopy analysis.
Healthy samples
Our healthy sample was 10 age-matched controls (4 males and 6 females), previously
collected and stored in our plasma repository. They were non-smokers, with CRP
levels within healthy ranges, and not on any anti-inflammatory medication.
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Lung CT scans
Amongst the COVID-19 patient sample 10 patients were admitted, but stabilized and
blood drawn and sent home for observation. Where patients were clinically deemed
as moderate or severely ill, CT scans of the patients were performed to determine the
severity of the lung pathology. We divided our sample into mild disease (no CT scan)
and moderate to severely ill. The CT scan and severity score (26) confirmed moderate
to severely ill patients according to the ‘ground glass’ opacities in the lungs.
Fluorescent Microscopy of patient whole blood and platelet poor plasma
Fluorescent (anomalous) amyloid signals present in PPP from COVID-19 patients and
healthy age-matched individuals were studied using PPP that was stored at -80°C.
On the day of analysis, PPP was thawed and incubated with thioflavin T (ThT; 5 µM
final concentration). Following this, the sample was incubated for 30 min (protected
from light) at room temperature. PPP smears were then created by transferring a small
volume (5 µl) of the stained PPP sample to a microscope slide (similar methods were
followed to create a blood smear). A cover slip was placed over the prepared smear
and viewed using a Zeiss AxioObserver 7 fluorescent microscope with a Plan-
Apochromat 63x/1.4 Oil DIC M27 objective. Unstained samples were also prepared
with both healthy and COVID-19 PPP, to assess any autofluorescence. Micrograph
analysis was done using ImageJ (version 2.0.0-rc-34/1.5a). The % area of amyloid
were calculated using the thresholding method. Statistical analysis was done using
Graphpad, Prism 8 (version 8.4.3).
Results
Age-matched COVID-19 (average age 49.9y) and healthy individuals (49.05y), were
used in this analysis (p= 0.065). Figure 1 shows representative CT scans of four of the
COVID-19 patients. Raw data is shared in
https://1drv.ms/u/s!AgoCOmY3bkKHirZOu5YKPlq1x5f1AQ?e=xmWGKm
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Figure 1A to D: Representative CT scans of a COVID-19 patient. Yellow arrows show
ground glass opacities.
Figure 2 to 4 show representative fluorescent micrographs of PPP from healthy and
COVID-19 individuals. In healthy PPP smears (Figure 2), very little ThT fluorescent
signal is visible, while in COVID-19 individuals (Figure 3), abundant amyloid signal is
noted. Note that these signals were as received; no thrombin was added to induce
clotting. Figure 4 shows the additional presence of fibrous or cellular deposits in the
PPP smears. From their appearance, some of these deposits seem to have originated
from endothelial cells. There have been reports of extensive endotheliopathy in
COVID-19 patients (27). Figure 5A and B show box plots of the % area of amyloid
signal calculated from representative micrographs of each individual. A nonparametric
one-way ANOVA test (Kruskal-Wallis test) between all three groups showed a highly
significant difference (p = <0.0001). However, a Mann-Whitney analysis between the
mild and the moderate to severe COVID-19 individuals showed no significant
difference (p = 0.554). Amyloid formation in plasma is therefore present in the early
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stages of COVID-19, when the patients are sufficiently unwell to visit the hospital and
in need of stabilization.
Figure 2 A to D): Representative fluorescent micrographs of platelet poor plasma
from healthy individuals. Some signals are very slight, as shown by the arrows in A).
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Figure 3A to H): Representative fluorescent micrographs of platelet poor plasma from
COVID-19 patients.
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Figure 4: Fibrous or cellular deposits in the plasma smears of COVID-19 patients.
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Figure 5A and B: Amyloid % area in platelet poor plasma smears with mean and SEM
(p = <0.0001). A) All controls and all COVID-19 patients. B) All controls vs 10 mild
and 10 moderate to severely ill patients.
Discussion
Strongly bound up with the coagulopathies accompanying severe COVID-19 disease
is the presence of hyperferritinaemia (in cases such as the present it is a cell damage
marker (28)) and a cytokine storm, (29-33) which usually occurs in the later phase of
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the disease (16). Excess iron has long been known to cause blood to clot into an
anomalous form (34), later shown to be amyloid in nature (18-24). These kinds of
phenomena seem to accompany essentially every kind of inflammatory disease (e.g.
(35)), but the amyloidogenic coagulopathies are normally assessed following the ex
vivo addition of thrombin to samples of plasma.
Many clinical features of COVID-19 are unprecedented, and here we demonstrate yet
another: the presence in platelet poor plasma to which thrombin has not been added
of amyloid microclots. This kind of phenomenon explains at once the extensive
microclotting that is such a feature of COVID-19 (8), and adds strongly to the view that
its prevention via anti-clotting agents should lie at the heart of therapy. Although
fluorescence microscopy is a specialized laboratory technique, TEG is a well-known
point of care technique, which is cheap and reliable. All told, the relative ease, speed
(40 minutes including 30 minutes ThT incubation time) and cheapness of the assay
we describe might be of considerable prognostic utility in assessing the clinical status
of COVID-19 patients.
Of course this must also be monitored (e.g. via Thromboelastography (36-39)) lest the
disease enters its later phase in which bleeding rather than clotting is the greater
danger (16). An important consideration is that TEG can be used to study the clotting
parameters of both whole blood and PPP. Whole blood TEG gives information on the
clotting potential affected by the presence of both platelets and fibrinogen, while PPP
TEG only presents evidence of the clotting potential of the plasma proteins (36-39).
Point-of-care devices and diagnostics like TEG are also particularly useful to assess
fibrinolysis. In COVID-19 patients, Wright and co-workers reported fibrinolysis
shutdown, confirmed by complete failure of clot lysis at 30 minutes on the TEG (40).
Thus TEG can therefore predict thromboembolic events in patients with COVID-19
(40). What we have shown here is that the clotting that is commonly seen in COVID-
19 patients is in an amyloid form; this alone would explain the complete shutdown of
fibrinolysis and the decreased ability to pass O2 into the blood that is such a feature of
the disease. Consequently, its prevention must lie at the heart of therapies.
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DECLARATIONS
Funding
We thank the Medical Research Council of South Africa (MRC) (Self-Initiated Research
Program: A0X331) for supporting this collaboration. DBK thanks the Novo Nordisk Foundation
for funding (grant NNF10CC1016517).
Competing interests
The authors declare that they have no competing interests.
Consent for publication
All authors approved submission of the paper.
Data sharing
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FIGURE LEGENDS
Figure 1A to D: Representative CT scans of a COVID-19 patient. Yellow arrows show
ground glass opacities.
Figure 2 A to D): Representative fluorescent micrographs of platelet poor plasma
from healthy individuals. Some signals are very slight, as shown by the arrows in A).
Figure 3A to H): Representative fluorescent micrographs of platelet poor plasma from
COVID-19 patients.
Figure 4: Fibrous or cellular deposits in the plasma smears of COVID-19 patients.
Figure 5A and B: Amyloid % area in platelet poor plasma smears with mean and SEM
(p = <0.0001). A) All controls and all COVID-19 patients. B) All controls vs 10 mild
and 10 moderate to severely ill patients.
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... Coagulopathies [84][85][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102], and especially the formation of extensive microclots in vivo, are a hallmark of both COVID [85,[103][104][105][106][107][108][109][110][111][112][113][114][115] and long COVID [116,117], and we have demonstrated that these microclots too are amyloid in character [108,109,116]. Importantly, the addition of purified, recombinant SARS-CoV-2 S1 spike protein to coagulation-competent normal plasma is sufficient to induce the formation of anomalous clots [118] that adopt amyloid states that are also resistant to fibrinolysis [108]. ...
... What is needed next? Many pieces of research-level evidence (especially [85,101,106,108,109,116,231,271]) suggest strongly that fibrin amyloid microclots, driven by the presence of the SARS-CoV-2 spike protein, are an inevitable accompaniment to (and a likely cause of ) Long COVID. A biologically coherent explanation [272,273] can link such observations with the other observable symptoms of Long COVID, and thus serve to satisfy the logic normally required [179,274,275] to provide a causative explanation. ...
Article
Full-text available
Post-acute sequelae of COVID (PASC), usually referred to as 'Long COVID' (a phenotype of COVID-19), is a relatively frequent consequence of SARS-CoV-2 infection, in which symptoms such as breathlessness, fatigue, 'brain fog', tissue damage, inflammation, and coagulopathies (dysfunctions of the blood coagulation system) persist long after the initial infection. It bears similarities to other post-viral syndromes, and to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Many regulatory health bodies still do not recognize this syndrome as a separate disease entity, and refer to it under the broad terminology of 'COVID', although its demographics are quite different from those of acute COVID-19. A few years ago, we discovered that fibrinogen in blood can clot into an anomalous 'amyloid' form of fibrin that (like other β-rich amyloids and prions) is relatively resistant to proteolysis (fibrinolysis). The result, as is strongly manifested in platelet-poor plasma (PPP) of individuals with Long COVID, is extensive fibrin amyloid microclots that can persist, can entrap other proteins, and that may lead to the production of various autoantibodies. These microclots are more-or-less easily measured in PPP with the stain thioflavin T and a simple fluorescence microscope. Although the symptoms of Long COVID are multifarious, we here argue that the ability of these fibrin amyloid microclots (fibrinaloids) to block up capillaries, and thus to limit the passage of red blood cells and hence O2 exchange, can actually underpin the majority of these symptoms. Consistent with this, in a preliminary report, it has been shown that suitable and closely monitored 'triple' anticoagulant therapy that leads to the removal of the microclots also removes the other symptoms. Fibrin amyloid microclots represent a novel and potentially important target for both the understanding and treatment of Long COVID and related disorders.
... SARS-CoV-2 can also drive multi-organ injury via stimulation of clotting cascades (Pretorius et al., 2020a) and related thromboinflammation, dysregulation of the renin-angiotensinaldosterone system, and endothelial cell damage (Grobler et al., 2020;Gupta et al., 2020). Infection-mediated endothelial injury and endothelialitis (marked by the presence of activated macrophages and neutrophils) can trigger excessive thrombin production, inhibit fibrinolysis, and activate complement pathways in a manner that leads to microvascular dysfunction and microthrombi deposition. ...
... However, SARS-CoV-2 also secretes products/proteins that modulate blood clotting cascades (Eslamifar et al., 2020). Pretorius et al. (2020aPretorius et al. ( ,b, 2021 identified fibrinolytic resistant amyloid microclots in COVID-19 plasma (Figure 4) and in plasma samples collected from 11 PASC subjects. This suggests that patients who suffer from both issues/insults might be disproportionally impacted by PASC symptoms that could result from blood inflammatory/coagulation cascades. ...
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The novel virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic of coronavirus disease 2019 (COVID-19). Across the globe, a subset of patients who sustain an acute SARS-CoV-2 infection are developing a wide range of persistent symptoms that do not resolve over the course of many months. These patients are being given the diagnosis Long COVID or Post-acute sequelae of COVID-19 (PASC). It is likely that individual patients with a PASC diagnosis have different underlying biological factors driving their symptoms, none of which are mutually exclusive. This paper details mechanisms by which RNA viruses beyond just SARS-CoV-2 have be connected to long-term health consequences. It also reviews literature on acute COVID-19 and other virus-initiated chronic syndromes such as post-Ebola syndrome or myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) to discuss different scenarios for PASC symptom development. Potential contributors to PASC symptoms include consequences from acute SARS-CoV-2 injury to one or multiple organs, persistent reservoirs of SARS-CoV-2 in certain tissues, re-activation of neurotrophic pathogens such as herpesviruses under conditions of COVID-19 immune dysregulation, SARS-CoV-2 interactions with host microbiome/virome communities, clotting/coagulation issues, dysfunctional brainstem/vagus nerve signaling, ongoing activity of primed immune cells, and autoimmunity due to molecular mimicry between pathogen and host proteins. The individualized nature of PASC symptoms suggests that different therapeutic approaches may be required to best manage care for specific patients with the diagnosis.
... We know that molecules such as LPS (e.g., [147,149,150]) and the spike protein of SARS-CoV-2 (e.g., [154,158,159,[163][164][165][166][167]173]) can cause microclots, such that any damage such molecules may cause to nerves may be indirect [174][175][176]. This said, it is reasonable that any damage to the membranes of nerves might be mediated via fibrinaloid microclots. ...
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Postural orthostatic tachycardia syndrome (POTS) is a common accompaniment of a variety of chronic, inflammatory diseases, including long COVID, as are small, insoluble, ‘fibrinaloid’ microclots. We here develop the argument, with accompanying evidence, that fibrinaloid microclots, through their ability to block the flow of blood through microcapillaries and thus cause tissue hypoxia, are not simply correlated with but in fact, by preceding it, may be a chief intermediary cause of POTS, in which tachycardia is simply the body’s exaggerated ‘physiological’ response to hypoxia. Similar reasoning accounts for the symptoms bundled under the term ‘fatigue’. Amyloids are known to be membrane disruptors, and when their targets are nerve membranes, this can explain neurotoxicity and hence the autonomic nervous system dysfunction that contributes to POTS. Taken together as a system view, we indicate that fibrinaloid microclots can serve to link POTS and fatigue in long COVID in a manner that is at once both mechanistic and explanatory. This has clear implications for the treatment of such diseases.
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Introduction The diagnosis of COVID-19 is normally based on the qualitative detection of viral nucleic acid sequences. Properties of the host response are not measured but are key in determining outcome. Although metabolic profiles are well suited to capture host state, most metabolomics studies are either underpowered, measure only a restricted subset of metabolites, compare infected individuals against uninfected control cohorts that are not suitably matched, or do not provide a compact predictive model. Objectives Here we provide a well-powered, untargeted metabolomics assessment of 120 COVID-19 patient samples acquired at hospital admission. The study aims to predict the patient’s infection severity (i.e., mild or severe) and potential outcome (i.e., discharged or deceased). Methods High resolution untargeted UHPLC-MS/MS analysis was performed on patient serum using both positive and negative ionization modes. A subset of 20 intermediary metabolites predictive of severity or outcome were selected based on univariate statistical significance and a multiple predictor Bayesian logistic regression model was created. Results The predictors were selected for their relevant biological function and include deoxycytidine and ureidopropionate (indirectly reflecting viral load), kynurenine (reflecting host inflammatory response), and multiple short chain acylcarnitines (energy metabolism) among others. Currently, this approach predicts outcome and severity with a Monte Carlo cross validated area under the ROC curve of 0.792 (SD 0.09) and 0.793 (SD 0.08), respectively. A blind validation study on an additional 90 patients predicted outcome and severity at ROC AUC of 0.83 (CI 0.74–0.91) and 0.76 (CI 0.67–0.86). Conclusion Prognostic tests based on the markers discussed in this paper could allow improvement in the planning of COVID-19 patient treatment.
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Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), also known as coronavirus disease 2019 (COVID-19)-induced infection, is strongly associated with various coagulopathies that may result in either bleeding and thrombocytopenia or hypercoagulation and thrombosis. Thrombotic and bleeding or thrombotic pathologies are significant accompaniments to acute respiratory syndrome and lung complications in COVID-19. Thrombotic events and bleeding often occur in subjects with weak constitutions, multiple risk factors and comorbidities. Of particular interest are the various circulating inflammatory coagulation biomarkers involved directly in clotting, with specific focus on fibrin(ogen), D-dimer, P-selectin and von Willebrand Factor (VWF). Central to the activity of these biomarkers are their receptors and signalling pathways on endothelial cells, platelets and erythrocytes. In this review, we discuss vascular implications of COVID-19 and relate this to circulating biomarker, endothelial, erythrocyte and platelet dysfunction. During the progression of the disease, these markers may either be within healthy levels, upregulated or eventually depleted. Most significant is that patients need to be treated early in the disease progression, when high levels of VWF, P-selectin and fibrinogen are present, with normal or slightly increased levels of D-dimer (however, D-dimer levels will rapidly increase as the disease progresses). Progression to VWF and fibrinogen depletion with high D-dimer levels and even higher P-selectin levels, followed by the cytokine storm, will be indicative of a poor prognosis. We conclude by looking at point-of-care devices and methodologies in COVID-19 management and suggest that a personalized medicine approach should be considered in the treatment of patients.
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Objectives To correlate a CT-based semi-quantitative score of pulmonary involvement in COVID-19 pneumonia with clinical staging of disease and laboratory findings. We also aimed to investigate whether CT findings may be predictive of patients’ outcome.Methods From March 6 to March 22, 2020, 130 symptomatic SARS-CoV-2 patients were enrolled for this single-center analysis and chest CT examinations were retrospectively evaluated. A semi-quantitative CT score was calculated based on the extent of lobar involvement (0:0%; 1, < 5%; 2:5–25%; 3:26–50%; 4:51–75%; 5, > 75%; range 0–5; global score 0–25). Data were matched with clinical stages and laboratory findings. Survival curves and univariate and multivariate analyses were performed to evaluate the role of CT score as a predictor of patients’ outcome.ResultsGround glass opacities were predominant in early-phase (≤ 7 days since symptoms’ onset), while crazy-paving pattern, consolidation, and fibrosis characterized late-phase disease (> 7 days). CT score was significantly higher in critical and severe than in mild stage (p < 0.0001), and among late-phase than early-phase patients (p < 0.0001). CT score was significantly correlated with CRP (p < 0.0001, r = 0.6204) and D-dimer (p < 0.0001, r = 0.6625) levels. A CT score of ≥ 18 was associated with an increased mortality risk and was found to be predictive of death both in univariate (HR, 8.33; 95% CI, 3.19–21.73; p < 0.0001) and multivariate analysis (HR, 3.74; 95% CI, 1.10–12.77; p = 0.0348).Conclusions Our preliminary data suggest the potential role of CT score for predicting the outcome of SARS-CoV-2 patients. CT score is highly correlated with laboratory findings and disease severity and might be beneficial to speed-up diagnostic workflow in symptomatic cases.Key Points • CT score is positively correlated with age, inflammatory biomarkers, severity of clinical categories, and disease phases. • A CT score ≥ 18 has shown to be highly predictive of patient’s mortality in short-term follow-up. • Our multivariate analysis demonstrated that CT parenchymal assessment may more accurately reflect short-term outcome, providing a direct visualization of anatomic injury compared with non-specific inflammatory biomarkers.
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Patients with coronavirus disease 2019 (COVID-19) have elevated D-dimer levels. Early reports describe high venous thromboembolism (VTE) and disseminated intravascular coagulation (DIC) rates, but data are limited. This multicenter, retrospective study described the rate and severity of hemostatic and thrombotic complications of 400 hospital-admitted COVID-19 patients (144 critically ill) primarily receiving standard-dose prophylactic anticoagulation. Coagulation and inflammatory parameters were compared between patients with and without coagulation-associated complications. Multivariable logistic models examined the utility of these markers in predicting coagulation-associated complications, critical illness, and death. The radiographically-confirmed VTE rate was 4.8% (95% CI, 2.9-7.3%) and the overall thrombotic complication rate was 9.5% (6.8-12.8%). The overall and major bleeding rates were 4.8% (2.9-7.3%) and 2.3% (1.0-4.2%). In the critically ill, radiographically-confirmed VTE and major bleeding rates were 7.6% (3.9-13.3%) and 5.6% (2.4-10.7%). Elevated D-dimer at initial presentation was predictive of coagulation-associated complications during hospitalization [D-dimer >2,500 ng/mL, adjusted OR for thrombosis, 6.79 (2.39-19.30), adjusted OR for bleeding, 3.56 (1.01-12.66)], critical illness, and death. Additional markers at initial presentation predictive of thrombosis during hospitalization included platelet count >450×109/L [adjusted OR, 3.56 (1.27-9.97)], C-reactive protein (CRP) >100 mg/L [adjusted OR, 2.71 (1.26-5.86)], and erythrocyte sedimentation rate >40 mm/h [adjusted OR, 2.64 (1.07-6.51)]. ESR, CRP, fibrinogen, ferritin, and procalcitonin were higher in patients with thrombotic complications than those without. DIC, clinically-relevant thrombocytopenia, and reduced fibrinogen were rare and were associated with significant bleeding manifestations. Given the observed bleeding rates, randomized trials are needed to determine any potential benefit of intensified anticoagulant prophylaxis in COVID-19 patients.
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Background COVID-19 is an ongoing global pandemic. Changes in haematological characteristics in patients with COVID-19 are emerging as important features of the disease. We aimed to explore the haematological characteristics and related risk factors in patients with COVID-19. Methods This retrospective cohort study included patients with COVID-19 admitted to three designated sites of Wuhan Union Hospital (Wuhan, China). Demographic, clinical, laboratory, treatment, and outcome data were extracted from electronic medical records and compared between patients with moderate, severe, and critical disease (defined according to the diagnosis and treatment protocol for novel coronavirus pneumonia, trial version 7, published by the National Health Commission of China). We assessed the risk factors associated with critical illness and poor prognosis. Dynamic haematological and coagulation parameters were investigated with a linear mixed model, and coagulopathy screening with sepsis-induced coagulopathy and International Society of Thrombosis and Hemostasis overt disseminated intravascular coagulation scoring systems was applied. Findings Of 466 patients admitted to hospital from Jan 23 to Feb 23, 2020, 380 patients with COVID-19 were included in our study. The incidence of thrombocytopenia (platelet count <100 × 10⁹ cells per L) in patients with critical disease (42 [49%] of 86) was significantly higher than in those with severe (20 [14%] of 145) or moderate (nine [6%] of 149) disease (p<0·0001). The numbers of lymphocytes and eosinophils were significantly lower in patients with critical disease than those with severe or moderate disease (p<0·0001), and prothrombin time, D-dimer, and fibrin degradation products significantly increased with increasing disease severity (p<0·0001). In multivariate analyses, death was associated with increased neutrophil to lymphocyte ratio (≥9·13; odds ratio [OR] 5·39 [95% CI 1·70–17·13], p=0·0042), thrombocytopenia (platelet count <100 × 10⁹ per L; OR 8·33 [2·56–27·15], p=0·00045), prolonged prothrombin time (>16 s; OR 4·94 [1·50–16·25], p=0·0094), and increased D-dimer (>2 mg/L; OR 4·41 [1·06–18·30], p=0·041). Thrombotic and haemorrhagic events were common complications in patients who died (19 [35%] of 55). Sepsis-induced coagulopathy and International Society of Thrombosis and Hemostasis overt disseminated intravascular coagulation scores (assessed in 12 patients who survived and eight patients who died) increased over time in patients who died. The onset of sepsis-induced coagulopathy was typically before overt disseminated intravascular coagulation. Interpretation Rapid blood tests, including platelet count, prothrombin time, D-dimer, and neutrophil to lymphocyte ratio can help clinicians to assess severity and prognosis of patients with COVID-19. The sepsis-induced coagulopathy scoring system can be used for early assessment and management of patients with critical disease. Funding National Key Research and Development Program of China.
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Background An important feature of severe acute respiratory syndrome coronavirus 2 pathogenesis is COVID-19-associated coagulopathy, characterised by increased thrombotic and microvascular complications. Previous studies have suggested a role for endothelial cell injury in COVID-19-associated coagulopathy. To determine whether endotheliopathy is involved in COVID-19-associated coagulopathy pathogenesis, we assessed markers of endothelial cell and platelet activation in critically and non-critically ill patients admitted to the hospital with COVID-19. Methods In this single-centre cross-sectional study, hospitalised adult (≥18 years) patients with laboratory-confirmed COVID-19 were identified in the medical intensive care unit (ICU) or a specialised non-ICU COVID-19 floor in our hospital. Asymptomatic, non-hospitalised controls were recruited as a comparator group for biomarkers that did not have a reference range. We assessed markers of endothelial cell and platelet activation, including von Willebrand Factor (VWF) antigen, soluble thrombomodulin, soluble P-selectin, and soluble CD40 ligand, as well as coagulation factors, endogenous anticoagulants, and fibrinolytic enzymes. We compared the level of each marker in ICU patients, non-ICU patients, and controls, where applicable. We assessed correlations between these laboratory results with clinical outcomes, including hospital discharge and mortality. Kaplan–Meier analysis was used to further explore the association between biochemical markers and survival. Findings 68 patients with COVID-19 were included in the study from April 13 to April 24, 2020, including 48 ICU and 20 non-ICU patients, as well as 13 non-hospitalised, asymptomatic controls. Markers of endothelial cell and platelet activation were significantly elevated in ICU patients compared with non-ICU patients, including VWF antigen (mean 565% [SD 199] in ICU patients vs 278% [133] in non-ICU patients; p<0·0001) and soluble P-selectin (15·9 ng/mL [4·8] vs 11·2 ng/mL [3·1]; p=0·0014). VWF antigen concentrations were also elevated above the normal range in 16 (80%) of 20 non-ICU patients. We found mortality to be significantly correlated with VWF antigen (r = 0·38; p=0·0022) and soluble thrombomodulin (r = 0·38; p=0·0078) among all patients. In all patients, soluble thrombomodulin concentrations greater than 3·26 ng/mL were associated with lower rates of hospital discharge (22 [88%] of 25 patients with low concentrations vs 13 [52%] of 25 patients with high concentrations; p=0·0050) and lower likelihood of survival on Kaplan–Meier analysis (hazard ratio 5·9, 95% CI 1·9–18·4; p=0·0087). Interpretation Our findings show that endotheliopathy is present in COVID-19 and is likely to be associated with critical illness and death. Early identification of endotheliopathy and strategies to mitigate its progression might improve outcomes in COVID-19. Funding This work was supported by a gift donation from Jack Levin to the Benign Hematology programme at Yale, and the National Institutes of Health.
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Damage to vessel lining may drive mysterious clotting disorders, inflammation.