Acknowledge the aerosol transmission route of SARS-CoV-2

Denying the aerosol transmission route is not going to get the world out of this pandemic
Since February 2020, I have been an advocate for the worldwide recognition of the aerosol transmission of SARS-CoV-2. Aerosols (aero-solutions) are a mix of micro droplets and solid particles with gasses, like the air surrounding us. Through breathing, tiny droplets are released into a gas cloud that travels through airflows. In indoor spaces, aerosols accumulate, saturating the air. This mechanism is known to contribute to the spread of infectious diseases, as is the case with Influenza, measles and SARS-CoV-1 (the coronavirus that was active during the 2003 epidemic). While airborne infection and aerosol transmission of viruses are not controversial among scientists (chemical engineers, biochemists and physicists), policy makers and health authorities fail to recognize the importance of aerosol transmission of SARS-CoV-2.

Instead, policies issued by the government and health authorities are based on the belief that infections only occur through fomites or through direct person-to-person droplet transmission. Remarkably, when droplet and fomite transmission fail to prove the exact transmission routes at work during superspreading events, the aerosol transmission route is rejected in advance, arguing that it is assumed that fomite and direct droplet transmission are the major transmission routes. Even more remarkable, health authorities discriminate between research on aerosol transmission routes of SARS-CoV-2 and research on droplet and fomite transmission. While preprints copied from the preprint server MedRxiv are deemed "supportive evidence" when these seem to confirm the predominance of fomites or direct (droplet) transmission, preprints from MedRxiv on airborne infection are rejected for 'not being peer reviewed yet'. Even more bizarre is that engineers, physicists and biochemists specialized in aerosol science are now pushed aside as "believers". People fail to understand that aerosols are minuscule parts of everyday life. It's not like they are insignificant because the bare human eye does not have the quality to spot them.

Aerosol transmission was reported to be the main transmission route in the Amoy Gardens Housing complex community during the 2003 SARS-CoV-1 epidemic (Evidence of Airborne Transmission of the SARS Virus, NEJM, 22 April 2004) and air distribution played a major role in the largest nosocomial SARS outbreak in Hong Kong (Role of air distribution in SARS transmission during the largest nosocomial outbreak in Hong Kong, Indoor air, April 2005, Volume 15, Issue 2). A more extensive read on cases of aerosol transmission of SARS-CoV-2, as well as insights in sets of genes imposing vulnerabilities on people who inherited these genetic traits, is published in the May edition of Human Genomics (COVID-19 vulnerability: the potential impact of genetic susceptibility and airborne transmission, Human Genomics, 2020; 14 :17). Repeatedly, since the 2003 outbreak of SARS-CoV-1, the global community has been urged to adopt measures to ensure ventilation rates and air filtration and to prevent aerosol generation during (medical and dental) procedures (Detection of Airborne SARS Coronavirus and Environmental Contamination in SARS Outbreak Units, Journal of Infectious Diseases, 1 May 2005, Volume 191, Issue 9).

No rigid divisive line should be drawn between transmission routes
Neither transmission route should be rejected, nor should there be drawn a sharp dividing line  between aerosol transmission and other transmission routes. Fomite transmission of faecal particles, urine and saliva contributes to the spread of SARS-CoV-2, as well as direct droplet transmission. There is more to this. Aerosols are even borne out of fomites ("airborne"). It starts with fomites like poop and saliva, which turn into aerosols generated by mechanisms like breathing, talking, using, drilling, flushing the toilet, draining down, ventilating, blowing into the air by mechanical climate control. Pathogens under both classifications (droplet and airborne) have the potential to be transmitted by aerosols, as should be recognized (Recognition of aerosol transmission of infectious agents: a commentary, BMC Infectious Diseases, 31 January 2019; 19; 101).

A recent test estimates that 1 minute of loud speaking generates at least 1,000 virion-containing droplet nuclei (particles) that remain airborne for more than 8 minutes. The droplets that were observed were regarded to be sufficiently small to reach the lower respiratory tract, implying an increased risk for infection (The airborne lifetime of small speech droplets and their potential importance in SARS-CoV-2 infection, Proceedings of the National Academy of Sciences of the United States of America, 13 May 2020).  

Large vs. small droplets: a definition too narrow
The point is, policies force people to maintain (social) distance of of approximately 5 feet, but maintaining distance does not protect against aerosol transmission. The distance provision is based on Wells' 1930 theory, which is now interpreted as a theory distinguishing between large and small droplets. In this view, small droplets would evaporate after exhaling/coughing/sneezing, as the environment is colder and drier than the lungs of the person exhaling the drops. Interestingly, research on airborne dispersion has centered on 'aggressive' forms of exhalation like sneezing and coughing for years, while it was already proven in 1946 that breathing and talking generate large amounts of aerosols (The size and duration of air-carriage of respiratory droplets and droplet-nuclei, Journal of Epidemiology and Infection, J.P. Duguid, September 1946).

The 'large drops vs. small drops'-debate is of relevance when it comes to measures preventing direct droplet transmission: ballistic research makes clear that medium and large droplets are transmitted mainly through the droplet-borne route. After dispersion into the air, aerosols are transmitted via the short-range and long-range airborne route. Regardless of the transmission route (direct droplets or airborne), surgical masks reduce infectiousness (Airborne spread of infectious agents in the indoor environment, American Journal of Infection Control, 2 September 2016, 44(9)).

The turbulent gas cloud does not respect social distance rules of 5 feet
Drops do not fly isolated through the air, but through a turbulent gas cloud. This is why the 1-2 meter/5 ft distance model falls short. The actual mechanism is called "multiphase turbulent gas cloud", which means that respiratory drops, mucus, nearby gases and moisture accumulate in a cloud. Depending on humidity and temperature of the environment and the size of the virus-carrying particles, this cloud can travel up to 8 meters through the air. Finally, particles settle on surfaces ánd remain up in the air (droplet nuclei = aerosols) for about 3 hours after evaporation from droplets to gas (Aerosol and Surface Stability of SARS-CoV-2 as compared to SARS-CoV-1, NEJM, 16 April 2020).

The distance rule is based on the assumption that air does not move though a space. This model created in 1934, called the 'Wells-Riley" model of well-mixed air, hypothesizes that there is an ideal homogeneous mixing of respiratory drops and an ideal homogeneous distribution of humans in any given space. Only ventilation rate is included in the well-mixed air model, while parameters of dose-response, air turbulence, flow direction, flow pattern and spatial heterogeneity should be included (Review and comparison between the Wells-Riley and dose-response approaches to risk assessment of infectious respiratory diseases, Indoor Air, Vol. 20 Issue 1, February 2010). This explains why some people sitting next to an infected person do not get infected, while people sitting at a greater distance can become infected through the movement of viral particles through air flows. An inter-personal distance of 2 m can only be considered to be reasonable when wearing face masks during public activities is compulsory (Airborne Transmission Route of COVID-19: Why 2 Meters/6 Feet of Inter-Personal Distance Could Not Be Enough, International Journal of Environmental Research and Public Health, MDPI, April 2020; 17(8)).
 
The Open Air Factor (OAF)

The "open-air factor" OAF refers to the influence of natural air flow and UV on the destabilization of viruses and other infections. Modern hospitals are generally worse at limiting the spread of viruses than hospitals dating from before 1950. Old-fashioned large windows that can be opened are conducive to limiting the spread of virus particles in the hospital (The open-air factor and infection control, Journal of Hospital Infection, April 9, 2019). In other words, being indoors is not ideal, poor ventilation is completely disastrous. A 2013 study confirms the suspicion that modern hospitals with small windows (that cannot be opened) and poor air conditioning accelerate the spread of SARS. Mechanical ventilation is not necessarily worse than natural airflows: the point is to achieve 'cross-ventilation', where air flows from the inside out (Roles of sunlight and natural ventilation for controlling infection: historical and current perspectives, Journal of Hospital Infection 84, June 20, 2013). A lack of SARS transmission among public hospital workers in Vietnam in 2003 was associated with a health policy of cross-ventilation, as well as a face mask policy (Lack of SARS Transmission among public hospital workers, Vietnam, Emerging Infectious Diseases, Februari 2004; 10(2)). A massive body of knowledge on the clinical management of SARS outbreaks is to be found in the Review 'Clinical management and infection control of SARS: lessons learned', Antiviral Research, Volume 100, Issue 2, November 2013.

But cross-ventilating a room is not sufficient to minimize the risk of aerosol transmission. Larger exhaled droplets can be moved upwards by heat, then spread and dispersed horizontally. Ventilation is determined by factors ventilation rate, flow direction and flow pattern (Ventilation control for airborne transmission of human exhaled bio-aerosols in buildings, Journal of Thoracic Disease, July 2018; 10(suppl. 19)). The spatial distribution of viral particles is dependent on airflow pattern. Ventilation dilution depends on ventilation rate. This explains why natural airflow, which has an effective ventilation rate, dilates viral particles. 

Using ventilation as means to reduce the risk of airborne transmission of SARS-CoV-2
I've mentioned that natural ventilation is preferable to ensure natural airflow. The ventilation rate of natural airflow is effective when it comes to the dilation of droplet nuclei. As a result, the concentration of viral nuclei in air is minimized. However, objects should not get in the way of the airflow. Another consideration is that people should avoid the course of the airflow, to reduce the risk of becoming infected with viral nuclei. 

Recirculation of air should be avoided, as air recirculation increases the risk of airborne transmission. The Open Air Factor is recommended: replace recirculation with Open Air if possible. 
Disinfection agents and radiation can speed up destabilization of SARS-CoV-2. UV-C is known to inactivate SARS-CoV-1 within 15 minutes; this might be similar with regards to SARS-CoV-2 (Inactivation of the coronavirus that induces SARS, SARS-CoV, Journal of Virological Methods, October 2004; 121(1)).

Further reading 

1. Judson SD, Munster VJ. Nosocomial transmission of emerging viruses via aerosol‐generating medical procedures. Viruses. 2019;11(10). doi:10.3390/v11100940

2. Tran K, Cimon K, Severn M, Pessoa‐Silva CL, Conly J. Aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: A
systematic review. PLoS One. 2012;7(4). doi:10.1371/journal.pone.0035797

3. Li Y, Huang X, Yu ITS, Wong TW, Qian H. Role of air distribution in SARS transmission during the largest nosocomial outbreak in Hong Kong. Indoor Air.
2005;15(2):83‐95. doi:10.1111/j.1600‐0668.2004.00317.x

4. Grosskopf K, Mousavi E. Bioaerosols in health‐care environments. ASHRAE J. 2014;56(8):22‐31.

5. Lindsley WG, Blachere FM, Thewlis RE, et al. Measurements of airborne influenza virus in aerosol particles from human coughs. PLoS One. 2010;5(11).
doi:10.1371/journal.pone.0015100

6. Moriyama M, Hugentobler WJ, Iwasaki A. Seasonality of Respiratory Viral Infections. Annu Rev Virol. 2020:1‐19. doi:10.1146/annurev‐virology‐012420‐022445

Denkbeeldige lockdown en schadelijk laconiek gedrag

Een denkbeeldige lockdown met overvolle winkelstraten
Er is geen lockdown geweest in Nederland. Mensen hebben zichzelf ingebeeld dat er een lockdown was opgelegd. Afgezien van de sluiting van kappers, scholen en horeca heeft de bevolking geen restricties opgelegd gekregen. Dat was te merken, getuige de vanaf maart 2020 aanhoudende drukte in winkels en op meubelboulevards. Waarom bestaat dan toch de hardnekkige misvatting dat de vrijheid van mensen de afgelopen maanden is ingeperkt door een lockdown?

Via sociale media is de leus "blijf zoveel mogelijk binnen" verspreid, waarmee mensen zichzelf hebben wijsgemaakt dat ze niet meer buiten mogen komen. Met "blijf zoveel mogelijk" thuis wordt uitgedrukt, dat voorkomen moet worden dat openbare ruimten frequent en onbeschermd worden bezocht, waarmee verspreiding van het virus vrij spel krijgt. Wat niet de bedoeling is, is dat mensen zichzelf in hun huis opsluiten. Het gedrag is tegenstrijdig: enerzijds leggen mensen zichzelf op dat ze zoveel mogelijk binnen moeten blijven, anderzijds is de drukte in de winkelstraten niet afgenomen.

Door degenen die zichzelf hebben wijsgemaakt dat de lockdown is opgelegd, wordt geklaagd over de (grondwettelijke) aantasting van de vrijheid. De discussie wordt gedramatiseerd door voor te wenden dat Nederland voor altijd een samenleving zal zijn waarin fysieke afstand moet worden gehouden tot vreemden. Het cliché luidt dat de samenleving op slot zou blijven tot er een vaccin beschikbaar is. De ernst van SARS-CoV-2 wordt gebagatelliseerd door te stellen dat de dood bij het leven hoort, dat er niet-risicogroepen bestaan en dat het virus vrijwel alleen gevaarlijk is voor zieken en ouderen. Het anti-maatregelenkamp stelt bovendien de economie en de noodzaak tot indamming van het virus lijnrecht tegenover elkaar, als twee elkaar uitsluitende waarden. Volgens het anti-maatregelenkamp moet de samenleving volledig worden opengesteld, zonder beschermende middelen zoals mondmaskers. Het mondkapje wordt geschuwd omdat mensen bang zijn dat hun uiterlijk er niet voordelig in uitkomt en omdat het mondkapje geen zin zou hebben.

Mondkapjes: de enige manier om aërosole transmissie te beperken
Vanuit het RIVM is de informatie over het dragen van mondmaskers tegenstrijdig. Het zou een vervangende maatregel zijn, wanneer het houden van 1,5 meter afstand niet mogelijk is. Daarbij wordt genegeerd dat de 1,5 meter afstand-regel geen nut heeft. SARS-CoV-2 wordt verspreid via aërosole transmissie. Dat wil zeggen dat het virus door een besmette persoon aan de lucht wordt afgegeven, door te ademen, te praten en te hoesten. Het virus blijft in kleine deeltjes in de lucht hangen in een gesloten ruimte. Ook als mensen 1,5 meter afstand houden, blijven deze virusdeeltjes in de lucht hangen. Daarom is het nodig om een gecertificeerd mondmasker te dragen dat virusdeeltjes uit de lucht kan filteren. Door allocatie van middelen (onder meer door het transporteren van mondmaskers naar China in februari 2020) heeft de overheid ervoor gezorgd dat voor zowel medici als voor burgers geen of onvoldoende gecertificeerde mondmaskers beschikbaar zijn. Het is dus juist dat het dragen van een mondkapje de drager zélf niet beschermt.

Het dragen van een mondmasker om te voorkomen dat men een ander beschermt, is weer een andere maatregel. Als iedereen verplicht een mondkapje draagt, ook niet-gecertificeerde exemplaren, dan is de kans op transmissie kleiner dan wanneer er géén mondkapjes in gesloten publieke ruimten worden gedragen. Het risico op transmissie zou verder worden gereduceerd wanneer wél gecertificeerde mondmaskers met een filter verkrijgbaar zouden zijn voor iedereen. Zolang die persoonlijke bescherming er niet is, is het niet veilig om in gesloten publieke ruimten te komen waar geïnfecteerden rond kunnen lopen.

Iedereen is een risicogroep voor SARS-2
Het meest hardnekkige misverstand is dat er "niet-risicogroepen" zouden bestaan. Risicogroepen zouden in deze opvatting ouderen en chronisch zieken zijn, die kunnen worden afgezonderd van de samenleving, zodat jonge mensen zonder onderliggend medisch lijden weer onbeschermd de samenleving in kunnen (dat is: bijeenkomsten in gesloten publieke ruimten, uitgaansleven, volle collegezalen en volle treinen).

In de eerste maanden van 2020 werd bekend dat de gemiddelde leeftijd van ernstig getroffen SARS-2-patiënten gemiddeld 52 jaar is (Clinical characteristics of Coronavirus disease 2019 in China, NEJM, 30 april 2020). Aan de voorwaarde "comorbiditeiten" is al snel voldaan, zo is een verhoogde bloeddruk een factor die wordt geassocieerd met een ernstig verloop van SARS. Het is echter niet duidelijk of sommige patiënten al een verhoogde bloeddruk hadden vóór infectie met SARS-CoV-2.

Evenals bij SARS-1 het geval was, is iedereen een risicogroep voor SARS-CoV-2. Dat heeft ermee te maken dat alle leeftijdscategorieën en ook mensen zonder aanwezig medisch lijden te maken kunnen krijgen met een cytokinestorm (een overdreven reactie van het immuunsysteem). Tromboinflammatie is daarnaast een kenmerkende complicatie van SARS-coronavirussen. Ook bij jongeren worden coagulatiestoornissen waargenomen, die worden veroorzaakt door de combinatie van een ontstekingsreactie en een cytokinecascade (een 'waterval' van eigen stoffen die helpen om een immuunreactie op gang te brengen).

Blootstelling aan het virus is geen mogelijkheid, gezien de ernst van de complicaties
Onafhankelijk van leeftijd en ernst van het ziekteverloop, ontstaat bij SARS-patiënten op langere termijn schade aan het longweefsel. Pulmonaire fibrose, een veelvoorkomende complicatie van SARS-coronavirussen, brengt mee dat 8 maanden na het herstel nog verdikkingen in het longweefsel worden gezien en dat de longcapaciteit permanent slechter is. Ook andere organen, waaronder de nieren en het hart, kunnen schade oplopen door besmetting met SARS-CoV-2.

Gezien deze complicaties is een bewust blootstelling aan het virus om "immuniteit" op te bouwen, een kansloze doelstelling. Gedoseerde blootstelling kan niet, omdat niet bekend is of een veilige minimale virale lading bestaat die bij kan dragen aan het opbouwen van immuniteit. Als één van deze voorwaarden niet kan worden vervuld (veilige dosis of effectiviteit voor het bereiken van immuniteit), dan is blootstelling aan het virus geen mogelijkheid.  

Waar het nu op aankomt
Dat gewacht moet worden tot het moment dat een vaccin beschikbaar is, is niet juist. Het treffen van maatregelen om verspreiding van het virus aan banden te leggen is nodig om:
- een geschikte behandeling voor tromboinflammatie als gevolg van SARS-CoV-2 beschikbaar te maken;
- immunomodulatie te ontwikkelen zonder het immuunsysteem zodanig plat te leggen dat het lichaam het virus niet meer kan bestrijden;
- andere behandelmethoden beschikbaar te hebben om immuniteit op te bouwen, bijvoorbeeld door plasma met antilichamen toe te dienen;
- het gen te vinden dat de cytokinestorm veroorzaakt en het exacte eiwit van SARS-CoV-2 te vinden dat het weefsel beschadigt en de cytokinestorm en tromboinflammatie bij de patiënt verergert.

Pathways to deterioration in SARS-CoV-2 (Part II): coagulation disorders/haemostatic imbalance: COVID is thromboinflammation and pulmonary fibrosis

Hemostatic imbalance in SARS-CoV-2
In this series, I discuss the haemostatic imbalance typically involved in SARS-CoV-2, the virus that causes COVID-19. As can be learned from the SARS-CoV-1 epidemic (2003) and its massive body of knowledge still expanding since 2003, the topic of deterioration should not mainly focus on "SARS as a lung disease"; under no condition the key role of coagulation disorders as a response to inflammation should be underestimated. 

 

The threefold mechanism involved in highly infectious diseases like SARS consists of haemostatic, inflammatory and thrombotic responses, which has become recognized only recently (The era of thromboinflammation: Platelets are dynamic sensors and effector cells during infectious diseases, Frontiers in Immunology, 13 September 2019). In this technical feature, I will discuss:

1.   Determining factors of SARS-CoV-2 associated thromboinflammation;
2.   Mechanisms underlying Thrombocytopenia;
2.1 Von Willebrand Factor- ADAMTS-13 (metalloprotease) mechanism in Thrombotic   Thrombocytopenic Purpura;
3.   Interaction of endothelial damage and platelet consumption;
3.1 NETs: Neutrophil Extracellular Traps: exaggeration of a normal inflammatory process?
4.   Dynamics of (pro)thrombin and fibrin;
4.1 Inflammatory effects of thrombin, promoting microvascular thrombosis, DIC and MOF;
4.2 Fibrinolysis dysregulates the barrier function of fibrin, resulting in accumulation of leukocytes associated with ischemia;
4.3 Urokinase pathway: the role of Serpine1 overexpression in fibrin clotting and inflammation;
4.4 Diffuse Alveolar Damage (DAD);
4.5 Pulmonary fibrosis;
5.   Rare cases of thrombosis: antiphospholipid antibodies (COVID-19);
6.   Treatment with LMWH in hypercoagulant patients

1. Determining factors of SARS-CoV-2 associated thromboinflammation
1.1 Prognostic factors for severity of SARS-1 and SARS-2 cases
In a cohort study concerning 191 severely ill SARS-CoV-2 patients, low lymphocyte counts, severe lymphopenia, leukocytosis (elevated white blood cells), elevated alanine aminotransferase (ALT, severely elevated by a damaged liver), lactate dehydrogenase, high-sensitivity cardiac troponin I, creatinine kinase, elevated D-dimer levels, serum ferritin, IL-6, prolonged prothrombin time and procalcitonin were observed in cases of severe deterioration (Clinical course and risk factors for adult inpatients with COVID-19 in Wuhan: China: a retrospective cohort study, The Lancet, 9 March 2020).

Increased D-dimer levels of more than double the upper limit of normal is a prognostic marker for the risk of venous thromboembolism (Pulmonary embolism in patients with COVID-19: Time to change the paradigm of CT, Thrombosis Research, June 2020). The level of inflammatory IL-6 was reported to be extremely high in critically ill COVID-19 patients (Detectable Serum SARS-CoV-2 viral load (RNAaemia) is closely correlated with drastically elevated interleukin 6 (IL-6) level in critically ill COVID-19 patients, Clinical Infectious Diseases, 17 April 2020).

Similarities are found in a 2004 SARS-Cov study. Predictive factors for respiratory failure were initial absolute neutrophil count (ANC), peak CK level, peak CRP level. peak LDH level and lowest lymphocyte count. Most patients had elevated C-reactive protein levels and lymphopenia, other common abnormal findings included leukopenia, thrombocytopenia and elevated levels of aminotransferase, lactate dehydrogenase and creatinine kinase (Clinical Manifestations, Laboratory Findings and Treatment Outcomes of SARS patients, Emerging Infectious Diseases, May 2004).

In a study involving 85 severe cases of SARS-CoV-2, 81,2% of patients had significantly low eosinophil (white blood cells) on admission, 60% had neutrophils above the normal range, 77,6% of patients had lymphocytes below the normal range and 78,8% of patients had albumin below the normal range. Elevated procalcitonin of more than 0,5 was associated with a death chance of 93%.It has been hypothesized that eosinophilopenia may be related to depletion of CD8 T-cells, rendering SARS-CoV-2 infected patients with lower levels of IL-5, an interleukin involved in proliferation of eosinophils (Clinical features of  85 fatal cases of COVID-19 from Wuhan: A retrospective observational study, 3 April 2020).

1.2 Most typical factors associated with SARS-CoV-2 thrombotic risk 
On admission, patients with a severe progression of SARS-CoV-2 present with elevation of D-dimer levels and fibrin/fibrinogen degradation products, but abnormalities in prothrombin time, partial thromboplastin time and platelet counts are not common. While platelet counts progressively decrease, no bleeding has been reported, regardless of DIC occuring. The hypothesis is posed that this indicates a local expression of DIC, pulmonary vascular thrombosis with subsequent activation of fibrinolysis. Pulmonary thrombosis could induce prothrombotic endothelial dysfunction, which causes an inflammation cascade via complement and cytokine release and blood coagulation with vascular microthrombosis that induces local consumption coagulopathy (Pulmonary thrombosis in 2019-nCoV pneumonia?, Journal of Thrombosis and Haemostasis, 15 April 2020).

Elevations in PT are limited, while aPTT is normal on admission. 10 days after admission, progressive DIC, decreased fibrinogen, increased D-dimer and increased PT have been reported. The level of inflammation on admission is indicated by elevated levels of IL-6, correlating with elevated fibrinogens (COVID-19 and its implications for thrombosis and anticoagulation, American Society for Hematology, 27 April 2020). Thrombocytopenia is reported in 12% of cases. Fibrinolytic shutdown occurs in sepsis. However, the pattern of prothrombic coagulopathy noticed in SARS-CoV-2 patients differs from what is noticed in sepsis, where thrombocyte count is usually decreased (The procoagulant pattern of patients with COVID-19 acute respiratory distress syndrome, Journal of Thrombosis and Haemostasis, 17 April 2020).

1.3 Platelet count: progressive decrease as a precursor for deterioration
Interestingly, some case studies show that on admission of patients with SARS-CoV-2, prothrombin time, platelet count and activated partial thromboplastin time are within the normal range, while D-dimer levels are typically elevated (Acute aorto-iliac and mesenteric arterial thromboses as presenting features of COVID-19, Letter to British Journal of Haematology, 30 April 2020). In 2003, the most prominent finding in severe clinical courses was thrombocytopenia. Platelet count had progressively decreased in 90% of the most severe cases, suggesting the occurrence of disseminated intravascular coagulation following damage to the pulmonary capillary membrane caused by inflammatory platelet aggregation and microthrombus formation (Prognostic factors for SARS: a clinical analysis of 165 cases, Clinical Infectious Diseases, Vol. 38 Issue 4, 15 Februari 2004).
 
Thrombocytopenia (low count of blood platelets that contribute to clotting following bleeding) and elevated D-dimer (fibrin degradation) levels can be explained by excessive activation of coagulation cascade and platelets. In addition to endothelial dysfunction, Von Willebrand Factor (VWF) activation, the release of tissue factors and activation of the Toll-like receptor (TLR) result in homeostatic imbalance. Platelets are key in inciting an inflammatory response through connection of white blood cells and clotting (The era of thromboinflammation: platelets are dynamic sensors and effector cells during infectious diseases, Frontiers in Immunology, 13 September 2019). Upon triggering an inflammatory response, Toll-like receptor 2 (TLR2) is known for its promotion of thrombosis (Stimulation of Toll-like receptor 2 in human platelets induces a thromboinflammatory response through activation of phosphoinositide 3-kinase, Circ Res. (2009) 104:346–54).

Markers to assess severity of SARS-CoV-2/COVID-19

2. Mechanisms underlying thrombocytopenia in SARS-CoV-2
Three mechanisms by which coronaviruses interfere with the hematopoietic system are hypothesized. They may interact.

1. The first hypothesis is that SARS-CoV-2, like other coronaviruses, enter bone marrow cells and platelets through aminopeptidase CD13, present on epithelial cells, subsequently inducing growth inhibition and apoptosis, which leads to inhibition of hematopoiesis (formation of blood cells and platelets), resulting in thrombocytopenia. Activation of the macrophage system (the recruitment and release of inflammatory cytokines) consumes red blood cells. Following the activation of T-cells (transporter cells), an inflammatory soup containing IL-6 causes immune damage to lung tissue. Damage to capillary tissue ruptures megakaryocytes (in which platelets are produced) and blocks platelets, impairing platelet release into the pulmonary system. It should be noted that IL-6, SARS-proteins ORF3a and ORF8a and a variety of cytokines contribute to epithelial and vascular permeability, further increasing the inflammatory cascade.

2. A second hypothesized mechanism is that antibodies are detected on platelet surfaces by the reticuloendothelial system (RES), a part of the immune system located in endothelial tissue. Platelet destruction is a result of platelets being coated by anti-platelet antibodies.

3. A third hypothesis explains common clinical findings in severe cases of SARS-CoV-2. Damaged pulmonary endothelial cells activates platelets in the lungs, aggregating microthrombi, followed by platelet consumption. This seems to be compatible with DIC seen in SARS-CoV-2 cases (Mechanism of thrombocytopenia in COVID-19 patients, Annals of Hematology, 30 March 2020).

Remarkably, when the Von Willebrand Factor is knocked out of mice, adenovirus-induced thrombocytopenia does not occur. Virus-induced thrombocytopenia most likely depends on the interaction between platelets and Von Willebrand Factor, a clotting factor involved in the adherence of platelets to the injured subendohelium (Adenovirus-induced thrombocytopenia: the role of the van Willebrand factor and P-selectin in mediating accelerated platelet clearance, Blood Vol. 109 Issue 7, 1 April 2007).

Activated endothelium upregulates VCAM-1, a protein mediating the adhesion of leukocytes to vascular endothelium. Viral inflammation activates endothelial cells, stimulates the generation of endothelial cell-derived Microparticles (MPs), which are associated with an elevated release of Ultra-Large molecular weight von Willebrand Factor (ULVWF) plasma multimers. Endothelial Microparticles are involved in the regulation of blood flow, inflammation, transport and coagulation (Endothelial Microparticle-Derived Reactive Oxygen Species: Role in endothelial signaling and vascular function, Oxidative Medicine and Cellular Longevity, 2016:5047954).

2.1 Von Willebrand Factor- ADAMTS-13 (metalloprotease) mechanism in Thrombotic Thrombocytopenic Purpura (TTP)
The hemostatic function of the Von Willebrand Factor (VWF), affixed to the subendothelium, is to recruit platelets to injured vessels by binding to the platelet GP Ib-IX-V complex. VWF is stored in megakaryocytes/platelets and in histamine-activated endothelial cells. Following stimulation of the endothelium, Ultra Large multimers of the VWF (ULVWF) are released, binding to platelets firmly.

The release of hyper-reactive ULVWF is moderated by ADAMTS-13, a metalloprotease with thrombospondin motif. ADAMTS-13 cleaves Von Willebrand Factors. If this mechanism fails due to deficiency of ADAMTS-13, thrombotic thrombocytopenic purpura (TTP) occurs. The acquired form of TTP is a result of antibodies directed against ADAMTS-13. An inherent 'weakness' of ADAMTS-13 is the absence of a transmembrane domain; a soluble form of ADAMTS-13 adheres to the A3 domain of VWF (ADAMTS-13 interacts with the endothelial cell-derived Ultra-large von Willebrand Factor, Journal of Biological Chemistry, 8 August 2003, Vol. 278, No.32).

ADAMTS-13 prevents formation of thrombi. This metalloprotease is key in downregulating thrombosis and inflammation. Deficiency of ADAMTS-13 does not constitute TTP or ischemic stroke by itself, but it does induce the prothromobotic state to be enhanced by other ADAM metalloproteases, cytokines and MMPs.

3. Interaction of endothelial damage and platelet consumption
Endothelial damage is associated with Multisystem Organ Failure (MOF), as recently reported in a severe case of COVID-19. Endothelial dysfunction is mentioned as a principal determinant of microvascular dysfunction, by shifting towards enhanced vasoconstriction with subsequent organ ischaemia, inflammation with tissue oedema and a pro-coagulant state. In addition, induction of apoptosis and pyroptosis is hypothesized to have a key role in endothelial cell injury, impairing microcirculation in vascular beds (Endothelial cell infection and endotheliitis in COVID-19, The Lancet, 20 April 2020).

Damaged lung tissue and pulmonary endothelial cells results in platelet aggregation in the lungs, while thrombi formations at the injured site might cause platelet consumption. Long term ventilation may result in pulmonary fibrosis. Further increased platelet consumption and decreased platelet production can result in thrombocytopenia (Thrombocytopenia in patients with SARS, Immune Hematology, April 2005; 10(2)).

The key role of ACE2 receptors as the entry site for SARS-CoV-2 is explanatory. Found on epithelial cells, the ACE2 receptor is a target for inflammatory cell infiltration and indirect endothelial cell apoptosis (cell death). Induction of cell death and pyroptosis (inflammatory programmed or caspase-1 cell death) is associated with microcirculatory dysfunction in vascular beds (Endothelial cell infection and endotheliitis in COVID-19, The Lancet, 20 April 2020).

3.1 NETs: Neutrophil Extracellular Traps: exaggeration of a normal inflammatory process?
Upon pathogen detection, activated platelets promote neutrophil extracellular traps (NETs). NETs contain chromatin, histone and granulate enzymes expelled by activated neutrophils. This process is called NETosis. P-selectin, derived from platelets, facilitates platelet-neutrophil interactions during the early stage of the NETosis process. Platelet GPIba and integrin aIIbb3 are mediators of  NETosis. The release of cathepsin G and serine protease (among which TMPRSS2) by activated neutrophils can cause an exagerrated activation of platelets, coagulation and thrombosis as well as endothelial damage (The era of thromboinflammation: Platelets are dynamic sensors and effector cells during infectious diseases, Frontiers in immunology, 13 September 2019).

Eosinophils, mast cells and macrophages are reported to be capable of releasing NETs. Noteworthy is that neutrophils undergo programmed cell death that must be distinguished from apoptosis and necrotic cell death (Regulation of Innate Immune Responses by Platelets, Frontiers in immunology, 2019; 10: 1320).

Uncontrolled NET formation contributes to arterial and venous thrombosis (Neutrophil Extracellular Traps: Villains and targets in arterial, venous and cancer-associated thrombosis, Arteriosclerosis, Thrombosis and Vascular Biology September 2019, Vol. 39, Issue 9). NET formations were observed at the site of superficially eroded plaques to contribute to thrombus progression (Platelet Interaction with Innate Immune Cells, Karger Transfusion Medicine and Hemotherapy, March 2016; 43(2)).

4. Dynamics of (pro)thrombin and fibrin
4.1 Inflammatory effects of thrombin promoting Microvascular thrombosis, DIC and MOF 
Disseminated intravascular coagulation (DIC) and deep venous thrombosis (DVT) are explicitly mentioned in a 1999 review concerning the mechanism between infectious diseases and coagulation disorders (Review: Infectious Diseases and Coagulation Disorders, The Journal of Infectious Diseases, 1 July 1999). Microvascular thrombi are known to form (Pathogenesis of disseminated intravascular coagulation in sepsis, JAMA, 1993 vol. 270) following the conversion of fibrinogen into fibrin. Microvascular thrombosis, multi-organ failure and hemorrhage occur due to consumption of coagulation factors and activation of the fibrinolytic system. While DIC is associated with both platelet and clotting factor consumption, hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) are not associated with consumption of clotting factors; HUS and TTP are characterized by thrombocytopenia (Par. 2.1 describes the mechanism of VWF and ADAMTS-13 underlying TTP).

The function of prothrombin is to enhance clotting by activating platelets and by converting fibrinogen to fibrin. Although thrombin is a necessary enzyme, thrombin also contributes to further inflammation. The controlling of thrombins by antithrombin III, tissue factor pathway inhibitor and protein C system is compromised by infections such as SARS-CoV-2, promoting microthrombosis, DIC and Multisystem Organ Failure. In addition to Deep Venous Thrombosis, high prevalence of acute pulmonary embolism has been reported (COVID-19 Complicated by Acute Pulmonary Embolism, Radiology: Cardiothoracic Imaging 2020:2(2):e200067).

4.2 Fibrinolysis dysregulates the barrier function of fibrin, resulting in accumulation of leukocytes and neutrophils associated with ischemia
Thrombin activation of endothelial and immune effector cells induces production of growth factors, chemokines and cytokines and alters adhesion. Thrombin stimulation of endothelial cells results in the expression of chemokines including IL-6, IL-8, Platelet Activating Factor (PAT) and MCP-1, Monocyte Chemoattractant Protein, proangiogenic mediators (growth factor-beta), proadhesive factors such as ICAM-1, an intercellular adhesion molecule and P-selectin. PPACK-alpha-thrombin enhances leukocyte recruitment to injured endothelial sites.

The binding of thrombin to platelet GPIbα reduces platelet activation and early leukocyte migration. Fibrin binds alpha-thrombin and acts as a physical barrier to leukocyte migration. Following fibrinolysis, the migration of leukocytes to the site of injury is extensive, suggesting that fibrin retards leukocyte trafficking. Inducing fibrinolysis by rt-PA (Plasminogen Activator) dysregulates the physical barrier activity of fibrin, resulting in enhanced leukocyte migration and neutrophil accumulation, associated with ischemia. The finding that plasminogen activator-induced fibrinolysis induces thromboinflammation by dysregulation of the physical barrier function of fibrin poses a therapeutic target (Thrombin-dependent intravascular leukocyte trafficking regulated by fibrin and the platelet receptors GPIb and PAR4, Nature Communications 6, Article 7835, July 2015).

4.3 Urokinase pathway: the role of Serpine1 overexpression in fibrin accumulation and inflammation
Patients with SARS have significantly lower counts of platelets and lymphocytes (Role of vascular cell adhesion molecules and leukocyte apoptosis in the lymphopenia and thrombocytopenia of patients with SARS, Microbes and Infection, January 2006, 8(1)). It should be noted, though, that it is still unclear whether apoptosis is responsible for the reduction of blood cells. The urokinase pathway could be key. The function of the urokinase system is to regulate fibrinolytic and procoagulative responses to prevent hemorrhage and vascular permeability.

A 2013 study reports that, following SARS-CoV-1 infection, excess fibrin was likely mediated by Serpine1-driven inhibition of the urokinase and tissue type plasminogen activators (PLAU and PLAT) and by blocking of plasmin activity by α2-plasmin inhibitor. SARS dysregulates the profibrinolytic signaling of the urokinase system and increases Serpine1 (also: PAI-1 or Plasminogen activator-1) expression. Fibrin accumulation stimulates profibrotic growth factors and cytokines. Collagen deposition and fibrosis are result of fibroblast. Fibrin and fibrin breakdown products enhance vascular permeability, stimulating migration of inflammatory cells and recruiting neutrophils to the lungs.

PLAT serves as an anticlotting agent
Tissue plasminogen activator (PLAT or tPA), inhibited by Serpine, serves as an anticlotting agent by promoting cleavage of plasminogen into plasmin and stimulating the breakdown of fibrin clots. This explains why lack of Serpine1 leads to hemorrhage (bleeding). Serpine1-knockout mice succumb to SARS-CoV infection faster than control groups, while viral load is unaffected by Serpine1 (Mechanisms of SARS Coronavirus-Induced Acute Lung Injury, American Society for Microbiology, July/August 2013, Volume 4 Issue 4).

A clotting problem due to overexpression of Serpine1
When working properly, the urokinase/coagulation system is balanced: upon detection of damage to the endothelium, cells induce te release of fibrin to the site of injury. During this stage in which the body needs to repair its tissue, Serpine1 prevents the premature breakdown of fibrin. Later on in the process, fibrin needs to be dissolved. This is where tPA/PLAT and plasminogen is bound to fibrin within the thrombus, to protect PLAT against inhibition by Serpine1, enabling plasmin generation and fibrinolysis (breakdown of fibrin clots). Inhibition of Nitric Oxide induces expression of Serpine1, which ultimately results in fibrosis. Overexpression is caused by factors such as the release of inflammatory cytokines, Ang II, Transforming Growth Factor-beta (TGF-beta), aldosterone and lipoproteins (Serpins in thrombosis, hemostasis and fibrinolysis, Journal of Thrombosis and Haemostasis, July 2007; 5).

4.4. Diffuse Alveolar Damage (DAD)
Diffuse Alveolar Damage (DAD) has been observed as a characteristic feature in severe cases of SARS-CoV-2 (Pulmonary Fibrosis and COVID-19: the potential role for antifibrotic therapy, The Lancet Respiratory Medicine, 15 May 2020). Alongside DAD, the presence of microthrombi in pulmonary arteries is reported (Thromboembolic Findings in COVID-19 Autopsies: Pulmonary Thrombosis or Embolism?, Annals of Medicine, 15 May 2020). Acute-phase DAD is characterized by hyaline membranes in the pulmonary alveoli.

In SARS-CoV-1 patients, exudative-phase DAD and increased macrophages, along with edema, hemorrhage and hyaline membrane formation were observed during the early stage of infection. 10 days post-infection, SARS-CoV-1 patients showed DAD occupying up to 100% of the lung, as well as pulmonary fibrosis resulting in long-term consistent loss of lung elasticity.

Hemorrhage indicates premature breakdown of fibrin products, indicated by vascular leakage into alveolar spaces and development of DAD. In mice models with severe cases of SARS-infection, an elevation of serum albumin was observed (Mechanisms of SARS-Coronavirus-Induced Acute Lung Injury, mBio Microbiology ASM, July/August 2013, Vol. 4 Issue 4, e00271-13); see also 'Serum prealbumin is a prognostic indicator in idiopathic pulmonary fibrosis', The Clinical Respiratory Journal, 18 May 2019..

4.5 Pulmonary fibrosis mechanisms
Impairment of STAT1, a key protein in interferon mediated immunity responses, causes SARS-CoV to induce an innate inflammatory cascade, including large amounts of macrophages, neutrophils and eosinophils (white blood cells). Excessive activation of M2 macrophages results in pulmonary fibrosis. In addition, impairment of ACE2 in the Renin-Angiotensin System (RAS) enables Ang II to induce pulmonary hypertension, increasing the risk of pulmonary fibrosis. While the RAS induces neutrophil recruitment to lung tissue, neutrophils, cytokines such as IL-6 and Tumor Necrosis Factor-alpha (TNF) and infected T cells can stimulate pulmonary fibrosis.

Upon detection of fluid, haemorrhage and fibrin in the alveoli, a coagulation cascade increases the release of factors, among which is F10 that cleaves prothrombin into thrombin (Blood clotting Factor 10. Thrombin activates fibrinogen to fibrin (The coagulation factors fibrinogen, thrombin and Factor XII in inflammatory disorders, Frontiers in Immunology, 2018:9:1731). The accumulation of blood clots incites fibrinolysis, a system to clear fibrin formations by cleaving plasmins into plasminogens. These mechanisms underlying fibrosis and fibrin clearance explain why tiny clots are found in tissue from SARS-CoV-2 infected patients. A 2015 review stresses the importance of attention for pulmonary fibrosis in emerging coronavirus infections (Molecular pathology of emerging coronavirus infections, Journal of Pathology 2015: 235).

5. Rare cases of thrombosis: antiphospholipid antibodies in patients with COVID-19
A case study of three patients admitted to the ICU mentions the presence of anticardiolipin IgA antibodies and anti-β₂-glycoprotein I IgA and IgB antibodies. These antiphospholipid antibodies target phospholipid proteins, which may rarely result in thrombosis (Coagulopathy and antiphospholipidantibodies in patients with COVID-19, NEJM, 2020;382:e38).

6. Treatment with LMWH- a Low Molecular Weight Heparin policy in hypercoagulant patients
Anticoagulant treatment with LMWH (heparin) is recommended in the early stage of the disease (Hypothesis for potential pathogenesis of SARS-CoV-2 infection- a review of immune changes in patients with viral pneumonia, Emerging Microbes and Infections, 2020; 9(1)). Heparin has anti-inflammatory properties (Anti-inflammatory effects of heparin and its derivates: a systemic review, Pharmacological Sciences, 12 May 2015). In patients with markedly elevated D-dimer levels, adjustment of LMWH is associated with lower 28-day mortality (Anticoagulant treatment is associated with decreased mortality risk in severe coronavirus disease 2019 patients with coagulopathy, Journal of Thrombosis and Haemostasis, 27 March 2020). The International Society on Thrombosis and Haemostasis' guidance prescribes that all patients requiring hospital admission should receive LMWH (Pulmonary embolism in patients with COVID-19: Time to change the paradigm of computed tomography, Thrombosis Research, June 2020; 190).

The risk of Heparin-Induced Thrombocytopenia (HIT) is a complicating factor. However,  thrombosis-associated thrombocytopenia must be distinguished from HIT. Thrombosis and thrombocytopenia are not paradoxical: what is observed is VTE with consumption of platelets early on in the course of the disease, before the administration of heparin. Even in patients with HIT who need anticoagulants, lepirudin and argatroban are considered safe thrombin inhibitors (Thrombocytopenia due to acute venous thromboembolism and its role in expanding the differential diagnosis of Heparin-Induced Thrombocytopenia, American Journal of Hematology 76:69-73 (2004). In addition to its anticoagulant properties, heparin is mentioned to have antiarrhytmic effects (Anticoagulant and antiarrhytmic effects of heparin in the treatment of COVID-19 patients, Journal of Thrombosis and Haemostasis, 14 May 2020).

 

Next feature: interactions of MMPs, notably MMP9, ADAMTS-13 and VWF in severe SARS-CoV-2 cases
In this "deterioration series", I have mentioned the involvement of Von Willebrand Factor platelet binding to the injured endothelium and the role of ADAMTS-13 in taming down the release of overactive ULVWF into the plasma. There is more to it. Upregulated MMPs are notable contributors to pulmonary fibrosis. In next feature, I will elaborate on the influence of overexpression of MMP-9 on thrombus formation and the role of ADAMTS-13 in thrombosis. While a lack of ADAMTS-13 does not consitute TTP or ischemic stroke by itself, deficiency of ADAMTS-13 induces a prothrombotic state to be enhanced by other metalloproteases and inflammatory cytokines.

Pathways to deterioration in SARS-CoV-2 I: is enhancement of ACE2 in the RAAS system key?

In this "Deterioration series", I will elaborate on the mechanisms underlying the severity of SARS-CoV-2 infection. I made this summary of mechanisms known to contribute to severe complications in COVID-19 patients. There is an ever growing body of knowledge since SARS-CoV-1 (2003), the first SARS-Coronavirus that was associatied with the same mechanisms as are observed in SARS-CoV-2. From this BoK and SARS-CoV-2 reports can be gathered that the following mechanisms are involved: 

Intertwined! Mechanisms of COVID/ SARS-CoV-2 pathogenesis

In this message, I will discuss the influence of SARS-CoV-2 on dysregulation of the Renin-Angiotensin-Aldosterone System (RAS/RAAS). Notably, disruption of ACE2 upregulates Angiotensin II (Ang II). Ang II in its turn increases blood pressure. Actions on the Ang II type I receptor (AT1) adversely affects the vascular wall and enhances oxidative stress, resulting in endothelial damage and endothelial cell apoptosis. Oxidative stress increases expression of plasminogen activator inhibitor type I, resulting in the recruitment and binding of inflammatory cells to the endothelium, which leads to inflammation and thrombosis. Aside from ACE2, there is ACE. Bradykinin is a substrate for ACE. Bradykinin has vasodilator and natriuretic properties. ACE inactivates bradykinin and is therefore known as kininase II. ACE inhibition increases the level of bradykinin, rendering the vasculature permeable. Thus, reduction of ACE adversely affects degradation of bradykinin, increasing the risk of 'leaking' vessels.

How ACE2 is affected by SARS-CoV-2 pathogens

The spike glycoprotein of SARS-CoV-2 is known to use human (and other mammal, with the exception of some rodents) Angiotensin Converting Enzyme 2-receptors. The ACE2-receptor is found in type II alveolar cells (AT2) of the lungs, esophagus epithelial cells, enterocytes from ileum and colon, nasal cavity, vascular endothelia, renal tissue (kidneys), epithelia of the small intestine, testes and on the epithelial cells of oral mucosa (High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa, International Journal of Oral Science, 24 February 2020), as well as in the brain stem and neural cortex. SARS invades ACE2-enriched cells, only to replicate itself very quickly in order to contract other cells. The infection of type II pneumocytes and release of virus in the respiratory tract with close proximity to the pulmonary capillary bed has been thought to allow systemic spread of virus to distant organs (ACE2 Receptor Expression and SARS Infection Depend on Differentiation of Human Airway Epithelia, Journal of Virology, December 2005). 

Uncertainty: is ARDS an adequate classification?
It is yet unclear what exactly constitutes sudden deterioration in critically ill SARS-CoV-2 patients. A sudden deterioration in patients with oxygen deprivation is labelled "Acute Respiratory Distress Syndrome" (ARDS). It is prone to doubt whether ARDS, characterized by bilateral infiltrates, hypoxaemia and dyspnea (Acute Lung Injury and ARDS: Pathophysiology and Treatment, Missouri Medicine, Jul-Aug 2010), is an adequate classification for the pathophysiology associated with SARS-CoV-2 (COVID-19 Pneumonia: ARDS or not?, Critical Care 24, Article number 154 (2020)). In general, the role of specific pathogens for development of ARDS is considered to be difficult to assess (Acute Respiratory Distress Syndrome and Pneumonia: A Comprehensive Review of Clinical Data, Clinical Infectious Diseases, Vol. 43 Issue 6, 15 September 2006).

Pathological findings of an early COVID case study: overactivation of T cells
In one case concerning pathological findings of COVID-19 associated with ARDS, oxygen saturation remained above 95% until the fourteenth day of illness. Day 14 of the illness, hypoxaemia worsened and oxygen saturation dropped below 60%, followed by cardiac arrest. Bilateral diffuse alveolar damage with cellular fibromyxoid exudates was observed, as well as peeling of pneumocytes and hyaline membrane formation in the right lung; the left lung displayed pulmonary oedema with hyaline formation. These findings were regarded suggestive of early-onset ARDS. Mononuclear inflammatory infiltrates dominated by lymphocytes were seen in both lungs. One of major contributions to deterioration in this case was likely an overactivation of T cells. While peripheral CD4 and CD8 T cells were reduced, they were hyperactivated. High proportions of HLA-DR (presenting antigen and providing a ligand to T cells) and CD38 (glycoprotein on the surface of immune cells) were seen. Concentrations of CCR6+ Th17 (T-helper cells) were high, CD8 T cells were found to harbor high concentrations of cytotoxic granulates (Pathological findings of COVID-19 associated with ARDS, The Lancet, February 18 2020). Similar observations were made in a 2005 study (ACE2 Receptor Expression and SARS Infection Depend on Differentiation of Human Airway Epithelia, Journal of Virology, December 2005).

Cytokine storm and invasion of the Central Nervous System
Cytokine storm, an overreaction of the hosts' immune system, is coined to explain the mechanisms behind sudden deterioration (The cytokine release syndrome of severe COVID-19 and IL-6R antagonist Tocilizumab may be the key, International Journal of Antimicrobial Agents, 29 March 2020; see also Cytokine release syndrome in severe COVID-19, AAAS, 17 April 2020). Another pathway could be the invasion of the Central Nervous System and involving of the brain stem, which could explain patients' oxygen starvation. Both pathways have recently been rejected by researchers, as supplied data is still insufficient to prove that either cytokine storm or CNS involvement plays a major role in oxygen starvation- beside multiple organ failure.

Multi-factor assessment
Foremost, it must be noted that a homogeneous kind of ARDS does not exist. ARDS is a definition to describe acute onset of hypoxaemia (ARDS subphenotypes: Understanding a heterogeneous syndrome, Critical Care, 24 March 2020). Likewise, immunity response in SARS-CoV-2 patients is heterogeneous: treatment of immunity response is not a 'one size fits all' matter. This is even further complicated by the fact that immunomodulation should not impair antiviral activity in the host.

What should and could be learned from SARS-1 (2003) when it comes to assessing medical factors?
A comprehensive, multi-factor assessment could be of use to explain SARS' pathways from invasion to deterioration. The body is a system in which the affected parts each contribute to severity of disease. Immunity and cardiovascular mechanisms are intertwined, therefore these mechanisms cannot be considered to contribute independently to deterioration. These systems are a basic necessity to keep the machine going. I'd say that SARS causes imbalance in homestasis, characterized by ACE2 impairment and imbalance of the RAS, damage to endothelial tissue, instigation of the inflammatory soup/cascade, thrombotic events- given that these mechanisms enhance each other into an overdrive mode. The interplay between inflammation and venous thromboembolism is not simply one of cause and consequence, but an intertwined interaction. For example, see: 'The role of inflammation in Venous Thromboembolism', Frontiers in Pediatrics, 23 May 2018, 2018:6:142. What SARS-CoV-1 has made clear, is that coagulation disorders occur frequently in severe cases. Endothelial damage may be an early stage cause of an inflammation cascade, followed by thrombotic events. In a 2004 SARS review, it was said that the 2003 SARS epidemic experience should warrant increased vigilance against stroke and other thrombotic SARS-related events in future outbreaks of coronaviruses (Large artery ischaemic stroke in SARS, Journal of Neurology 251, October 2004).

I'll get to what has been intriguing me in the context of other disease case studies (cardiovascular) and what caught my attention again during the early phase of the SARS-CoV-2 pandemic: that is the plausible role of ACE2 as part of the RAAS system and the detrimental loss of ACE2 during infection with SARS-CoV-2.

The RAAS system and ACE
The Renin-Angiotensin-Aldosterone System (RAAS) regulates blood pressure and fluid balance. The RAS generates angiotensin II (Ang II), which binds to receptors in the brain, kidneys, vasculature and immune system. Angiotensinogen (Agt) is a substrate of renin. Renin cleaves Agt to Angiotensin I (Ang I), subsequently to be cleaved by ACE to Ang II. Renin is primarily expressed in the kidneys. Mast cells are involved in the release of renin. It was found in 2006 that release of renin by cardiac mast cells can be induced by ischemia (Classical Renin-Angiotensin System in Kidney Physiology, Comprehensive Physiology, Vol. 4 Issue 3, July 2014).

Bradykinin is a substrate for ACE. Bradykinin has vasodilator and natriuretic properties. ACE inactivates bradykinin and is therefore known as kininase II. ACE inhibitors increase the level of bradykinin (Unraveling the pivotal role of Bradykinin in ACE inhibitor activity, American Journal of Cardiovascular Drugs, 3 June 2016). The inhibition of ACE is associated with angioedema (Effect of bradykinin receptor antagonism on ACE inhibitor-associated angioedema, Journal of Allergy and Clinical Immunology, July 2017, Vol. 140 Issue 1).

ACE-AngII should be in balance with ACE2-Ang 1-7
ACE2 cleaves a residue from Ang I to form Ang 1-9 and converts Ang II to vasodilator Ang 1-7 (NCBI: gene ID), which blocks Ang II and inhibits ACE (ACE2, a new regulator of the renin-angiotensin system, Trends in Endocrinology and Metabolism, May 2004). In a 2008 study, it was proposed that loss of ACE2 expression and locally increased Ang II production triggered leakage of pulmonary blood vessels after SARS infection. Penninger mentions the catalytic inactivation of bradykinin metabolites by ACE2 (The discovery of angiotensin-converting enzyme 2 and its role in acute lung injury in mice, Experimental Physiology, 25 April 2008). Impairment of ACE2 during infection with SARS is likely the cause of disproportionate levels of bradykinin, which could lead to leakage.

Ang II is known to play a central role in endothelial dysfunction. Not only does Ang II increase blood pressure via vasoconstriction (the narrowing of blood vessels), actions on the Ang II type I receptor (AT1) adversely affects the vascular wall and enhances oxidative stress, resulting in endothelial damage and endothelial cell apoptosis. Oxidative stress increases expression of plasminogen activator inhibitor type I, resulting in the recruitment and binding of inflammatory cells to the endothelium, which leads to inflammation and thrombosis (A review of the role of bradykinin and nitric oxide in the cardioprotective action of Angiotensin-Converting Enzyme Inhibitors: Focus on Perindopril, Cardiology and Therapy 8, 1 October 2019).

Enhancement of ACE2 could be key (New agents modulating the renin-angiotensin-aldosterone system- Will there be a new therapeutic option?, Experimental Biology and Medicine, 19 July 2016). A recent follow-up of the 2008 study by Penninger proposes human recombinant ACE2 for another mechanism that seems plausible: the 2020 study shows inhibition of the virus by hrsACE2 (Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade hrsACE2, Cell Journal Pre-Proof, April 2020). Previously, a similar therapy was proposed involving recombinant human ACE2, rhACE2, to decrease plasma Ang II levels and increase Ang 1-7 and 1-5 (Recombinant human ACE2: acing out Ang II in ARDS therapy, Critical Care, 13 December 2017).

Next feature
In next feature, I will discuss the prevalence of thrombotic events and pulmonary embolism associated with SARS-CoV-1 (2003) and SARS-CoV-2. Twenty years of SARS has made clear that coagulation disorders occur in SARS cases- even patients who are casually deemed "not to be at risk" are in fact at risk of thrombotic events. Keep in mind that all of these mechanisms, from ACE2 impairment and dysregulation of the RAS, inflammation, thrombosis and thrombocytopenia (coagulation disorders) and cytokine storm are intertwined; these mechanisms and pathologies contribute to deterioration in severe cases of COVID-19.

The RAS and its relation to inflammation and coagulation disorders in COVID-19

 

In summary: slides

 

De onderschatting van aërosole transmissie is in strijd met de basale wetten van de fysica

Waarom is de 1,5 meter afstand-regel onvoldoende?  
De 1,5 meter afstand-regel die door nationale overheden en WHO wordt aangehouden als richtlijn, is gebaseerd op achterhaalde onderzoeken. Richtlijnen die inhouden dat de veilige afstand maximaal 2 meter is, houden er geen rekening mee dat ademhalingsdruppels zich via wolken over langere afstanden verplaatsen. De "1,5 meter-afstand" is gebaseerd op een theorie van Wells uit 1930. De uitleg van het model is dat onderscheid moet worden gemaakt tussen grote en kleine druppels. Grote druppels zouden direct neerdalen. Kleine druppels zouden direct na uitademing/hoesten/niezen verdampen, omdat de omgeving kouder en droger is dan de longen, keel en neus van de persoon die de druppels uitademt. Daarbij worden alleen 'agressieve' vormen van uitademing, zoals hoesten en niezen, tot uitgangspunt genomen. Dit is een te beperkte opvatting: sinds 1946 is algemeen bekend dat ademhalen en praten uitstoot van grote hoeveelheden aërosolen veroorzaken (The size and the duration of air-carriage of respiratory droplets and droplet-nuclei, Journal of Epidemiology and Infection, J.P. Duguid, september 1946).

Druppels vliegen niet geïsoleerd door de lucht, maar via een turbulent gas cloud
Het model voor 1,5 meter afstand, gebaseerd op de theorie van Wells, schiet tekort omdat druppels zich niet zomaar door het luchtledige, ofwel geïsoleerd door de lucht vliegen, maar via een wolk van gassen en vocht worden verplaatst. Het werkelijke mechanisme wordt "multiphase turbulent gas cloud genoemd", waarmee wordt bedoeld dat ademhalingsdruppels, slijm, gassen uit de nabije omgeving en vocht zich verzamelen in de wolk. Afhankelijk van de luchtvochtigheid en temperatuur in de omgeving en de grootte van de virusdragende deeltjes, kan deze wolk wel 8 meter door de lucht reizen. Uiteindelijk dalen deeltjes neer op oppervlakken en blijven zij na evaporatie (verdamping van druppels tot gas) ongeveer 3 uur in de lucht hangen (droplet nuclei of aërosolen).

In een voorpublicatie waarin de aërosole transmissie van SARS-CoV-2 (verspreiding van het virus via de lucht) is onderzocht binnen ziekenhuisafdelingen, wordt vastgesteld dat de gemiddelde afstand van verspreiding van het virus 4 meter is (Aerosol and Surface Distribution of Severe Acute Respiratory Syndrome Coronavirus 2 in Hospital Wards, Wuhan, China, 2020, Emerging Infectious Diseases, Volume 26, Number 7, Juli 2020). De conclusie van deze studie is dat thuisisolatie ongeschikt is, omdat overige gezinsleden niet over professionele beschermingsmaterialen beschikken.

Ademhalen zorgt al voor het verplaatsen van deeltjes over 8 meter afstand
Simpelweg ademen kan ervoor zorgen dat virusdeeltjes zich over een afstand van 8 meter door de lucht verplaatsen. Het is begrijpelijk dat een "8 meter afstand-regel" niet haalbaar is in een drukbevolkte omgeving. Een veilige afstand kan om die reden niet los worden gezien van andere maatregelen. Het dragen van een mondkapje en het desinfecteren van oppervlakken zijn maatregelen die bijdragen aan het beperken van het risico op infectie.

De beperking van epidemiologisch onderzoek (in vergelijking met chemisch, biologisch en aërosolenonderzoek) is dat de reproductiefactor R0 wordt berekend op basis van de aanname dat mensenpopulaties en ademhalingsdruppels homogeen vermengd raken. Dit is een te beperkte berekeningswijze, omdat factoren als mechanische of natuurlijke ventilatie, invloeden van de buitenlucht en UV, temperatuur en luchtvochtigheid, maar ook de combinatie van druppeltransmissie en transmissie via aërosolen moeten worden meegerekend in het vaststellen van de reproductiefactor (What aerosol physics tells us about airborne pathogen transmission, Journal of Aerosol Science and Technology, 31 maart 2020).

1,5 meter afstand: dit model gaat ervan uit dat lucht zich niet verplaatst
Een laatste opmerking over de toereikendheid van een 1,5 meter afstand-samenleving. Sociale distantie van 1,5 meter is gebaseerd op de gedachte dat de lucht zich niet door een ruimte verplaatst. Dit model gaat uit van de 'ideale' homogene vermenging van ademhalingsdruppels en aërosolen en de homogene verdeling van mensenpopulaties in een ruimte ("Wells-Riley": well mixed air, model uit 1934). De 1,5 meter-regel negeert luchtstromen door een kamer. Luchtstromen kunnen virale deeltjes over grote afstand verplaatsen. Zo kan het gebeuren dat mensen die dicht bij elkaar zitten, elkaar niet kunnen besmetten, terwijl iemand op grote afstand door de verplaatsing van virale deeltjes via luchtstromen wél besmet raakt (The coronavirus pandemic and aerosols: Does COVID-19 transmit via expiratory particles?, Journal of Aerosol Science and Technology, 3 april 2020). Bovendien is het mechanisme van dispersie van invloed: door de generatie van en spreiding via aërosolen worden druppels door de ruimte gedistribueerd.

Bron:
Turbulent Gas Clouds and Respiratory Pathogen Emissions: Potential Implications for Reducing Transmission of COVID-19, JAMA Insights, 26 maart 2020

Welke belangrijke onderzoeksvragen moeten nog beantwoord worden?
1. Is aërosole transmissie of transmissie van SARS-CoV-2 via geïnfecteerde oppervlakken, in staat om cellen in vivo te infecteren?
Dit is een belangrijke kwestie, omdat nog niemand de vraag heeft beantwoord of dierlijke cellen daadwerkelijk besmet worden via de lucht of via objecten. Weliswaar hebben Van Doremalen en de onderzoekers aan het Nebraska University Center aangetoond dat het virus 3 uur in de lucht kan blijven hangen en in luchtsamples gemonsterd kan worden, maar deze resultaten zeggen nog niets over de besmetting van een dierlijke cel.

De Duitse viroloog Streeck heeft recentelijk opgemerkt dat niemand via winkelkarretjes of een deurklink besmet raakt. Die stelling heeft hij niet onderbouwd. Het gaat om een hypothese die door sommigen wordt gepresenteerd als 'resultaat'. Zijn veldonderzoek zal over drie weken worden afgerond. Het is onjuist dat Streeck beweert dat het virus niet overleeft op voorwerpen, omdat virale deeltjes bewezen tot 72 uur lang stabiel blijven op oppervlakken. Het contactonderzoek dat door hem is geïnitieerd is weliswaar nuttig om te achterhalen in welke situaties/omgeving personen mogelijk geïnfecteerd zijn, maar contactonderzoek zegt niets over de wijze van transmissie (The coronavirus pandemic and aerosols: Does COVID-19 transmit via expiratory particles?, Journal of Aerosol Science and Technology, 3 april 2020). Onderzoek naar de daadwerkelijke infectie van dierlijke cellen via de lucht en oppervlakken is dus dringend nodig!

2. Wat is de gemiddelde virale lading (viral titer) in het ademhalingsvocht en in de uitgestoten aërosolen?
Om de vorige en volgende vraag te kunnen beantwoorden, zal per onderdeel van het respiratoire systeem (longen, stembanden, keel, neus) de gemiddelde virale lading van het ademhalingsvocht en de door de luchtwegen uitgestoten partikels moeten worden gemonsterd.

3. Wat is de minimale besmettelijke dosis SARS-CoV-2?
De drempelwaarde van de besmettelijke dosis SARS moet worden berekend om te kunnen onderzoeken of dierlijke cellen daadwerkelijk via de verspreiding van aërosolen en via contact met geïnfecteerde oppervlakken plaatsvindt.

4. Hoe moet de "open-air factor" worden meegewogen in het bepalen van de reproductiefactor R0? 
De "open-air factor" OAF refereert aan de invloed van natuurlijke luchtstromen en UV op de destabilisering van virussen en andere infecten. Moderne ziekenhuizen zijn over het algemeen slechter in het beperken van de verspreiding van virussen dan ziekenhuizen uit de periode vóór 1950. Ouderwetse grote ramen die geopend kunnen worden zijn bevorderend voor de beperking van verspreiding van virusdeeltjes in het ziekenhuis (The open-air factor and infection control, Journal of Hospital Infection, 9 april 2019). Met andere woorden: binnen zijn is niet ideaal, slechte ventilatie is helemaal funest. Een studie uit 2013 bevestigt het vermoeden dat moderne ziekenhuizen met kleine ramen (die niet open kunnen) en slechte airco de verspreiding van SARS bespoedigen. Mechanische ventilatie is niet per definitie slechter dan natuurlijke luchtstromen: het gaat erom dat 'cross-ventilatie' wordt bereikt, waarbij de lucht van binnen naar buiten stroomt (Roles of sunlight and natural ventilation for controlling infection: historical and current perspectives, Journal of Hospital Infection 84, 20 juni 2013).

In 2012 is Nederland legendarisch geworden door een ventilatieblunder, waarbij 67% van de mechanisch geventileerde huizen over vieze filters beschikte en meer dan 50% van de woningen van sterk vervuilde lucht bleek te worden voorzien. Grotendeels hing deze ventilatieblunder samen met het feit dat Nederlanders weigeren om zich aan gebruiksvoorschriften te houden.

De reproductiefactor R0 zoals die nu door epidemiologen wordt berekend, moet de OAF meewegen om een realistisch beeld te krijgen van de invloed van mechanische en natuurlijke ventilatiemethoden op transmissie onder grote groepen mensen.

1,5 meter afstand is onvoldoende

Nog te beantwoorden onderzoeksvragen over transmissie van SARS-CoV-2

FAQ Coronavirus on symptoms, viabililty on surfaces and in aerosols, prolonged faecal shedding and the need for serological testing

What symptoms of SARS-CoV-2 are common?
Of 99 patients, 83% presented with fever, 82% with a cough and 31% with shortness of breath. Symptoms such as muscle ache, confusion, headache, sore throat, diarrhoea, nausea and vomiting were also observed. The NHS, RIVM and other health autorities seem to present rhinorrhoea and sneezing as main symptoms of COVID. Rhinorrhoea was observed in 4% of COVID cases (Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, The Lancet, 30 January 2020).

Another case study, Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study, confirms the abovementioned. Of 30 patients, the most common symptom was fever (96%), followed by cough (22%) and dyspnoea (17%). In severe cases, serum alkaline phosphatase was significantly higher than in mild cases. Multifocal ground-glass lung opacities were seen in 74%.

According to a study involving 191 patients the median time from illness onset until discharge is 22 days. The median duration of viral shedding for survivors is 20 days. Among 29 patients receiving lopinavir/ritonavir treatment, the median time of viral shedding is 22 days. In critical patients, the median duration of viral shedding is 24 days, the longest viral shedding observed being 30 days (Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study, The Lancet, published online on 9 March 2020).

How viable is SARS-CoV-2 on materials and in aerosols?
Viability refers to the time a virus can 'survive' on materials and in aerosols. Airborne transmission is the transmission route of viral particles through air. In aerosols, SARS-CoV-2 proved to remain stable up to 3 hours. The virus remains stable for up to 72 hours on plastic, up to 48 hours on stainless steel,  24 hours on cardboard and 4 hours on copper (Aerosol and Surface Stability of SARS-CoV-2 as compared to SARS-CoV-1, The New England Journal of Medicine, 17 March 2020).

Air samples taken from airborne infection isolation rooms in Singapore were all negative (Air, Surface, Environmental and Personal Protective Equipment Contamination by SARS-CoV-2 From a Symptomatic Patient, JAMA, 4 March 2020), indicating that inducing air flow by regular ventilation of rooms is important. A preprint claims that air samples taken at the Nebraska Medical Center contained SARS-CoV-2. The highest concentration was recovered from an air handling grate. Air samples were found to be 63.2% positive by RT-PCR, while none of the patients were observed to cough. The highest airborne concentration was observed while a patient was receiving oxygen through a nasal cannula (Transmission potential of SARS-CoV-2 in viral shedding observed at the University of Nebraska Medical Center, preprint available via MedRxiv).

Caution is recommended. What has to be proven yet is:
1. The infection of in vivo cells through airborne transmission;
2. Given that airborne transmission proves to be a route to infect hosts' cells, how concentration of viable loads, expelled through aerosols, relates to severity of SARS-CoV-2.

Why is hand hygiene important?
The fecal-oral route and respiratory droplet-oral route are two possible transmission routes. Note that contamination through surfaces and faeces is not the only transmission route, but the fecal route must not be understated as a shedding route of SARS-CoV-2 (Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes, Emerging Microbes and Infections, 17 February 2020).

One study supplied evidence that some SARS-CoV-2-infected patients can harbor the virus in the intestines during the early stage of the infection, whereas in SARS-infected patients, intestinal infection was observed during the late stage of the infection. A study involving 1099 patients confirms that asymptomatic people might be shedding virus through stool. Eight out of ten children tested positive on rectal swabs, even after testing negative on nasopharyngael surveillance (COVID-19: faecal-oral transmission?, Nature Reviews Gastroenterology & Hepatology, 25 March 2020).

There is evidence for prolonged presence of SARS-CoV-2 in feces. For 5 weeks after the patients' respiratory samples tested negative for viral RNA, virus was detected on faecal sampling. One patient had positive faecal samples for 33 days after testing negative on respiratory sampling; another patient had a prolonged faecal shedding for 47 days after first symptom onset (Prolonged presence of SARS-CoV-2 viral RNA in faecal samples, The Lancet Gastroenteroloy & Hepatology, 19 March 2020).

Is routine surveillance sufficient?
No: routine surveillance typically involves oral sampling, which is not accurate in respect of the difference in viral loads during different stages of a SARS-CoV-2 infection. In a January 2020 molecular study, of the patients with viremia blood, none tested oral or anal positive, indicating that patients should not be discharged on oral negative swabbing alone. This confirms the necessity of serological testing using IgM and IgG (Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes, Emerging Microbes and Infections, 17 February 2020).

Moreover, in a SARS-CoV-2 infected patient with mild symptoms, ASC peaked on day 8 after onset. Antibodies are observed until day 20 (Breadth of concomitant immune responses prior to patient recovery: a case report of non-severe COVID-19, Nature Medicine, 16 March 2020). Rebound of viral load after 5 days was observed in one cohort study (Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2, The Lancet, 23 March 2020).

Thus: a policy that demands discharging a patient on oral negative swabbing is contrary to findings concerning patients testing positive on viremia blood sampling. Add a health policy of anal swabbing and serological testing. One should be aware of the fact that feces of a SARS-CoV-2 patient might test positive until a median of 5 weeks after onset. Awareness and adjustment of health policies should reduce the risk of contamination with faecal shedding of SARS-CoV-2.