First Diagnostic Test for Long Covid, Detecting Spike Protein in Blood, to Launch in September. Or is it ‘Long Vaccine’?

Authors{ WILL JONES 3 SEPTEMBER 2022 

The first diagnostic designed to identify patients with Long Covid has received CE-IVD marking in Europe, meaning it is ready for its formal launch in countries accepting the designation this month. It works by looking for “patterns of inflammatory marker expression” in the blood, and in particular for the Covid spike protein persisting in white blood cells. Business Wire has the story.

The simple blood test can help to objectively diagnose patients suffering from Post-Acute Sequelae of COVID-19 (PASC), commonly known as Long Covid. Developed by IncellDx, the test will be available to prescribers and patients in September through one of the world’s largest providers of diagnostic services.

A CE Mark indicates that the incellKINE Long Covid In Vitro Diagnostic fulfills the requirements of relevant European product directives and meets all the requirements of the relevant recognised European harmonised performance and safety standards…

The CE marking is supported by data from a validation study conducted by one of the world’s largest providers of diagnostic services, showing the test provides greater than 90% accuracy across Covid strains. The test was developed based on clinical studies published in the peer reviewed journal Frontiers in Immunology, which showed that IncellDx researchers generated credible, objective disease scores for Long Covid using machine learning and artificial intelligence to measure and analyse sets of inflammatory markers called cytokines and chemokines. The studies also demonstrated that patients with previous COVID-19 infection and lingering symptoms were found to have a distinct immunologic profile characterised by patterns of inflammatory marker expression. In a subsequent publication, IncellDx found SARS CoV-2 S1 spike protein in monocytic reservoirs of Long Covid patients up to 15 months after acute infection. These papers can be found here and here.

Patterson added, “Long Covid presents a significant diagnostic and treatment challenge for patients. Many of the symptoms that are associated with long Covid, including fatigue, brain fog, shortness of breath, insomnia, and a wide range of cardiovascular issues, can easily be mistaken for other conditions like post-Lyme, ME-CFS, Fibromyalgia, or even the common cold. Having an effective – and importantly an objective – tool to diagnose the condition is absolutely essential. An objective test that can detect immune signatures specific to Long Covid is vital for effective diagnosis and to enable patients to seek effective treatment.”

Patients who have or think they may have Long Covid can learn more and register for a test here.

One question is how it will distinguish Long Covid from ‘Long Vaccine’. The same research team behind this test also investigated whether vaccination produced a similar syndrome characterised by lingering spike protein, immune inflammation and the typical symptoms. They found it did: spike protein persistence from vaccination appeared, they said, to be a “major contributor” of symptoms similar to Long Covid post-vaccination. Further, given that the spike protein “alone delivered by vaccination can cause similar pathologic features”, they concluded it may be a “major contributor” of Long Covid symptoms post-infection as well. In other words, Long Covid after infection may be being caused or prolonged by spike protein from the vaccine rather than the infection. It’s not clear if the new diagnostic test will distinguish between these causes (or if that’s possible).

Neurotoxic amyloidogenic peptides in the proteome of SARS-COV2: potential implications for neurological symptoms in COVID-19

Authors : Mirren CharnleySaba IslamGuneet K. BindraJeremy EngwirdaJulian RatcliffeJiangtao ZhouRaffaele MezzengaMark D. HulettKyunghoon HanJoshua T. Berryman & Nicholas P. Reynolds  Nature Communications volume 13, Article number: 3387 (2022)  July 2022

Abstract

COVID-19 is primarily known as a respiratory disease caused by SARS-CoV-2. However, neurological symptoms such as memory loss, sensory confusion, severe headaches, and even stroke are reported in up to 30% of cases and can persist even after the infection is over (long COVID). These neurological symptoms are thought to be produced by the virus infecting the central nervous system, however we don’t understand the molecular mechanisms triggering them. The neurological effects of COVID-19 share similarities to neurodegenerative diseases in which the presence of cytotoxic aggregated amyloid protein or peptides is a common feature. Following the hypothesis that some neurological symptoms of COVID-19 may also follow an amyloid etiology we identified two peptides from the SARS-CoV-2 proteome that self-assemble into amyloid assemblies. Furthermore, these amyloids were shown to be highly toxic to neuronal cells. We suggest that cytotoxic aggregates of SARS-CoV-2 proteins may trigger neurological symptoms in COVID-19.

Introduction

The disease caused by viral infection with severe acute respiratory syndrome (SARS)-COV-2 is known as COVID-19 and whilst predominantly a respiratory disease affecting the lungs it has a remarkably diverse array of symptoms. These include a range of moderate to severe neurological symptoms reported in as many as 30% of patients, which can persist for up to 6 months after infection1. These symptoms include memory loss, sensory confusion (e.g., previously pleasant smells become fixed as unpleasant), cognitive and psychiatric issues, severe headaches, brain inflammation and haemorrhagic stroke1,2,3,4,5. COVID-19-related anosmia and phantosmia have been shown to correlate with a persistence of virus in the olfactory mucosa and in the olfactory bulb of the brain, and with persistent inflammation; however, negative evidence for continuing viral replication has also been shown for long-term anosmia6. Furthermore, there is evidence that SARS-CoV-2 is neuroinvasive with either the full virus7,8 or viral proteins8 being found in the CNS of mouse models and the post-mortem brain tissue of COVID-19 patients. Whilst the neuroinvasiveness of SARS-CoV-2 is apparent the molecular origin of the associated neurological symptoms is as yet unknown, although they are similar to hallmarks of amyloid-related neurodegenerative diseases such as Alzheimer’s (AD)9,10, and Parkinson’s11. For instance, impaired olfactory identification ability and mild cognitive impairment have also been reported in the early stages of AD and prodromal AD12.

A number of in vitro studies have shown that proteins from SARS-CoV-2 can detrimentally affect a variety of cell types including kidney, liver and immune cells13,14. Furthermore, experiments on brain organoids show that SARS-CoV-2 can infect neuronal cells resulting in cell death15. Combined these papers point to a potential cytotoxic cause of neurological symptoms in COVID-19.

Proteins from the Zika virus16 and also the coronavirus responsible for the SARS outbreak in 2003 (SARS-COV-1)17 have been shown to contain sequences that have a strong tendency to form amyloid assemblies. As the proteome of SARS-CoV-1 and SARS-CoV-2 possess many similarities18, we propose amyloid nanofibrils formed from proteins in SARS-CoV-2 may be implicated in the neurological symptoms in COVID-19. Therefore, amyloid-forming proteins from the SARS-CoV-2 virus in the CNS of COVID-19 infected patients could have similar cytotoxic and inflammatory functions to amyloid assemblies that are the molecular hallmarks of amyloid-related neurodegenerative diseases such as AD (Aβ, Tau) and Parkinson’s (α-synuclein). The worst-case scenario given the present observations is that of the progressive neurological amyloid disease being triggered by COVID-19. To the authors’ knowledge, there has so far been no documented example of this; however, it has been noted that up-regulation of Serum amyloid A protein driven by inflammation in COVID-19 seems like a probable trigger for the systemic (non-neurological) amyloid disease AA amyloidosis19, which is already known to be a concomitant of inflammatory disease in general.

If the proteome of SARS-COV-2 does contain amyloid-forming sequences, this raises the question, what is their function? It is known that viral genomes evolve rapidly and are highly constrained by size; therefore, every component is typically functional either to help the virus replicate or to impede the host immune system. To this end, there are several potential roles for amyloid assemblies in pathogens generally20 and specifically in coronaviruses such as SARS-CoV-2. The simplest is that amyloid is an inflammatory stimulus21, and proinflammatory cytokines can up-regulate the expression of the spike protein receptor ACE-2 such that intercellular transmissibility of SARS-CoV-2 is increased. Alternatively Tayeb-Fligelman et al.22 found that the nucleocapsid protein in SARS-CoV-2, which is responsible for packaging RNA into the virion, contains a number of highly amyloidogenic short peptide sequences within its intrinsically disordered regions22. It has been shown that the self-assembly of these peptides is enhanced in the presence of viral RNA, during liquid–liquid phase separation (LLPS is an important stage in the viral replication cycle)23,24. These findings suggest amyloids may play an important role in RNA binding and packaging during the viral replication cycle. Finally, it is also possible that amyloid assemblies in coronaviruses might have a role in inhibiting the action of the host antiviral response similar to a discussed role for amyloid in other viruses. Pham et al.25 observed that amyloid aggregates from murine cytomegalovirus can interfere with RIPK3 kinase activation and potentially inhibit its antiviral immune signalling capabilities.

In this study, we choose to focus on a selection of proteins from the SARS-CoV-2 proteome known as the open reading frames (ORFs). These ORF proteins were chosen as they have no obvious roles in viral replication26, perhaps freeing them up to have yet uncharacterised roles in disrupting the host antiviral responses. By sequence and length, they appear to be largely unstructured, making them good candidates for amyloid formation in vivo. We performed a bioinformatic screening of the ORF proteins to look for potential amyloidogenic peptide sequences. This analysis was used to select two sub-sequences, one each from ORF6 and ORF10, for synthesis. The synthesised peptides were both found to rapidly self-assemble into amyloid assemblies with a variety of polymorphic morphologies. Cytotoxicity assays on neuronal cell lines showed these peptide assemblies to be highly toxic at concentrations as low as 0.0005% (0.04 mg mL−1).

Since commencing this work, others have found that ORF6 is the most cytotoxic single protein of the SARS-CoV-2 proteome, showing localisation to membranes when overexpressed in human and primate immune cell lines13. In contrast, ORF10 has been reported as an unimportant gene with very low expression and no essential role in virus replication26; however, the functions of immune suppression or inflammation promotion via amyloid formation would be non-essential, if present, and should not necessarily require transcription in large volumes, making ORF10 an intriguing second candidate for the present study. It is also interesting that ORF10 and ORF8 are the only two coded proteins present in SARS-CoV-2 which do not have a homologue in SARS-CoV-127, perhaps suggesting a unique amyloid etiology for COVID-19. While long-term consequences from SARS-CoV-1 infection were severe, including tiredness, depression, and impaired respiration, few or zero unequivocally neurological post-viral symptoms were recorded from the (admittedly quite small) set of documented cases28.

Results and discussion

Amyloid aggregation prediction algorithms identified two short peptides from ORF6 and ORF10 that are likely to form amyloids

Figure 1 shows selected output from bioinformatics tools applied to predict the amyloidogenicity of peptide sequences within larger polypeptides. Application of the ZIPPER tool to ORF6 provides more than ten choices of six-residue windows of the sequence predicted to be highly amyloidogenic (Fig. 1a), while ORF10 shows only three such highly amyloidogenic sequence windows (Fig. 1b). To narrow down our search for candidate peptides we also used the TANGO algorithm, for ORF6 there are two regions that are predicted to be highly aggregation prone, I14LLIIMR and D30YIINLIIKNL. The region I14-R20 overlaps almost perfectly with the hexapeptide I14LLIIM identified by ZIPPER. The region 30–40 also contains multiple hits in ZIPPER, but as this study was limited to two candidate peptides we chose ILLIIM as our first candidate as it closely resembles the sequence ILQINS from Hen Egg White Lysozyme that has previously been seen to be highly amyloidogenic (the mutation TFQINS in human lysozyme is disease-linked)29,30,31. Looking now at the TANGO plots for ORF10 the main aggregation-prone sequence is residues F11TIYSLLLC, although there are no high stability hexapeptides in this sequence predicted by ZIPPER. The octapeptide R24NYIAQVD was chosen due to its zwitterionic residue pair R-D which should strongly enhance interpeptide association, despite being too far apart in the sequence to trigger the highly local bioinformatics algorithms. Encouragingly ZIPPER also predicts the hexapeptide NYIAQV contained within RNYIAQVD to be highly amyloidogenic. Based on the outputs from ZIPPER and TANGO and also on the experience in making and studying amyloid, we selected RNYIAQVD and ILLIIM to be synthesised and their amyloid-forming capability investigated.

figure 1
Fig. 1: Output from amyloid assembly prediction software for SARS-CoV-2 ORF6 and ORF10 sequences.

Nanoscale imaging reveals both peptide sequences self-assemble into polymorphic amyloid assemblies

Atomic force microscopy (AFM) imaging of the two peptide assemblies revealed that both peptides assembled at 37 °C almost immediately at 1 mg mL−1 (Supplementary Figs. 1 and 2) into a highly polymorphic mixture of nanofibrous and crystalline structures (Supplementary Fig.  3). For both peptides, the dominant polymorph was needle-like crystalline assemblies as seen in Fig. 2. In an attempt to ensure any observed polymorphism was not due to a heterogeneous mixture of insoluble peptide seeds we added warm PBS (90 °C) to the lyophilised peptides, and maintained this elevated temperature for at least 3 h in order dissolve as much of the monomeric peptide as possible. Self-assembly was subsequently initiated by slowly reducing the temperature, using a previously developed protocol32. This method produced less polymorphism resulting in the needle-like crystalline polymorph being overwhelmingly dominant, however a number of twisted fibrillar polymorphs were still present for RNYIAQVD (Fig. 2i and Supplementary Fig. 4a). To facilitate repeatable quantitative analysis of the biochemical and biophysical properties of the assemblies we used the slow cooling assembly method for all further experiments.

figure 2
Fig. 2: Atomic force and transmission electron microscopy images of peptide assemblies at 5 mg mL−1 incubated for 24 h.

AFM and transmission electron microscopy (TEM) imaging of assemblies formed at either 1 or 5 mg mL−1 for 24 h revealed that assemblies from both peptides tend to stack on top of each other forming multi-laminar structures (Fig. 2a–d and Supplementary Figs. 5 and 6). Evidence of lateral assembly of the needles was also observed but this appears to happen more frequently in the ILLIIM assemblies (Fig. 2g) compared to RNYIAQVD. ILLIIM tends to form very large (2–3 μm in width) multi-laminar crystalline assemblies (Fig. 2g), whereas RNYIAQVD predominantly forms long linear needle-like structures. The apparent lower tendency of RNYIAQVD to form large two-dimensional lateral assemblies can be explained by the polymorphism seen in this peptide, which does not promote translational symmetry (i.e., extended crystals). Figure 2i and Supplementary Fig. 4a both clearly show that in addition to the flat needle-like crystals seen elsewhere, RNYIAQVD can also form non-planar partially twisted fibrillar assemblies. This polymorphism observed in RNYIAQVD assemblies may reduce the ability of the crystals to laterally associate and stack into multi-laminar species, simply because of a mismatch in planarity between two adjacent assemblies, the molecular basis for this polymorphism is briefly discussed in the next section of the manuscript. AFM was further used to investigate the height of the individual assemblies of both ILLIIM and RNYIAQVD. Figure 2a, b shows a line section through a multi-laminar RNYIAQVD assembly with two distinct layers with a step height of 5.5 nm between each layer. Similarly for ILLIIM (Fig. 2c and Supplementary Fig. 5), we see multi-laminar stacking with individual step heights that vary between 4 and 12 nm. Turning to RNYIAQVD we see single crystals with step heights varying between 5 and 20 nm (Supplementary Fig. 6). Together this heterogeneous distribution of crystal heights provides further evidence for the polymorphic nature of both the ILLIIM and RNYIAQVD assemblies.

Quantitative analysis of the distribution of assembly heights and contour lengths was performed using the freely available software tool FiberApp33. The analysis of assembly height distribution was taken from the z-axis (height) of the AFM images, both peptide assemblies show a heterogeneous distribution of fibril heights due to the previously observed tendency of both assemblies to form polymorphic multi-laminar stacks (Fig. 2f, h). Analysis of the distribution of the contour length of the two assemblies showed a biphasic distribution of lengths for both fibrils with two broad sub-populations centred around 1 and 3 µm (Fig. 2j, l). The sub-population at 3 µm was seen to be much larger for the RNYIAQVD peptide (Fig. 2j) compared to the ILLIIM (Fig. 2l). This population of longer fibrils correlates with the observation from Fig. 2 that for RNYIAQVD longer, thinner assemblies are favoured (self-assembly via fibril extension) over the wider shorter assemblies more commonly seen for ILLIIM (assembly via lateral association of protofilaments). Analysis of the persistence length of the fibrils (Supplementary Fig. 7) showed that whilst both peptides formed very straight linear assemblies, the persistence length of RNYIAQVD (λ = 41.92 µm) is greater than that of ILLIIM (λ = 31.96 µm).

To further investigate the polymorphic nature of the assembly of these peptides, we investigated the structures formed from 1:1 mixtures of the two peptides. Interestingly when mixed prior to assembly the peptides form a wide range of polymorphic structures exceeding that of either peptide assembled individually. Supplementary Fig. 8 shows a selection of some of the polymorphs formed, especially interesting are the large flat structures with well-defined edges that seem almost to interlock (Supplementary Fig. 8c). Such well-ordered 2D crystals were never observed for either peptide individually, and provide clear evidence of co-crystallisation. At this stage we have no evidence for the biological relevance of this co-crystallisation; however, as the ORF proteins from which these peptides are identified are themselves very small proteins (ORF6 is 61 amino acids in length), it is feasible that these small proteins may undergo similar co-crystallisation during their viral replication cycle facilitating an as yet unknown biological function.

X-ray scattering, spectroscopic characterisation, fluorescent microscopy and molecular modelling confirm the amyloid nature of the assemblies

Figure 3a shows the radially averaged 1D small-angle X-ray scattering (SAXS) plots for ILLIIM and RNYIAQVD at the lower concentration studied (at the higher concentration, sedimentation made recording X-ray scattering spectra impossible). In the central part of the scattering curve, the ILLIIM assemblies produced a slope with a q−2 dependence which is consistent with the form factor of an infinite 2D surface30, and is most likely arising due to the broader lateral dimensions observed by AFM and TEM for ILLIIM compared to RNYIAQVD. RNYIAQVD, however, displays a q−4 dependence in the central part of the scattering curve, appearing more towards the high-q limit. Porod’s law indicates that q−4 scaling (at high q but still less than 0.1 Å−1) is consistent with any aggregates having sharp surfaces but does not otherwise specify shape34. The data from the SAXS plots provide supporting evidence that the laterally associated amyloid assemblies seen by AFM and TEM are not artefacts induced either by the dehydration (AFM), applying vacuum conditions (TEM) or the mica (AFM) or carbon (TEM) substrates used, but a genuine structure observed also in bulk.

figure 3
Fig. 3: Spectroscopic analysis of the secondary structure of the peptide assemblies.

Figure 3b shows the CD spectra of mature assemblies, of both peptides. Assemblies of ILLIIM display a quite simple spectrum indicating the dominance of β-sheets, with a minimum between 225 and 230 nm and a strong maximum at 205 nm29. The CD spectra of RNYIAQVD possess a well-defined minimum at 203 nm and a distinct shoulder at around 215 nm.

To further investigate the predicted secondary structure of both peptide assemblies we employed the secondary structure analysis software BeStSel (Supplementary Table 1)35,36. As expected from the classic shape of the spectra the predicted secondary structure of ILLIIM at 5 mg mL−1 is exclusively made up of β-sheets (41.8%) and β-turns (58.2%). At these high concentrations, the composition of these β-sheets is shown to be exclusively left twisted, whilst at lower concentrations (1 mg mL−1) a more complex mixture of right, left and non-twisted (relaxed) β-sheets are predicted. At both concentrations, the CD spectra of RNYIAQVD again suggest the secondary structure is dominated by β-sheets; however, now they appear to be exclusively in the form of higher energy right-twisted β-sheets, similar to that observed in the highly strained structure of other amyloidogenic ultra-short peptides30. This additional strain introduced by β-sheets opposing the left-handed chirality seen in natural amino acids may explain the additional polymorphism and twisted microstructures seen in the AFM and TEM images of the RNYIAQVD assemblies (Fig. 2i and Supplementary Fig. 4a)29. The BeStSel fitting algorithm predicted the remainder of the RNYIAQVD secondary structure is composed of α-helical structure and further backbone conformations that could not be assigned (Supplementary Table 1). Part-helical CD spectra do not necessarily imply helical structure, especially considering that a single octapeptide cannot literally be 19% helix (two residues). Backbone conformation as reported by CD correlates through sheet structure to the assembled tertiary structure but no single level of organisation exclusively dictates any other, this is especially true in the case of coupling the twist of a peptide strand to the overall twist of the aggregate, which can relax to meet surface and shape-driven constraints through intersheet and interchain as well as intrachain degrees of freedom29.

To further investigate the conformation of the amyloid assemblies formed we utilised the conformation-specific antibody A11 and the fluorescent probe thioflavin T (ThT). The former binds specifically to non-fibrillar amyloid oligomers and the latter is a commonly used molecular probe that becomes highly fluorescent when binding to amyloid assemblies37. As expected, when ILLIIM and RNYIAQVD assemblies were stained with ThT both demonstrated clearly visible fluorescent emission at 590 nm, providing further evidence of their amyloid nature (Supplementary Fig. 9a, b). Conversely, A11 exhibited no positive binding; specifically, the level of fluorescence observed was similar to the background staining seen in the negative controls, as confirmed by similar fluorescent intensities for both assemblies (Supplementary Fig. 9e, f) and the negative controls (Supplementary Fig. 9d, h). Higher levels of A11 binding were seen for the positive control that consisted of phenylalanine assemblies known to form oligomeric species38 (Supplementary Fig. 9g), with fluorescent intensities for these assemblies over 4 times greater than for ILLIIM or RNYIAQVD. Combined these data confirm the amyloid nature of the two ORF peptide fragments and suggest that non-fibrillar oligomeric amyloid species are absent.

The amyloid nature of the two assemblies is further confirmed by the wide-angle X-ray scattering (WAXS) spectra (Fig. 3d) of the peptide assemblies which possessed a number of strongly diffracting Bragg peaks. Both peptides have a clear peak at 1.38 Å−1 corresponding to a d-spacing of 4.6 Å which is indicative of an amyloid assembly composed of extended β-sheets39. It is worth noting that the apparent intrastrand spacing of ILLIIM assemblies is very slightly lower than the typically reported values (4.7–4.8 Å). This may be explained by the BeStCell analysis of the ILLIIM assemblies at 1 mg mL−1, which suggests that the β-sheets in these assemblies are composed of a complex mixture of left-handed, strained right-handed and relaxed β-sheets (Supplementary Table 1); therefore, it is perhaps not surprising that the observed average intersheet spacing very slightly differs from that which is commonly reported. Furthermore, Lomont et al.40 report that the observed intrastrand spacing from a range of amyloid crystal structures can vary by as much as 0.45 Å. ILLIIM also has a very strong Bragg reflection at 0.58 Å−1 (11 Å) corresponding to a typical intersheet spacing given moderately bulky hydrophobic sidechains forming a steric zipper. RNYIAQVD has a number of well-defined Bragg peaks between 0.3 and 0.75 Å−1 that are consistent with a mixture of first and second-order reflections corresponding to an amyloid-like 3D symmetry. Typical reflections arising from the combinations of the longer two axes of the unit cell of short peptide amyloid crystals arise in the 0.3–0.75 Å−1 region with a qualitatively similar appearance to the pattern from RNYIAQVD, although in this case the peaks could not be individually assigned.

Discovery of sub-Å resolution structures from solution WAXS is highly challenging; however, given the simple nature of the scattering from the ILLIIM system, it was possible to produce an atomistic model matching the positions of the observed peaks, although not their sharpness. Physically, peak sharpness increases with the ordering length scale, indicating that some structures in the solution were larger than could be managed in the simulation. The sheet-like shape factor and the presence of peaks at roughly 2π/4.6 and 2π/11 Å−1 indicate assembly in solution dominated by the hydrogen bonding axis (with the typical parallel β sheet period of ≈ 4.7 Å) and by a sidechain-sidechain hydrophobic zipper interface. A metastable candidate structure of size 6 × 50 × 1 peptides was constructed following this geometry (see Methods) and found to reproduce the observed WAXS and to fully exclude water at the steric zipper (Fig. 4). The q−2 dependence of ILLIIM scattering at low q in solution (Fig. 3a) is consistent with a 2D sheet-like structure similar to that produced in the modelling. Initial assembly into sheets is also consistent with the eventual formation of multi-laminar structures as shown in the AFM (Fig. 2), as well as with the tendency of ILLIIM in particular to form lateral assemblies of needle microcrystals (Fig. 2g, k vs. e, i). Atomistic details of the interaction of the 2D sheet-like oligomer structure of Fig. 4 with neuronal cell membrane are difficult to predict and would be an interesting subject for further work. However, the juxtaposition of hydrophobic sidechains with polar termini in the ILLIIM fragment (or with titratable residues in the longer fragment E13ILLIIMR, which unfortunately could not be synthesised) has a length of approximately 10 Å, comparable to the polar-hydrophobic-polar length scale of 40 Å for the two leaflets of the eukaryotic cell membrane, indicating a potential for planar aggregates of, in particular, four sheets in thickness (four peptides end-to-end, linked in the middle by salt bridges) to disrupt the cell membrane.

figure 4
Fig. 4: Molecular dynamics simulations of the ORF6 fragment, showing a proposed molecular unit cell that corresponds to the Bragg reflections from the WAXS.

The ThT stain, which becomes highly fluorescent upon binding to β-sheet rich amyloid assemblies was used to assess the assembly kinetics of both ILLIIM and RNYIAQVD (Fig. 3c). Both peptides show rapid kinetics with significant assembly occurring almost instantaneously and reaching a plateau after 30–60 min. Longer amyloidogenic polypeptides typically show a distinct lag phase in their assembly kinetics; however, this was not observed in these sequences. This apparent lack of a lag phase in the assembly kinetics behaviour is typical of amyloidogenic short peptides, which have been previously seen to assemble very rapidly29,41. This is highly likely to be due to a lack of additional non-amyloidogenic amino acid sequences acting as a kinetic barrier to amyloid formation. The ThT signal for ILLIIM at 5 mg mL−1 plateaus at about 300 a.u., this is slightly stronger than the maximum signal generated from mature fibrils of the somewhat homologous peptide ILQINS, which was around 250 a.u29,30,31,42, suggesting that the amyloidogenicity of the two peptides is comparable. RNYIAQVD, whilst showing similar ThT values at low concentrations (1 mg mL−1), generated a ThT signal nearly 3 times as large at 5 mg mL−1 suggesting that the assembly of this peptide is highly concentration dependent. For reasons yet unknown, it seems that RNYIAQVD appears to reach a maximum ThT value and then begin to drop, this can be seen at both concentrations but is most obvious at the higher concentration. This could be due to a reversible self-assembly as seen in other functional amyloids39,41,43,44, or to self-quenching of the amyloid bound aromatic ThT molecules, or simply to a reduction of exposed ThT-binding sites as larger aggregates with a lower surface area to volume ratio come to dominate the solution.

Cytotoxicity of both studied peptides also began to drop slightly (without statistical significance) at the highest concentrations tested (vide infra, Fig. 5), together with the drop in ThT response this supports the existence of a kinetically or thermodynamically available aggregate structure with reduced ‘amyloid activity’. This is reminiscent of strongly amyloid correlated diseases such as AD, where the toxicity of amyloid can vary dramatically, with the relationship between the amount of amyloid deposited to the progress of the disease being idiosyncratic and highly non-linear45. Combined the CD spectroscopy (Fig. 3b), the ThT spectroscopy (Fig. 3c) and confocal microscopy (Supplementary Fig. 9), the presence of the Bragg peaks corresponding to the intra- and inter-β-sheet spacings (Fig. 3d) and the molecular modelling (Fig. 4) confirm beyond doubt the β-sheet rich, amyloid nature of these two fragments.

figure 5
Fig. 5: Cell metabolic and viability assays of ILLIIM and RNYIAQVD assemblies over a range of concentrations.

ILLIIM and RNYIAQVD peptide assemblies are both highly toxic towards the neuroblastoma cell line SH-SY5Y

Given the physical evidence and the discussions referred to in the introduction of various means by which SARS-CoV-2 and other viral infections could enhance their fitness (to the detriment of the host) by the production of amyloidogenic peptides, we hypothesised that the SARS-CoV-2 viral transcript fragments ILLIIM and RNYIAQVD are toxic to human neurons. This is in particular supported by the previously reported neuroinvasive capabilities of SARS-CoV-27,8, the noted similarities of the symptoms to a (hopefully transient form of) AD5 and the previous detection of amyloid assemblies driven by other viruses20. To investigate this further we performed a number of cytotoxicity assays of the two peptide sequences against a human-derived neuroblastoma cell line (SH-SY5Y) often used as a model cell line for studying Parkinson’s and other neurodegenerative diseases46. Using an MTT assay we found that cells grown in the presence of both peptide assemblies possessed much lower viability after 48 h incubation. Concentrations as low as 0.04 and 0.03 mM (for RNYIAQVD and ILLIIM, respectively) were seen to reduce the viability of cultured cells after 48 h to <50% (IC50) compared to the cells  cultured without the peptides (Fig. 5a, b). This toxicity in relation to concentration is similar to that reported for Aβ4247 although expression levels and time-scales (sudden for COVID versus chronic for AD) are likely to be very different.

To gain further insight into the mechanism of cell death occurring in the peptide exposed cells, we performed a detailed flow cytometry analysis using the apoptotic stain Annexin V and the viability dye 7-AAD. Figure 5c shows representative flow cytometry plots; cells can be identified as viable (bottom left quadrant), viable but undergoing early apoptosis (bottom right), non-viable and necrotic (top left) or non-viable due to late-stage apoptosis (top right). The percentages of cells in these quadrants are roughly equal for all conditions tested except in the case of late-stage apoptosis where we see a large increase in the cells exposed to the peptide assemblies (a 6.25-fold increase for RNYIAQVD at 2.5 mg mL−1). Quantification over a range of concentrations showed that on average cells exposed to both ILLIIM and RNYIAQVD had a 3–5-fold increase in late-stage apoptosis compared to SH-SY5Y cells cultured in the absence of peptide assemblies (Fig. 5d, e). No evidence of increasing necrosis was seen in any of the samples, suggesting that the amyloid assemblies are triggering programmed cell death via an apoptotic pathway. This triggering of late-stage apoptosis in the cells was more pronounced for ILLIIM than for RNYIAQVD, showing statistically significant increases in apoptotic cells at concentrations as low as 0.04 mg mL−1 for ILLIIM compared to 0.15 mg mL−1 for RNYIAQVD. This increase in apoptosis down to low concentration provides convincing evidence, especially for ILLIIM, that the amyloid aggregates are responsible for this toxicity, as at these low concentrations we would expect very little un-assembled peptide to exist. The mechanisms of cell death in neurodegenerative diseases are complex and can vary between different diseases48, and here we provide evidence that induction of apoptosis may be an important mechanism of neuronal death in COVID-19. Intriguingly, the conserved protein ORF6 from SARS-CoV-1 (not SARS-CoV-2) has previously been shown to induce apoptosis49. Furthermore, we performed a series of cell counting experiments and demonstrated that after 48 h incubation we saw statistically significant decreases in cell number for both peptides at concentrations as low as 0.04 mg mL−1 for ILLIIM and 0.32 mg mL−1 for RNYIAQVD. These results confirm that in addition to the cytotoxic nature of the peptide assemblies, they significantly reduce cell number especially in the case of ILLIIM. The significant increase in apoptosis and reduction in cell number seen for ILLIIM correlates with the work of Lee et al. who have previously shown that the ORF6 protein (that contains the ILLIIM sequence) is the most cytotoxic protein in the proteome of SARS-CoV-213. Combined with our data, this suggests that this toxicity might be due to the amyloidogenic nature of this short protein.

Previous research has shown that the polymorphism, size distribution and the morphology of amyloid aggregates can have a large influence on their cytotoxicity. Marshall et al.50 showed that a range of crystal-forming assemblies formed from short peptide sequences show surprisingly little toxicity to the same neuroblastoma cell line used in this study. Our TEM and AFM images (Fig. 2) confirm that the assemblies formed by the sequences identified from ORF6 and ORF10 look very similar to the assemblies in Marshall et al.50 but the SARS-CoV-2-related peptides are significantly more toxic, suggesting a specific mechanism of toxicity for these assemblies. Xue et al.51 showed that shorter amyloid assemblies from a range of different proteins/peptides have increased the ability to disrupt the bilayer of unilamellar vesicles and provide a greater cytotoxic effect on neuroblastoma cells. Mocanu et al.52 showed a dose-dependent cytotoxic effect in epithelial cells for long-thin lysozyme amyloid fibrils, and a threshold dependent mechanism for the larger laterally associated fibrils. We see similar effects to both Xue et al. and Mocanu et al. suggesting that the observed toxicity of the assemblies may be related to their aspect ratio. We observed that ILLIIM assemblies are both more toxic, wider (Fig. 2h) and shorter than their RNYIAQVD counterparts (Fig. 2k), this is shown schematically in Fig. 6. Similarly to Mocanu et al.52 we see that the long-thin RNYIAQVD fibrils show a clear dose-dependent increase in apoptosis (Fig. 5e), and the laterally associated ILLIIM fibrils show similarly high levels of apoptosis induction at all concentrations above a threshold of 0.04 mg mL−1 (Fig. 5d).

figure 6
Fig. 6: Amyloid assemblies formed from ORF6 and ORF10 fragments cause cell death to neurons via an apoptotic pathway.

There is a wealth of literature suggesting that in neurodegenerative diseases like Alzheimer’s and Parkinson’s amyloid oligomers are the main toxic culprits and mature amyloid fibrils are a more inert assembly end-point. This is seemingly at odds with our data; however, there have also been multiple studies that show mature assemblies can also display significant toxicity37,53,54. Alternatively, it may be the nature of the amyloids species seen here that differs from amyloids in neurodegenerative diseases; the amyloids seen here appear to be largely crystalline (especially in the case of ILLIIM). Previous work has shown that amyloid crystals are deeper in the free energy landscape compared to twisted protofilaments and amyloid ribbons30,55, representing a global energy minima. AFM and TEM data have shown that these stable amyloid crystals are the dominant polymorph for ILLIIM (Fig. 2c, g, k) and that RNYIAQVD shows examples of higher energy (partially) twisted fibrils (Fig. 2i and Supplementary Figs. 4a and 6). Therefore, we hypothesise that the low energy ILLIIM crystalline assemblies are more slowly metabolised and cells are exposed for longer timeframes to the cytotoxic effect compared to RNYIAQVD assemblies. To date, there have been few investigations into the toxicity of amyloid crystals compared to other more commonly reported amyloid species. For the reasons above, the toxic nature of these amyloid assemblies warrants further investigations into the potential presence of amyloid aggregates from SARS-CoV-2 in the CNS of COVID-19 patients, and the potential role of amyloids in the neurological symptoms observed.

In conclusion, using a bioinformatics approach we identified two strongly amyloidogenic sub-sequences from the ORF6 and ORF10 sections of the SARS-COV-2 proteome. Nanoscale imaging, X-ray scattering, molecular modelling, spectroscopy and kinetic assays revealed that these self-assembled structures are amyloid in nature, and screening against neuronal cells revealed that they are highly toxic (approximately as toxic as the toxic amyloid assemblies in AD) to a cell line frequently used as a neurodegenerative diseases model. The neuroinvasive nature of SARS-COV-2 has been established previously7,8; therefore, it is entirely plausible that amyloid assemblies either from these ORF proteins or other viral proteins could be present in the CNS of COVID-19 patients. The cytotoxicity and protease-resistant structure of these assemblies may result in their persistent presence in the CNS of patients post-infection that could partially explain the lasting neurological symptoms of COVID-19, especially those that are novel in relation to other post-viral syndromes such as that following the original SARS-CoV-1. The outlook in relation to triggering of progressive neurodegenerative disease remains uncertain. Given the typically slow progress of neurodegenerative disease if such a phenomenon exists, it will most probably take some time to become evident epidemiologically.

Methods

Amyloid prediction algorithms

The online amyloid prediction algorithms TANGO and ZIPPER were used to predict peptide sequences with a tendency to form β-rich amyloid assemblies. TANGO is an algorithm that predicts aggregation nucleating regions in unfolded polypeptide chains56. It works on the assumption that the aggregating regions are buried in the hydrophobic core of the natively folded protein. ZIPPER is an algorithm that predicts hexapeptides within larger polypeptide sequences that have a strong energetic drive to form the two complementary β-sheets (known as a steric zipper) that give rise to the spine of an amyloid fibril57. Both methods are physically motivated but rely on statistically determined potentials.

Self-assembly of peptides

NH2-ILLIIM-CO2H and Ac-RNYIAQVD-NH2 (>95% pure) were purchased from GL Biochem Ltd (Shanghai, China). Ideally, it would have been preferred to have both peptides capped (N-terminus: Acetyl and C-terminus: Amide), as they would better represent small fragments of a larger peptide sequence. Due to the fact ILLIIM contains no charged sidechains, synthesising capped sequences to high purity would have been very challenging, therefore only the RNYIAQVD sequence remained capped and the ILLIIM sequence had regular carboxyl and amino termini. To ensure that all peptide seeds were fully dissolved before self-assembly was initiated the peptides were solubilised in warmed PBS (90 °C) at either 1 or 5 mg mL−1 The warmed peptide solutions were vortexed vigorously and held at 90 °C for 3 h to ensure maximum dissolution. After the second round of vortexing, the peptide suspensions were cooled slowly. This protocol has been previously used to maximise a homogenous starting population of monomeric peptide32. Alternatively, self-assembly was carried out at a constant temperature of 37 °C without pre-solubilising the peptides in hot PBS.

Atomic force microscopy (AFM)

AFM imaging was performed on a Bruker Multimode 8 AFM and a Nanoscope V controller. Tapping mode imaging was used throughout, with antimony (n)-doped silicon cantilevers having approximate resonant frequencies of 525 or 150 kHz and spring constants of either 200 or 5 Nm−1 (RTESPA-525, Bruker or RTESPA-150, Bruker). No significant differences were observed between cantilevers. 50 µL aliquots of the peptide (either at 1 or 5 mg mL−1) were drop cast onto freshly cleaved muscovite mica disks (10 mm diameters) and incubated for 20 min before gently rinsing in MQ water and drying under a nitrogen stream. All images were flattened using the first order flattening algorithm in the nanoscope analysis software and no other image processing occurred. Statistical analysis of the AFM images was performed using the open-source software FiberApp33 from datasets of no less than 900 fibres.

Transmission electron microscopy (TEM)

Copper TEM grids with a formvar-carbon support film (GSCU300CC-50, ProSciTech, Qld, Australia) were glow discharged for 60 s in an Emitech k950x with k350 attachment. Then, 5 µL drops of sample suspension were pipetted onto each grid, allowed to adsorb for at least 30 s and blotted with filter paper. Two drops of 2% uranyl acetate were used to negatively stain the particles with excess negative stain removed by blotting with filter paper after 10 s each. Grids were then allowed to dry before imaging. Grids were imaged using a Joel JEM-2100 (JEOL (Australasia) Pty Ltd) transmission electron microscope equipped with a Gatan Orius SC 200 CCD camera (Scitek Australia).

Small- and wide-angle X-ray scattering (SAXS/WAXS)

SAXS/WAXS experiments were performed at room temperature on the SAXS/WAXS beamline at the Australian Synchrotron. Peptide assemblies in PBS prepared at both 1 and 5 mg mL−1 were loaded into a 96-well plate held on a robotically controlled xy stage and transferred to the beamline via a quartz capillary connected to a syringe pump. Data from the 5 mg mL−1 assemblies were discarded due to sedimentation of the assemblies preventing reliable sample transfer into the capillaries. The experiments used a beam wavelength of λ = 1.03320 Å−1 (12.0 keV) with dimensions of 300 µm × 200 µm and a typical flux of 1.2 × 1013 photons per second. 2D diffraction images were collected on a Pilatus 1M detector. SAXS experiments were performed at q ranges between 0.002 and 0.25 Å−1 and WAXS experiments were performed at a q range between 0.1 and 2 Å−1. These overlapping spectra provide a total q range of 0.002–2.2 Å−1. Spectra were recorded under flow (0.15 mL min−1) to prevent X-ray damage from the beam. Multiples of approximately 15 spectra were recorded for each time point (exposure time = 1 s) and averaged spectra are shown after background subtraction against PBS in the same capillary.

Circular dichroism spectroscopy

CD spectroscopy was performed using an AVIV 410-SF CD spectrometer. Spectra were collected between 190 and 260 nm in PBS using 1 mm quartz cuvettes with a step size of 0.5 nm and 2 s averaging time. Data were analysed using the BeStSel (Beta Structure Selection) method of secondary structure determination35.

Atomistic modelling

Atomistic models were constructed using the Nucleic Acid Builder58. Simulations were run in explicit water (TIP3P59) using the ff15ipq forcefield60 and the pmemd time integrator61. In order to hold the unit cell geometry to values consistent with the observed scattering, the alpha carbon of the central residue of each chain was subjected to a restraining force with spring constant 2 kcal mol−1 Å−2. Periodic boundaries were applied to the system such that it formed a truncated octahedron, which was relaxed during equilibration to a volume of 10,310 nm3, giving a density of 0.973 reference with 323,535 water molecules. The system state after 10 ns of equilibration was stripped of water molecules more than 10 Å from any non-hydrogen solute atom, and passed to CRYSOL3 for calculation of orientationally averaged scattering profile given the example state (including the ordered waters from the explicit solvent shell, and also including an approximate treatment of ordered water beyond this shell)42,62.

Thioflavin T amyloid kinetic assays

Peptide assemblies were made up to concentrations of 1 or 5 mg mL−1 suspensions containing 25 µM ThT in PBS. The first fluorescence measurement (t = 0) was recorded immediately after sample preparation. All the samples were then stored at room temperature and fluorescence intensity was recorded at different time points. Measurements were performed in triplicate using a ClarioStar fluorimeter equipped with a 96-well plate reader (excitation wavelength: 440 nm, emission wavelength: 482 nm).

Cell line and cultures

Human-derived neuroblastoma cells (SH-SY5Y, ATCC Product Number: CRL-2266) were cultured in DMEM-F12 (Invitrogen) medium supplemented with 10% (v/v) foetal calf serum (FCS), 100 UmL−1 penicillin and 100 µgmL−1 streptomycin (Invitrogen, Carlsbad, CA). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2.

Immunofluorescent and thioflavin T microscopy

ThT staining was performed by incubating the amyloid assemblies with a 25 µM solution of ThT in PBS for 15 min, in an 8-well Labtek II chamber (Nunc). For the antibody stain the same assemblies were incubated in a 1:200 dilution of the A11 polyclonal antibody raised in rabbit (Invitrogen, REF: AHB0052, LOT:VF299837) in 2% BSA in PBS for 1 h. Following this the wells were carefully washed in PBS and a solution of the 2° antibody (Goat-Anti Rabbit IgG-Alexa Fluor 647, Product A21244, Lot #: 1871168, Molecular Probes) at 1:1000 dilution in PBS was incubated with the peptides for 1 h. Finally the assemblies were once again washed in PBS before being imaged via laser scanning confocal microscopy using a FV3000 microscope (Olympus) and 60× objective lens (1.35 NA Oil Plan Apochromat) using the following settings: ThT channel λex = 450 nm, λem = 490 nm, Alexa Fluora 647 channel λex = 650 nm, λem = 665 nm. The same imaging settings were used for all samples and the negative controls (peptide assemblies + 2° antibody only) were used to determine the level of background fluorescence. For positive controls, amyloid assemblies of phenylalanine were used under concentrations known to readily form oligomeric amyloid assemblies38, which were shown here to bind strongly to the A11 antibody (Supplementary Fig. 9e, f).

Cell viability assay

Cells were seeded into 96-well plates at 1 × 105 cells per mL and incubated for 24 h to ensure good attachment to the surface. A stock solution of peptide assemblies (10 mg mL−1) was serially diluted into DMEM-F12 media 2.5–0.02 mg mL−1 or 3.3–0.027 mM for ILLIIM and 2.45–0.02 mM for RNYIAQVD) and seeded onto the SH-SY5Y cells and incubated for 48 h, cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) as described previously63. Equivalent MTT assays were performed on cells cultured in the same ratios of PBS to media, but in the absence of peptide, assemblies to confirm that the culture conditions were non-toxic (Supplementary Fig. 10). Absorbance readings of untreated control wells in 100% cell culture media were designated as 100% cell viability. Statistical analysis was performed by one-way ANOVA tests with Tukey comparison in the software GraphPad (Prism) ***p < 0.001. Flow cytometry assays to determine cell viability were performed in a similar manner to the MTT assays. Briefly, to determine the effect of the peptides on cellular viability SH-SY5Y cells were cultured in the presence of the peptides for 48 h, harvested and stained with the apoptosis stain Annexin V for 10 min on ice (Cat No. 550474, BD Biosciences, 5 µL in 100 µL of 2% FCS in PBS). Samples were diluted with 300 µL of 2% FCS in PBS and stained with the viability dye 7-AAD (559925 BD Biosciences, 5 µL per sample) and analysed using flow cytometry (FACS Aria III; BD Biosciences). Cell counts were performed manually using a hemocytometer, with tryphan blue to differentiate non-viable cells.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The authors declare that all the data supporting the findings of this study are provided in the Supplementary Information and Source Data file.

Code availability

All code used in this study is either free (NAB 1.3, pymol 2, TANGO 2.2 and ZIPPER) or commercially available (pmemd 19, CRYSOL3 3.0.3).

References

  1. Frontera, J. A. et al. A prospective study of long-term outcomes among hospitalized COVID-19 patients with and without neurological complications. J. Neurol. Sci. 426, 117486 (2021).CAS PubMed PubMed Central Article Google Scholar 
  2. Ellul, M. A. et al. Neurological associations of COVID-19. Lancet Neurol. 19, 767–783 (2020).CAS PubMed PubMed Central Article Google Scholar 
  3. Hampshire, A. et al. Cognitive deficits in people who have recovered from COVID-19. eClinicalMedicine 39, 101044 (2021).
  4. Oxley, T. J. et al. Large-vessel stroke as a presenting feature of COVID-19 in the young. N. Engl. J. Med. 382, e60 (2020).PubMed Article Google Scholar 
  5. Yang, A. C. et al. Dysregulation of brain and choroid plexus cell types in severe COVID-19. Nature 595, 565–571 (2021).ADS CAS PubMed PubMed Central Article Google Scholar 
  6. de Melo, G. D. et al. COVID-19-related anosmia is associated with viral persistence and inflammation in human olfactory epithelium and brain infection in hamsters. Sci. Transl. Med. 13, eabf8396 (2021) .
  7. Matschke, J. et al. Neuropathology of patients with COVID-19 in Germany: a post-mortem case series. Lancet Neurol. 19, 919–929 (2020).CAS PubMed PubMed Central Article Google Scholar 
  8. Song, E. et al. Neuroinvasion of SARS-CoV-2 in human and mouse brainNeuroinvasion of SARS-CoV-2 in humans and mice. J. Exp. Med. 218, e20202135 (2021).CAS PubMed PubMed Central Article Google Scholar 
  9. Kinney, J. W. et al. Inflammation as a central mechanism in Alzheimer’s disease. Alzheimer’s Dement.: Transl. Res. Clin. Interventions 4, 575–590 (2018).Article Google Scholar 
  10. Tolppanen, A.-M. et al. Incidence of stroke in people with Alzheimer disease. Neurology 80, 353 (2013).PubMed Article Google Scholar 
  11. Wang, Q., Liu, Y. & Zhou, J. Neuroinflammation in Parkinson’s disease and its potential as therapeutic target. Transl. Neurodegeneration 4, 19 (2015).Article CAS Google Scholar 
  12. Tu, L. et al. Association of odor identification ability with amyloid-β and tau burden: a systematic review and meta-analysis. Front. Neurosci. 14, 586330 (2020).
  13. Lee, J.-G. et al. Characterization of SARS-CoV-2 proteins reveals Orf6 pathogenicity, subcellular localization, host interactions and attenuation by Selinexor. Cell Biosci. 11, 58 (2021).CAS PubMed PubMed Central Article Google Scholar 
  14. Morais da Silva, M. et al. Cell death mechanisms involved in cell injury caused by SARS-CoV-2. Rev. Med. Virol. 32, e2292 (2022).
  15. Ramani, A. et al. SARS-CoV-2 targets neurons of 3D human brain organoids. EMBO J. 39, e106230 (2020).CAS PubMed PubMed Central Article Google Scholar 
  16. Saumya, K. U., Gadhave, K., Kumar, A. & Giri, R. Zika virus capsid anchor forms cytotoxic amyloid-like fibrils. Virology 560, 8–16 (2021).CAS PubMed Article Google Scholar 
  17. Ghosh, A. et al. Self-assembly of a nine-residue amyloid-forming peptide fragment of SARS corona virus E-protein: mechanism of self aggregation and amyloid-inhibition of hIAPP. Biochemistry 54, 2249–2261 (2015).CAS PubMed Article Google Scholar 
  18. Rangan, R. et al. RNA genome conservation and secondary structure in SARS-CoV-2 and SARS-related viruses: a first look. RNA 26, 937–959 (2020).CAS PubMed PubMed Central Article Google Scholar 
  19. Galkin, A. P. Hypothesis: AA amyloidosis is a factor causing systemic complications after coronavirus disease. Prion 15, 53–55 (2021).CAS PubMed PubMed Central Article Google Scholar 
  20. Shanmugam, N. et al. Microbial functional amyloids serve diverse purposes for structure, adhesion and defence. Biophysical Rev. 11, 287–302 (2019).CAS Article Google Scholar 
  21. Patel, N. S. et al. Inflammatory cytokine levels correlate with amyloid load in transgenic mouse models of Alzheimer’s disease. J. Neuroinflammation 2, 9 (2005).PubMed PubMed Central Article CAS Google Scholar 
  22. Tayeb-Fligelman, E. et al. Inhibition of amyloid formation of the Nucleoprotein of SARS-CoV-2. Preprint at bioRxiv 2021.
  23. Chen, H. et al. Liquid–liquid phase separation by SARS-CoV-2 nucleocapsid protein and RNA. Cell Res. 30, 1143–1145 (2020).CAS PubMed Article Google Scholar 
  24. Savastano, A., Ibáñez de Opakua, A., Rankovic, M. & Zweckstetter, M. Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates. Nat. Commun. 11, 6041 (2020).ADS CAS PubMed PubMed Central Article Google Scholar 
  25. Pham, C. L. et al. Viral M45 and necroptosis-associated proteins form heteromeric amyloid assemblies. EMBO Rep. 20, e46518 (2019).PubMed Article CAS Google Scholar 
  26. Pancer, K. et al. The SARS-CoV-2 ORF10 is not essential in vitro or in vivo in humans. PLOS Pathog. 16, e1008959 (2020).CAS PubMed PubMed Central Article Google Scholar 
  27. Xu, J. et al. Systematic comparison of two animal-to-human transmitted human coronaviruses: SARS-CoV-2 and SARS-CoV. Viruses 12, 244 (2020).
  28. Ngai, J. C. et al. The long-term impact of severe acute respiratory syndrome on pulmonary function, exercise capacity and health status. Respirology 15, 543–550 (2010).PubMed PubMed Central Article Google Scholar 
  29. Lara, C. et al. ILQINS hexapeptide, identified in lysozyme left-handed helical ribbons and nanotubes, forms right-handed helical ribbons and crystals. J. Am. Chem. Soc. 136, 4732–4739 (2014).CAS PubMed Article Google Scholar 
  30. Reynolds, N. P. et al. Competition between crystal and fibril formation in molecular mutations of amyloidogenic peptides. Nat. Commun. 8, 1338 (2017).ADS PubMed PubMed Central Article CAS Google Scholar 
  31. Zanjani, A. A. H. et al. Kinetic control of parallel versus antiparallel amyloid aggregation via shape of the growing aggregate. Sci. Rep. 9, 15987 (2019).ADS PubMed PubMed Central Article CAS Google Scholar 
  32. Zaguri, D. et al. Nanomechanical properties and phase behavior of phenylalanine amyloid ribbon assemblies and amorphous self-healing hydrogels. ACS Appl. Mater. Interfaces 12, 21992–22001 (2020).CAS PubMed Article Google Scholar 
  33. Usov, I. & Mezzenga, R. FiberApp: an open-source software for tracking and analyzing polymers, filaments, biomacromolecules, and fibrous objects. Macromolecules 48, 1269–1280 (2015).ADS CAS Article Google Scholar 
  34. Sinha, S. K., Sirota, E. B., Garoff, S. & Stanley, H. B. X-ray and neutron scattering from rough surfaces. Phys. Rev. B 38, 2297–2311 (1988).ADS CAS Article Google Scholar 
  35. Micsonai, A. et al. BeStSel: a web server for accurate protein secondary structure prediction and fold recognition from the circular dichroism spectra. Nucleic Acids Res. 46, W315–W322 (2018).CAS PubMed PubMed Central Article Google Scholar 
  36. Micsonai, A. et al. Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proc. Natl Acad. Sci. USA 112, E3095–E3103. (2015).CAS PubMed PubMed Central Article Google Scholar 
  37. Adler-Abramovich, L. et al. Phenylalanine assembly into toxic fibrils suggests amyloid etiology in phenylketonuria. Nat. Chem. Biol. 8, 701–706 (2012).CAS PubMed Article Google Scholar 
  38. Do, T. D., Kincannon, W. M. & Bowers, M. T. Phenylalanine oligomers and fibrils: the mechanism of assembly and the importance of tetramers and counterions. J. Am. Chem. Soc. 137, 10080–10083 (2015).CAS PubMed Article Google Scholar 
  39. Dharmadana, D., Reynolds, N. P., Conn, C. E. & Valéry, C. Molecular interactions of amyloid nanofibrils with biological aggregation modifiers: implications for cytotoxicity mechanisms and biomaterial design. Interface Focus 7, 20160160 (2017).PubMed PubMed Central Article Google Scholar 
  40. Lomont, J. P. et al. Not all β-sheets are the same: amyloid infrared spectra, transition dipole strengths, and couplings investigated by 2D IR spectroscopy. J. Phys. Chem. B 121, 8935–8945 (2017).CAS PubMed PubMed Central Article Google Scholar 
  41. Dharmadana, D., Reynolds, N. P., Conn, C. E. & Valéry, C. pH-dependent self-assembly of human neuropeptide hormone GnRH into functional amyloid nanofibrils and hexagonal phases. ACS Appl. Bio Mater. 2, 3601–3606 (2019).CAS PubMed Article Google Scholar 
  42. Zanjani, A. A. H. et al. Amyloid evolution: antiparallel replaced by parallel. Biophysical J. 118, 2526–2536 (2020).ADS CAS Article Google Scholar 
  43. Dharmadana, D. et al. Heparin assisted assembly of somatostatin amyloid nanofibrils results in disordered precipitates by hindrance of protofilaments interactions. Nanoscale 10, 18195–18204 (2018).CAS PubMed Article Google Scholar 
  44. Dharmadana, D. et al. Human neuropeptide substance P self-assembles into semi-flexible nanotubes that can be manipulated for nanotechnology. Nanoscale 12, 22680–22687 (2020).CAS PubMed Article Google Scholar 
  45. Morris, G. P., Clark, I. A. & Vissel, B. Inconsistencies and controversies surrounding the amyloid hypothesis of Alzheimer’s disease. Acta Neuropathologica Commun. 2, 135 (2014).Google Scholar 
  46. Xicoy, H., Wieringa, B. & Martens, G. J. M. The SH-SY5Y cell line in Parkinson’s disease research: a systematic review. Mol. Neurodegeneration 12, 10 (2017).Article CAS Google Scholar 
  47. Krishtal, J. et al. Toxicity of amyloid-β peptides varies depending on differentiation route of SH-SY5Y cells. J. Alzheimers Dis. 71, 879–887 (2019).CAS PubMed Article Google Scholar 
  48. Moujalled, D., Strasser, A. & Liddell, J. R. Molecular mechanisms of cell death in neurological diseases. Cell Death Differ. 28, 2029–2044 (2021).PubMed PubMed Central Article Google Scholar 
  49. Kopecky-Bromberg, S. A. et al. Severe acute respiratory syndrome coronavirus open reading frame (ORF) 3b, ORF 6, and nucleocapsid proteins function as interferon antagonists. J. Virol. 81, 548–557 (2007).CAS PubMed Article Google Scholar 
  50. Marshall, K. E., Marchante, R., Xue, W.-F. & Serpell, L. C. The relationship between amyloid structure and cytotoxicity. Prion 8, 192–196 (2014).CAS PubMed Central Article Google Scholar 
  51. Xue, W.-F. et al. Fibril fragmentation enhances amyloid cytotoxicity. J. Biol. Chem. 284, 34272–34282 (2009).CAS PubMed PubMed Central Article Google Scholar 
  52. Mocanu, M.-M. et al. Polymorphism of hen egg white lysozyme amyloid fibrils influences the cytotoxicity in LLC-PK1 epithelial kidney cells. Int. J. Biol. Macromolecules 65, 176–187 (2014).CAS Article Google Scholar 
  53. Novitskaya, V., Bocharova, O. V., Bronstein, I. & Baskakov, I. V. Amyloid fibrils of mammalian prion protein are highly toxic to cultured cells and primary neurons. J. Biol. Chem. 281, 13828–13836 (2006).CAS PubMed Article Google Scholar 
  54. El Moustaine, D. et al. Amyloid features and neuronal toxicity of mature prion fibrils are highly sensitive to high pressure. J. Biol. Chem. 286, 13448–13459 (2011).CAS PubMed PubMed Central Article Google Scholar 
  55. Adamcik, J. & Mezzenga, R. Amyloid polymorphism in the protein folding and aggregation energy landscape. Angew. Chem. Int. Ed. 57, 8370–8382 (2018).CAS Article Google Scholar 
  56. Fernandez-Escamilla, A.-M., Rousseau, F., Schymkowitz, J. & Serrano, L. Prediction of sequence-dependent and mutational effects on the aggregation of peptides and proteins. Nat. Biotechnol. 22, 1302–1306 (2004).CAS PubMed Article Google Scholar 
  57. Thompson, M. J. et al. The 3D profile method for identifying fibril-forming segments of proteins. Proc. Natl Acad. Sci. USA 103, 4074–4078 (2006).ADS CAS PubMed PubMed Central Article Google Scholar 
  58. Macke, T. J. & Case, D. A. Modeling unusual nucleic acid structures. In Molecular Modeling of Nucleic Acids (eds Leontis, N. B. & SantaLucia, J.) Vol. 682, 379–393 (American Chemical Society, 1997).
  59. Jorgensen, W. L. et al. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983).ADS CAS Article Google Scholar 
  60. Debiec, K. T. et al. Further along the road less traveled: AMBER ff15ipq, an original protein force field built on a self-consistent physical model. J. Chem. Theory Comput. 12, 3926–3947 (2016).CAS PubMed PubMed Central Article Google Scholar 
  61. Salomon-Ferrer, R. et al. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 2. Explicit solvent particle mesh Ewald. J. Chem. Theory Comput. 9, 3878–3888 (2013).CAS PubMed Article Google Scholar 
  62. Franke, D. et al. ATSAS 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions. J. Appl. Crystallogr. 50, 1212–1225 (2017).CAS PubMed PubMed Central Article Google Scholar 
  63. Poon, I. K. H. et al. Phosphoinositide-mediated oligomerization of a defensin induces cell lysis. eLife 3, e01808 (2014).PubMed PubMed Central Article CAS Google Scholar 
  64. Varrette, S., Bouvry, P., Cartiaux, H. & Georgatos, F. Management of an academic HPC cluster: the UL experience. In 2014 International Conference on High Performance Computing & Simulation (HPCS), 21–25 July 2014, 959–967 (2014).

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Acknowledgements

N.P.R. would like to acknowledge The La Trobe Institute of Molecular Sciences (LIMS) for the receipt of a Nicholas Hoogenraad fellowship, and the CASS foundation for partially funding this work through a philanthropic grant (#10053, ‘Determining the role of protein aggregation in COVID-19’). N.P.R. would also like to acknowledge that Fig. 6 was created using Biorender.com. The authors thank Dr Susi Seibt for assistance on the SAXS/WAXS beamline at the Australian Synchrotron. This research was undertaken, in part, on the SAXS/WAXS beamline at the Australian Synchrotron, part of ANSTO. Molecular dynamics calculations made use of the HPC service of the University of Luxembourg64. The project was part-funded by grant C20/MS/14588607 of the Fonds Nationale de la Recherche, Luxembourg.

Nature Communications volume 13, Article number: 3387 (2022) 

COVID-19: A Global Threat to the Nervous System

Authors: Igor J. Koralnik MD,Kenneth L. Tyler MD 07 June 2020 Annals of NeurologyVolume 88, Issue 1 p. 1-11

Abstract

In less than 6 months, the severe acute respiratory syndrome-coronavirus type 2 (SARS-CoV-2) has spread worldwide infecting nearly 6 million people and killing over 350,000. Initially thought to be restricted to the respiratory system, we now understand that coronavirus disease 2019 (COVID-19) also involves multiple other organs, including the central and peripheral nervous system. The number of recognized neurologic manifestations of SARS-CoV-2 infection is rapidly accumulating. These may result from a variety of mechanisms, including virus-induced hyperinflammatory and hypercoagulable states, direct virus infection of the central nervous system (CNS), and postinfectious immune mediated processes. Example of COVID-19 CNS disease include encephalopathy, encephalitis, acute disseminated encephalomyelitis, meningitis, ischemic and hemorrhagic stroke, venous sinus thrombosis, and endothelialitis. In the peripheral nervous system, COVID-19 is associated with dysfunction of smell and taste, muscle injury, the Guillain-Barre syndrome, and its variants. Due to its worldwide distribution and multifactorial pathogenic mechanisms, COVID-19 poses a global threat to the entire nervous system. Although our understanding of SARS-CoV-2 neuropathogenesis is still incomplete and our knowledge is evolving rapidly, we hope that this review will provide a useful framework and help neurologists in understanding the many neurologic facets of COVID-19. ANN NEUROL 2020;88:1–11 ANN NEUROL 2020;88:1–11

The novel coronavirus, now called severe acute respiratory syndrome-coronavirus type 2 (SARS-CoV-2), is the agent of coronavirus disease 2019 (COVID-19), that was first diagnosed on December 8, 2019, in a patient in the city of Wuhan in central China. Common symptoms of COVID-19 include fevercoughfatigue, and shortness of breath. Whereas most affected individuals have no or minor symptoms, some go on to develop pneumonia, acute respiratory distress syndrome (ARDS), and succumb from multiple organ failure. On January 30, 2020, the World Health Organization (WHO) declared it a Public Health Emergency of international concern. It has been estimated that the number of infected individuals during the early epidemic doubled every 2.4 days, and the R0 value, or number of people that can be infected by a single individual, may be as high as 4.7 to 6.6.1 After spreading throughout China, the disease took hold in Europe and the United States, and in view of this alarming development and the rapid growth of cases, public health officials in many jurisdictions ordered people to shelter in place beginning with the state of California on March 19, 2020. As of May 29, 2020, there have been 5.88 million confirmed cases in 188 countries and 363,000 reported deaths, and most countries are in various phases of relaxing quarantine requirements while continuing some social distancing measures.

What are coronaviruses and what makes SARS-CoV-2 so contagious? Coronaviruses, which have a diameter of approximately 100 nm, are named after their crown-like appearance on electron microscopy. They infect many animal species and are part of the family of Coronaviridae that contain four distinct Genera. Coronaviruses are positive strand, single stranded ribonucleic acid (+ss-RNA) viruses. They have the largest genome of all RNA viruses, approximately 30 kilobases in length. The full sequence of SARS-CoV-2 was published on January 7, 2020, and revealed that it is was a β-coronavirus, similar to other human coronaviruses that are responsible for 15% of all cases of acute viral nasopharyngitis, also known as “common cold.”2 However, SARS-CoV-2 contains unique sequences, including a polybasic cleavage site in the spike protein, which is a potential determinant of increased transmissibility.3

Coronaviruses have caused deadly outbreaks in the past. The first one caused by SARS-CoV, occurred in China in 2003 and affected approximately 8,000 people, with a 10% mortality rate. The Middle-East Respiratory Syndrome (MERS) outbreak began in Saudi Arabia in 2012, and affected 2,500 individuals with a 35% mortality rate. SARS-CoV-2 has approximately 80% sequence homology with SARS-CoV, but 96% homology with a bat coronavirus and 92% with a pangolin coronavirus, suggesting it arouse in animals and then spread between species to humans. The spike protein of SARS-CoV-2 binds to its cellular receptor, the angiotensin converting enzyme 2 (ACE2), which also acts as receptor for SARS-CoV. Viral entry occurs after proteolytic cleavage of the spike protein by the transmembrane protease TMPRSS2. ACE2 is expressed abundantly in lung alveolar cells, but also in many cell types and organs in the body, including the cerebral cortex, digestive tract, kidney, gallbladder, testis, and adrenal gland.4

Experience with the neurological complications of MERS and SARS provides a framework for considering both reported and potential neurological complications with SARS-CoV-2 and COVID-19.510 In both MERS and SARS, significant neurological complications were fortunately extremely rare. Reported cases of neurological disease suggests a minimum incidence of ~1:200 cases (MERS) -1:1,000 cases (SARS). It is important to recognize, however, that the total number of confirmed cases of MERS and SARS together is only ~10,500 cases. It is likely that the sheer numeracy of COVID-19 compared to MERS and SARS, with nearly 6 million cases reported worldwide to date, will bring out a broader spectrum of neurological manifestations. In MERS and SARS neurological disease could be considered in three major categories: (1) the neurological consequences of the associated pulmonary and systemic diseases, including encephalopathy and stroke, (2) direct central nervous system (CNS) invasion by virus, including encephalitis, and (3) postinfectious and potentially immune-mediated complications, including Guillain-Barre syndrome (GBS) and its variants and acute disseminated encephalomyelitis (ADEM).

Neurological Complications of Systemic COVID-19

In a review of 214 patients hospitalized in 3 dedicated COVID-19 hospitals in Wuhan, China, 36% of patients had nerurologic.11 These were further subdivided into those thought to reflect CNS, peripheral nervous system (PNS), and skeletal muscle injury. Overall, 25% of patients had symptoms considered as evidence of CNS dysfunction, including dizziness (17%), headache (13%), impaired consciousness (7.5%), acute cerebrovascular disease (3%), ataxia (0.5%), and seizures (0.5%). Confirming this low incidence of seizures, no cases of status epilepticus or new onset seizures were reported in a large cohort of over 304 hospitalized patients with COVID-19 in Hubei Province, China,12 although there have been isolated case reports describing seizures at presentation in both adult and pediatric patients with COVID-19.1314

In the series by Mao and colleagues,11 the patients were subdivided based on the severity of their pneumonia and pulmonary impairment, and among those with “severe” disease (n = 88) the incidence of CNS symptoms was higher (31%) compared to the non-severe group (21%), although the results were not statistically significant (p = 0.09). Although all the categorized CNS symptoms occurred more frequently in patients with severe disease compared to non-severe disease, only impaired consciousness (15% in severe vs 2% in non-severe, p < 0.001) and acute cerebrovascular disease (5.7% vs 0.8%; p = 0.03) were significantly different between the two groups. Diagnostic studies were limited, but the impairment of consciousness seems most consistent with encephalopathy. Not surprisingly, when compared to those with non-severe disease, the severe cohort were older (58 ± 15 years vs 49 ± 15 years), and more likely to have comorbidities, including hypertension, diabetes, malignancy, cardiac, cerebrovascular, or kidney disease (48% vs 33%; p = 0.03). The severe group also had more evidence of systemic inflammation, including elevated C-reactive protein (CRP; median 37 mg/L) and D-dimer (median 0.9 mg/L) compared to non-severe cases, and were also more likely to have evidence of hepatic (elevated alanine and aspartate aminotransferases) and renal (elevated BUN and creatinine) dysfunction.

A second survey of 58 hospitalized patients (median age 63 years) with COVID-19 ARDS at Strasbourg University Hospital found that 69% of patients had agitation, 67% had corticospinal tract signs, and 36% had a “dysexecutive” syndrome with difficulty in concentration, attention, orientation, and following commands.15 All patients studied (11/11) had evidence of frontal hypoperfusion on arterial spin label and dynamic susceptibility-weighted perfusion magnetic resonance imaging (MRI). Only seven patients had a cerebrospinal fluid (CSF) examination, none had a pleocytosis, and none had SARS-CoV-2 RNA detected by reverse transcriptase-polymerase chain reaction (RT-PCR). One patient did have elevated immunoglobulin G (IgG) levels and “mildly” elevated total protein. CSF specific oligoclonal bands (OCBs) were not detected, but one patient had “mirror pattern” OCBs in CSF and serum.

In a study of MRI abnormalities in patients in the intensive care unit (ICU) with COVID-19, 21% (50/235) of patients developed neurological symptoms.16 In this group of neurologically symptomatic patients, only 27 had MRIs performed, and of these 44% (12/27) had new acute findings. Surprisingly, 56% (15/27) had no new MRI changes. The most common new abnormalities were multifocal areas of cortical fluid-attenuated inversion recovery (FLAIR) signal (10/12), accompanied in three patients by areas of increased FLAIR signal in the subcortical and deep white matter. One patient each had new transverse sinus thrombosis and acute middle cerebral artery infarction. Five of the 10 patients with cortical FLAIR abnormalities had a CSF examination, and none of these patients had a pleocytosis elevated IgG index, or OCBs (0/3 tested), although 4 patients had an elevated protein (mean 80 mg/dl; range = 60–110). RT-PCR for SARS-CoV-2 was negative in all 5 cases tested. In another MRI series of critically ill patients on mechanical ventilation, many were found to have confluent T2 hyperintensities and restricted diffusion in the deep and subcortical white matter, in some cases, accompanied by punctate microhemorrhages in the juxtacortical and callosal white matter that resembled findings seen in delayed post-hypoxic leukoencephalopathy.17

The mechanism of encephalopathy in COVID-19 remains to be determined. From available studies, COVID-19 encephalopathy seems to be more common in patients with more severe disease, associated comorbidities, evidence of multi-organ system dysfunction, including hypoxemia, and renal and hepatic impairment, and elevated markers of systemic inflammation. Virus is not detected in CSF by RT-PCR and pleocytosis is usually absent. Some patients may have altered perfusion detectable by MRI, others have leukoencephalopathy with or without punctate microhemorrhages. This group needs to be distinguished from patients with encephalitis (who have a pleocytosis) and postinfectious immune-mediated encephalitis (see below).

In a series of five consecutive patients with COVID-19 with delayed awakening post-mechanical ventilation for ARDS, MRI showed enhancement of the wall of basal skull arteries without enlargement of the vessel wall or stenosis. Toxic-metabolic derangements and seizures were ruled out, CSF SARS-CoV-2 RT-PCR was negative in all and they showed marked improvement in alertness 48 to 72 hours after treatment with methylprednisolone 0.5 g/days iv for 5 days. These findings suggest that an endothelialitis rather than a vasculitis was responsible for the encephalopathy.18 Direct infection of endothelial cells by SARS-CoV-2 and associated endothelial inflammation has been demonstrated histologically in postmortem specimens from a variety of organs, which did not include the brain.19

However, in an autopsy series, including examination of the brain, of 20 patients with COVID-19, six had microthrombi and acute infarctions and two focal parenchymal infiltrates of T-lymphocytes, whereas the others mainly had minimal inflammation and slight neuronal loss without acute hypoxic–ischemic changes in most cases. There was no evidence of meningoencephalitis, microglial nodules, or viral inclusions, including in the olfactory bulbs and brainstem, and no demyelination. ACE2 was expressed in lung and brain capillaries. All cases had evidence of systemic inflammation.20

A second major manifestation of systemic COVID-19 disease is acute cerebrovascular disease. In the study by Mao and colleagues,11 this was present in 6 of the 214 (3%) hospitalized cases, but 5 of the 6 events occurred in those with severe disease (incidence 6%; p = 0.03 vs non-severe disease).11 Five of the six reported events were ischemic strokes, and one was hemorrhagic. In the review of cases at Strasbourg University Hospital,15 3 of 13 (23%) had cerebral ischemic stroke. In a single center retrospective study from China of 221 patients hospitalized with COVID-19, 13 had acute strokes, including 11 ischemic, 1 hemorrhagic, and 1 venous sinus thrombosis.21 The stroke patients were older, had more comorbidities, including diabetes, hypertension, and a prior stroke, and elevated inflammatory markers, including D-dimer and CRP. Another review of six consecutive patients with COVID-19 admitted to the National Hospital in Queen Square with stroke, noted that occlusions typically involved large vessels and often occurred in multiple vascular territories.22 In 5 of 6 cases, the strokes occurred 8 to 24 days after onset of COVID-19 symptoms. All patients had a highly prothrombotic state with very high D-dimer levels and elevated ferritin. Five of the six patients had detectable lupus anticoagulant, suggesting another potential prothrombotic mechanism for stroke in COVID-19. Anticardiolipin IgA and antiphospholipid IgA and IgM antibodies directed against β2-glycoprotein-1 were also found in three patients with COVID-associated multiple territory large vessel infarctions.23 Finally, a postmortem MRI study showed subcortical micro- and macro-bleeds (two decedents), cortico-subcortical edematous changes evocative of posterior reversible encephalopathy syndrome (PRES; one decedent), and nonspecific deep white matter changes (one decedent).24

Although initial reports emphasized acute cerebrovascular disease in older patients with COVID-19, a recent report described five cases of large vessel stroke as a presenting feature of COVID-19 in younger individuals, two of whom lacked classic stroke risk factors.25 These patients ranged in age from 33 to 49 years. Two of the five patients had diabetes, one of whom had had a mild prior stroke, and one had hypertension and dyslipidemia. The infarcts involved large vessel territories, including the middle cerebral artery (3), posterior cerebral artery (1), and internal carotid artery (1). Two patients had preceding COVID-19 symptoms, including fever, chills, cough, and headache; one patient had only lethargy. Surprisingly, two of the five patients had no COVID-19-related symptoms preceding their stroke presentation. These five patients had elevated prothrombin (range = 12.8–15.2 seconds) and activated partial thromboplastin times (range = 25–42.7 seconds), elevated fibrinogen (range = 370–739 mg/dl), D-dimer (range = 52–13,800 ng/ml) and ferritin (range = 7–1,564 ng/ml) consistent with a hypercoagulable state and the presence of disseminated intravascular coagulation (DIC).

COVID-19 cerebrovascular disease seems to be predominantly ischemic and to involve large vessels. In older individuals, it reflects the underlying severity of systemic disease as well as the hyperinflammatory state, whereas in younger patients, it seems to be due to hypercoagulopathy. Children with a Kawasaki disease-like multisystem inflammatory syndrome (MIS) have recently been described.2627 Patients with Kawasaki disease can develop cerebral vasculopathy and forms of neurological involvement, and in one series of 10 COVID-19 associated cases of MIS, two patients had meningeal symptoms.27 As noted, in addition to hypercoagulable states, SARS-CoV-2 can infect and injure endothelial cells. However, it remains to be determined whether virus-induced injury to endothelial cells (a vasculopathy) or even true vasculitis contributes to COVID-19 related cerebrovascular syndromes, and this determination will require additional detailed vessel imaging and neuropathological analyses. Similarly, the number of cases is too small to determine the comparative therapeutic benefit, if any, of antiplatelet or anticoagulant drugs or immunomodulatory therapies in COVID-19 associated neurovascular syndromes.

Neuroinvasion by SARS-CoV-2

In contrast to encephalopathy, in which evidence for direct invasion by virus of the CNS is absent, encephalitis occurs when direct invasion of the CNS by virus produces tissue injury and neurological dysfunction. Evidence for direct invasion of the CNS was seen in patients with SARS. Xu and colleagues described a fatal case in a 39-year-old man with delirium that progressed to somnolence and coma.10 At postmortem, the SARS-CoV antigen was detected in brain tissue by immunohistochemistry (IHC) and viral RNA by in situ hybridization (ISH). SARS-CoV virions were seen by transmission electron microscopy of brain tissue inoculated cell culture. In a postmortem analysis of four patients with SARS, low level infection of cerebral neurons with SARS-CoV (1–24% of cells) was seen in the cerebrum in all four cases by IHC and ISH, although none of the cases had virus detected in the cerebellum.28

By definition, encephalitis is an inflammatory process, with supportive evidence, including the presence of a CSF pleocytosis and elevated protein. However, in studies of transgenic mice expressing the human SARS-CoV receptor, ACE2, infection with SARS-CoV was associated with viral entry into the CNS, spread within the CNS, and neuronal injury with relatively limited inflammation.29 This suggests the possibility that, in some cases of SARS-CoV-2 CNS invasion, that signs of inflammation could be modest or even absent. Regardless of the presence or absence of inflammation, diagnostic studies may show evidence of either a generalized or focal CNS process, including areas of attenuation on computed tomography (CT), hyperintense signal on FLAIR, or T2-weighted sequences on MRI, and focal patterns, including seizures on electroencephalogram (EEG). Definitive evidence supporting direct viral invasion would include a positive CSF RT-PCR for SARS-CoV-2, demonstration of intrathecal synthesis of SARS-CoV-2-specific antibodies, or detection of SARS-CoV-2 antigen or RNA in brain tissue obtained at biopsy or autopsy.

Cases meeting strict criteria for encephalitis resulting from direct SARS-CoV-2 are currently extremely rare, although several plausible case reports have now surfaced. Moriguchi et al described a 24-year-old man with COVID-19 disease who developed nuchal rigidity, progressively decreased consciousness (Glasgow Coma Scale [GCS] = 6), and generalized seizures.30 CSF showed a slight mononuclear predominant pleocytosis (12 cells/μl3) and elevated opening pressure (>320 mm H20). Neuroimaging showed hippocampal and mesial temporal increased FLAIR signal and the CSF RT-PCR was positive for SARS-CoV-2. Unfortunately, studies to exclude other viral etiologies of encephalitis were limited. A second case involved a 41-year-old woman with headache, fever, a new onset seizure, and photophobia and nuchal rigidity, followed by hallucinations and disorientation. A head CT scan was normal and MRI was not performed. An EEG showed generalized slowing. The CSF examination showed a lymphocytic pleocytosis (70 cells/μl; 100% lymphocytes), and elevated protein (100 mg/dl), and a positive SARS-CoV-2 RT-PCR.3132

Several cases have emerged in which patients had inflammatory features consistent with encephalitis, but who did not have evidence of direct viral CNS invasion. Bernard-Valnet et al reported on two patients with “meningoencephalitis concomitant to SARS-CoV2.”33 These patients had nuchal rigidity, altered mental status, mild CSF lymphocytic pleocytosis (17–21 cells/μl3 on initial lumbar puncture [LP]), and mildly elevated CSF protein (46–47 mg/dl). However, in both patients, the MRI was normal and neither patient had a positive CSF RT-PCR for SARS-CoV-2. Similarly, Pilotto et al describe a 60-year-old man with COVID-19 who developed confusion, irritability, and then apathy progressing to “akinetic mutism” with nuchal rigidity.34 The CSF showed a mild lymphocytic pleocytosis (18 cells/μl3) and elevated protein (70 mg/dl). An EEG showed generalized slowing with an anterior predominance. The CT and MRI were normal, and CSF RT-PCR was negative twice for SARS-CoV-2. Although treated with a wide variety of medications, this patient showed improvement coincident to administration of high dose methylprednisolone.34 Another study reported on six critically ill patients with severe ARDS, elevated inflammatory markers, and depressed consciousness and/or agitation, who were considered to have “autoimmune meningoencephalitis.”35 No patient had a CSF pleocytosis but five had elevated CSF protein (52–131 mg/dL) and three had an MRI that showed cortical hyperintensities with sulcal effacement. There were no controls but patients were felt to have responded to plasma exchange. In one report, a patient with neuropsychiatric symptoms and COVID-19 had a “hematic” CSF tap with 960 “red and white blood cells” and an elevated protein (65 mg/dL) and detectable N-methyl-D-aspartate (NMDA) receptor antibodies. This currently isolated case also raises the possibility that COVID-19 may trigger auto-antibody production.36

The available studies suggest that SARS-CoV-2 can rarely produce a true encephalitis or meningoencephalitis with associated evidence of direct viral invasion of the CNS. The failure to detect virus in CSF in the other reported cases, despite evidence of inflammation as evidenced by CSF pleocytosis and elevated protein, raises the possibility that some cases of COVID-19 encephalitis may occur in the absence of direct virus invasion, and could potentially result from immune-mediated inflammatory mechanisms (see below). It is important to realize that techniques, including detection of intrathecal SARS-CoV-2 antibody synthesis or of viral antigen or nucleic acid in brain tissue, may establish evidence for viral invasion when CSF RT-PCR studies are negative. For example, detection of intrathecal antibody synthesis is significantly more sensitive than CSF nucleic acid amplification tests for diagnosis of both West Nile Virus neuroinvasive disease and Enterovirus (EV)-D68 associated acute flaccid myelitis (AFM).3739 In the case of EV-D68-associated AFM, nasopharyngeal and throat swabs are frequently positive for virus by RT-PCR when obtained early after disease onset, yet, CSF RT-PCR tests are only positive in a small minority (<3%) of cases.40 The sensitivity of SARS-CoV-2 RT-PCR in properly performed nasopharyngeal swabs for detection of acute COVID-19 is high, but data are currently too limited to evaluate sensitivity of this technique in CSF in patients with neurological disease.

Post-Infectious and Immune-Mediated Complications of SARS-CoV-2

The identification of postinfectious complications of SARS-CoV-2 would be expected to temporally lag behind those resulting from acute infection. Occasional cases of GBS and its variants and of ADEM were reported after MERS and SARS.579 Reports are now emerging of similar associations with COVID-19 and GBS, and with GBS variants, including the Miller-Fisher syndrome.4146 The largest series to date, describes five patients.47 In this series, all patients developed GBS 5 to 10 days following COVID-19 symptom onset. The clinical presentation included bilateral multi-limb flaccid weakness with areflexia. Three patients had associated respiratory failure and two had associated facial weakness. MRI showed caudal root nerve enhancement in two cases and enhancement of the facial nerve in a third case. The CSF was normocellular in all five cases, and had an elevated protein consistent with albuminocytological dissociation in three cases. Electrophysiological studies showed reduced compound motor amplitudes and prolonged distal latencies, and the overall pattern was felt to be consistent with demyelination in two cases and axonal neuropathy in three cases. Fibrillation potentials were seen by electromyography (EMG) acutely in three patients and later in a fourth patient. None of the patients had SARS-CoV-2 detected in the CSF by RT-PCR. Antiganglioside antibodies were absent in the three tested patients. All patients received intravenous immunoglobulin (ivIG) and one plasma exchange, although improvement was noted in only two cases (one “mild improvement” only).

Cases of acute necrotizing encephalopathy (ANE) have been reported in COVID-19.4849 One patient was a 50-year-old woman with COVID-19 confirmed by nasopharyngeal RT-PCR who developed altered mental status and MRI and CT findings typical of ANE, including bilateral thalamic lesions. Unfortunately, CSF studies were limited and CSF RT-PCR testing for SARS-CoV-2 was not performed. A second case occurred in a 59-year-old woman with aplastic anemia who developed seizures and reduced consciousness 10 days after onset of her COVID-19 symptoms.49 The mechanism behind ANE remains unknown, and either direct viral or postinfectious inflammatory processes have been postulated to play a role, and many cases have been reported after upper respiratory infections, including influenza. Some patients have mutations in RAN binding protein-2 (RANBP2), indicating that host genetic factors may also play a role in susceptibility.

Rare cases of ADEM were associated with MERS.6 The first case of “COVID-19 associated disseminated encephalomyelitis” was reported in a 40-year-old woman.50 This individual had COVID-19 symptoms followed 11 days later by dysarthria, dysphagia, facial weakness, and a gaze preference. A chest X-ray showed pneumonia and nasopharyngeal RT-PCR was positive for SARS-CoV-2. Head CT showed multiple areas of patchy hypoattenuation and an MRI showed areas of increased FLAIR and T2 signal in the subcortical and deep white matter that were felt to be consistent with demyelination. Her CSF was normal. A second reported case was in a 54-year-old woman who developed seizures and neurological deterioration (GCS = 12) and had chest X-ray lesions consistent with COVID-19 and a positive nasopharyngeal RT-PCR for SARS-CoV-2.51 Her MRI showed multiple periventricular T2 hyperintense, nonenhancing, lesions in the white matter of the cerebrum, brainstem, and spinal cord consistent with multifocal demyelination. Her CSF studies were unremarkable, including a negative CSF RT-PCR for SARS CoV-2. She was treated with high dose dexamethasone and her symptoms gradually resolved. A single case of acute flaccid myelitis has also been described in COVID-19.52 This patient developed upper limb weakness and a flaccid areflexic lower limb paralysis, urinary and bowel incontinence, and a T10 sensory level. Unfortunately, neither spine imaging nor CSF studies were available so the mechanism remains unknown. The most convincing example of ADEM-like pathology associated with COVID-19 was in a 71-year-old man who developed symptoms immediately following coronary bypass graft surgery that progressed to respiratory failure and a hyperinflammatory state. A postmortem examination showed brain swelling and disseminated hemorrhagic lesions and subcortical white matter pathology with perivenular myelin injury but also necrotic blood vessels and perivascular inflammation. The lesions had features of both acute hemorrhagic leukoencephalitis and of acute disseminated encephalomyelitis.53

The rarity of postinfectious potentially immune-mediated cases following COVID-19 other than GBS and its variants, and the general paucity of details, makes their status unclear. The cases of ADEM-like illness are hard to distinguish from some of the patients with acute encephalopathy and associated MRI white matter lesions, but can be differentiated from cases of encephalitis by the absence of CSF pleocytosis. GBS is a common neurological disease even in the absence of COVID-19, and identifying the magnitude of the COVID-19 risk and association will require better epidemiological data. However, the 5 cases of GBS occurring in a population of 1,000 to 1,200 patients with COVID-19 seen over a 1 month period by Toscano et al in Northern Italy suggest an incidence that is much higher than that can be expected in the general population (~1/100,000 person-years).54 The mechanism of pathogenesis will need to be identified, and the efficacy of conventional therapies, including ivIG and plasma exchange, evaluated.

Other COVID-19 Related Neurological Disorders

One of the more striking reported symptom manifestations in patients with COVID-19 is loss or perturbation of smell (anosmia or hyposmia) and/or taste (dysgeusia). The frequency of these symptoms, their specificity as a potential diagnostic clue for COVID-19 infection as opposed to influenza or other symptomatologic similar diseases, and their implication for understanding viral pathogenesis all remain uncertain. In the Wuhan COVID-19 series, impairment of smell was noted in 5% and of taste in 6% of the 214 hospitalized patients.11 It is likely that the frequency was under-represented due to incomplete evaluations in these hospitalized sick patients. A later study of 31 patients, suggested that disorders of taste occurred in 81% of COVID-19 cases (46% anosmia, 29% hyposmia, and 6% dysosmia) and disorders of taste in 94% (ageusia 45%, hypogeusia 23%, and dysgeusia 26%).55 The average duration of smell and taste disorders in the COVID-19 cases was 7.1 ± 3.1 days. A multicenter European study of 417 cases with “mild-to-moderate” COVID-19 disease found a similarly high frequency of olfactory dysfunction (86%), with 80% of those affected having anosmia and 20% hyposmia.56 Approximately 70% of patients had recovered within 8 days of symptom onset. It has been suggested that olfactory and/or gustatory dysfunction may be indicative of neuro-invasion and provide a route from the nasopharynx or oropharynx to cardiorespiratory centers in the medulla, based on studies of transgenic mice expressing the human SARS virus receptor (ACE2) and infected with SARS-CoV, however, no evidence supporting host entry via this pathway yet exists in man.29 The transient nature of the dysfunction in most patients would seem to make direct viral infection and subsequent killing of olfactory or gustatory neurons unlikely. MRI of the olfactory bulb was normal in one RT-PCR confirmed patient with anosmia.57

In the Wuhan COVID-19 series, 11% of patients were reported to have evidence of skeletal muscle injury (defined as a creatine kinase [CK] >200 U/L and skeletal muscle pain).11 Injury was significantly more common in patients with “severe” disease (19%) compared to non-severe disease (5%; p < 0.001). Unfortunately, almost no clinical details were provided beyond the presence of associated muscle pain. Subsequently two reports have emerged of rhabdomyolysis as either a presenting feature or a late complication of COVID-19.5859 One patient had limb pain and weakness with a peak CK of ~12,000 U/L and myoglobulin >12,000 μg/L, and the other had a peak CK of 13,581 U/L. Neither patient had muscle biopsy performed. The mechanism of injury remains to be determined.

Immunopathogenesis of SARS-CoV-2 and Implication for Management and Treatment of Neurologic Manifestations

One of the most puzzling features of SARS-CoV-2 infection is that it is asymptomatic or associated with minor symptoms in approximately 80% of patients, especially children and young adults, whereas 20% will develop COVID-19 with various degrees of severity. Can knowledge gathered on SARS-CoV inform us about the immunopathogenesis of SARS-CoV-2? A successful production of type I interferon (IFN) response is a key first line defense for suppressing replication of many neurotropic viruses at the site of entry and dissemination. SARS-CoV suppresses type I IFN response and downstream signaling using multiple strategies, and this dampening is closely associated with disease severity.60

Because SARS-CoV-2 shares an overall genomic similarity of 80% with SARS-CoV and uses the same receptor, it is reasonable to expect that the innate immune mechanisms involved in pathogenesis will be similar for the two viruses. SARS-CoV has developed multiple strategies to evade the innate immune response in order to optimize its replication capacity.61 It seems likely that SARS-CoV-2 uses the same strategy. The magnitude of the immune response against SARS-CoV-2 needs to be precisely calibrated to control viral replication without triggering immunopathogenic injury. A hyperinflammatory response likely plays a major role in ARDS and, in a subset of children, may contribute to the development of a Kawasaki-like multisystem inflammatory disorder.20 In a mouse model of SARS, rapid SARS-CoV replication and delay in IFN-I signaling led to inflammatory monocyte–macrophage accumulation, resulting in elevated lung cytokine/chemokine levels and associated vascular leakage and lethal pneumonia. This “cytokine storm,” in turn, was associated with a decrease in T cell counts and suboptimal T cell responses to SARS-CoV infection.62

The same pattern is found in 522 patients with COVID-19, where the number of total T cells, CD4+ and CD8+ T cells, were dramatically reduced, especially in those requiring ICU care, and T cell numbers were negatively correlated to serum IL-6, IL-10, and TNF-α concentration. Conversely, patients in the disease resolution period showed reduced IL-6, IL-10, and TNF-α levels and restored T cell counts.63 These data were corroborated by other groups who also noticed a decrease in type 1 interferon response in severely affected patients.6465 It has been suggested that reduced and delayed IFN gamma production (“too little and too late”) in the lungs and depletion of both CD4+ and CD8+ T cells may combine to potentiate viral injury, by reducing control of viral replication and enhancing the upregulation of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-10 (“cytokine storm”), and that it may be the immune dysregulation as much or more than the direct viral infection that results in pulmonary epithelial cell injury, and similar mechanisms could be operative in the CNS.66

What are the possible mechanisms for the apparent immune dysregulation seen in those patients and could they have a role in the neuropathogenesis of COVID-19? The source of cytokines found in the serum in unclear, but they could be produced by lung macrophages. IL-6 could also come from infected neurons, as seen in a transgenic mouse model of SARS-Cov.29 A high level of circulating cytokines, in turn, could lead to lymphocytopenia. TNF-α, a pro-inflammatory cytokine, may cause T cell apoptosis via interacting with its receptor, TNFR1, which expression is increased in aged T cells.6768 IL-6, that has both pro-inflammatory and anti-inflammatory properties, contributes to host defense in response to infections. However, continual synthesis of IL-6 has been shown to play a pathological role in chronic inflammation and infection.6970 IL-10, an inhibitory cytokine that prevents T cell proliferation, can also induce T cell exhaustion. Interestingly, patients with COVID-19 have high levels of the PD-1 and Tim-3 exhaustion markers on their T cells.63 In turn, decreased numbers of CD4+ and CD8+ T lymphocytes will considerably weaken the cellular immune response to SARS-CoV-2 in severe cases, allowing further viral replication. This can be compounded by the use of corticosteroids. Of note, a study in convalescent patients with SARS-CoV showed that CD8+ T cell responses were more frequent and had a greater magnitude of response than CD4+ T cells.71 Finally, one autopsy series of patients with COVID-19 showed histological features suggestive of secondary hemophagocytic lymphohistiocytosis (sHLH), also known as macrophage activation syndrome. This syndrome is characterized by an imbalance of innate and adaptive immune responses with aberrant activation of macrophages, and a blunted adaptive immune response.20

This dysregulated immune response may have a role in the pathogenesis of the COVID-19 encephalopathy. High levels of circulating pro-inflammatory cytokines can cause a confusion and alteration of consciousness, whereas a weakened T cell response may be unable to eliminate virus-infected cells in the brain causing further neurologic dysfunction. Careful studies of the CSF cytokine profile and T cell response to SARS-CoV-2 as well as postmortem studies, including CNS and muscle tissues, are urgently needed to better understand the neuropathogenesis of COVID-19. These will help inform whether therapeutic strategies aimed at blocking pro-inflammatory cytokines, including the IL-6 inhibitors tocilizumab and sarilumab, could have a beneficial effect on encephalopathy or whether corticosteroids that dampened the adaptive cellular immune response to viruses are contra-indicated. As we strive to find medications to counter the deleterious inflammatory state triggered by SARS-CoV-2, lessons can also be learned from COVID-19 outcomes in patients with neurological diseases, such as multiple sclerosis or myasthenia gravis, treated with immunomodulatory therapies.

Although we are only starting to grasp the complexity of SARS-CoV-2 biology, it is already apparent that COVID-19 causes a global threat to the entire nervous system, both through its worldwide distribution and multifactorial pathogenic mechanisms (Fig). As we hope for a vaccine or a cure, neurologists will play an important role in diagnosing, investigating, and treating the many neurologic manifestations of COVID-19 (Table).72

Details are in the caption following the image
FIGURE 1Open in figure viewerPowerPointMechanisms of severe acute respiratory syndrome-coronavirus type 2 (SARS-CoV-2) neuropathogenesis. SARS-CoV-2 pathogenic effects on the nervous system are likely multifactorial, including manifestations of systemic disease, direct neuro-invasion of the central nervous system (CNS), involvement of the peripheral nervous system (PNS) and muscle, as well as through a postinfectious, immune-mediated mechanism. MOF = multi-organ failure; GBS = Guillain-Barre syndrome. *CNS inflammation (CSF pleocytosis and proteinorrachia) with no evidence of direct viral infection of CNS; §direct evidence of viral invasion (reverse transcriptase-polymerase chain reaction positive [RT-PCR+], biopsy); ADEM = acute disseminated encephalomyelitis; ANE = acute necrotizing encephalopathy. [Color figure can be viewed at www.annalsofneurology.org]

TABLE 1. Neurologic Conditions Associated with SARS-CoV-2 Infection

Disease entityPresentationSupportive Neurodiagnostic testingPathogenesis
EncephalopathyAltered mental statusMRI: non-specificEEG: abnormal (slow)CSF: nl cells and ProCSF SARS-CoV-2 RT-PCR: NEGMultiple organ failureHypoxemiaSystemic InflammationEndothelialitis
EncephalitisAltered mental status and CNS dysfunctionMRI: non-specific (? WM changes)EEG: abnormal (slow, +focal)CSF: pleocytosis & elev. ProCSF SARS-CoV-2 RT-PCR: NEGCNS inflammation
Viral encephalitisAltered mental status and CNS dysfunctionMRI: new abnormalityEEG: abnormal (slow, ±focal)CSF: Pleocytosis and elev. ProCSF SARS-CoV-2 RT-PCR: POSBrain Tissue: POS (Ag or RNA)Brain parenchymal neuro-invasion
Viral meningitisHeadache, nuchal rigidityMRI: meningeal enhancement, CSF: pleocytosis & elev. ProCSF SARS-CoV-2 RT PCR: POSSubarachnoid invasion
StrokeFocal motor or sensory deficitMRI: ischemia or bleed, abnormal coagulation factors, increased inflammatory markersCoagulopathy
Anosmia/ageusiaOlfactory or taste dysfunctionAbnormal smell/taste tests? Peripheral vs central neuro-invasion
ADEMHeadache, acute neurologic symptomsMRI: hyperintense FLAIR lesions with variable enhancementPostinfectious
Guillain-Barre syndromeFlaccid muscle weaknessCSF: increased protein, nl WBC CSF SARS-CoV-2 RT-PCR: NEGEMG/NCS: abnormalPostinfectious
Muscle injuryMyalgiaCK elevatedMyopathy or myositis?
  • ADEM = acute disseminated encephalomyelitis; CNS = central nervous system; CK= creatinine kinase; CSF = cerebrospinal fluid; EEG = electroencephalogram; EMG = electromyogram; FLAIR = fluid-attenuated inversion recovery; MRI = magnetic resonance imaging; NCS = nerve conduction study; NEG = negative; POS = positive; pro = protein; RT-PCR = reverse transcriptase-polymerase chain reaction; SARS-CoV-2 = severe acute respiratory syndrome-coronavirus type 2; WBC = white blood cell; WM = white matter.

Why are boosted Americans testing positive for COVID more than those without extra shot?

Authors: Julia Marnin, McClatchy Washington Bureau June 7, 2022

Since late February, Americans who have gotten a booster shot appear to be testing positive for COVID-19 more often than those vaccinated without the extra shot, according to Centers for Disease Control and Prevention data.

This is based on numbers up until the week of April 23, which is the most recently released CDC data comparing case rates of those boosted, vaccinated and unvaccinated against the coronavirus. Ultimately, the numbers, which are updated monthly, showed those unvaccinated had the highest case rates overall.

Meanwhile, about 119 out of 100,000 boosted individuals tested positive for COVID-19 during the week of April 23, according to CDC data. In comparison, 56 out of 100,000 individuals vaccinated with only a primary series tested positive.

But why are the case rates higher for boosted individuals than for those vaccinated without a booster?

Dr. Sheela Shenoi, an infectious disease doctor and assistant professor at Yale School of Medicine, told McClatchy News over the phone that “there’s no biological reason that people who have had (the vaccine) and boosters are going to be at increased risk for COVID.”

“These numbers are not telling us the whole truth,” Shenoi said.

The CDC wrote in a summary accompanying its data that “several factors likely affect crude case rates” and this makes “interpretation of recent trends difficult.”

Here are some potential factors to keep in mind, according to health experts, when looking at the data.

At-home testing

“The wide availability of at-home tests has substantially muddied the waters, because these do not necessarily show up in official figures,” Bill Hanage, an associate professor of epidemiology at Harvard University’s T.H. Chan School of Public Health and a co-director for the Center for Communicable Disease Dynamics, told McClatchy News in a statement.

“Individuals receiving boosters may be more likely to have their cases counted,” Hanage said.

Hanage said this is because “just in being boosted, they are displaying ‘health seeking’ behavior” and “they are more likely to have contact with healthcare and get a test that ends up in official stats.”

In the U.S., more than 221 million people are fully vaccinated and more than 103 million of those people have received their first booster dose as of June 7, according to the CDC.

Those vaccinated without a booster “are more likely young, and so less likely to be severely ill in general,” Hanage said. “If they do a rapid test, they may not report it. They may not even do a test.”

Dr. Wafaa El-Sadr, a professor of epidemiology and medicine at Columbia University’s Mailman School of Public Health and director of ICAP, told McClatchy News over the phone that the wide availability of self testing “has completely changed the picture overall.”

“We don’t know the number of tests that are done and we don’t know how many are positive, how many are negative,” El-Sadr said. “So it’s a whole kind of black box that makes looking at case rates really very unreliable.”

Behaviors

Shenoi said it’s possible that individual behaviors might influence why CDC data shows those with booster shots are testing positive more than those vaccinated with a primary vaccine series.

Those boosted may feel more comfortable and safe, according to Shenoi, and as a result, they might be taking less COVID-19 precautions such as masking and social distancing “because they feel like they’re protected by the booster.”

Over the past few months, Shenoi said the country has seen “people getting infections, although overwhelmingly mild, thankfully, and that may correlate with people feeling that they’re protected and engaging in kind of their normal activities where they may be more exposed to other people with COVID and facilitating spread.”

El-Sadr said the issue with the CDC case rate data is that it depends “very much on behaviors, whether it be testing behavior” or “the characteristics of people who are boosted versus people who are not boosted.”

Prior infections and those at higher risk

It’s possible that people who have gotten their primary vaccine series but not a booster “are more likely to have been recently infected during the first omicron wave,” Hanage said.

With that “additional immunity from that infection,” they are less likely to be infected now, he added.

Dr. Peter Gulick, an associate professor of medicine at Michigan State University and the director of its Internal Medicine Osteopathic Residency Program, told McClatchy News in a statement that “one thing to consider is the group that gets the boosters.”

He described this group as older, immunocompromised individuals who are at a higher risk when it comes to COVID-19 breakthrough infections because of their “only partial response (to the) vaccines.”

El-Sadr also said those who are boosted may be at a higher risk of testing positive for COVID-19.

Because of this, “you can’t really attribute their higher risk of getting infected with COVID to the booster.”

The CDC’s late April case rate data was recorded when COVID-19 cases were trending upward in the U.S. due to the omicron variant and its subvariants.

The omicron variant, which is highly infectious and generally causes less severe symptoms compared with other variants, continues to dominate cases in the U.S. as of June 4, according to the CDC.

Shenoi said she predicts cases are going to continue to rise in the summer based on how infectious the variant and its subvariants are and how Americans appear ready to “move on and get back to their normal lives.”

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The Impact of Initial COVID-19 Episode Inflammation Among Adults on Mortality Within 12 Months Post-hospital Discharge

Authors: Arch G. Mainous III1,2*Benjamin J. Rooks1 and Frank A. Orlando1 May 12, 2022 Frontiers in Medicine

Background: Inflammation in the initial COVID-19 episode may be associated with post-recovery mortality. The goal of this study was to determine the relationship between systemic inflammation in COVID-19 hospitalized adults and mortality after recovery from COVID-19.

Methods: An analysis of electronic health records (EHR) for patients from 1 January, 2020 through 31 December, 2021 was performed for a cohort of COVID-19 positive hospitalized adult patients. 1,207 patients were followed for 12 months post COVID-19 episode at one health system. 12-month risk of mortality associated with inflammation, C-reactive protein (CRP), was assessed in Cox regressions adjusted for age, sex, race and comorbidities. Analyses evaluated whether steroids prescribed upon discharge were associated with later mortality.

Results: Elevated CRP was associated other indicators of severity of the COVID-19 hospitalization including, supplemental oxygen and intravenous dexamethasone. Elevated CRP was associated with an increased mortality risk after recovery from COVID-19. This effect was present for both unadjusted (HR = 1.60; 95% CI 1.18, 2.17) and adjusted analyses (HR = 1.61; 95% CI 1.19, 2.20) when CRP was split into high and low groups at the median. Oral steroid prescriptions at discharge were found to be associated with a lower risk of death post-discharge (adjusted HR = 0.49; 95% CI 0.33, 0.74).

Discussion: Hyperinflammation present with severe COVID-19 is associated with an increased mortality risk after hospital discharge. Although suggestive, treatment with anti-inflammatory medications like steroids upon hospital discharge is associated with a decreased post-acute COVID-19 mortality risk.

Introduction

The impact of coronavirus disease 2019 (COVID-19) has been immense. In terms of directly measured outcomes, as of February, 2022, worldwide more than 5.9 million people have died from directly linked COVID-19 episodes. More than 950,000 direct deaths from COVID-19 have been documented in the United States (1). Some evidence has suggested that some patients with COVID-19 may be at risk for developing health problems after the patient has recovered from the initial episode (24). Common sequelae that have been noted are fatigue, shortness-of-breath, and brain fog. Perhaps more concerningly, in addition to these symptoms, several studies have shown that following recovery from the initial COVID-19 episode, some patients are at risk for severe morbidity and mortality (58). Patients who have recovered from COVID-19 are at increased risk for hospitalization and death within 6–12 months after the initial episode. This morbidity and mortality is typically not listed or considered as a COVID-19 linked hospitalization or death in the medical records and thus are underreported as a post-acute COVID-19 sequelae.

The reason for this phenomenon of severe outcomes as post-acute sequelae of COVID-19 is not well understood. Early in COVID-19 episode, the disease is primarily driven by the replication of SARS-CoV-2. COVID-19 also exhibits a dysregulated immune/inflammatory response to SARS-CoV-2 that leads to tissue damage. The downstream impact of the initial COVID-19 episode is consistently higher in people with more severe acute infection (569). Cytokine storm, hyperinflammation, and multi-organ failure have also been indicated in patients with a severe COVID-19 episode (10). Cerebrospinal fluid samples indicate neuroinflammation during acute COVID-19 episodes (11). Moreover, even 40–60 days post-acute COVID-19 infection there is evidence of a significant remaining inflammatory response in patients (12). Thus, it could be hypothesized that the hyperinflammation that some COVID-19 patients have during the initial COVID-19 episode creates a systemic damage to multiple organ systems (1314). Consequently, that hyperinflammation and the corresponding systemic damage to multiple organ systems may lead to severe post-acute COVID-19 sequelae.

Following from this hyperinflammation, the use of steroids as anti-inflammatory treatments among patients with high inflammation during the initial COVID-19 episode may do more than just help in the initial episode but may act as a buffer to the downstream morbidity and mortality from the initial COVID-19 episode (1415).

The purpose of this study was to examine the relationship between substantial systemic inflammation, as measured by C-reactive protein (CRP), with post-acute COVID-19 sequelae among patients hospitalized with COVID-19. This 12-month mortality risk was examined in a longitudinal cohort of patients who tested positive for COVID-19 as determined by Polymerase Chain Reaction (PCR) testing within a large healthcare system.

Methods

The data for this project comes from a de-identified research databank containing electronic health records (EHR) of patients tested for or diagnosed with COVID-19 in any setting in the University of Florida (UF) Health system. Usage of the databank for research is not considered human subjects research, and IRB review was not required to conduct this study.

Definition of Cohort

The cohort for this study consisted of all adult patients aged 18 and older who were tested for COVID-19 between January 01, 2020 and December 31, 2021 within the UF Health system, in any encounter type (ambulatory, Emergency Department, inpatient, etc.). Although a patient in the cohort could have had a positive test administered in any of these settings, a patient was only included into the cohort if that patient experienced a hospitalization for COVID-19. Since this study included data from the early stages of the pandemic before consistent coding standards for documenting COVID-19 in the EHR had been established, a patient was considered to have been hospitalized for COVID-19 if they experienced any hospitalization within 30 days of a positive test for COVID-19. The databank contained EHR data for all patients in the cohort current through December 31, 2021. COVID-19 diagnosis was validated by PCR. Baseline dates for COVID-19 positive patients were established at the date of their earliest recorded PCR-confirmed positive COVID-19 test. Each patient was only included once in the analysis. For patients with multiple COVID-19 tests, if at least one test gave a positive result, the patient was classified as COVID-19 positive, and the date of their earliest positive COVID-19 test result was used as their baseline date. Patients who did not have a positive COVID-19 test were not included in the analysis. Patients were tested in the context of seeking care for COVID-19; the tests were not part of general screening and surveillance.

Only patients with at least 365 days of follow-up time after their baseline date were retained in the cohort. Patients with more than 365 days of follow-up were censored at 365 days. The cohort was also left censored at the 30-day mark post-hospital discharge to ensure that health care utilization was post-acute and not part of the initial COVID-19 episode of care (e.g., not a readmission).

Inflammation

C-reactive protein (CRP) was used as the measure of inflammation in this study. The UF Health laboratory measured CRP in serum using latex immunoturbidimetry assay. CRP measures were sourced from patient EHR data. The cohort was restricted to only include patients with at least one CRP measurement within their initial COVID-19 episode of care (between the date of their initial positive COVID-19 test and the left-hand censoring date). For patients with multiple measurements of CRP, the maximum value available was used.

Steroids

Intravenous dexamethasone during their initial COVID-19 hospitalization was assessed. Prescriptions for oral steroids (tablets of dexamethasone) that were prescribed either at or post-hospital discharge for their initial COVID-19 episode of care were included into the analysis. Prescriptions were identified using RxNorm codes available in each patient’s EHR.

Severity of Initial COVID-19 Hospitalization

We also measured the severity of the initial episode of COVID-19 hospitalization. This severity should track with the level of inflammation in the initial COVID-19 episode. We used the National Institutes of Health’s “Therapeutic Management of Hospitalized Adults With COVID-19” disease severity levels and definitions (16). The recommendations are based on four ascending levels: hospitalized but does not require supplemental oxygen, hospitalized and requires supplemental oxygen, hospitalized and requires supplemental oxygen through a high-flow device or noninvasive ventilation, hospitalized and requires mechanical ventilation or extracorporeal membrane oxygenation. For this study, because of the general conceptual model of severity moving from no supplemental oxygen to supplemental oxygen to mechanical ventilation, we collapsed the two supplemental non-mechanical ventilation oxygen into one intermediate category of severity.

Outcome Variables

The primary outcome investigated in this study was the 365-day all-cause mortality. Mortality data was sourced both from EHR data and the Social Security Death Index (SSDI), allowing for the assessment of deaths which occurred outside of UF’s healthcare system. When conflicting dates of death were observed between the EHR and SSDI, the date recorded in the patient’s medical record was used. Patients who died within their 365-day follow-up window were censored at the date of their recorded death. The cause of death was not available in the EHR based database and was not routinely and reliably reported in either the SSDI or EHR. We were unable to estimate the cause of death.

Comorbidities

Comorbidities and demographic variables which could potentially confound the association between inflammation represented by CRP and mortality post-acute COVID-19 were collected at baseline for each member of the cohort. Demographic variables included patient age, race, ethnicity, and sex. The Charlson Comorbidity Index was also calculated, accounting for the conditions present for each patient at their baseline. The Charlson Comorbidity Index was designed to be used to predict 1-year mortality and is a widely used measure to account for comorbidities (17).

Analysis

CRP was evaluated using descriptive statistics. We performed a median split of the CRP levels and defined elevated inflammation as a CRP level at or above the median and levels below the median as low inflammation. Additionally, as a way to examine greater separation between high and low inflammation, we segmented CRP levels into tertiles and categorized elevated inflammation as the top tertile and compared it to the first tertile by chi-square tests.

CRP level was also cross classified by severity of COVID-19 hospitalization and associations between the two variables were assessed using one-way ANOVA tests.

Kaplan-Meier curves comparing the survival probabilities of the high and low inflammation groups were created and compared using a log-rank test. Hazard ratios for the risk of death for post-acute COVID-19 complications by COVID-19 status were determined using Cox proportional hazard models. We obtained hazard ratios for mortality based on tertile and median splits of CRP. These analyses were then modified to control for age, sex, race, ethnicity, and the Charlson Comorbidity Index.

Additional analyses stratified by use of steroids were performed to compare the strength of the association between inflammation and death. The proportional hazards assumption was confirmed by inspection of the Schoenfeld residual plots for each variable included in the models and testing of the time-dependent beta coefficients. Analyses were conducted using the survival package in R v4.0.5.

Results

A total of 1,207 patients were included in the final cohort (Table 1). The characteristics of the patients are featured in Table 1. The mean CRP rises with the severity of illness in these COVID-19 inpatients. The mean CRP in the lowest severity (no supplemental oxygen) is 59.4 mg/L (SD = 61.8 mg/L), while the mean CRP in the intermediate severity group (supplemental oxygen) is 126.9 mg/L (SD = 98.6 mg/L), and the mean CRP in the highest severity group (ventilator or ECMO) is 201.2 mg/L (SD = 117.0 mg/L) (p < 0.001). Similarly, since dexamethasone is only recommended for the most severe patients with COVID-19, patients with dexamethasone had higher CRP (158.8 mg/L; SD = 114.9 mg/L) than those not on Dexamethasone (102.8 mg/L; SD = 90/8 mg/L) (p < 0.001).TABLE 1

Table 1. Characteristics of the patients in the cohort.

Figure 1 presents the Kaplan-Meier curves comparing the risk of mortality by inflammation over time. A log-rank test indicated there was a statistically significant difference in survival probabilities between the two groups (p = 0.002).FIGURE 1

Figure 1. All-cause mortality Kaplan-Meier curve comparing individuals with median or greater vs. below median C-reactive protein levels. Log rank test = p.002.

Table 2 shows the relationship between levels of inflammation and mortality post-recovery from COVID-19. In both unadjusted and adjusted analyses, elevated inflammation has a significantly increased risk compared to those with low inflammation in the initial COVID-19 episode. This finding of higher inflammation during the initial COVID-19 hospitalization and increased mortality risk after recovery was similar when CRP was split at the median and when the third tertile of CRP was compared to the first tertile of CRP. The proportional hazards assumption was met when the Schoenfeld plots.TABLE 2

Table 2. All-cause mortality hazard ratios by inflammation and steroid use.

We examined the hypothesized relationship that potentially decreasing inflammation in COVID-19 patients with an initial severe episode may have beneficial downstream effects on post-acute COVID-19 sequelae. Oral steroid prescriptions at discharge among these hospitalized COVID-19 patients were found to be associated with a lower risk of death post-discharge (Table 2).

Discussion

The results of this study reaffirm the importance of post-acute COVID-19 sequelae. This study is the first to show the impact of inflammation in the initial COVID-19 hospitalization episode on downstream mortality after the patient has recovered. This expands our understanding of post-acute COVID-19 sequelae by providing a better concept of why certain patients have post-acute COVID-19 mortality risk.

Previous studies have shown that patients who are hospitalized with COVID-19 have an increased risk of mortality 12 months after recovery (5). Those findings suggest that prevention of COVID-19 hospitalizations is of paramount importance. However, some patients will be hospitalized. The finding that elevated inflammation during the initial hospitalization episode is associated with mortality risk after recovery suggests that it may be worthwhile treating the viral episode but also consider treating the hyperinflammation. The NIH recommendations for care of COVID-19 hospitalized patients recommend steroids only for patients who need supplemental oxygen (16). The finding that the use of steroids prescribed upon discharge from the hospital and the corresponding reduced risk of mortality indicate that treating inflammation after the acute COVID-19 episode may act as a buffer to the downstream mortality risk from the initial COVID-19 episode (1415). Perhaps this requires a reconceptualization of COVID-19 as both an acute disease and potentially a chronic disease because of the lingering risks. Future research is needed to see if ongoing treatment for inflammation in a clinical trial has positive benefits.

There are several strengths and limitations to this study. The strengths of this study include the PCR validated COVID-19 tests at baseline for the cohort. Further, the linked electronic health record allows us to look not only at health care utilization like hospitalizations and both inpatient and outpatient medication but also laboratory tests like CRP levels. The cohort also allows us to have a substantial follow-up time.

In terms of limitations, the first that needs to be considered is that the analysis was based on hospitalized patients seen in one health system with a regional catchment area. Although more than 1200 hospitalized patients with PCR validated COVID-19 diagnoses were included in the analysis, and the cohort was followed for 12 months, the primary independent variable was systemic inflammation which should not be substantially affected by region of the country. Second, the data are observational. Thus, the analyses related to steroids and downstream mortality require a clinical trial to confirm these suggestive findings. Third, we did not have death certificates available to us to compute cause of death. The Social Security Death Index in partnership with the EHR allows us to be confident that the patient died and so we have a strong measure of all-cause mortality but we were unable to determine specific causes of death within this database. Fourth, although there are a variety of other markers of inflammation (e.g., D dimer, IL 6), CRP is one of the most robust measures of systemic inflammation. Moreover, it is much more widely used and was the most prevalent marker among the patients in the study.

In conclusion, hyperinflammation present with severe COVID-19 is associated with an increased mortality risk after hospital discharge. Although suggestive, treatment with anti-inflammatory medications like steroids upon hospital discharge is associated with a decreased post-acute COVID-19 mortality risk. This suggests that treating inflammation may also benefit other post-acute sequelae like long COVID. A reconceptualization of COVID-19 as both an acute and chronic condition may be useful.

References

1. COVID-19 Dashboard by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University (JHU). Available online at: https://coronavirus.jhu.edu/map.html (accessed February 28, 2022).

Google Scholar

2. Bell ML, Catalfamo CJ, Farland LV, Ernst KC, Jacobs ET, Klimentidis YC, et al. Post-acute sequelae of COVID-19 in a non-hospitalized cohort: results from the Arizona CoVHORT. PLoS ONE. (2021) 16:e0254347. doi: 10.1371/journal.pone.0254347

PubMed Abstract | CrossRef Full Text | Google Scholar

3. Garrigues E, Janvier P, Kherabi Y, Le Bot A, Hamon A, Gouze H, et al. Post-discharge persistent symptoms and health-related quality of life after hospitalization for COVID-19. J Infect. (2020) 81:e4–6. doi: 10.1016/j.jinf.2020.08.029

PubMed Abstract | CrossRef Full Text | Google Scholar

4. Alkodaymi MS, Omrani OA, Fawzy NA, Shaar BA, Almamlouk R, Riaz M, et al. Prevalence of post-acute COVID-19 syndrome symptoms at different follow-up periods: a systematic review and meta-analysis. Clin Microbiol Infect. (2022). doi: 10.1016/j.cmi.2022.01.014. [Epub ahead of print].

PubMed Abstract | CrossRef Full Text | Google Scholar

5. Mainous AG 3rd, Rooks BJ, Wu V, Orlando FA. COVID-19 post-acute sequelae among adults: 12 month mortality risk. Front Med. (2021) 8:778434. doi: 10.3389/fmed.2021.778434

PubMed Abstract | CrossRef Full Text | Google Scholar

6. Mainous AG 3rd, Rooks BJ, Orlando FA. Risk of New Hospitalization Post-COVID-19 Infection for Non-COVID-19 Conditions. J Am Board Fam Med. (2021) 34:907–13. doi: 10.3122/jabfm.2021.05.210170

PubMed Abstract | CrossRef Full Text | Google Scholar

7. Bhaskaran K, Rentsch CT, Hickman G, Hulme WJ, Schultze A, Curtis HJ, et al. Overall and cause-specific hospitalisation and death after COVID-19 hospitalisation in England: a cohort study using linked primary care, secondary care, and death registration data in the OpenSAFELY platform. PLoS Med. (2022) 19:e1003871. doi: 10.1371/journal.pmed.1003871

PubMed Abstract | CrossRef Full Text | Google Scholar

8. Al-Aly Z, Xie Y, Bowe B. High-dimensional characterization of post-acute sequelae of COVID-19. Nature. (2021) 594:259–64. doi: 10.1038/s41586-021-03553-9

PubMed Abstract | CrossRef Full Text | Google Scholar

9. Xie Y, Bowe B, Al-Aly Z. Burdens of post-acute sequelae of COVID-19 by severity of acute infection, demographics and health status. Nat Commun. (2021) 12:6571. doi: 10.1038/s41467-021-26513-3

PubMed Abstract | CrossRef Full Text | Google Scholar

10. Wong RSY. Inflammation in COVID-19: from pathogenesis to treatment. Int J Clin Exp Pathol. (2021) 14:831–44.

Google Scholar

11. Spudich S, Nath A. Nervous system consequences of COVID-19. Science. (2022) 375:267–9. doi: 10.1126/science.abm2052

PubMed Abstract | CrossRef Full Text | Google Scholar

12. Doykov I, Hällqvist J, Gilmour KC, Grandjean L, Mills K, Heywood WE. ‘The long tail of Covid-19’ – The detection of a prolonged inflammatory response after a SARS-CoV-2 infection in asymptomatic and mildly affected patients. F1000Res. (2020) 9:1349. doi: 10.12688/f1000research.27287.1

PubMed Abstract | CrossRef Full Text | Google Scholar

13. Theoharides TC. Could SARS-CoV-2 spike protein be responsible for long-COVID syndrome? Mol Neurobiol. (2022) 59:1850–61. doi: 10.1007/s12035-021-02696-0

PubMed Abstract | CrossRef Full Text | Google Scholar

14. Cron RQ, Caricchio R, Chatham WW. Calming the cytokine storm in COVID-19. Nat Med. (2021) 27:1674–5. doi: 10.1038/s41591-021-01500-9

PubMed Abstract | CrossRef Full Text | Google Scholar

15. Keller MJ, Kitsis EA, Arora S, Chen JT, Agarwal S, Ross MJ, et al. Effect of systemic glucocorticoids on mortality or mechanical ventilation in patients with COVID-19. J Hosp Med. (2020) 15:489–93. doi: 10.12788/jhm.3497

PubMed Abstract | CrossRef Full Text | Google Scholar

16. National Institutes of Health. Therapeutic Management of Hospitalized Adults With COVID-19. (2021). https://www.covid19treatmentguidelines.nih.gov/management/clinical-management/hospitalized-adults–therapeutic-management/ (accessed February 28, 2022).

Google Scholar

17. Charlson ME, Carrozzino D, Guidi J, Patierno C. Charlson comorbidity index: a critical review of clinimetric properties. Psychother Psychosom. (2022) 91:8–35. doi: 10.1159/000521288

PubMed Abstract | CrossRef Full Text | Google Scholar

Acute Mesenteric Ischemia in COVID-19 Patients

Authors: Dragos Serban 1,2,*† , Laura Carina Tribus 3,4,†, Geta Vancea 1,5,† , Anca Pantea Stoian, Ana Maria Dascalu 1,* Andra Iulia Suceveanu 6Ciprian Tanasescu 7,8, Andreea Cristina Costea 9 Mihail Silviu Tudosie 1, Corneliu Tudor 2, Gabriel Andrei Gangura 1,10, Lucian Duta 2 and Daniel Ovidiu Costea 6,11,

Abstract:

Acute mesenteric ischemia is a rare but extremely severe complication of SARS-CoV-2 infection. The present review aims to document the clinical, laboratory, and imaging findings, management, and outcomes of acute intestinal ischemia in COVID-19 patients. A comprehensive search was performed on PubMed and Web of Science with the terms “COVID-19” and “bowel ischemia” OR “intestinal ischemia” OR “mesenteric ischemia” OR “mesenteric thrombosis”. After duplication removal, a total of 36 articles were included, reporting data on a total of 89 patients, 63 being hospitalized at the moment of onset. Elevated D-dimers, leukocytosis, and C reactive protein (CRP) were present in most reported cases, and a contrast-enhanced CT exam confirms the vascular thromboembolism and offers important information about the bowel viability. There are distinct features of bowel ischemia in non-hospitalized vs. hospitalized COVID-19 patients, suggesting different pathological pathways. In ICU patients, the most frequently affected was the large bowel alone (56%) or in association with the small bowel (24%), with microvascular thrombosis. Surgery was necessary in 95.4% of cases. In the non-hospitalized group, the small bowel was involved in 80%, with splanchnic veins or arteries thromboembolism, and a favorable response to conservative anticoagulant therapy was reported in 38.4%. Mortality was 54.4% in the hospitalized group and 21.7% in the non-hospitalized group (p < 0.0001). Age over 60 years (p = 0.043) and the need for surgery (p = 0.019) were associated with the worst outcome. Understanding the mechanisms involved and risk factors may help adjust the thromboprophylaxis and fluid management in COVID-19 patients.

1. Introduction Acute mesenteric ischemia (AMI) is a major abdominal emergency, characterized by a sudden decrease in the blood flow to the small bowel, resulting in ischemic lesions of the intestinal loops, necrosis, and if left untreated, death by peritonitis and septic shock. In nonCOVID patients, the etiology may be mesenteric arterial embolism (in 50%), mesenteric arterial thrombosis (15–25%), venous thrombosis (5–15%), or less frequent, from nonocclusive causes associated with low blood flow [1]. Several systemic conditions, such as arterial hypertension, atrial fibrillation, atherosclerosis, heart failure, or valve disease are risk factors for AMI. Portal vein thrombosis and mesenteric vein thrombosis can be seen with celiac disease [2], appendicitis [3], pancreatitis [4], and, in particular, liver cirrhosis and hepatocellular cancer [5]. Acute intestinal ischemia is a rare manifestation during COVID-19 disease, but a correct estimation of its incidence is challenging due to sporadic reports, differences in patients’ selection among previously published studies, and also limitations in diagnosis related to the strict COVID-19 regulations for disease control and difficulties in performing imagistic investigations in the patients in intensive care units. COVID-19 is known to cause significant alteration of coagulation, causing thromboembolic acute events, of which the most documented were pulmonary embolism, acute myocardial infarction, and lower limb ischemia [6]. Gastrointestinal features in COVID-19 disease are relatively frequently reported, varying from less than 10% in early studies from China [7,8] to 30–60%, in other reports [9,10]. In an extensive study on 1992 hospitalized patients for COVID-19 pneumonia from 36 centers, Elmunzer et al. [7] found that the most frequent clinical signs reported were mild and self-limited in up to 74% of cases, consisting of diarrhea (34%), nausea (27%), vomiting (16%), and abdominal pain (11%). However, severe cases were also reported, requiring emergency surgery for acute bowel ischemia or perforation [5,8]. The pathophysiology of the digestive features in COVID-19 patients involves both ischemic and non-ischemic mechanisms. ACE2 receptors are present at the level of the intestinal wall, and enterocytes may be directly infected by SARS-CoV-2. The virus was evidenced in feces and enteral walls in infected subjects [4,11–13]. In a study by Xu et al., rectal swabs were positive in 8 of 10 pediatric patients, even after the nasopharyngeal swabs became negative [14]. However, the significance of fecal elimination of viral ARN is still not fully understood in the transmission chain of the SARS-CoV-2 infection. On the other hand, disturbance of lung-gut axis, prolonged hospitalization in ICU, and the pro coagulation state induced by SARS-CoV-2 endothelial damage was incriminated for bowel ischemia, resulting in intestinal necrosis and perforation [8,9,15]. Early recognition and treatment of gastrointestinal ischemia are extremely important, but it is often challenging in hospitalized COVID-19 patients with severe illness. The present review aims to document the risk factors, clinical, imagistic, and laboratory findings, management, and outcomes of acute intestinal ischemic complications in COVID-19 patients. 2. Materials and Methods A comprehensive search was performed on PubMed and Web of Science with the terms “COVID-19” AND (“bowel ischemia” OR “intestinal ischemia” OR “mesenteric ischemia” OR “mesenteric thrombosis”). All original papers and case reports, in the English language, for which full text could be obtained, published until November 2021, were included in the review. Meeting abstracts, commentaries, and book chapters were excluded. A hand search was performed in the references of the relevant reviews on the topic. 2.1. Data Extraction and Analysis The review is not registered in PROSPERO. A PRISMA flowchart was employed to screen papers for eligibility (Figure 1) and a PRISMA checklist is presented as a Supple- J. Clin. Med. 2022, 11, 200 3 of 22 mentary File S1. A data extraction sheet was independently completed by two researchers, with strict adherence to PRISMA guidelines. J. Clin. Med. 2022, 11, 200 3 2.1. Data Extraction and Analysis The review is not registered in PROSPERO. A PRISMA flowchart was employedscreen papers for eligibility (Figure 1) and a PRISMA checklist is presented as a Supmentary File S1. A data extraction sheet was independently completed by two researchwith strict adherence to PRISMA guidelines. Figure 1. PRISMA 2020 flowchart for the studies included in the review. The relevant data abstracted from these studies are presented in Tables 1–3. COV19 diagnosis was made by PCR assay in all cases. All patients reported with COVIDdisease and mesenteric ischemia were documented in terms of age, sex, comorbidittime from SARS-CoV-2 infection diagnosis, presentation, investigations, treatment, outcome. A statistical analysis of the differences between acute intestinal ischemia in pviously non-hospitalized vs. previously hospitalized patients was performed. The pottial risk factors for an adverse vital prognosis were analyzed using SciStat® softw(www.scistat.com (accessed on 25 November 2021)). Papers that did not provide sufficient data regarding evaluation at admission, domentation of SARS-CoV-2 infection, or treatment were excluded. Patients suffering frother conditions that could potentially complicate intestinal ischemia, such as liver cirrsis, hepatocellular carcinoma, intraabdominal infection (appendicitis, diverticulitis), pcreatitis, and celiac disease were excluded. Any disagreement was solved by discussioFigure 1. PRISMA 2020 flowchart for the studies included in the review. The relevant data abstracted from these studies are presented in Tables 1–3. COVID-19 diagnosis was made by PCR assay in all cases. All patients reported with COVID-19 disease and mesenteric ischemia were documented in terms of age, sex, comorbidities, time from SARS-CoV-2 infection diagnosis, presentation, investigations, treatment, and outcome. A statistical analysis of the differences between acute intestinal ischemia in previously nonhospitalized vs. previously hospitalized patients was performed. The potential risk factors for an adverse vital prognosis were analyzed using SciStat® software (www.scistat.com (accessed on 25 November 2021)). Papers that did not provide sufficient data regarding evaluation at admission, documentation of SARS-CoV-2 infection, or treatment were excluded. Patients suffering from other conditions that could potentially complicate intestinal ischemia, such as liver cirrhosis, hepatocellular carcinoma, intraabdominal infection (appendicitis, diverticulitis), pancreatitis, and celiac disease were excluded. Any disagreement was solved by discussion. J. Clin. Med. 2022, 11, 200 4 of 22 Table 1. Patients with intestinal ischemia in retrospective studies on hospitalized COVID-19 patients. Study No of Patients with Gastrointestinal Ischemia (Total No of COVID-19 Patients in ICU) Sex (M; F) Age (Mean) BMI Time from Admission to Onset (Days) Abdominal CT Signs Intraoperative/Endoscopic Findings Treatment Outcomes Kaafarani HMA [16] 5 (141); 3.8% 1;3 62.5 32.1 51.5 (18–104) days NA Cecum-1—patchy necrosis Cecum_ileon-1 Small bowel-3; yellow discoloration on the antimesenteric side of the small bowel; 1 case + liver necrosis Surgical resection NA Kraft M [17] 4 (190); 2.1% NA NA NA NA NA Bowel ischemia + perforation (2) Bowel ischemia + perforation (1) MAT+massive bowel ischemia (1) Right hemicolectomy (2) Transverse colectomy (1) Conservative, not fit for surgery Recovery (3) Death (1) Yang C [18] 20 (190 in ICU; 582 in total); 10.5% 15:5 69 31.2 26.5 (17–42) Distension Wall thickness Pneumatosis intestinalis Perforation SMA or celiac thrombosis no info Right hemicolectomy 7(35%) Sub/total colectomy12 (60%) Ileocecal resection 1(5%) Recovery (11) Death (9) Hwabejire J [19] 20 13:7 58.7 32.5 13 (1–31) Pneumatosis intestinalis 42% Portal venous gas (33%) Mesenteric vessel patency 92% large bowel ischemia (8) small bowel ischemia (4) both (8) yellow discoloration of the ischemic bowel resection of the ischemic segment abdomen left open + second look (14) Recovery (10) Death (10) O’Shea A [20] 4 (142); 2.8% NA NA NA NA bowel ischemia, portal vein gas, colic pneumatosis NA NA NA Qayed E [21] 2 (878); 0.22% NA NA NA NA NA diffuse colonic ischemia (1) Small + large bowel ischemia and pneumatosis (1) Total colectomy (1) Extensive resection (1) Recovery (1) Death (1) NA: not acknowledged; MAT: mesenteric artery thrombosis; SMA: superior mesenteric artery. J. Clin. Med. 2022, 11, 200 5 of 22 Table 2. Case reports and case series presenting gastrointestinal ischemia in hospitalized COVID-19 patients under anticoagulant medication. Article Sex Age Comorbidities Time from COVID-19 Diagnosis; Time from Admission (Days) ICU; Type of Ventilation Clinical Signs at Presentation Leukocytes (/mm3 ) CRP (mg/L) Lactat mmol/L Ferritin (ng/mL) LDH (U/L) Thrombocytes (/mm3) D-Dimers (ng/mL) Abdominal CT Signs Treatment Outcome Azouz E [22] M 56 none 1; 2 (hospitalized for acute ischemic stroke) No info abdominal pain and vomiting No info – – – – – – Multiple arterial thromboembolic complications: AMS, right middle cerebral artery, a free-floating clot in the aortic arch Anticoagulation (no details), endovascular thrombectomy Laparotomy + resection of necrotic small bowel loops No info Al Mahruqi G [23] M 51 none 26; 24 yes, intubated Fever, metabolic acidosis, required inotropes 30,000 – 7 687 – – 2.5 Non-occlusive AMI Hypoperfused small bowel, permeable aorta, SMA, IMA + deep lower limb thrombosis enoxaparin 40 mg/day from admission; surgery refused by family death Ucpinar BA [24] F 82 Atrial fibrillation, hypertension, chronic kidney disease 3; 3 no – 14,800 196 5.1 – – – 1600 SMA thrombosis; distended small bowel, with diffuse submucosal pneumatosis portomesenteric gas fluid resuscitation; continued ceftriaxone, enoxaparin 0.4cc twice daily; not operable due to fulminant evolution Death Karna ST [25] F 61 DM, hypertension 4; 4 Yes, HFNO diffuse abdominal pain with distention 21,400 421.6 1.4 – – 464,000 No thrombosis of the distal SMA with dilated jejunoileal loops and normal enhancing bowel wall. Iv heparin 5000 ui, followed by 1000 ui, Ecospin and clopidogrel Laparotomy after 10 days with segmental enterectomy of the necrotic bowel Death by septic shock and acute renal failure Singh B [26] F 82 Hypertension, T2DM 32; 18 Yes, Ventilator support severe diffuse abdominal distension and tenderness 22,800 308 2.5 136 333 146,000 1.3 SMA—colic arteries thrombosis pneumatosis intestinalis affecting the ascending colon and cecum laparotomy, ischemic colon resection, ileostomy; heparin in therapeutic doses preand post-surgery slow recovery J. Clin. Med. 2022, 11, 200 6 of 22 Table 2. Cont. Article Sex Age Comorbidities Time from COVID-19 Diagnosis; Time from Admission (Days) ICU; Type of Ventilation Clinical Signs at Presentation Leukocytes (/mm3 ) CRP (mg/L) Lactat mmol/L Ferritin (ng/mL) LDH (U/L) Thrombocytes (/mm3) D-Dimers (ng/mL) Abdominal CT Signs Treatment Outcome Nakatsutmi K [27] F 67 DM, diabetic nephropathy requiring dialysis, angina, postresection gastric cancer 16; 12 ICU, intubation hemodynamic deterioration, abdominal distension 15,100 32.14 – – – – 26.51 edematous transverse colon; abdominal vessels with sclerotic changes laparotomy, which revealed vascular micro thrombosis of transverse colon—right segment resection of the ischemic colonic segment, ABTHERA management, second look, and closure of the abdomen after 24 h death Dinoto E [28] F 84 DM, hypertension, renal failure 2; 2 no Acute abdominal pain and distension; 18,000 32.47 – – 431 – 6937 SMA origin stenosis and occlusion at 2 cm from the origin, absence of bowel enhancement Endovascular thrombectomy of SMA; surgical transfemoral thrombectomy and distal superficial femoral artery stenting Death due to respiratory failure Kiwango F [29] F 60 DM, hypertension 12; 3 no Sudden onset abdominal pain 7700 – – – – – 23.8 Not performed Not performed due to rapid oxygen desaturation Massive bowel acute ischemia death J. Clin. Med. 2022, 11, 200 7 of 22 Table 3. Case reports and case series presenting gastrointestinal ischemia in non-hospitalized COVID-19 patients. Article Sex Age Comorbidities Time from COVID-19 Diagnosis (Days) Clinical Signs at Presentation Leukocyte Count (/mm3 ) CRP (mg/L) Lactate mmol/L Ferritin (ng/mL) LDH (U/L) Thrombocytes (/mm3 ) D-Dimers (ng/mL) Abdominal CT Signs Treatment Outcome Sevella, P [30] M 44 none 10 Acute abdominal pain constipation, vomiting 23,400 – – – 1097 360,000 1590 Viable jejunum, ischemic bowel, peritoneal thickening with fat stranding; free fluid in the peritoneal cavity LMWH 60 mg daily Piperacillin 4g/day Tazobactam 500 mg/day Extensive small bowel + right colon resection death Nasseh S [31] M 68 no info First diagnosis epigastric pain and diarrhea for 4 days 17,660 125 – – – – 6876 terminal segment of the ileocolic artery thrombosis; thickening of the right colon wall and the last 30 cm of the small bowl unfractionated heparin laparoscopy -no bowel resection needed recovery Aleman W [32] M 44 none 20 severe abdominopelvic pain 36,870 – – 456.23 – 574,000 263.87 absence of flow at SMV, splenic, portal vein; Small bowel loop dilatation and mesenteric fat edema enoxaparin and pain control medication 6 days, then switched to warfarin 6 months recovery Jeilani M [33] M 68 Alzheimer disease, COPD 9 Sharp abdominal pain +distension 12,440 307 – – – 318,000 897 a central venous filling defect within the portal vein extending to SMV; no bowel wall changes LMWH, 3 months recovery Randhawa J [34] F 62 none First diagnosis right upper quadrant pain and loss of appetite for 14 days Normal limits – – – 346 – – large thrombus involving the SMV, the main portal vein with extension into its branches Fondaparinux 2.5. mg 5 days, then warfarin 4 mg (adjusted by INR), 6 months recovery Cheung S [35] M 55 none 12 (discharged for 7 days) Nausea, vomiting and worsening generalized abdominal pain with guarding 12,446 – 0.68 – – – – low-density clot, 1.6 cm in length, causing high-grade narrowing of the proximal SMA continuous heparin infusion continued 8 h postoperative, Laparotomy with SMA thromboembolectomy and enterectomy (small bowel) recovery J. Clin. Med. 2022, 11, 200 8 of 22 Table 3. Cont. Article Sex Age Comorbidities Time from COVID-19 Diagnosis (Days) Clinical Signs at Presentation Leukocyte Count (/mm3 ) CRP (mg/L) Lactate mmol/L Ferritin (ng/mL) LDH (U/L) Thrombocytes (/mm3 ) D-Dimers (ng/mL) Abdominal CT Signs Treatment Outcome Beccara L [36] M 52 none 22 (5 days after discharge and cessation prophylactic LWMH) vomiting and abdominal pain, tenderness in epigastrium and mesogastrium 30,000 222 – – – – – arterial thrombosis of vessels efferent of the SMA with bowel distension Enterectomy (small bowel) LMWH plus aspirin 100 mg/day at discharge recovery Vulliamy P [37] M 75 none 14 abdominal pain and vomiting for 2 days 18,100 3.2 – – – 497,000 320 intraluminal thrombus was present in the descending thoracic aorta with embolic occlusion of SMA Catheter-directed thrombolysis, enterectomy (small bowel) recovery De Barry O [38] F 79 none First diagnosis Epigastric pain, diarrhea, fever for 8 days, acute dyspnea 12600 125 5.36 – – – – SMV, portal vein, SMA, and jejunal artery thrombosis Distended loops, free fluid anticoagulation Resection of affected colon+ ileum, SMA thrombolysis, thrombectomy death Romero MCV [39] M 73 smoker, DM, hypertension 14 severe abdominal pain, nausea. fecal emesis, peritoneal irritation 18,000 – – – – 120,000 >5000 RX: distention of intestinal loops, inter-loop edema, intestinal pneumatosis enoxaparin (60 mg/0.6 mL), antibiotics (no info) enterectomy, anastomotic fistula, reintervention death Posada Arango [40] M F F 62 22 65 None Appendectomy 7 days before left nephrectomy, 5 3 15 colicative abdominal pain at food intake; unsystematized gastrointestinal symptoms; abdominal pain in the upper hemiabdomen 20,100 – – – – – – – – 1536 – – 534 – – – – – – – – Case 1: thrombus in distal SMA and its branches, intestinal loops dilatation, hydroaerical levels, free fluid thrombosis of SMV Case 2: SMV thrombosis and adiacent fat edema Case 3: thrombi in the left jejunal artery branch with infarction of the corresponding jejunal loops Case 1: Laparotomy: extensive jejunum + ileum ischemia; surgery could not be performed Case 2: Anticoagulation analgesic and antibiotics Case 3: segmental enterectomy Case 1: death Case 2: recovery Case 3: recovery J. Clin. Med. 2022, 11, 200 9 of 22 Table 3. Cont. Article Sex Age Comorbidities Time from COVID-19 Diagnosis (Days) Clinical Signs at Presentation Leukocyte Count (/mm3 ) CRP (mg/L) Lactate mmol/L Ferritin (ng/mL) LDH (U/L) Thrombocytes (/mm3 ) D-Dimers (ng/mL) Abdominal CT Signs Treatment Outcome Pang JHQ [41] M 30 none First diagnosis colicky abdominal pain, vomiting – – – – – – 20 SMV thrombosis with diffuse mural thickening and fat stranding of multiple jejunal loops conservative, anticoagulation with LMWH 1mg/kc, twice daily, 3 months; readmitted and operated for congenital adherence causing small bowel obstruction recovery Lari E [42] M 38 none First diagnosis abdominal pain, nausea, intractable vomiting, and shortness of breath Mild leukocytosis – 2.2 – – – 2100 extensive thrombosis of the portal, splenic, superior, and inferior mesenteric veins + mild bowel ischemia Anticoagulation, resection of the affected bowel loop No info Carmo Filho A [43] M 33 Obesity (BMI: 33), other not reported 7 severe low back pain radiating to the hypogastric region – 58.2 – 1570 – – 879 enlarged inferior mesenteric vein not filled by contrast associated with infiltration of the adjacent adipose planes enoxaparin 5 days, followed by long term oral warfarin recovery Hanif M [44] F 20 none 8 abdominal pain and abdominal distension 15,900 62 – 1435.3 825 633,000 2340 not performed evidence of SMA thrombosis; enterectomy with exteriorization of both ends recovery Amaravathi U [45] M 45 none 5 Acute epigastric and periumbilical pain – Normal value 1.3 324.3 – – 5.3 SMA and SMV thrombus i.v. heparin; Laparotomy with SMA thrombectomy; 48 h Second look: resection of the gangrenous bowel segment No info Al Mahruqi G [23] M 51 none 4 generalized abdominal pain, nausea, vomiting 16,000 – – 619 – – 10 SMA thrombosis and non-enhancing proximal ileal loops consistent with small bowel ischemia unfractionated heparin, thrombectomy + repeated resections of the ischemic bowel at relook (jejunum+ileon+cecum) Case 2: recovery J. Clin. Med. 2022, 11, 200 10 of 22 Table 3. Cont. Article Sex Age Comorbidities Time from COVID-19 Diagnosis (Days) Clinical Signs at Presentation Leukocyte Count (/mm3 ) CRP (mg/L) Lactate mmol/L Ferritin (ng/mL) LDH (U/L) Thrombocytes (/mm3 ) D-Dimers (ng/mL) Abdominal CT Signs Treatment Outcome Goodfellow M [46] F 36 RYGB, depression, asthma 6 epigastric pain, irradiating back, nausea 9650 1.2 0.7 – – – – abrupt cut-off of the SMV in the proximal portion; diffuse infiltration of the mesentery, wall thickening of small bowel IV heparin infusion, followed by 18,000 UI delteparin after 72 h recovery Abeysekera KW [26] M 42 Hepatitis B 14 right hypochondrial pain, progressively increasing for 9 days – – – – – – – enhancement of the entire length of the portal vein and a smaller thrombus in the mid-superior mesenteric vein, mural edema of the distal duodenum, distal small bowel, and descending colon factor Xa inhibitor apixaban 5 mg ×2/day, 6 months – recovery RodriguezNakamura RM [27] M F 45 42 -vitiligo -obesity 14 severe mesogastric pain, nausea, diaphoresis 16,400 18,800 367 239 – – 970 – – – 685,000 – 1450 14,407 Case 1: SMI of thrombotic etiology with partial rechanneling through the middle colic artery, and hypoxic-ischemic changes in the distal ileum and the cecum Case 2: thrombosis of the portal and mesenteric veins and an abdominopelvic collection in the mesentery with gas Case 1: resection with entero-enteral anastomosis; rivaroxaban 10 mg/day, 6 months Case 2: Loop resection, entero-enteral manual anastomosis, partial omentectomy, and cavity wash (fecal peritonitis) Case 1: Recovery Case 2: death Plotz B [47] F 27 SLE with ITP First diagnosis acute onset nausea, vomiting, and non-bloody diarrhea – – – – – – 5446 diffuse small bowel edema enoxaparin, long term apixaban at discharge recovery J. Clin. Med. 2022, 11, 200 11 of 22 Table 3. Cont. Article Sex Age Comorbidities Time from COVID-19 Diagnosis (Days) Clinical Signs at Presentation Leukocyte Count (/mm3 ) CRP (mg/L) Lactate mmol/L Ferritin (ng/mL) LDH (U/L) Thrombocytes (/mm3 ) D-Dimers (ng/mL) Abdominal CT Signs Treatment Outcome Chiu CY [48] F 49 Hypertension, DM, chronic kidney disease 28 diffuse abdominal pain melena and hematemesis – – – – – – 12,444 distended proximal jejunum with mural thickening laparotomy, proximal jejunum resection no info Farina D [49] M 70 no info 3 abdominal pain, nausea 15,300 149 – – – – – acute small bowel hypoperfusion, SMA thromboembolism not operable due to general condition Death SMA: superior mesenteric artery; SMV: superior mesenteric vein; DM: diabetes mellitus; T2DM: type 2 diabetes mellitus; AMI: acute mesenteric ischemia; IMV: inferior mesenteric vein; RYGB: Roux-en-Y gastric bypass (bariatric surgery). J. Clin. Med. 2022, 11, 200 12 of 22 2.2. Risk of Bias The studies analyzed in the present review were comparable in terms of patient selection, methodology, therapeutic approach, and the report of final outcome. However, there were differences in the reported clinical and laboratory data. The sample size was small, most of them being case reports or case series, which may be a significant source of bias. Therefore, studies were compared only qualitatively. 3. Results After duplication removal, a total of 36 articles were included in the review, reporting data on a total of 89 patients. Among these, we identified 6 retrospective studies [16–21], documenting intestinal ischemia in 55 patients admitted to intensive care units (ICU) with COVID-19 pneumonia for whom surgical consult was necessary (Table 1). We also identified 30 case reports or case series [22–51] presenting 34 cases of acute bowel ischemia in patients positive for SARS-CoV-2 infection in different clinical settings. 8 cases were previously hospitalized for COVID-19 pneumonia and under anticoagulant medication (Table 2). In 26 cases, the acute ischemic event appeared as the first symptom of COVID-19 disease, or in mild forms treated at home, or after discharge for COVID -19 pneumonia and cessation of the anticoagulant medication (Table 3). 3.1. Risk Factors of Intestinal Ischemia in COVID-19 Patients Out of a total of 89 patients included in the review, 63 (70.7%) were hospitalized for severe forms of COVID-19 pneumonia at the moment of onset. These patients were receiving anticoagulant medication when reported, consisting of low molecular weight heparin (LMWH) at prophylactic doses. The incidence of acute intestinal ischemia in ICU patients with COVID-19 varied widely between 0.22–10.5% (Table 1). In a study by O’Shea et al. [20], 26% of hospitalized patients for COVID-19 pneumonia who underwent imagistic examination, presented results positive for coagulopathy, and in 22% of these cases, the thromboembolic events were with multiple locations. The mean age was 56.9 years. We observed a significantly lower age in non-hospitalized COVID-19 patients presenting with acute intestinal ischemia when compared to the previously hospitalized group (p < 0.0001). There is a slight male to female predominance (M:F = 1:68). Obesity might be considered a possible risk factor, with a reported mean BMI of 31.2–32.5 in hospitalized patients [16,18,19]. However, this association should be regarded with caution, since obesity is also a risk factor for severe forms of COVID-19. Prolonged stay in intensive care, intubation, and the need for vasopressor medication was associated with increased risk of acute bowel ischemia [8,18,19]. Diabetes mellitus and hypertension were the most frequent comorbidities encountered in case reports (8 in 34 patients, 23%), and 7 out of 8 patients presented both (Table 4). There was no information regarding the comorbidities in the retrospective studies included in the review. 3.2. Clinical Features in COVID-19 Patients with Acute Mesenteric Ischemia Abdominal pain, out of proportion to physical findings, is a hallmark of portomesenteric thrombosis, typically associated with fever and leukocytosis [4]. Abdominal pain was encountered in all cases, either generalized from the beginning, of high intensity, or firstly localized in the epigastrium or the mezogastric area. In cases of portal vein thrombosis, the initial location may be in the right hypochondrium, mimicking biliary colic [26,34]. Fever is less useful in COVID-19 infected patients, taking into consideration that fever is a general sign of infection, and on the other hand, these patients might be already under antipyretic medication. J. Clin. Med. 2022, 11, 200 13 of 22 Table 4. Demographic data of the patients included in the review. Nr. of Patients 89 M 48 (61.5% *) F 30 (38.5% *) NA 11 The first sign of COVID-19 6 (6.7%) Home treated 17 (19.1%) Hospitalized • ICU 63 (70.7%) 58 (92% of hospitalized patients) Discharged 3 (3.3%) Time from diagnosis of COVID-19 infection • Non-Hospitalized • Hospitalized (*when mentioned) 8.7 ± 7.4 (1–28 days) 9.6 ± 8.3 (1–26 days) Time from admission in hospitalized patients 1–104 days Age (mean) • Hospitalized • Non-hospitalized 59.3 ± 12.7 years 62 ± 9.6 years. (p < 0.0001) 52.8 ± 16.4 years. BMI 31.2–32.5 Comorbidities • Hypertension • DM • smokers • Atrial fibrillation • COPD • Cirrhosis • RYGB • Vitiligo • Recent appendicitis • Operated gastric cancer • Alzheimer disease • SLE 8 7 2 1 2 1 1 1 1 1 1 1 *: percentage calculated in known information group; BMI: body mass index; COPD: chronic obstructive pulmonary disease; SLE: systemic lupus erythematosus. Other clinical signs reported were nausea, anorexia, vomiting, and food intolerance [23,31,38,45]. However, these gastrointestinal signs are encountered in 30–40% of patients with SARS-CoV-2 infection. In a study by Kaafarani et al., up to half of the patients with gastrointestinal features presented some degrees of intestinal hypomotility, possibly due to direct viral invasion of the enterocytes and neuro-enteral disturbances [16]. Physical exam evidenced abdominal distension, reduced bowel sounds, and tenderness at palpation. Guarding may be evocative for peritonitis due to compromised vascularization of bowel loops and bacterial translocation or franc perforation [35,39]. A challenging case was presented by Goodfellow et al. [25] in a patient with a recent history of bariatric surgery with Roux en Y gastric bypass, presenting with acute abdominal pain which imposed the differential diagnosis with an internal hernia. Upcinar et al. [24] reported a case of an 82-years female that also associated atrial fibrillation. The patient was anticoagulated with enoxaparin 0.4 cc twice daily before admission and continued the anticoagulant therapy during hospitalization for COVID-19 pneumonia. Bedside echocardiography was performed to exclude atrial thrombus. Although SMA was reported related to COVID-19 pneumonia, atrial fibrillation is a strong risk factor for SMA of non-COVID-19 etiology. J. Clin. Med. 2022, 11, 200 14 of 22 In ICU patients, acute bowel ischemia should be suspected in cases that present acute onset of digestive intolerance and stasis, abdominal distension, and require an increase of vasopressor medication [19]. 3.3. Imagistic and Lab Test Findings D-dimer is a highly sensitive investigation for the prothrombotic state caused by COVID-19 [45] and, when reported, was found to be above the normal values. Leukocytosis and acute phase biomarkers, such as fibrinogen and CRP were elevated, mirroring the intensity of inflammation and sepsis caused by the ischemic bowel. However, there was no significant statistical correlation between either the leukocyte count (p = 0.803) or D-dimers (p = 0.08) and the outcome. Leucocyte count may be within normal values in case of early presentation [34]. Thrombocytosis and thrombocytopenia have been reported in published cases with mesenteric ischemia [30,35,42,46,50]. Lactate levels were reported in 9 cases, with values higher than 2 mmol/L in 5 cases (55%). LDH was determined in 6 cases, and it was found to be elevated in all cases, with a mean value of 594+/−305 U/L. Ferritin is another biomarker of potential value in mesenteric ischemia, that increases due to ischemia-reperfusion cellular damage. In the reviewed studies, serum ferritin was raised in 7 out of 9 reported cases, with values ranging from 456 to 1570 ng/mL. However, ferritin levels were found to be correlated also with the severity of pulmonary lesions in COVID-19 patients [52]. Due to the low number of cases in which lactate, LDH, and ferritin were reported, no statistical association could be performed with the severity of lesions or with adverse outcomes. The location and extent of venous or arterial thrombosis were determined by contrastenhanced abdominal CT, which also provided important information on the viability of the intestinal segment whose vascularity was affected. Radiological findings in the early stages included dilated intestinal loops, thickening of the intestinal wall, mesenteric fat edema, and air-fluid levels. Once the viability of the affected intestinal segment is compromised, a CT exam may evidence pneumatosis as a sign of bacterial proliferation and translocation in the intestinal wall, pneumoperitoneum due to perforation, and free fluid in the abdominal cavity. In cases with an unconfirmed diagnosis of COVID-19, examination of the pulmonary basis during abdominal CT exam can add consistent findings to establish the diagnosis. Venous thrombosis affecting the superior mesenteric vein and or portal vein was encountered in 40.9% of reported cases of non-hospitalized COVID-19 patients, and in only one case in the hospitalized group (Table 5). One explanation may be the beneficial role of thrombotic prophylaxis in preventing venous thrombosis in COVID-19 patients, which is routinely administrated in hospitalized cases, but not reported in cases treated at home with COVID-19 pneumonia. In ICU patients, CT exam showed in most cases permeable mesenteric vessels and diffuse intestinal ischemia affecting the large bowel alone (56%) or in association with the small bowel (24%), suggesting pathogenic mechanisms, direct viral infection, small vessel thrombosis, or “nonocclusive mesenteric ischemia” [16]. 3.4. Management and Outcomes The management of mesenteric ischemia includes gastrointestinal decompression, fluid resuscitation, hemodynamic support, anticoagulation, and broad antibiotics. Once the thromboembolic event was diagnosed, heparin, 5000IU iv, or enoxaparin or LMWH in therapeutic doses was initiated, followed by long-term oral anticoagulation and/or anti-aggregating therapy. Favorable results were obtained in 7 out of 9 cases (77%) of splanchnic veins thrombosis and in 2 of 7 cases (28.5%) with superior mesenteric artery thrombosis. At discharge, anticoagulation therapy was continued either with LMWH, for a period up to 3 months [33,36,41], either, long term warfarin, with INR control [32,34,41] or apixaban 5 mg/day, up to 6 months [26,47]. No readmissions were reported. J. Clin. Med. 2022, 11, 200 15 of 22 Table 5. Comparative features in acute intestinal ischemia encountered in previously hospitalized and previously non-hospitalized COVID-19 patients. Parameter Hospitalized (63) NonHospitalized (26) p * Value Type of mesenteric ischemia: • Arterial • Venous • Mixt (A + V) • Diffuse microthrombosis • Multiple thromboembolic locations • NA 5 (14.7% *) 1 (2.9%) 0 30 (88.2%) 2 (5.8%) 29 10 (38.4%) 11 (42.3%) 2 (7.6%) 3 (11.5%) 1 (3.8%) 0 p < 0.0001 Management: • Anticoagulation therapy only • Endovascular thrombectomy • Laparotomy with ischemic bowel resection • None (fulminant evolution) 0 2 (1 + surgery) (3%) 60 (95.4%) 2 (3%) 10 (38.4%) 2 (+surgery) 15 (57.6%) 1 (3.8%) p < 0.0001 Location of the resected segment: • Colon • Small bowel • Colon+small bowel • NA 35 (56%) 10 (16%) 15 (24%) 6 0 12 (80%) 3 (20%) 0 p < 0.0001 Outcomes: • Recovery • Death • NA 26 (46.4%) 30 (54.4%) 7 17 (79.3%) 5 (21.7%) 3 p = 0.013 * calculated for Chi-squared test. Antibiotic classes should cover anaerobes including F. necrophorum and include a combination of beta-lactam and beta-lactamase inhibitor (e.g., piperacillin-tazobactam), metronidazole, ceftriaxone, clindamycin, and carbapenems [4]. In early diagnosis, during the first 12 h from the onset, vascular surgery may be tempted, avoiding the enteral resection [25,53]. Endovascular management is a minimally invasive approach, allowing quick restoration of blood flow in affected vessels using techniques such as aspiration, thrombectomy, thrombolysis, and angioplasty with or without stenting [40]. Laparotomy with resection of the necrotic bowel should be performed as quickly as possible to avoid perforation and septic shock. In cases in which intestinal viability cannot be established with certainty, a second look laparotomy was performed after 24–48 h [43] or the abdominal cavity was left open, using negative pressure systems such as ABTHERA [51], and successive segmentary enterectomy was performed. Several authors described in acute bowel ischemia encountered in ICU patients with COVID-19, a distinct yellowish color, rather than the typical purple or black color of ischemic bowel, predominantly located at the antimesenteric side or circumferentially with affected areas well delineated from the adjacent healthy areas [18,19]. In these cases, patency of large mesenteric vessels was confirmed, and the histopathological reports J. Clin. Med. 2022, 11, 200 16 of 22 showed endothelitis, inflammation, and microvascular thrombosis in the submucosa or transmural. Despite early surgery, the outcome is severe in these cases, with an overall mortality of 45–50% in reported studies and up to 100% in patients over 65 years of age according to Hwabejira et al. [19]. In COVID-19 patients non hospitalized at the onset of an acute ischemic event, with mild and moderate forms of the disease, the outcome was less severe, with recovery in 77% of cases. We found that age over 60 years and the necessity of surgical treatment are statistically correlated with a poor outcome in the reviewed studies (Table 6). According to the type of mesenteric ischemia, the venous thrombosis was more likely to have a favorable outcome (recovery in 80% of cases), while vascular micro thombosis lead to death in 66% of cases. Table 6. Risk factors for severe outcome. Parameters Outcome: Death p-Value Age • Age < 60 • Age > 60 27.2% 60% 0.0384 * 0.043 ** Surgery • No surgery • surgery 0% 60% 0.019 ** Type of mesenteric ischemia • Arterial • Venous • Micro thrombosis 47% 20% 66% 0.23 ** D dimers Wide variation 0.085 * 0.394 ** Leucocytes Wide variation (9650–37,000/mmc) 0.803 0.385 ** * One-way ANOVA test; ** Chi-squared test (SciStat® software, www.scistat.com (accessed on 25 November 2021)). 4. Discussions Classically, acute mesenteric ischemia is a rare surgical emergency encountered in the elderly with cardiovascular or portal-associated pathology, such as arterial hypertension, atrial fibrillation, atherosclerosis, heart failure, valve disease, and portal hypertension. However, in the current context of the COVID-19 pandemic, mesenteric ischemia should be suspected in any patient presenting in an emergency with acute abdominal pain, regardless of age and associated diseases. Several biomarkers were investigated for the potential diagnostic and prognostic value in acute mesenteric ischemia. Serum lactate is a non-specific biomarker of tissue hypoperfusion and undergoes significant elevation only after advanced mesenteric damage. Several clinical trials found a value higher than 2 mmol/L was significantly associated with increased mortality in non-COVID-patients. However, its diagnostic value is still a subject of debate. There are two detectable isomers, L-lactate, which is a nonspecific biomarker of anaerobic metabolism, and hypoxia and D-lactate, which is produced by the activity of intestinal bacteria. Higher D-lactate levels could be more specific for mesenteric ischemia due to increased bacterial proliferation at the level of the ischemic bowel, but the results obtained in different studies are mostly inconsistent [53,54]. Several clinical studies found that LDH is a useful biomarker for acute mesenteric ischemia, [55,56]. However, interpretation of the results may be difficult in COVID-19 patients, as both lactate and LDH were also found to be independent risk factors of severe forms of COVID-19 [57,58]. The diagnosis of an ischemic bowel should be one of the top differentials in critically ill patients with acute onset of abdominal pain and distension [50,59]. If diagnosed early, the J. Clin. Med. 2022, 11, 200 17 of 22 intestinal ischemia is potentially reversible and can be treated conservatively. Heparin has an anticoagulant, anti-inflammatory, endothelial protective role in COVID-19, which can improve microcirculation and decrease possible ischemic events [25]. The appropriate dose, however, is still a subject of debate with some authors recommending the prophylactic, others the intermediate or therapeutic daily amount [25,60]. We found that surgery is associated with a severe outcome in the reviewed studies. Mucosal ischemia may induce massive viremia from bowel epithelium causing vasoplegic shock after surgery [25]. Moreover, many studies reported poor outcomes in COVID-19 patients that underwent abdominal surgery [61,62]. 4.1. Pathogenic Pathways of Mesenteric Ischemia in COVID-19 Patients The intestinal manifestations encountered in SARS-CoV-2 infection are represented by inflammatory changes (gastroenteritis, colitis), occlusions, ileus, invaginations, and ischemic manifestations. Severe inflammation in the intestine can cause damage to the submucosal vessels, resulting in hypercoagulability in the intestine. Cases of acute cholecystitis, splenic infarction, or acute pancreatitis have also been reported in patients infected with SARS-CoV-2, with microvascular lesions as a pathophysiological mechanism [63]. In the study of O’Shea et al., on 146 COVID-19 hospitalized patients that underwent CT-scan, vascular thrombosis was identified in 26% of cases, the most frequent location being in lungs [20]. Gastrointestinal ischemic lesions were identified in 4 cases, in multiple locations (pulmonary, hepatic, cerebellar parenchymal infarction) in 3 patients. The authors raised awareness about the possibility of underestimation of the incidence of thrombotic events in COVID-19 patients [20]. Several pathophysiological mechanisms have been considered, and they can be grouped into occlusive and non-occlusive causes [64]. The site of the ischemic process, embolism or thrombosis, may be in the micro vascularization, veins, or mesenteric arteries. Acute arterial obstruction of the small intestinal vessels and mesenteric ischemia may appear due to hypercoagulability associated with SARS-CoV-2 infection, mucosal ischemia, viral dissemination, and endothelial cell invasion vis ACE-2 receptors [65,66]. Viral binding to ACE2Receptors leads to significant changes in fluid-coagulation balance: reduction in Ang 2 degradation leads to increased Il6 levels, and the onset of storm cytokines, such as IL-2, IL-7, IL-10, granulocyte colony-stimulating factor, IgG -induced protein 10, monocyte chemoattractant protein-1, macrophage inflammatory protein 1-alpha, and tumor necrosis factor α [67], but also in the expression of the tissue inhibitor of plasminogen -1, and a tissue factor, and subsequently triggering the coagulation system through binding to the clotting factor VIIa [68]. Acute embolism in small vessels may be caused by the direct viral invasion, via ACE-2 Receptors, resulting in endothelitis and inflammation, recruiting immune cells, and expressing high levels of pro-inflammatory cytokines, such as Il-6 and TNF-alfa, with consequently apoptosis of the endothelial cells [69]. Capillary viscometry showed hyperviscosity in critically ill COVID-19 patients [70,71]. Platelet activation, platelet–monocyte aggregation formation, and Neutrophil external traps (NETs) released from activated neutrophils, constitute a mixture of nucleic DNA, histones, and nucleosomes [59,72] were documented in severe COVID-19 patients by several studies [70,71,73]. Plotz et al. found a thrombotic vasculopathy with histological evidence for lectin pathway complement activation mirroring viral protein deposition in a patient with COVID19 and SLE, suggesting this might be a potential mechanism in SARS-CoV-2 associated thrombotic disorders [47]. Numerous alterations in fluid-coagulation balance have been reported in patients hospitalized for COVID-19 pneumonia. Increases in fibrinogen, D-dimers, but also coagulation factors V and VIII. The mechanisms of coagulation disorders in COVID-19 are not yet fully elucidated. In a clinical study by Stefely et al. [68] in a group of 102 patients with severe disease, an increase in factor V > 200 IU was identified in 48% of cases, the levels determined being statistically significantly higher than in non-COVID mechanically J. Clin. Med. 2022, 11, 200 18 of 22 ventilated or unventilated patients hospitalized in intensive care. This showed that the increased activity of Factor V cannot be attributed to disease severity or mechanical ventilation. Additionally, an increase in factor X activity was shown, but not correlated with an increase in factor V activity, but with an increase in acute phase reactants, suggesting distinct pathophysiological mechanisms [74]. Giuffre et al. suggest that fecal calcoprotein (FC) may be a biomarker for the severity of gastrointestinal complications, by both ischemic and inflammatory mechanisms [75]. They found particularly elevated levels of FC to be well correlated with D-dimers levels in patients with bowel perforations, and hypothesized that the mechanism may be related to a thrombosis localized to the gut and that FC increase is related to virus-related inflammation and thrombosis-induced ischemia, as shown by gross pathology [76]. Non-occlusive mesenteric ischemia in patients hospitalized in intensive care units for SARS-CoV-2 pneumonia requiring vasopressor medication may be caused vasospastic constriction [19,64,65]. Thrombosis of the mesenteric vessels could be favored by hypercoagulability, relative dehydration, and side effects of corticosteroids. 4.2. Question Still to Be Answered Current recommendations for in-hospital patients with COVID-19 requiring anticoagulation suggest LMWH as first-line treatment has advantages, with higher stability compared to heparin during cytokine storms, and a reduced risk of interaction with antiviral therapy compared to oral anticoagulant medication [77]. Choosing the adequate doses of LMWH in specific cases—prophylactic, intermediate, or therapeutic—is still in debate. Thromboprophylaxis is highly recommended in the absence of contraindications, due to the increased risk of venous thrombosis and arterial thromboembolism associated with SARS-CoV-2 infection, with dose adjustment based on weight and associated risk factors. Besides the anticoagulant role, some authors also reported an anti-inflammatory role of heparin in severe COVID-19 infection [66,78,79]. Heparin is known to decrease inflammation by inhibiting neutrophil activity, expression of inflammatory mediators, and the proliferation of vascular smooth muscle cells [78]. Thromboprophylaxis with enoxaparin could be also recommended to ambulatory patients with mild to moderate forms of COVID-19 if the results of prospective studies show statistically relevant benefits [80]. In addition to anticoagulants, other therapies, such as anti-complement and interleukin (IL)-1 receptor antagonists, need to be explored, and other new agents should be discovered as they emerge from our better understanding of the pathogenetic mechanisms [81]. Several studies showed the important role of Il-1 in endothelial dysfunction, inflammation, and thrombi formation in COVID-19 patients by stimulating the production of Thromboxane A2 (TxA2) and thromboxane B2 (TxB2). These findings may justify the recommendation for an IL-1 receptor antagonist (IL-1Ra) which can prevent hemodynamic changes, septic shock, organ inflammation, and vascular thrombosis in severe forms of COVID-19 patients [80–82]. 5. Conclusions Understanding the pathological pathways and risk factors could help adjust the thromboprophylaxis and fluid management in COVID-19 patients. The superior mesenteric vein thrombosis is the most frequent cause of acute intestinal ischemia in COVID-19 nonhospitalized patients that are not under anticoagulant medication, while non-occlusive mesenteric ischemia and microvascular thrombosis are most frequent in severe cases, hospitalized in intensive care units. COVID-19 patients should be carefully monitored for acute onset of abdominal symptoms. High-intensity pain and abdominal distension, associated with leukocytosis, raised inflammatory biomarkers and elevated D-dimers and are highly suggestive for mesenteric ischemia. The contrast-enhanced CT exam, repeated, if necessary, offers valuable information regarding the location and extent of the acute ischemic event. Early diagnosis and treatment are essential for survival.

J. Clin. Med. 2022, 11, 200 19 of 22 Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jcm11010200/s1, File S1: The PRISMA 2020 statement. Author Contributions: Conceptualization, D.S., L.C.T. and A.M.D.; methodology, A.P.S., C.T. (Corneliu Tudor); software, G.V.; validation, A.I.S., M.S.T., D.S. and L.D.; formal analysis, A.C.C., C.T. (Ciprian Tanasescu); investigation, G.A.G.; data curation, D.O.C.; writing—original draft preparation, L.C.T., A.M.D., G.V., D.O.C., G.A.G., C.T. (Corneliu Tudor); writing—review and editing, L.D., C.T. (Ciprian Tanasescu), A.C.C., D.S., A.P.S., A.I.S., M.S.T.; visualization, G.V. and L.C.T.; supervision, D.S., A.M.D. and D.S. have conducted the screening and selection of studies included in the review All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. References 1. Bala, M.; Kashuk, J.; Moore, E.E.; Kluger, Y.; Biffl, W.; Gomes, C.A.; Ben-Ishay, O.; Rubinstein, C.; Balogh, Z.J.; Civil, I.; et al. Acute mesenteric ischemia: Guidelines of the World Society of Emergency Surgery. World J. Emerg. Surg. 2017, 12, 38. [CrossRef] 2. Dumic, I.; Martin, S.; Salfiti, N.; Watson, R.; Alempijevic, T. Deep Venous Thrombosis and Bilateral Pulmonary Embolism Revealing Silent Celiac Disease: Case Report and Review of the Literature. Case Rep. Gastrointest. Med. 2017, 2017, 5236918. [CrossRef] [PubMed] 3. Akhrass, F.A.; Abdallah, L.; Berger, S.; Sartawi, R. Gastrointestinal variant of Lemierre’s syndrome complicating ruptured appendicitis. IDCases 2015, 2, 72–76. [CrossRef] 4. Radovanovic, N.; Dumic, I.; Veselinovic, M.; Burger, S.; Milovanovic, T.; Nordstrom, C.W.; Niendorf, E.; Ramanan, P. Fusobacterium necrophorum subsp. necrophorum Liver Abscess with Pylephlebitis: An Abdominal Variant of Lemierre’s Syndrome. Case Rep. Infect. Dis. 2020, 2020, 9237267. [CrossRef] 5. Sogaard, K.K.; Astrup, L.B.; Vilstrup, H.; Gronbaek, H. Portal vein thrombosis; risk factors, clinical presentation and treatment. BMC Gastroenterol. 2007, 7, 34. [CrossRef] [PubMed] 6. Moradi, H.; Mouzannar, S.; Miratashi Yazdi, S.A. Post COVID-19 splenic infarction with limb ischemia: A case report. Ann. Med. Surg. 2021, 71, 102935. [CrossRef] [PubMed] 7. Elmunzer, B.J.; Spitzer, R.L.; Foster, L.D.; Merchant, A.A.; Howard, E.F.; Patel, V.A.; West, M.K.; Qayed, E.; Nustas, R.; Zakaria, A.; et al. North American Alliance for the Study of Digestive Manifestations of COVID-19. Digestive Manifestations in Patients Hospitalized With Coronavirus Disease 2019. Clin. Gastroenterol. Hepatol. 2021, 19, 1355–1365.e4. [CrossRef] 8. Guan, W.J.; Ni, Z.Y.; Hu, Y. Clinical characteristics of coronavirus disease 2019 in China. N. Engl. J. Med. 2020, 382, 1708–1720. [CrossRef] 9. Estevez-Cerda, S.C.; Saldaña-Rodríguez, J.A.; Alam-Gidi, A.G.; Riojas-Garza, A.; Rodarte-Shade, M.; Velazco-de la Garza, J.; Leyva-Alvizo, A.; Gonzalez-Ruvalcaba, R.; Martinez-Resendez, M.F.; Ortiz de Elguea-Lizarraga, J.I. Severe bowel complications in SARS-CoV-2 patients receiving protocolized care. Rev. Gastroenterol. Mex. Engl. Ed. 2021, 86, 378–386. [CrossRef] 10. Redd, W.D.; Zhou, J.C.; Hathorn, K.E. Prevalence and characteristics of gastrointestinal symptoms in patients with SARS-CoV-2 infection in the United States: A multicenter cohort study. Gastroenterology 2020, 159, 765–767.e2. [CrossRef] 11. Hajifathalian, K.; Krisko, T.; Mehta, A. Gastrointestinal and hepatic manifestations of 2019 novel coronavirus disease in a large cohort of infected patients from New York: Clinical implications. Gastroenterology 2020, 159, 1137–1140.e2. [CrossRef] 12. Kotfis, K.; Skonieczna-Zydecka, K. COVID-19: Gastrointestinal symptoms and potential sources of SARS-CoV-2 transmission. ˙ Anaesthesiol. Intensive Ther. 2020, 52, 171–172. [CrossRef] 13. Xiao, F.; Tang, M.; Zheng, X. Evidence for gastrointestinal infection of SARS-CoV-2. Gastroenterology 2020, 158, 1831–1833. [CrossRef] [PubMed] 14. Xu, Y.; Li, X.; Zhu, B.; Liang, H.; Fang, C.; Gong, Y.; Guo, Q.; Sun, X.; Zhao, D.; Shen, J.; et al. Characteristics of pediatric SARS-CoV-2 infection and potential evidence for persistent fecal viral shedding. Nat. Med. 2020, 26, 502–505. [CrossRef] [PubMed] 15. Ludewig, S.; Jarbouh, R.; Ardelt, M.; Mothes, H.; Rauchfuß, F.; Fahrner, R.; Zanow, J.; Settmacher, U. Bowel Ischemia in ICU Patients: Diagnostic Value of I-FABP Depends on the Interval to the Triggering Event. Gastroenterol. Res. Pract. 2017, 2795176. [CrossRef] 16. Kaafarani, H.; El Moheb, M.; Hwabejire, J.O.; Naar, L.; Christensen, M.A.; Breen, K.; Gaitanidis, A.; Alser, O.; Mashbari, H.; Bankhead-Kendall, B.; et al. Gastrointestinal Complications in Critically Ill Patients With COVID-19. Ann. Surg. 2020, 272, e61–e62. [CrossRef] 17. Kraft, M.; Pellino, G.; Jofra, M.; Sorribas, M.; Solís-Peña, A.; Biondo, S.; Espín-Basany, E. Incidence, features, outcome and impact on health system of de-novo abdominal surgical diseases in patients admitted with COVID-19. Surg. J. R. Coll. Surg. Edinb. Irel. 2021, 19, e53–e58. [CrossRef] 18. Yang, C.; Hakenberg, P.; Weiß, C.; Herrle, F.; Rahbari, N.; Reißfelder, C.; Hardt, J. Colon ischemia in patients with severe COVID-19: A single-center retrospective cohort study of 20 patients. Int. J. Colorectal Dis. 2021, 36, 2769–2773. [CrossRef] J. Clin. Med. 2022, 11, 200 20 of 22 19. Hwabejire, J.O.; Kaafarani, H.M.; Mashbari, H.; Misdraji, J.; Fagenholz, P.J.; Gartland, R.M.; Abraczinskas, D.R.; Mehta, R.S.; Paranjape, C.N.; Eng, G.; et al. Bowel Ischemia in COVID-19 Infection: One-Year Surgical Experience. Am. Surg. 2021, 87, 1893–1900. [CrossRef] [PubMed] 20. O’shea, A.; Parakh, A.; Hedgire, S.; Lee, S.I. Multisystem assessment of the imaging manifestations of coagulopathy in hospitalized patients with coronavirus. Am. J. Roentgenol. 2021, 216, 1088–1098. [CrossRef] [PubMed] 21. Qayed, E.; Deshpande, A.R.; Elmunzer, B.J.; North American Alliance for the Study of Digestive Manifestations of COVID-19. Low Incidence of Severe Gastrointestinal Complications in COVID-19 Patients Admitted to the Intensive Care Unit: A Large, Multicenter Study. Gastroenterology 2021, 160, 1403–1405. [CrossRef] [PubMed] 22. Azouz, E.; Yang, S.; Monnier-Cholley, L.; Arrivé, L. Systemic arterial thrombosis and acute mesenteric ischemia in a patient with COVID-19. Intensive Care Med. 2020, 46, 1464–1465. [CrossRef] [PubMed] 23. Al Mahruqi, G.; Stephen, E.; Abdelhedy, I.; Al Wahaibi, K. Our early experience with mesenteric ischemia in COVID-19 positive patients. Ann. Vasc. Surg. 2021, 73, 129–132. [CrossRef] [PubMed] 24. Ucpinar, B.A.; Sahin, C. Superior Mesenteric Artery Thrombosis in a Patient with COVID-19: A Unique Presentation. J. Coll Physicians Surg. Pak. 2020, 30, 112–114. [CrossRef] 25. Karna, S.T.; Panda, R.; Maurya, A.P.; Kumari, S. Superior Mesenteric Artery Thrombosis in COVID-19 Pneumonia: An Underestimated Diagnosis—First Case Report in Asia. Indian J. Surg. 2020, 82, 1235–1237. [CrossRef] 26. Abeysekera, K.W.; Karteszi, H.; Clark, A.; Gordon, F.H. Spontaneous portomesenteric thrombosis in a non-cirrhotic patient with SARS-CoV-2 infection. BMJ Case Rep. 2020, 13, e238906. [CrossRef] 27. Rodriguez-Nakamura, R.M.; Gonzalez-Calatayud, M.; Martinez Martinez, A.R. Acute mesenteric thrombosis in two patients with COVID-19. Two cases report and literature review. Int. J. Surg. Case Rep. 2020, 76, 409–414. [CrossRef] 28. Dinoto, E.; Ferlito, F.; La Marca, M.A.; Mirabella, D.; Bajardi, G.; Pecoraro, F. Staged acute mesenteric and peripheral ischemia treatment in COVID-19 patient: Case report. Int. J. Surg. Case Rep. 2021, 84, 106105. [CrossRef] 29. Kiwango, F.; Mremi, A.; Masenga, A.; Akrabi, H. Intestinal ischemia in a COVID-19 patient: Case report from Northern Tanzania. J. Surg. Case Rep. 2021, 2021, rjaa537. [CrossRef] 30. Sevella, P.; Rallabhandi, S.; Jahagirdar, V.; Kankanala, S.R.; Ginnaram, A.R.; Rama, K. Acute Mesenteric Ischemia as an Early Complication of COVID-19. Cureus 2021, 13, e18082. [CrossRef] 31. Nasseh, S.; Trabelsi, M.M.; Oueslati, A.; Haloui, N.; Jerraya, H.; Nouira, R. COVID-19 and gastrointestinal symptoms: A case report of a Mesenteric Large vessel obstruction. Clin. Case Rep. 2021, 9, e04235. [CrossRef] [PubMed] 32. Alemán, W.; Cevallos, L.C. Subacute mesenteric venous thrombosis secondary to COVID-19: A late thrombotic complication in a nonsevere patient. Radiol. Case Rep. 2021, 16, 899–902. [CrossRef] [PubMed] 33. Jeilani, M.; Hill, R.; Riad, M.; Abdulaal, Y. Superior mesenteric vein and portal vein thrombosis in a patient with COVID-19: A rare case. BMJ Case Rep. 2021, 14, e244049. [CrossRef] 34. Randhawa, J.; Kaur, J.; Randhawa, H.S.; Kaur, S.; Singh, H. Thrombosis of the Portal Vein and Superior Mesenteric Vein in a Patient With Subclinical COVID-19 Infection. Cureus 2021, 13, e14366. [CrossRef] [PubMed] 35. Cheung, S.; Quiwa, J.C.; Pillai, A.; Onwu, C.; Tharayil, Z.J.; Gupta, R. Superior Mesenteric Artery Thrombosis and Acute Intestinal Ischemia as a Consequence of COVID-19 Infection. Am. J. Case Rep. 2020, 21, e925753. [CrossRef] 36. Beccara, L.A.; Pacioni, C.; Ponton, S.; Francavilla, S.; Cuzzoli, A. Arterial Mesenteric Thrombosis as a Complication of SARS-CoV-2 Infection. Eur. J. Case Rep. Intern. Med. 2020, 7, 001690. [CrossRef] [PubMed] 37. Vulliamy, P.; Jacob, S.; Davenport, R.A. Acute aorto-iliac and mesenteric arterial thromboses as presenting features of COVID-19. Br. J. Haematol. 2020, 189, 1053–1054. [CrossRef] 38. De Barry, O.; Mekki, A.; Diffre, C.; Seror, M.; El Hajjam, M.; Carlier, R.Y. Arterial and venous abdominal thrombosis in a 79-year-old woman with COVID-19 pneumonia. Radiol. Case Rep. 2020, 15, 1054–1057. [CrossRef] 39. Romero, M.D.C.V.; Cárdenas, A.M.; Fuentes, A.B.; Barragán, A.A.S.; Gómez, D.B.S.; Jiménez, M.T. Acute mesenteric arterial thrombosis in severe SARS-Co-2 patient: A case report and literature review. Int. J. Surg. Case Rep. 2021, 86, 106307. [CrossRef] 40. Posada-Arango, A.M.; García-Madrigal, J.; Echeverri-Isaza, S.; Alberto-Castrillón, G.; Martínez, D.; Gómez, A.C.; Pinto, J.A.; Pinillos, L. Thrombosis in abdominal vessels associated with COVID-19 Infection: A report of three cases. Radiol. Case Rep. 2021, 16, 3044–3050. [CrossRef] 41. Pang, J.H.Q.; Tang, J.H.; Eugene-Fan, B. A peculiar case of small bowel stricture in a coronavirus disease 2019 patient with congenital adhesion band and superior mesenteric vein thrombosis. Ann. Vasc. Surg. 2021, 70, 286–289. [CrossRef] 42. Lari, E.; Lari, A.; AlQinai, S. Severe ischemic complications in COVID-19-a case series. Int. J. Surg. Case Rep. 2020, 75, 131–135. [CrossRef] [PubMed] 43. Carmo Filho, A.; Cunha, B.D.S. Inferior mesenteric vein thrombosis and COVID-19. Rev. Soc. Bras. Med. Trop. 2020, 53, e20200412. [CrossRef] 44. Hanif, M.; Ahmad, Z.; Khan, A.W.; Naz, S.; Sundas, F. COVID-19-Induced Mesenteric Thrombosis. Cureus 2021, 13, e12953. [CrossRef] 45. Amaravathi, U.; Balamurugan, N.; Muthu Pillai, V.; Ayyan, S.M. Superior Mesenteric Arterial and Venous Thrombosis in COVID-19. J. Emerg. Med. 2021, 60, e103–e107. [CrossRef] [PubMed] 46. Goodfellow, M.; Courtney, M.; Upadhyay, Y.; Marsh, R.; Mahawar, K. Mesenteric Venous Thrombosis Due to Coronavirus in a Post Roux-en-Y Gastric Bypass Patient: A Case Report. Obes. Surg. 2021, 31, 2308–2310. [CrossRef] [PubMed] J. Clin. Med. 2022, 11, 200 21 of 22 47. Plotz, B.; Castillo, R.; Melamed, J.; Magro, C.; Rosenthal, P.; Belmont, H.M. Focal small bowel thrombotic microvascular injury in COVID-19 mediated by the lectin complement pathway masquerading as lupus enteritis. Rheumatology 2021, 60, e61–e63. [CrossRef] 48. Chiu, C.Y.; Sarwal, A.; Mon, A.M.; Tan, Y.E.; Shah, V. Gastrointestinal: COVID-19 related ischemic bowel disease. J. Gastroenterol. Hepatol. 2021, 36, 850. [CrossRef] [PubMed] 49. Farina, D.; Rondi, P.; Botturi, E.; Renzulli, M.; Borghesi, A.; Guelfi, D.; Ravanelli, M. Gastrointestinal: Bowel ischemia in a suspected coronavirus disease (COVID-19) patient. J. Gastroenterol. Hepatol. 2021, 36, 41. [CrossRef] 50. Singh, B.; Mechineni, A.; Kaur, P.; Ajdir, N.; Maroules, M.; Shamoon, F.; Bikkina, M. Acute Intestinal Ischemia in a Patient with COVID-19 Infection. Korean J. Gastroenterol. 2020, 76, 164–166. [CrossRef] 51. Nakatsutsumi, K.; Endo, A.; Okuzawa, H.; Onishi, I.; Koyanagi, A.; Nagaoka, E.; Morishita, K.; Aiboshi, J.; Otomo, Y. Colon perforation as a complication of COVID-19: A case report. Surg. Case Rep. 2021, 7, 175. [CrossRef] 52. Carubbi, F.; Salvati, L.; Alunno, A.; Maggi, F.; Borghi, E.; Mariani, R.; Mai, F.; Paoloni, M.; Ferri, C.; Desideri, G.; et al. Ferritin is associated with the severity of lung involvement but not with worse prognosis in patients with COVID-19: Data from two Italian COVID-19 units. Sci. Rep. 2021, 11, 4863. [CrossRef] 53. Isfordink, C.J.; Dekker, D.; Monkelbaan, J.F. Clinical value of serum lactate measurement in diagnosing acute mesenteric ischaemia. Neth. J. Med. 2018, 76, 60–64. [PubMed] 54. Montagnana, M.; Danese, E.; Lippi, G. Biochemical markers of acute intestinal ischemia: Possibilities and limitations. Ann. Transl. Med. 2018, 6, 341. [CrossRef] 55. Matsumoto, S.; Sekine, K.; Funaoka, H.; Yamazaki MShimizu, M.; Hayashida, K.; Kitano, M. Diagnostic performance of plasma biomarkers in patients with acute intestinal ischaemia. Br. J. Surg. 2014, 101, 232–238. [CrossRef] [PubMed] 56. Soni, N.; Bhutra, S.; Vidyarthi, S.H.; Sharma, V. Role of serum lactic dehydrogenase, glutamic oxaloacetic transaminase, creatine phosphokinase, alkaline phospatase, serum phosphorus in the cases of bowel Ischaemia in acute abdomen. Int. Surg. J. 2017, 4, 1997–2001. [CrossRef] 57. Han, Y.; Zhang, H.; Mu, S.; Wei, W.; Jin, C.; Tong, C.; Song, Z.; Zha, Y.; Xue, Y.; Gu, G. Lactate dehydrogenase, an independent risk factor of severe COVID-19 patients: A retrospective and observational study. Aging 2020, 12, 11245–11258. [CrossRef] 58. Carpenè, G.; Onorato, D.; Nocini, R.; Fortunato, G.; Rizk, J.G.; Henry, B.M.; Lippi, G. Blood lactate concentration in COVID-19: A systematic literature review. Clin. Chem. Lab. Med. 2021. advance online publication. [CrossRef] 59. Singh, B.; Kaur, P.; Maroules, M. Splanchnic vein thrombosis in COVID-19: A review of literature. Dig. Liver Dis. 2020, 52, 1407–1409. [CrossRef] 60. Jagielski, M.; Pi ˛atkowski, J.; Jackowski, M. Challenges encountered during the treatment of acute mesenteric ischemia. Gastroenterol. Res. Pract. 2020, 5316849. [CrossRef] [PubMed] 61. Rasslan, R.; Dos Santos, J.P.; Menegozzo, C.; Pezzano, A.; Lunardeli, H.S.; Dos Santos Miranda, J.; Utiyama, E.M.; Damous, S. Outcomes after emergency abdominal surgery in COVID-19 patients at a referral center in Brazil. Updates Surg. 2021, 73, 763–768. [CrossRef] 62. Lei, S.; Jiang, F.; Su, W.; Chen, C.; Chen, J.; Mei, W.; Zhan, L.Y.; Jia, Y.; Zhang, L.; Liu, D.; et al. Clinical characteristics and outcomes of patients undergoing surgeries during the incubation period of COVID-19 infection. EClinicalMedicine 2020, 21, 100331. [CrossRef] 63. Serban, D.; Socea, B.; Badiu, C.D.; Tudor, C.; Balasescu, S.A.; Dumitrescu, D.; Trotea, A.M.; Spataru, R.I.; Vancea, G.; Dascalu, A.M.; et al. Acute surgical abdomen during the COVID 19 pandemic: Clinical and therapeutic challenges. Exp. Ther. Med. 2021, 21, 519. [CrossRef] [PubMed] 64. Patel, S.; Parikh, C.; Verma, D.; Sundararajan, R.; Agrawal, U.; Bheemisetty, N.; Akku, R.; Sánchez-Velazco, D.; Waleed, M.S. Bowel ischaemia in COVID-19: A systematic review. Int. J. Clin. Pract. 2021, 75, e14930. [CrossRef] [PubMed] 65. Yantiss, R.K.; Qin, L.; He, B.; Crawford, C.V.; Seshan, S.; Patel, S.; Wahid, N.; Jessurun, J. Intestinal Abnormalities in Patients With SARS-CoV-2 Infection: Histopathologic Changes Reflect Mechanisms of Disease. Am. J. Surg. Pathol. 2021, 46, 89–96. [CrossRef] [PubMed] 66. McGonagle, D.; Bridgewood, C.; Ramanan, A.V.; Meaney, J.F.M.; Watad, A. COVID-19 vasculitis and novel vasculitis mimics. Lancet Rheumatol. 2021, 3, e224–e233. [CrossRef] 67. Huang, C.; Wang, Y.; Li, X. Clinical features of patients infected with 2019 novel coronavirus in Wuhan. China Lancet 2020, 395, 497–506. [CrossRef] 68. Avila, J.; Long, B.; Holladay, D.; Gottlieb, M. Thrombotic complications of COVID-19. Am. J. Emerg. Med. 2021, 39, 213–218. [CrossRef] 69. Varga, Z.; Flammer, A.J.; Steiger, P.; Haberecker, M.; Andermatt, R.; Zinkernagel, A.S.; Mehra, M.R.; Schuepbach, R.A.; Ruschitzka, F.; Moch, H. Endothelial cell infection and endotheliitis in COVID-19. Lancet 2020, 395, 1417–1418. [CrossRef] 70. Maier, C.L.; Truong, A.D.; Auld, S.C.; Polly, D.M.; Tanksley, C.L.; Duncan, A. COVID-19-associated hyperviscosity: A link between inflammation and thrombophilia? Lancet 2020, 395, 1758–1759. [CrossRef] 71. Miyara, S.J.; Becker, L.B.; Guevara, S.; Kirsch, C.; Metz, C.N.; Shoaib, M.; Grodstein, E.; Nair, V.V.; Jandovitz, N.; McCannMolmenti, A.; et al. Pneumatosis Intestinalis in the Setting of COVID-19: A Single Center Case Series From New York. Front. Med. 2021, 8, 638075. [CrossRef] [PubMed] J. Clin. Med. 2022, 11, 200 22 of 22 72. Panigada, M.; Bottino, N.; Tagliabue, P.; Grasselli, G.; Novembrino, C.; Chantarangkul, V.; Pesenti, A.; Peyvandi, F.; Tripodi, A. Hypercoagulability of COVID-19 patients in intensive care unit: A report of thromboelastography findings and other parameters of hemostasis. J. Thromb. Haemost. 2020, 18, 1738–1742. [CrossRef] 73. Hottz, E.D.; Azevedo-Quintanilha, I.G.; Palhinha, L.; Teixeira, L.; Barreto, E.A.; Pão, C.R.; Righy, C.; Franco, S.; Souza, T.M.; Kurtz, P.; et al. Platelet activation and platelet-monocyte aggregate formation trigger tissue factor expression in patients with severe COVID-19. Blood J. Am. Soc. Hematol. 2020, 136, 1330–1341. [CrossRef] 74. Stefely, J.A.; Christensen, B.B.; Gogakos, T.; Cone Sullivan, J.K.; Montgomery, G.G.; Barranco, J.P.; Van Cott, E.M. Marked factor V activity elevation in severe COVID-19 is associated with venous thromboembolism. Am. J. Hematol. 2020, 95, 1522–1530. [CrossRef] 75. Giuffrè, M.; Di Bella, S.; Sambataro, G.; Zerbato, V.; Cavallaro, M.; Occhipinti, A.A.; Palermo, A.; Crescenti, A.; Monica, F.; Luzzati, R.; et al. COVID-19-Induced Thrombosis in Patients without Gastrointestinal Symptoms and Elevated Fecal Calprotectin: Hypothesis Regarding Mechanism of Intestinal Damage Associated with COVID-19. Trop. Med. Infect. Dis. 2020, 5, 147. [CrossRef] [PubMed] 76. Giuffrè, M.; Vetrugno, L.; Di Bella, S.; Moretti, R.; Berretti, D.; Crocè, L.S. Calprotectin and SARS-CoV-2: A Brief-Report of the Current Literature. Healthcare 2021, 9, 956. [CrossRef] [PubMed] 77. Buso, G.; Becchetti, C.; Berzigotti, A. Acute splanchnic vein thrombosis in patients with COVID-19: A systematic review. Dig. Liver Dis. 2021, 53, 937–949. [CrossRef] 78. Thachil, J. The versatile heparin in COVID-19. J. Thromb. Haemost. 2020, 18, 1020–1022. [CrossRef] 79. Poterucha, T.J.; Libby, P.; Goldhaber, S.Z. More than an anticoagulant: Do heparins have direct anti-inflammatory effects? Thromb. Haemost. 2017, 117, 437–444. [CrossRef] 80. Wang, M.K.; Yue, H.Y.; Cai, J.; Zhai, Y.J.; Peng, J.H.; Hui, J.F.; Hou, D.Y.; Li, W.P.; Yang, J.S. COVID-19 and the digestive system: A comprehensive review. World J. Clin. Cases 2021, 9, 3796–3813. [CrossRef] 81. Manolis, A.S.; Manolis, T.A.; Manolis, A.A.; Papatheou, D.; Melita, H. COVID-19 Infection: Viral Macro- and Micro-Vascular Coagulopathy and Thromboembolism/Prophylactic and Therapeutic Management. J. Cardiovasc. Pharmacol. Ther. 2021, 26, 12–24. [CrossRef] [PubMed] 82. Conti, P.; Caraffa, A.; Gallenga, C.E.; Ross, R.; Kritas, S.K.; Frydas, I.; Younes, A.; Di Emidio, P.; Ronconi, G.; Toniato, E. IL-1 induces throboxane-A2 (TxA2) in COVID-19 causing inflammation and micro-thrombi: Inhibitory effect of the IL-1 receptor antagonist (IL-1Ra). J. Biol. Regul. Homeost. Agents 2020, 34, 1623–1627. [CrossRef] [PubMed]

Review of Mesenteric Ischemia in COVID-19 Patients

Authors: Amit GuptaOshin SharmaKandhala SrikanthRahul MishraAmoli Tandon & Deepak Rajput  Indian Journal of Surgery (2022) Published: 

Abstract

The new coronavirus (COVID-19) infection, first detected in Wuhan, China in 2019 has become a pandemic that has spread to nearly every country in the world. Through October 11, 2021, more than 23 billion confirmed cases and 4.8 million fatalities were reported globally. The bulk of individuals afflicted in India during the first wave were elderly persons. The second wave, however, resulted in more severe diseases and mortality in even younger age groups due to mutations in the wild virus. Symptoms may range from being asymptomatic to fatal acute respiratory distress syndrome (ARDS). In addition to respiratory symptoms, patients may present with gastrointestinal symptoms such as stomach pain, vomiting, loose stools, or mesenteric vein thrombosis. The frequency of patients presenting with thromboembolic symptoms has recently increased. According to certain studies, the prevalence of venous thromboembolism among hospitalized patients ranges from 9 to 25%. It was also shown that the incidence is significantly greater among critically sick patients, with a prevalence of 21–31%. Although the exact origin of thromboembolism is unknown, it is considered to be produced by several altered pathways that manifest as pulmonary embolism, myocardial infarction, stroke, limb gangrene, and acute mesenteric ischemia. Acute mesenteric ischemia (AMI) is becoming an increasingly prevalent cause of acute surgical abdomen in both intensive care unit (ICU) and emergency room (ER) patients. Mesenteric ischemia should be evaluated in situations with unexplained stomach discomfort. In suspected situations, appropriate imaging techniques and early intervention, either non-surgical or surgical, are necessary to avert mortality. The purpose of this article is to look at the data on acute mesenteric ischemia in people infected with COVID-19.

Introduction

Aside from the respiratory system, the gastrointestinal system is the most common site of SARS-COV-2 infection. This might be because enterocyte and vascular endothelial membranes have large amounts of angiotensin-converting enzyme receptor 2, a membrane integral protein. As a result, the COVID virus induces direct enterocyte invasion as well as indirect endothelial injury-induced thrombosis/intestinal ischemia in the bowel [1]. ICU patients are more prone than non-ICU patients to suffer acute mesenteric ischemia. This might be because, in addition to the direct viral activity on vascular endothelium, ICU patients have extra persistent pro-inflammatory effects. Cases have been observed even among individuals who have recovered from infection [2]. A rising number of cases of acute mesenteric ischemia in COVID-19 patients have been reported in the literature since the outbreak of this pandemic (list of reported cases are summarized in the Table 1). AMI risk was shown to be increased with age, male sex, and comorbidities such as hypertension, obesity, and diabetes mellitus. Because of delayed clinical manifestation, AMI-related mortality is quite significant, with 60–80% [3].Table 1 Summary of the cases reported on mesenteric ischemia in COVID-19 patientsFull size table

Case summary

A 55 years old man with no known comorbidity presented to the emergency department of our institute with severe pain abdomen and multiple episodes of vomiting. He reported the recent recovery from the non-complicated COVID-related illness. He did not report any intake of anticoagulants. On clinical examination, abdomen was unremarkable. X-ray chest, x-ray erect abdomen, and ultrasound abdomen were unremarkable. Mesenteric ischemia was suspected and the patient was subjected to CT angiography abdomen, which revealed thrombus at the origin of the superior mesenteric artery and impending gangrene of the small bowel (Fig. 1). Emergency laparotomy was done and intraoperatively found the gangrenous bowel involving the distal jejunum and almost the entire ileum sparing the terminal ileum (Fig. 2). Resection of the gangrenous small bowel and end jejunostomy was done. Later, he was given ICU care, but unfortunately, the patient succumbed to multi-organ dysfunction syndrome.

figure 1
Fig. 1
figure 2
Fig. 2

Pathophysiology

Although the specific etiology of hypercoagulable state and subsequent mesenteric ischemia in COVID-19 patients is unknown, these thromboembolic events can be related to alterations in all three Virchow triad characteristics (vascular endothelial injury, hypercoagulability, and stasis). A variety of variables complicate the etiology of thrombus development, one of which is vascular endothelial injury. Capillary permeability, hemostasis, and fibrinolysis are all maintained by the vascular endothelium (Fig. 3). Direct invasion causes endothelial cells to be damaged and lysed, resulting in an imbalance between pro and anticoagulant states [4]. Furthermore, vascular endothelial cells displayed morphological changes such as cellular expansion, retraction, and intercellular connection breakage [5]. The elevated levels of pro-inflammatory markers, von Willebrand factor, tissue factor, fibrinogen, and circulating microvesicles in the COVID-19 patients explain their hypercoagulability [6]. Antiphospholipid antibodies are elevated in some situations [7]. Patients who are critically ill, on limited oxygen support, and mechanical breathing are less mobilized, which increases the risk of deep venous thrombosis [3].

figure 3
Fig. 3

These mesenteric vascular thromboses cause acute hypoxia in the intestinal wall, which stimulates the renin-angiotensin system, causing mesenteric vasospasm and an elevated risk of hypoxic injury. SARS-COV binds to ACE 2 receptors in intestinal cells, causing cell lysis [8]. As a result, both hypoxia and direct invasion can trigger intestinal cell death. The loss of this epithelial barrier function in the gut promotes increased contact with enteric bacteria/endotoxins and viral particle penetration into the circulation [5]. The hypoxia continues, resulting in transmural infarction, perforation, and peritonitis. In one example of mesenteric ischemia induced by invasive mucormycosis, the presence of fungal components in the mesenteric microcirculation was documented [2]. See the flow chart summarizing the pathophysiology of mesenteric ischemia in covid-19 infection.

Clinical Presentation

Patients with mesenteric ischemia may exhibit a range of symptoms, from nonspecific complaints to peritonitis-like symptoms. Most of the patients developed symptoms a few days after being discharged successfully with proper symptomatic inpatient care. Although the respiratory symptoms predominate mesenteric ischemia presents with nonspecific abdominal symptoms such as loose stools, abdominal pain, nausea, vomiting, abdominal distension, and bleeding per rectum may occur in addition to the usual clinical presentation with respiratory features [6]. When opposed to arterial thrombosis, venous thrombosis has a delayed onset of symptoms. At first, sudden onset pain in the abdomen may be the sole symptom, and it may develop after 5–14 days. Abdominal clinical examination is nonyielding in the majority of cases. Abdominal signs would not develop unless the bowel gangrene or bowel perforation with peritonitis occurs [9].

Investigations

Blood investigations

Despite extensive study on the subject of acute mesenteric ischemia, the associated biomarkers were shown to be neither sensitive nor selective [10]. Elevated lactic acid levels and fibrin degradation products like D-dimer have low specificity and remain elevated in severe COVID-19 without AMI. However, biomarkers associated with hypercoagulable conditions aid in the initiation of preventive treatment and, to a lesser extent, in the management of COVID-related thrombotic events. Increased biomarkers of inflammation and infection include leukopenia (due to corticosteroid usage) and other signs such as C-reactive protein, procalcitonin, and IL-6. D-dimer, ferritin, prothrombin time, and lactate dehydrogenase are additional significant markers. The severity of increased lactate dehydrogenase and ferritin levels is associated with high mortality[8].

Radiological imaging

In the emergency room, an X-ray of the abdomen and an ultrasound are helpful for early examinations. X-ray of the erect abdomen helps in initial assessment in cases presented with features of obstruction or perforation. Ultrasound in the early phase may show SMA occlusion and bowel spasm or ultrasound findings in the early stages of acute mesenteric ischemia may appear normal [11]. In the intermediate phase, USG is not useful because of the presence of a large amount of gas-filled intestinal loops. In the late phase, USG may reveal fluid-filled lumen, bowel wall thinning, evidence of extra-luminal fluid, decreased or absent peristalsis. Therefore, USG may be helpful in the diagnosis of advanced bowel obstruction, gangrene, and perforation with peritoneal collection [12]. Ultrasonography revealed some other important features with distended and sludge-filled gall bladder with bile stasis. Portal venous gas also can be detected on ultrasonography which can be better characterized with the help of computed tomography [13].

Computed tomography

The gold standard investigation is CT angiography. CT observations commonly encountered in acute mesenteric ischemia secondary to COVID-19 includes thrombus in the aorta/SMA/portal circulation, augmentation of the bowel wall, thickness of the bowel wall with distention(> 3 cm), edema, and stranding of the mesentery, pneumatosis intestinalis or portal venous gas suggesting bowel wall ischemia, and non-enhancing thick bowel wall seen in bowel infarction, bowel perforation secondary to bowel infarction may present discontinuity of bowel wall with localized air collection. One should remember that pneumatosis intestinalis may also occur due to mechanical ventilation. Pneumoperitoneum occurs when there is severe intestinal necrosis and perforation. There were additional reports of nonspecific features such as a dilated gut with a fluid-filled lumen, distended gallbladder with bile stasis, features of solid organ ischemia, and pancreatitis [14]. MRI, despite its accessibility, has drawbacks such as a longer acquisition time and lower resolution than CT angiography [12].

Management

A summary of cases of acute mesenteric ischemia has been tabulated (Table 1). Management of acute mesenteric ischemia in COVID-19 includes the following:

  • Supportive measures: Crystalloid rehydration and empirical antibacterial treatment should begin before angiography or any surgical resection. Comorbidity management, hemodynamic support in unstable patients, and electrolyte balance correction are all critical components of patient care [10].
  • Anticoagulation: There is insufficient data in 19 patients to warrant thromboprophylaxis. According to the Tang et al. study, low-dose heparin prophylaxis decreased thrombotic events and mortality in those with D-dimer levels over 3 mg/ml. Despite the increased risk of bleeding, mesenteric ischemia should be treated with intraoperative and postoperative anticoagulation [15].
  • Revascularisation: Revascularization with catheter-directed thrombolysis and thrombectomy by percutaneous/surgical intervention can be explored in instances where there is no indication of significant intestinal ischemia. Catheter-directed thrombolysis with unfractionated heparin and recombinant tissue plasminogen activators can accomplish this. Because of the increased risk of re-thrombosis, vascular clearance is not indicated in instances of superior mesenteric vein thrombus [15].
  • Resection of the gangrenous bowel: Depending on clinical suspicion, a CT angiography examination of mesenteric vasculature and bowel health can be performed, and an emergency exploration call should be placed. Intraoperatively, if the patient is normotensive, has no sepsis or peritonitis, and the remaining bowel viability is unquestionable, the gangrenous bowel is to be removed, and the remaining bowel can be considered for re-anastomosis. In unfavorable circumstances, a stoma should be created following gangrenous bowel resection [11]. The margin dissection in venous thrombosis should be broader than in arterial thrombosis. To assure the bowel’s survivability, abdominal closure should be temporary, and a relook laparotomy should be done 48 h later. Histopathological examination of the resected intestine may indicate patchy or widespread necrotic changes from mucosa to transmural thickness. In the submucosal vasculature, fibrin-containing microthrombi with perivascular neutrophilic infiltration is observed.
  • Management of short bowel syndrome: The therapy varies depending on the length of colon left after excision of infarcted bowel caused by mesenteric ischemia.
  • Medical- In severe diarrhea, fluid and electrolyte loss must be replaced. TPN for feeding and histamine-2 receptor antagonists or PPIs for stomach acid secretion reduction. Loperamide and diphenoxylate are anti-motility medicines that delay small intestine transit whereas Octreotide reduces the volume of gastrointestinal secretions.
  • Non-transplant surgical therapy- Done to improve the absorption capacity of the remaining intestine by restoring intestinal continuity. Increased nutrient and fluid absorption is the goal. Segmental reversal of the small bowel, fabrication of small intestinal valves, and electrical pacing of the small bowel are all procedures used to delay intestinal transit. Longitudinal intestinal lengthening and tailoring technique (LILT) and serial transverse arthroplasty process are two intestinal lengthening procedures (STEP).
  • Intestinal transplantation- Life-threatening problems such as liver failure, thrombosis of major central veins, frequent episodes of severe dehydration, and catheter-related sepsis are reasons for intestinal transplantation [16].

Prognosis

Acute mesenteric ischemia has a poor prognosis, and life is reliant on prompt diagnosis and treatment. If detected within 24 h, the likelihood of survival is 50%, but it declines to 30% beyond that [17].In operated cases, COVID infection acts as an independent risk factor and is responsible for higher mortality [18].

Conclusion

SARS-COV-2 infection even though initially thought to be respiratory infection; later cases detected presenting with multisystem involvement. The presentation may vary from asymptomatic or mildly symptomatic to severe respiratory distress syndrome or thromboembolic phenomenon requiring ICU care. The exact mechanism of thromboembolism is not established. However, the increasing number of acute mesenteric ischemia is quite alarming. The treating physician should be overcautious in patients presenting with abdominal symptoms either currently affected or recovered from COVID-related illness. In high-risk patients, early start of prophylactic anticoagulation may be beneficial. Earlier intervention is known acute mesenteric ischemia cases with operative or minimally invasive procedures may give higher survival benefits. It mandates more research to determine the causes of thromboembolism, as well as preventive and therapeutic anticoagulation in these individuals.

References

  1. Jin B, Singh R, Ha SE, Zogg H, Park PJ, Ro S (2021) Pathophysiological mechanisms underlying gastrointestinal symptoms in patients with COVID-19. World J Gastroenterol. Baishideng Publishing Group Co 27:2341–52CAS Article Google Scholar 
  2. Jain M, Tyagi R, Tyagi R, Jain G (2021) Post-COVID-19 gastrointestinal invasive mucormycosis. Indian J Surg 22:1–3
  3. Kerawala AA, Das B, Solangi A (2021) Mesenteric ischemia in COVID-19 patients: a review of current literature. World J Clin Cases 9(18):4700–4708Article Google Scholar 
  4. Kichloo A, Dettloff K, Aljadah M, Albosta M, Jamal S, Singh J et al (2020) COVID-19 and hypercoagulability: a review. Clin Appl Thromb 26
  5. Parry AH, Wani AH, Yaseen M (2020) Acute mesenteric ischemia in severe Coronavirus-19 (COVID-19): possible mechanisms and diagnostic pathway. Acad Radiol 27(8):1190Article Google Scholar 
  6. Cheung S, Quiwa JC, Pillai A, Onwu C, Tharayil ZJ, Gupta R (2020) Superior mesenteric artery thrombosis and acute intestinal ischemia as a consequence of COVID-19 infection. Am J Case Rep 21:1–3Google Scholar 
  7. Zhang Y, Xiao M, Zhang S, Xia P, Cao W, Jiang W et al (2020) Coagulopathy and antiphospholipid antibodies in patients with Covid-19. N Engl J Med. 382(17):e38Article Google Scholar 
  8. Al Mahruqi G, Stephen E, Abdelhedy I, Al WK (2021) Our early experience with mesenteric ischemia in COVID-19 positive patients. Ann Vasc Surg 73:129–132Article Google Scholar 
  9. Karna ST, Panda R, Maurya AP, Kumari S (2020) Superior mesenteric artery thrombosis in COVID-19 Pneumonia: an underestimated diagnosis—first case report in Asia. Indian J Surg 82(6):1235–1237Article Google Scholar 
  10. Singh B, Kaur P (2021) COVID-19 and acute mesenteric ischemia: a review of literature. Hematol Transfus Cell Ther 43(1):112–116Article Google Scholar 
  11. Janež J, Klen J (2021) Multidisciplinary diagnostic and therapeutic approach to acute mesenteric ischaemia: a case report with literature review. SAGE Open Med Case Rep 9:2050313X2110048Article Google Scholar 
  12. Mc W (2010) Acute mesenteric ischemia: diagnostic approach and surgical treatment. Semin Vasc Surg 23(1):9–20Article Google Scholar 
  13. Bhayana R, Som A, Li MD, Carey DE, Anderson MA, Blake MA et al (2020) Abdominal imaging findings in COVID-19: Preliminary observations. Radiology 297(1):E207–E215
  14. Keshavarz P, Rafiee F, Kavandi H, Goudarzi S, Heidari F, Gholamrezanezhad A (2021) Ischemic gastrointestinal complications of COVID-19: a systematic review on imaging presentation. Clin Imaging 73:86–95Article Google Scholar 
  15. Bergqvist D, Svensson PJ (2010) Treatment of mesenteric vein thrombosis. Semin Vasc Surg 23(1):65–68Article Google Scholar 
  16. Seetharam P, Rodrigues G (2011) Short bowel syndrome: a review of management options. Saudi J Gastroenterol 17(4):229–235Article Google Scholar 
  17. Krothapalli N, Jacob J (2021) A rare case of acute mesenteric ischemia in the setting of COVID-19 infection. Cureus 13(3):0–4Google Scholar 
  18. Haffner MR, Le HV, Saiz AM, Han G, Fine J, Wolinsky P et al (2021) Postoperative In-hospital morbidity and mortality of patients with COVID-19 infection compared with patients without COVID-19 infection. JAMA Netw Open 4(4):10–13Article Google Scholar 
  19. Ucpinar BA, Sahin C (2020) Superior mesenteric artery thrombosis in a patient with COVID-19: a unique presentation. J Coll Physicians Surg Pak 30(10):S112–S114Google Scholar 
  20. Khesrani LS, Chana k, Sadar FZ, Dahdouh A, Ladjadj Y, Bouguermouh D (2020) Intestinal ischemia secondary to Covid-19. J Pediatr Surg Case Rep 61:101604Article Google Scholar 
  21. Norsa L, Valle C, Morotti D, Bonaffini PA, Indriolo A, Sonzogni A (2020) Intestinal ischemia in the COVID-19 era. Dig Liver Dis 52(10):1090–1091CAS Article Google Scholar 
  22. Rodriguez-Nakamura RM, Gonzalez-Calatayud M, Martinez Martinez AR (2020) Acute mesenteric thrombosis in two patients with COVID-19. Two cases report and literature review. Int J Surg Case Rep 76:409–14Article Google Scholar 
  23. VartanogluAktokmakyan T, Tokocin M, Meric S, Celebi F (2021) Is mesenteric ischemia in COVID-19 patients a surprise? Surg Innov 28(2):236–238Article Google Scholar 
  24. Levolger S, Bokkers RPH, Wille J, Kropman RHJ, de Vries JPPM (2020) Arterial thrombotic complications in COVID-19 patients. J Vasc Surg Cases Innov Tech 6(3):454–459Article Google Scholar 
  25. Thuluva SK, Zhu H, Tan MML, Gupta S, Yeong KY, Wah STC et al (2020) A 29-year-old male construction worker from india who presented with left-sided abdominal pain due to isolated superior mesenteric vein thrombosis associated with SARS-CoV-2 infection. Am J Case Rep 21:1–5Article Google Scholar 
  26. Lari E, Lari A, AlQinai S, Abdulrasoul M, AlSafran S, Ameer A et al (2020) Severe ischemic complications in Covid-19—a case series. Int J Surg Case Rep 75(June):131–135Article Google Scholar 
  27. Singh B, Mechineni A, Kaur P, Ajdir N, Maroules M, Shamoon F et al (2020) Acute intestinal ischemia in a patient with COVID-19 infection. Korean J Gastroenterol 76(3):164–166Article Google Scholar 
  28. De Roquetaillade C, Chousterman BG, Tomasoni D, Zeitouni M, Houdart E (2020) Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID- 19. The COVID-19 resource centre is hosted on Elsevier Connect , the company ’ s public news and information. (January)
  29. Sehhat S, Talebzadeh H, Hakamifard A, Melali H, Shabib S, Rahmati A et al (2020) Acute mesenteric ischemia in a patient with COVID-19: a case report. Arch Iran Med 23(9):639–643Article Google Scholar 
  30. Beccara LA, Pacioni C, Ponton S, Francavilla S, Cuzzoli A (2020) Arterial mesenteric thrombosis as a complication of SARS-CoV-2 infection. Eur J Case Rep Intern Med 7(5).
  31. Ignat M, Philouze G, Aussenac-belle L (2020) Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID- 19 . The COVID-19 resource centre is hosted on Elsevier Connect , the company ’ s public news and information. (Jan)
  32. Farina D, Rondi P, Botturi E, Renzulli M, Borghesi A, Guelfi D et al (2021) Gastrointestinal: bowel ischemia in a suspected coronavirus disease (COVID-19) patient. J Gastroenterol Hepatol 36(1):41CAS Article Google Scholar 
  33. Azouz E, Yang S, Monnier-Cholley L, Arrivé L (2020) Systemic arterial thrombosis and acute mesenteric ischemia in a patient with COVID-19. Intensive Care Med 46(7):1464–1465CAS Article Google Scholar 
  34. Vulliamy P, Jacob S, Davenport RA (2020) Acute aorto-iliac and mesenteric arterial thromboses as presenting features of COVID-19. Br J Haematol 189(6):1053–1054CAS Article Google Scholar 
  35. Bianco F, Ranieri AJ, Paterniti G, Pata F, Gallo G (2020) Acute intestinal ischemia in a patient with COVID-19. Tech Coloproctol 24(11):1217–1218CAS Article Google Scholar 
  36. Filho A do C, Cunha B da S (2020) Case report – inferior mesenteric vein thrombosis and COVID-19. 2020060282
  37. Mitchell JM, Rakheja D, Gopal P (2021) SARS-CoV-2-related hypercoagulable state leading to ischemic enteritis secondary to superior mesenteric artery thrombosis. Clin Gastroenterol Hepatol 19(11):e111CAS Article Google Scholar 
  38. English W, Banerjee S (2020) Coagulopathy and mesenteric ischaemia in severe SARS-CoV-2 infection. ANZ J Surg 90(9):1826Article Google Scholar 
  39. de Barry O, Mekki A, Diffre C, Seror M, El Hajjam M, Carlier RY (2020) Arterial and venous abdominal thrombosis in a 79-year-old woman with COVID-19 pneumonia. Radiol Case Rep 15(7):1054–1057Article Google Scholar 
  40. Kraft M, Pellino G, Jofra M, Sorribas M, Solís-Peña A, Biondo S, Espín-Basany E (2021) Incidence, features, outcome and impact on health system of de-novo abdominal surgical diseases in patients admitted with COVID-19. Surg J R Coll Surg Edinb Irel 19:e53–e58Google Scholar 
  41. Besutti G, Bonacini R, Iotti V, Marini G, Riva N, Dolci G et al (2020) Abdominal visceral infarction in 3 patients with COVID-19. Emerg Infect Dis 26(8):1926–1928CAS Article Google Scholar 
  42. Kielty J, Duggan WP, O’Dwyer M (2020) Extensive pneumatosis intestinalis and portal venous gas mimicking mesenteric ischaemia in a patient with SARS-CoV-2. Ann R Coll Surg Engl 102(6):E145–E147CAS Article Google Scholar 
  43. Pang JHQ, Tang JH, Eugene-Fan B (2021) A peculiar case of small bowel stricture in a coronavirus disease 2019 patient with congenital adhesion band and superior mesenteric vein thrombosis. Ann Vasc Surg 70:286–289Article Google Scholar 
  44. Osilli D, Pavlovica J, Mane R, Ibrahim M, Bouhelal A, Jacob S (2020) Case reports: mild COVID-19 infection and acute arterial thrombosis. J Surg Case Rep (9):1–3

Consequences of COVID-19 for the Pancreas

Authors: Urszula Abramczyk,1,*Maciej Nowaczyński,2Adam Słomczyński,2Piotr Wojnicz,2Piotr Zatyka,2 and Aleksandra Kuzan1 Int J Mol Sci. 2022 Jan; 23(2): 864.Published online 2022 Jan 13. doi: 10.3390/ijms23020864

Abstract

Although coronavirus disease 2019 (COVID-19)-related major health consequences involve the lungs, a growing body of evidence indicates that COVID-19 is not inert to the pancreas either. This review presents a summary of the molecular mechanisms involved in the development of pancreatic dysfunction during the course of COVID-19, the comparison of the effects of non-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on pancreatic function, and a summary of how drugs used in COVID-19 treatment may affect this organ. It appears that diabetes is not only a condition that predisposes a patient to suffer from more severe COVID-19, but it may also develop as a consequence of infection with this virus. Some SARS-CoV-2 inpatients experience acute pancreatitis due to direct infection of the tissue with the virus or due to systemic multiple organ dysfunction syndrome (MODS) accompanied by elevated levels of amylase and lipase. There are also reports that reveal a relationship between the development and treatment of pancreatic cancer and SARS-CoV-2 infection. It has been postulated that evaluation of pancreatic function should be increased in post-COVID-19 patients, both adults and children.

1. Effects of Severe Acute Respiratory Syndrome-Related Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome-Related Coronavirus (MERS-CoV) on the Pancreas

Coronaviruses are enveloped, single- and positive-stranded RNA viruses that infect birds and mammals. In humans, coronaviruses cause respiratory tract infection, usually the common cold, but they can also cause severe respiratory illness including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), caused by severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), respectively [1]. Coronaviruses tend to cause epidemics and even pandemics. The first coronavirus pandemic was the SARS outbreak in 2002–2003 [2]. With the experience gained during the SARS pandemic, it was possible to more quickly identify subsequent outbreaks of the MERS epidemic in 2012 [3]. The pathomechanism of both viruses is very similar—they even both use transmembrane protease serine 2 (TMPRSS2), except SARS-CoV uses angiotensin-converting enzyme 2 (ACE2) as its receptor, whereas MERS uses dipeptidyl peptidase-4 (DPP4) [4,5]. Moreover, there is a difference in terms of the severity and frequency of symptoms, which was observed in MERS patients as more frequent hospitalization in the intensive care unit (ICU) compared to SARS patients [2] (Table 1). Diabetes was one of the significant and independent predictors for developing severe SARS-CoV and MERS-CoV [6,7,8]. In MERS, no viral antigen was detected in any tissue other than pneumocytes [7], despite multiple organ dysfunction syndrome in critically ill patients. In SARS-CoV, the presence of the virus was detected not only in respiratory epithelial cells, but also in small intestinal and colonic epithelial cells, in which it also revealed replication features [9]. It is known that the ACE2 receptor is also present in tissues such as the heart, kidney, and pancreas [8,9]. According to some authors, the presence of the receptor is sufficient for tissue entry and pathogenic activity, although other researchers do not support this thesis [9,10]. Yang et al. were some of the first researchers who hypothesized that SARS coronavirus enters islets using ACE2 as its receptor and damages islets causing acute diabetes [8]. Yang’s study revealed that SARS-CoV had a much higher affinity for pancreatic islet cells than for pancreatic exocrine cells, which was consistent with the hyperglycemia observed in some patients and rarely reported acute pancreatitis (AP) [8]. Furthermore, insulin-dependent diabetes mellitus (IDDM) and high fasting blood glucose values were observed in some inpatients [8]. A 3-year follow-up revealed that both abnormalities were transient, which may be indicative of only temporary damage to the pancreatic islets [8]. However, another reason (different from that given by Young et al.) for high fasting blood glucose value in patients may result from increased stress hormones release. Cortisol, catecholamines, growth hormone, and glucagon, which are released during infection, fever, and trauma, can lead to hyperglycemia to the same degree as SARS-CoV can [11]. No information was found in the literature about a direct impact of the MERS virus on the pancreas or on glycemia during or after infection. This may be due to an insufficiently detailed analysis of the available data during previous studies that oscillated primarily, for laboratory tests, between complete blood count (CBC), lactate dehydrogenase (LDH), urea, and creatinine analysis. A summary of SARS-CoV, MERS, and SARS-CoV-2 is shown in Table 1.Table 1. The summary of characteristics of SARS and MERS coronaviruses. Dipeptidyl peptidase-4 (DPP4), transmembrane protease serine 2 (TMPRSS2), hospitalization in the intensive care unit (ICU), and cathepsin L (CTSL).

Table

In 2019, a new coronavirus named SARS-CoV-2 was identified, causing COVID-19. This virus has many characteristics that are analogous to SARS-CoV, for example, ACE2 is also used as its receptor [12]. Patients with diabetes are among those with the most severe forms of COVID-19 and related mortality; insights from recent experience can guide future management [17], particularly for the consequences on the pancreas. As the COVID-19 pandemic has been ongoing for nearly two years, this study aims to collect data concerning the impact of SARS-CoV-2 on the pancreas and analyze them to estimate the future health consequences of COVID-19 in populations.

2. Pancreatic Damage during Diabetes Mellitus and COVID-19

Pancreas tissue damage may cause to the lack of control over normal blood glucose levels in the body. Type 1 diabetes (T1D) is caused by insulin deficiency due to βcell dysfunction of immunologic or idiopathic cause. In contrast, β pancreatic cells in type 2 diabetes (T2D) become depleted over time due to compensatory insulin secretion caused by insulin resistance. There is also type 3 diabetes (T3D), which is described as diabetes associated with the development of Alzheimer’s disease [18]. It should not be confused with type 3c (pancreatogenic) diabetes, which relates to the exocrine and digestive functions of the pancreas. The issue concerning the impairing effect of hyperglycemia (glucotoxicity) on the secretory function of the islets of Langerhans has also been increasingly raised. In addition to endocrine dysfunction, some diabetic patients may also develop moderate exocrine pancreatic insufficiency (EPI), in which pancreatic enzyme secretion is impaired. EPI can be observed in almost all patients with type 3c (pancreatogenic) diabetes (secondary to pancreatic pathology), whereas the prevalence of this dysfunction in patients with T1D or T2D is 40% and 27%, respectively [19].With the ongoing SARS-CoV-2 pandemic, patients with reduced normal pancreatic function are at high risk for COVID-19 requiring hospitalization. In particular, elevated blood glucose levels in patient with and without diabetes makes them at high risk of mortality [20]. Hyperglycemia impairs the immune response (e.g., by reducing the activity of macrophages and polymorphonuclear leukocytes), which in addition influences the excessive cytokine response, and thus has a strong proinflammatory effect.The receptors for ACE2, which are also present in the pancreas, are a target of SARS-CoV-2 in the body, which may result in acute failure of both the islets of Langerhans and exocrine cells [15]. Infection-induced, transient β cell dysfunction may cause an uncontrolled hyperglycemic state, especially in patients whose pancreas is already affected by diabetes mellitus. Persistent hyperglycemia usually predisposes to severe COVID-19 and to viral infection complicated by secondary infections. The aforementioned risk can be found in T1D, T2D, and gestational diabetes mellitus (GDM). In T2D patients, the much more frequent coexistence of other risk factors such as atherosclerosis, hypertension, and obesity should be taken into consideration, which usually implies a worse prognosis for the course of COVID-19 [21,22]. In GDM, SARS-CoV-2 infection not only increases the risk of more severe course of the disease in a patient, but may also result in diabetic fetopathy or, in more advanced pregnancies, increase the risk of future pathologies involving glucose metabolism (such as T2D) in a child [23].

3. Pancreatic Damage in Patients without Pre-Existing Diabetes Infected with SARS-CoV-2

It has been postulated that, either by direct invasion of pancreatic cells by the virus or by indirect mechanisms described below, SARS-CoV-2 has a destructive effect on the pancreas and can lead to insulin deficiency and development of T1D [24].If the hypothesis that SARS-CoV-2 infection causes hyperglycemia is true, increased statistics of new T1D cases should be observed. Indeed, there are publications that describe such a phenomenon. For instance, Unsworth et al. and Kamrath et al. describe an increase in new-onset T1D in children during the COVID-19 pandemic [16,25]. Although pancreatic β cell damage induced transient hyperglycemia in SARS-CoV, it is still unclear whether β cell damage is transient or permanent in SARS-CoV-2 [22]. This information appears to be of great importance because COVID-19 in children is frequently considered “harmless”. Therefore, it is reasonable to sensitize parents to the fact that the consequences of COVID-19 may be potentially dangerous for their children.Below you will find the proposed molecular mechanisms that may participate in pancreatic damage that causes carbohydrate metabolism disorders.

4. Etiology Associated with ACE2, TMPRSS2, and Na+/H+ Exchanger

As previously mentioned, SARS-CoV infection of host cells is facilitated by ACE2, but also by the transmembrane protease serine 2 (TMPRSS2) and other host cell proteases such as cathepsin L (CTSL) [13].ACE2 is an enzyme that is expressed to varying degrees in most cells of the human body [14,26,27]. This enzyme catalyzes the conversion of angiotensin II to angiotensin 1–7, taking part in the maintenance of body homeostasis by influencing the regulation of blood pressure and water–electrolyte balance through the renin–angiotensin–aldosterone (RAA) system [28]. Moreover, ACE2/angiotensin (1–7) stimulates insulin secretion, reduces insulin resistance, and increases pancreatic βcell survival [27,28].In addition to the key role it plays in maintaining body homeostasis, ACE2 is now also the best-studied target for SARS-CoV-2 S glycoprotein, enabling infection of host cells [27,29]. ACE2 in the pancreas is expressed mainly within the pericytes of pancreatic microvessels and to a lesser extent on the surface of the islets of Langerhans, including pancreatic β cells [30]. SARS-CoV-2 shows 10–20 times more activity against ACE2 than SARS-CoV, which significantly increases the infectivity of SARS-CoV-2 [31,32]. Furthermore, studies indicate that SARS-CoV may also downregulate ACE2 expression in cells. This causes an imbalance between ACE and ACE2, consequently leading to blood pressure disorders and systemic inflammation [27,33,34]. Due to the 79% genetic similarity between SARS-CoV and SARS-CoV-2 [35], it is speculated that ACE2 expression may also be downregulated during SARS-CoV-2 infection, causing i.a. MODS observed in COVID-19 [27].During cell infection by SARS-CoV-2, in addition to the role played by ACE2, it is also appropriate to consider the significant pathogenic role of TMPRSS2 that is necessary for the preparation of S glycoprotein by its cleavage, thereby enabling fusion of the virus with the host cell [36,37]. The S1 and S2 domains can be distinguished in the SARS-CoV-2 S glycoprotein. The S1 domain is involved in binding to the ACE2 receptor and then TMPRSS2 intersects with the S protein, including at the boundary of the S1 and S2 domains and within the S2 domain, which enables the virus–cell fusion [38,39]. According to studies, TMPRSS2 expression is significantly increased in obese patients, which may contribute to the poorer prognosis that is observed during COVID-19 in this patient group [40]. Moreover, obese patients are frequently already burdened with problems such as insulin resistance at baseline, while the presence of ACE2 and TMPRSS2 within the pancreas as a binding site for SARS-CoV-2 may exacerbate insulin resistance causing problems in terms of diabetes management in COVID-19 patients.There are also other mechanisms by which COVID-19 may affect the development of hyperglycemia. It is reported that the virus may also affect the glucose regulation through the Na+/H+ exchanger and lactate pathways. The mechanism is that angiotensin II, which accumulates during infection, contributes to insulin resistance and—by activating the Na+/H+ exchanger in the pancreas—it leads to hypoxia and extracellular acidification, which, through the accumulation of calcium and sodium ions in the cells and the production of reactive oxygen species, damages pancreatic tissue [41]. Simultaneously, the concentration of lactate increases, which in COVID-19 infection is intensively released, among other things, from adipose tissue, and then monocarboxylate transporters transport lactate and H+ ion inward in the cell, which increases Na+/H+ exchanger activation, further disrupting pancreatic homeostasis [41].

5. The Etiology Associated with a Systemic Proinflammatory Environment, Immune System Aggression, and Production of Novel Autoantigens

A broad spectrum of proinflammatory cytokines, such as IL-2, IL-6, IL-7, IL-8, interferon-γ, and Tumor Necrosis Factor α (TNF-α), is released during, in particular severe, COVID-19 infection [42,43,44]. Based on current studies, it is reasonable to suspect that these cytokines are released in response to the binding of the virus to ACE2 receptors that are also located in the pancreas [9,42]. The cause of pancreatic damage during COVID-19 is the cytokine storm that plays a key role in this case, because in both acute pancreatitis (AP) and severe COVID-19, elevated levels of the aforementioned interleukins are associated with the severity of these both disease entities. Particular attention should be paid to IL-6, because it is suspected to play a key role in the pathogenesis of AP as well as acute respiratory distress syndrome (ARDS) that is the most common and most severe clinical manifestation of COVID-19. In COVID-19-induced ARDS, IL-6 levels are correlated with disease-related mortality [45,46,47]. At the same time, high IL-6 levels correlate with an increased risk of developing severe pancreatitis [48,49].The production of neutralizing antibodies is also an important response of the body in the course of COVID-19 [50,51,52]. It has been observed that early seroconversion and very high antibody titers occur in patients with severe SARS-CoV-2 infection [53,54]. The available literature details a mechanism called antibody-dependent enhancement (ADE), which is associated with a pathological response of the immune system [53]. ADE exploits the existence of FcRS receptors located on various cells of the immune system, for example, macrophages and B lymphocytes [53]. This relationship may lead to a likely bypass of the classical viral infection pathway by ACE2, and virus–antibody complexes may stimulate macrophages to overproduce cytokines including significant IL-6 [53,55].Molecular mimicry may be also one of potential causes of pancreatic cell damage [56]. There are similarities in the protein structure of the virus and β-pancreatic cells, which may induce cross-reactivity and lead to autoimmunity [56]. Furthermore, viral infection may also lead to increased cytokine secretion by surrounding dendritic cells and activation of naive T cells in genetically predisposed individuals [56].

6. Pancreatitis in COVID-19

Although the impact of the discussed coronavirus-induced disease on exocrine function is not fully understood, available literature is not able to unambiguously determine whether the tissue damage leading to AP occurs as a result of direct SARS-CoV-2 infection [57] or as a result of systemic MODS with increased levels of amylase and lipase [42]. Liu et al.’s study involving 121 COVID-19 patients with a mean age of 57 years and a variable course of infection proved above-normal levels of amylase and lipase in 1–2% of patients with moderate COVID-19 infection and in 17% of patients with severe COVID-19 infection. This may support the hypothesis that SARS-CoV-2-induced disease has a destructive effect not only on the endocrine portion of this gland, but also on the exocrine one [15].However, elevated levels of pancreatic enzymes in question do not have to mean the destruction of pancreatic cells—after all, such a situation may occur during kidney failure or diarrhea in the course of COVID-19. Furthermore, there remains the question of the effect of drugs administered during SARS-CoV-2 infection on changes in pancreatic function [42], discussed further in this article.According to the International Association of Pancreatology (IAP) and the American Pancreatic Association (APA), the diagnosis of AP is based on meeting two out of three of the following criteria: clinical (epigastric pain), laboratory (serum amylase or lipase > 3 × upper limit of normal), and/or imaging criteria (computed tomography, magnetic resonance imaging, ultrasound) [58]. Pancreatic lipase is considered as a potential marker of SARS-CoV-2 severity with concomitant AP. In Hemant Goyal et al.’s study, as many as 11.7% out of 756 COVID-19 patients had hyperlipidemia and they were three times more likely to have severe COVID-19 [59]. Those with higher lipase levels—17% out of 83 patients—required hospitalization [60]. However, it is difficult to distinguish whether these patients required hospitalization for severe systemic COVID-19 infection or for pancreatitis in the course of COVID-19 infection.AP in the course of COVID-19 was analyzed in different age groups; however, some studies only involve children [61]. Compared to pancreatic islet cells, cells of the exocrine pancreatic ducts are more abundant in ACE2 and TMPRSS2 that are necessary for the virus to penetrate the cell [62]. Infection of these cells may be one of the causes of AP [63]. Infections, both bacterial and viral, are one of the causes of AP. The definitive mechanism of how viral infections affect pancreatic cells is not known; however, a study by Maria K Smatti et al. found that there is infection of pancreatic islet cells and replication of the virus within them, ultimately resulting in autoimmune reactions that eventually affect both diabetes and AP in a negative way [64]. For non-SARS-CoV-2 patients, the etiology of AP is known and confirmed in most cases, although 69% of those undergoing infection do not have definite etiology of AP while meeting the AP-Atlanta criteria for diagnosis [65].Hegyi et al. show the mechanism of MODS formation during COVID-19 infection and AP [66]. This is lipotoxicity, involving an interstitial increase in pancreatic lipase levels, which leads to the breakdown of triacylglycerols contained in adipose tissue cells and the release of unsaturated fatty acids. These in turn exert a toxic effect on mitochondria causing the release of cytokines, which results in a cytokine storm.There is also a hypothesis, which claims that AP can develop because of blood circulatory centralization resulting from uncontrolled cytokine storm created by SARS-CoV-2 infection [67]. There exist reports that say that pancreatic ischemia may be the cause of different degrees of acute pancreatitis [68,69]. This statement can be supported by the reports that state that pancreatic blood reperfusion inhibits the development of AP and accelerate pancreas recovery [70].Another mechanism of developing AP during COVID-19 may be a coagulation cascade activation caused by active inflammatory process due to SARS-CoV-2 infection [71]. The ongoing inflammatory process causes not only hemostasis imbalance for blood clotting, but it also leads to intensification of coagulation by removing epithelial cell protein C receptor (EPCR) from epithelial by the means of inflammatory mediators and thrombin [71]. This means that both processes intensify each other. Simultaneously, it was proved that COVID-19 predisposes patients to venous thromboembolism resulting from excessive inflammation, platelet activation, and endothelial dysfunction [72]. It is also important to notice that AP is inherently connected with a coagulation cascade activation, increased fibrinolysis and, hence, higher level of D-dimers [73]. Acute pancreatitis severity may depend on hemostasis imbalance; local coagulation results in mild AP whereas, in more severe AP cases, the imbalance may lead to development of disseminated intravascular coagulation (DIC) [74]. These observations have been supported by the results of experimental studies showing that the inhibition of coagulation reduces the development of AP [75,76,77] and exhibits therapeutic effect in this disease [78,79]. Additionally it is worth noticing that infection-related hyperglycemia has powerful inflammation-promoting effects on the organism (especially when organism is under stress), thus increasing the number of inflammatory mediators [74]. Unfortunately, it is impossible to decide which process is dominant in causing AP in COVID-19 patients: local inflammation caused by SARS-CoV-2 or systemic hemostasis imbalance.Clinical reports on low molecular weight heparin (LMWH) treatment in AP seem to emphasize a more significant role of hemostasis imbalance in causing AP [74,80,81]. Heparin is extremely significant in the treatment of COVID19 patients due to its properties, mainly its similarity to heparan sulphate, which appears in a respiratory tract, its interactions with SARS-CoV-2 S protein, leading to viral adhesion inhibiting to the cell membrane [82], and its anti-inflammatory effects. Thanks to these properties, heparin may not only show its therapeutic effect as the anticoagulant, but also its protective role in acute pancreatitis or respiratory inflammations [83,84,85].

7. Drugs Used against SARS-CoV-2 Infection (Glucocorticoids, Lopinavir, Ritonavir, Remedesivir, Interferon-β1 (IFN-β1), and Azithromycin) Induce Pancreatic β Cell Damage

Statistical analyses revealed a significantly higher incidence of AP with the concomitant systemic use of glucocorticosteroids (GCS) [86]. In one study analyzing the development of drug-induced AP, dexamethasone, was classified as type IB—there was one case report in which administration of this drug-induced AP occurred; however, other causes of pancreatitis such as alcohol consumption could not be excluded [87]. Other GCS such as hydrocortisone, prednisone, and prednisolone were used in patients with mild to moderate AP; however, they cannot be classified into any group because they are frequently used together with other drugs that cause AP [86,87]. However, it has been determined that GCS independently increase the risk of AP, and patients with residual AP risk factors during GCS treatment should be more monitored for the development of AP [23]. Javier A. Cienfuegos et al. additionally observed that one of mechanisms of AP formation in COVID-19 patients may be GCS administered at the time of admission to the ICU with severe respiratory failure [88]. Because GCS were used in severe COVID-19 cases, it is difficult to say what true reason for AP was—either a severe course of COVID-19 or GCS application or both.GCS are used in the treatment of many diseases due to their immunosuppressive and anti-inflammatory nature. They induce diabetes in previously healthy patients as well as significantly exacerbate diabetes in diabetic patients [89,90]. Diabetes develops in these patients likely due to pancreatic β cell dysfunction, decreased insulin secretion, and increased insulin resistance in other tissues, which may depend on the timing and the dose of GCS used [89,91]. Long-acting or intermediate-acting insulin alone or combined with short-acting insulin should be used during the treatment [90]. At the same time, no advantage was found over the use of oral hypoglycemics [92]. Certainly, patients after long-term GCS therapy will need further observation for diabetes.Lopinavir/ritonavir was classified in the previously mentioned study as a type IV drug—medications reported with little information [87]. Both drugs are included in the group of antiretrovirals that act as protease inhibitors, and they are primarily used for HIV infection. Although Lopinavir is an active drug, it is not used alone. There have been reports about the occurrence of AP during the use of protease inhibitors in question, which is also described in the Summary of Product Characteristics (SmPC) of products approved by Committee for Medicinal Products for Human Use (CHMP). It has been proved that the use of lopinavir/ritonavir causes hyperglycemia [93,94].Remdesivir is an adenosine analogue with antiviral activity. There are single reports about the occurrence of pancreatitis as a result of the use of the aforementioned medication [95,96]. At the same time, it should be noted that other nucleoside-derivative drugs may cause pancreatitis [97].The current state of knowledge does not clearly indicate the therapeutic benefit of interferon-β in the treatment of COVID-19 patients [98,99]. To date, only single cases suggesting induction of pancreatitis by interferon-β have been reported. Based on this, Badalov et al. classified interferon into type III [87].There are few reports about the development of AP due to the use of azithromycin [100]. In the previously mentioned study by Badalov et al., two macrolide antibiotics were classified as type II and III. Unfortunately, there are no direct data concerning azithromycin. Interestingly, there were cases of patients with concomitant symptoms of AP and viral pneumonia caused by SARS-CoV-2 who were treated with azithromycin, which resulted in complete resolution of symptoms for both conditions [96,101]. Based on available data, the risk of azithromycin-induced AP is low.There is no clear evidence that azithromycin affects blood glucose levels in humans. However, it is known for its prokinetic effects, which may be helpful in patients who suffer from diabetic gastroparesis [102]). The incidence of hypo- and hyperglycemic episodes was not proved to be significant for azithromycin [103]; however, the risk of dysglycemia is emphasized [94]. In the SmPC, where azithromycin is the main ingredient, it is not possible to establish a causal relationship between the occurrence of pancreatitis and taking medications (Zithromax) based on the available data. In contrast, glycemic disturbances were not indicated as side effects (Zithromax) [104].Hydroxychloroquine has been extensively promoted for COVID-19 due to its anti-inflammatory and antiviral action; yet, the use of this agent in diabetes deserves particular attention for its documented hypoglycemic action, and its benefit on COVID-19 is controversial, although there is large usage [105].Table 2 shows a comparison of the side effects of medications in question.Table 2. Side effects of medications used in SARS-CoV-2 infection in the area of pancreatic effects and hyperglycemia.

Table

8. COVID-19, Pancreas, and Glycation

In T2D diabetics, oxidative stress leading to pancreatic damage may be stimulated by, among other things, the intense glycation that accompanies hyperglycemia [24]. Glycation is a non-enzymatic process involving reducing sugar and amino groups of proteins, which contributes to the formation of advanced glycation end products (AGEs). These products have significantly altered biochemical properties relative to the substrates, including proteins that have altered conformation, increased rigidity, resistance to proteolysis, etc. [106,107].Part of the pathomechanism involved in facilitating coronavirus infection in diabetics may be due to glycation of ACE2 and SARS-CoV-2 spike protein [108,109].An interesting hypothesis is that COVID-19 has a worse prognosis in patients with intense glycation, and thus high tissue AGE content. Glycated hemoglobin (HbA1c) is a commonly used diagnostic tool that estimates intensity of glycation. The parameter is not only a marker of long-term persistent hyperglycemia, but an active participant in immune processes, as HbA1c levels are associated with NK cell activity [110].Zhang et al.’s retrospective cohort study concerning COVID-19 patients revealed that glycated hemoglobin correlates negatively with saturation (SaO2) and positively with C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and fibrinogen (Fbg). It was concluded that determination of HbA1c levels may be helpful in assessing inflammation, hypercoagulability, and prognosis of COVID-19 patients [111].According to the meta-analysis by Chen et al. (2020), Hba1c levels were slightly higher in patients with severe COVID-19 compared to patients with mild COVID-19; however, this correlation was not statistically significant. However, it is of great importance to note that only two studies analyzing HbA1c in COVID-19 patients were included in this analysis because only these studies were available in May 2020 [112].Glycation plays its physiological effects not only directly by changing the properties of various proteins, but also indirectly through various receptors. RAGE is the most common receptor for AGEs. Binding of RAGE to its ligands activates a proinflammatory response primarily by mitogen-activated protein kinase (MAPK) and nuclear factor κβ (NFκβ) pathways. This interaction was proved to be significant in the pathogenesis of cancer, diabetes mellitus, and other inflammatory disorders [113]. RAGE was found to be expressed in the pancreas, and S100P-derived RAGE antagonistic peptide (RAP) reduces pancreatic tumor growth and metastasis [113]. The implications of this fact may also apply to the etiology and treatment of COVID-19. It has been postulated that targeting RAGE by various antagonists of this receptor may inhibit damage to various organs including the pancreas [114].

9. COVID-19 vs. Pancreatic Cancer

Immunosuppression as a treatment effect, elevated cytokine levels, altered expression of receptors for SARS-CoV-2, and a prothrombotic state in patients with various types of cancer may exacerbate the effects of COVID-19 [115].Focusing on pancreatic cancer, it can be observed that the pathomechanism of both diseases—COVID-19 and tumorigenesis in the pancreas—overlap in several molecular mechanisms. As mentioned above, SARS-CoV-2 infection of host cells is facilitated by ACE-2, TMPRSS2, and CTSL. Cathepsin L is upregulated in a wide variety of cancers, including pancreatic adenocarcinoma [13]. TMPRSS2 upregulation in pancreatic cancers is moderate, whereas ACE-2 is overexpressed in some cancers, including pancreatic carcinomas [115]. Interestingly, ACE2 upregulation seems to be associated with favorable survival in pancreatic cancer [116], and it is known that SARS-CoV-2 reduces ACE2 expression [22]. Furthermore, the above-mentioned RAGE may also participate in both pancreatic cancer development and SARS-CoV-2 infection. RAGE facilitates neutrophil extracellular trap (NET) formation in pancreatic cancer [117]. In conclusion, pancreatic cancer predisposes to an increased risk of COVID-19 and its more severe course, and coronavirus infection may contribute to pancreatic cancer.It also seems important how the COVID-19 epidemic has affected the treatment of patients with pancreatic cancer of SARS-CoV-2-independent etiology. According to the study by Pergolini et al., care of patients with pancreatic cancer can be disrupted or delayed, particularly in the context of treatment selection, postoperative course, and outpatient care [118].A separate issue is how patients after pancreatoduodenectomy respond to SARS-CoV-2 infection. A case series reported by Bacalbasa reveal that patients who develop SARS-CoV-2 infection postoperatively require re-admission in the ICU and a longer hospital stay; however, these infections are not fatal [119]. Although the analysis was performed on single cases, it is concluded that these results are an argument to perform elective oncological surgeries [119].There are also reports that chemotherapy in pancreatic cancer patients who become ill between treatment series can be successfully completed after a complete cure of the infection [120]. Guidelines for, e.g., prioritization and treatment regimens regarding pancreatic cancer treatment in the era of the pandemic, are developed and described, for example, by Catanese et al. or Jones et al. [121,122].

10. Conclusions

Evidence shows that SARS-CoV-2 infection contributes to damage within the pancreas. The mechanisms that are involved in this include but are not limited to direct cytopathic effect of SARS-CoV-2 replication and systemic and local inflammatory response [123]. At the current state of knowledge, it is certain that the virus attacks the endocrine portion of the pancreas as well as, to a much lesser extent, the exocrine portion. It has been shown that a bidirectional relationship between COVID-19 and diabetes exists; indeed, diabetes is associated with COVID-19 severity and mortality but, at the same time, patients with COVID-19 have shown new onset of diabetes [124]. SARS-CoV-2 virus infection not only directly affects glycemic levels, but also exacerbates already existing hyperglycemia through its negative impact on the functional competence of the islets of Langerhans. It cannot be excluded that the real cause of exocrine dysfunction of this gland is the negative effect of the drugs used for treatment of the infection. As the pandemic progresses, special attention should be given to the evaluation of chronic and acute pancreatic diseases, including pancreatic cancer, so that faster diagnosis enables faster implementation of treatment.

Author Contributions

Conceptualization, A.K.; investigation, U.A., M.N., A.S., P.W., P.Z. and A.K.; resources, U.A., M.N., A.S., P.W., P.Z. and A.K.; writing—original draft preparation, U.A., M.N., A.S., P.W., P.Z. and A.K.; visualization, U.A.; supervision, A.K. All authors have read and agreed to the published version of the manuscript.

References

  1. Zhang, S.F.; Tuo, J.L.; Huang, X.B.; Zhu, X.; Zhang, D.M.; Zhou, K.; Yuan, L.; Luo, H.J.; Zheng, B.J.; Yuen, K.Y.; et al. Epidemiology characteristics of human coronaviruses in patients with respiratory infection symptoms and phylogenetic analysis of HCoV-OC43 during 2010–2015 in Guangzhou. PLoS ONE 201813, e0191789. [Google Scholar] [CrossRef]
  2. De Wit, E.; Van Doremalen, N.; Falzarano, D.; Munster, V.J. SARS and MERS: Recent insights into emerging coronaviruses. Nat. Rev. Microbiol. 201614, 523–534. [Google Scholar] [CrossRef]
  3. Zaki, A.M.; van Boheemen, S.; Bestebroer, T.M.; Osterhaus, A.D.M.E.; Fouchier, R.A.M. Isolation of a Novel Coronavirus from a Man with Pneumonia in Saudi Arabia. N. Engl. J. Med. 2012367, 1814–1820. [Google Scholar] [CrossRef] [PubMed]
  4. Song, Z.; Xu, Y.; Bao, L.; Zhang, L.; Yu, P.; Qu, Y.; Zhu, H.; Zhao, W.; Han, Y.; Qin, C. From SARS to MERS, thrusting coronaviruses into the spotlight. Viruses 201911, 59. [Google Scholar] [CrossRef]
  5. Shirato, K.; Kawase, M.; Matsuyama, S. Middle East Respiratory Syndrome Coronavirus Infection Mediated by the Transmembrane Serine Protease TMPRSS2. J. Virol. 201387, 12552. [Google Scholar] [CrossRef]
  6. Azhar, E.I.; Hui, D.S.C.; Memish, Z.A.; Drosten, C.; Zumla, A. The Middle East Respiratory Syndrome (MERS). Infect. Dis. Clin. N. Am. 202033, 891–905. [Google Scholar] [CrossRef]
  7. Arabi, Y.M.; Balkhy, H.H.; Hayden, F.G.; Bouchama, A.; Luke, T.; Baillie, J.K.; Al-Omari, A.; Hajeer, A.H.; Senga, M.; Denison, M.R.; et al. Middle East Respiratory Syndrome. N. Engl. J. Med. 2017376, 584–594. [Google Scholar] [CrossRef]
  8. Yang, J.K.; Lin, S.S.; Ji, X.J.; Guo, L.M. Binding of SARS coronavirus to its receptor damages islets and causes acute diabetes. Acta Diabetol. 201047, 193–199. [Google Scholar] [CrossRef] [PubMed]
  9. Leung, W.K.; To, K.; Chan, P.K.; Chan, H.L.; Wu, A.K.; Lee, N.; Yuen, K.Y.; Sung, J.J. Enteric involvement of severe acute respiratory syndrome-associated coronavirus infection. Gastroenterology 2003125, 1011–1017. [Google Scholar] [CrossRef]
  10. Shi, X.; Gong, E.; Gao, D.; Zhang, B.; Zheng, J.; Gao, Z.; Zhong, Y.; Zou, W.; Wu, B.; Fang, W.; et al. Severe acute respiratory syndrome associated coronavirus is detected in intestinal tissues of fatal cases. Am. J. Gastroenterol. 2005100, 169–176. [Google Scholar] [CrossRef]
  11. Buczkowska, E.O. Alterations of blood glucose homeostasis during septic or injury stress-hyperglycemia. Wiad Lek. 200255, 731–744. [Google Scholar] [PubMed]
  12. Zippi, M.; Hong, W.; Traversa, G.; Maccioni, F.; De Biase, D.; Gallo, C.; Fiorino, S. Involvement of the exocrine pancreas during COVID-19 infection and possible pathogenetic hypothesis: A concise review. Infez. Med. 202028, 507–515. [Google Scholar]
  13. Katopodis, P.; Anikin, V.; Randeva, H.S.; Spandidos, D.A.; Chatha, K.; Kyrou, I.; Karteris, E. Pan-cancer analysis of transmembrane protease serine 2 and cathepsin L that mediate cellular SARS‑CoV‑2 infection leading to COVID-19. Int. J. Oncol. 202057, 533–539. [Google Scholar] [CrossRef] [PubMed]
  14. Zou, X.; Chen, K.; Zou, J.; Han, P.; Hao, J.; Han, Z. Single-cell RNA-seq data analysis on the receptor ACE2 expression reveals the potential risk of different human organs vulnerable to 2019-nCoV infection. Front. Med. 202014, 185–192. [Google Scholar] [CrossRef] [PubMed]
  15. Liu, F.; Long, X.; Zhang, B.; Zhang, W.; Chen, X.; Zhang, Z. ACE2 Expression in Pancreas May Cause Pancreatic Damage after SARS-CoV-2 Infection. Clin. Gastroenterol. Hepatol. 202018, 2128–2130. [Google Scholar] [CrossRef]
  16. Unsworth, R.; Wallace, S.; Oliver, N.S.; Yeung, S.; Kshirsagar, A.; Naidu, H.; Kwong, R.M.W.; Kumar, P.; Logan, K.M. New-Onset Type 1 Diabetes in Children During COVID-19: Multicenter Regional Findings in the U.K. Diabetes Care 202043, e170–e171. [Google Scholar] [CrossRef] [PubMed]
  17. Stoian, A.P.; Banerjee, Y.; Rizvi, A.A.; Rizzo, M. Diabetes and the COVID-19 Pandemic: How Insights from Recent Experience Might Guide Future Management. Metab. Syndr. Relat. Disord. 202018, 173–175. [Google Scholar] [CrossRef]
  18. Nguyen, T.T.; Ta, Q.T.H.; Nguyen, T.K.O.; Nguyen, T.T.D.; Giau, V. Van Type 3 Diabetes and Its Role Implications in Alzheimer’s Disease. Int. J. Mol. Sci. 202021, 3165. [Google Scholar] [CrossRef]
  19. Pezzilli, R.; Andriulli, A.; Bassi, C.; Balzano, G.; Cantore, M.; Fave, G.D.; Falconi, M.; Group, L.F. the E.P.I. collaborative (EPIc) Exocrine pancreatic insufficiency in adults: A shared position statement of the Italian association for the study of the pancreas. World J. Gastroenterol. 201319, 7930–7946. [Google Scholar] [CrossRef]
  20. Abramczyk, U.; Kuzan, A. What Every Diabetologist Should Know about SARS-CoV-2: State of Knowledge at the Beginning of 2021. J. Clin. Med. 202110, 1022. [Google Scholar] [CrossRef]
  21. Apicella, M.; Campopiano, M.C.; Mantuano, M.; Mazoni, L.; Coppelli, A.; Del Prato, S. COVID-19 in people with diabetes: Understanding the reasons for worse outcomes. Lancet Diabetes Endocrinol. 20209, 782–792. [Google Scholar] [CrossRef]
  22. Boddu, S.K.; Aurangabadkar, G.; Kuchay, M.S. New onset diabetes, type 1 diabetes and COVID-19. Diabetes Metab. Syndr. 202014, 2211–2217. [Google Scholar] [CrossRef] [PubMed]
  23. Sadr-Azodi, O.; Mattsson, F.; Bexlius, T.S.; Lindblad, M.; Lagergren, J.; Ljung, R. Association of oral glucocorticoid use with an increased risk of acute pancreatitis: A population-based nested case-control study. JAMA Intern. Med. 2013173, 444–449. [Google Scholar] [CrossRef]
  24. Hayden, M.R. An Immediate and Long-Term Complication of COVID-19 May Be Type 2 Diabetes Mellitus: The Central Role of β-Cell Dysfunction, Apoptosis and Exploration of Possible Mechanisms. Cells 20209, 2475. [Google Scholar] [CrossRef]
  25. Kamrath, C.; Mönkemöller, K.; Biester, T.; Rohrer, T.R.; Warncke, K.; Hammersen, J.; Holl, R.W. Ketoacidosis in Children and Adolescents with Newly Diagnosed Type 1 Diabetes During the COVID-19 Pandemic in Germany. JAMA 2020324, 801–804. [Google Scholar] [CrossRef] [PubMed]
  26. Hamming, I.; Timens, W.; Bulthuis, M.; Lely, A.; Navis, G.; Goor, H. van Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J. Pathol. 2004203, 631–637. [Google Scholar] [CrossRef]
  27. Ni, W.; Yang, X.; Yang, D.; Bao, J.; Li, R.; Xiao, Y.; Hou, C.; Wang, H.; Liu, J.; Yang, D.; et al. Role of angiotensin-converting enzyme 2 (ACE2) in COVID-19. Crit. Care 202024, 422. [Google Scholar] [CrossRef] [PubMed]
  28. Santos, R.A.S.; Sampaio, W.O.; Alzamora, A.C.; Motta-Santos, D.; Alenina, N.; Bader, M.; Campagnole-Santos, M.J. The ACE2/Angiotensin-(1–7)/MAS Axis of the Renin-Angiotensin System: Focus on Angiotensin-(1–7). Physiol. Rev. 201898, 505–553. [Google Scholar] [CrossRef]
  29. Li, W.; Moore, M.J.; Vasilieva, N.; Sui, J.; Wong, S.K.; Berne, M.A.; Somasundaran, M.; Sullivan, J.L.; Luzuriaga, K.; Greenough, T.C.; et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 2003426, 450–454. [Google Scholar] [CrossRef] [PubMed]
  30. Fignani, D.; Licata, G.; Brusco, N.; Nigi, L.; Grieco, G.E.; Marselli, L.; Overbergh, L.; Gysemans, C.; Colli, M.L.; Marchetti, P.; et al. SARS-CoV-2 Receptor Angiotensin I-Converting Enzyme Type 2 (ACE2) Is Expressed in Human Pancreatic β-Cells and in the Human Pancreas Microvasculature. Front. Endocrinol. 202011, 596898. [Google Scholar] [CrossRef] [PubMed]
  31. Wrapp, D.; Wang, N.; Corbett, K.S.; Goldsmith, J.A.; Hsieh, C.-L.; Abiona, O.; Graham, B.S.; McLellan, J.S. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 2020367, 1260–1263. [Google Scholar] [CrossRef]
  32. Glowacka, I.; Bertram, S.; Herzog, P.; Pfefferle, S.; Steffen, I.; Muench, M.O.; Simmons, G.; Hofmann, H.; Kuri, T.; Weber, F.; et al. Differential Downregulation of ACE2 by the Spike Proteins of Severe Acute Respiratory Syndrome Coronavirus and Human Coronavirus NL63. J. Virol. 201084, 1198–1205. [Google Scholar] [CrossRef] [PubMed]
  33. Haga, S.; Yamamoto, N.; Nakai-Murakami, C.; Osawa, Y.; Tokunaga, K.; Sata, T.; Yamamoto, N.; Sasazuki, T.; Ishizaka, Y. Modulation of TNF-α-converting enzyme by the spike protein of SARS-CoV and ACE2 induces TNF-α production and facilitates viral entry. Proc. Natl. Acad. Sci. USA 2008105, 7809–7814. [Google Scholar] [CrossRef] [PubMed]
  34. Oudit, G.Y.; Kassiri, Z.; Jiang, C.; Liu, P.P.; Poutanen, S.M.; Penninger, J.M.; Butany, J. SARS-coronavirus modulation of myocardial ACE2 expression and inflammation in patients with SARS. Eur. J. Clin. Investig. 200939, 618–625. [Google Scholar] [CrossRef] [PubMed]
  35. Muniangi-Muhitu, H.; Akalestou, E.; Salem, V.; Misra, S.; Oliver, N.S.; Rutter, G.A. COVID-19 and Diabetes: A Complex Bidirectional Relationship. Front. Endocrinol. 202011, 758. [Google Scholar] [CrossRef]
  36. Baughn, L.B.; Sharma, N.; Elhaik, E.; Sekulic, A.; Bryce, A.H.; Fonseca, R. Targeting TMPRSS2 in SARS-CoV-2 Infection. Mayo Clin. Proc. 202095, 1989–1999. [Google Scholar] [CrossRef] [PubMed]
  37. Matsuyama, S.; Nao, N.; Shirato, K.; Kawase, M.; Saito, S.; Takayama, I.; Nagata, N.; Sekizuka, T.; Katoh, H.; Kato, F.; et al. Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells. Proc. Natl. Acad. Sci. USA 2020117, 7001–7003. [Google Scholar] [CrossRef]
  38. Thunders, M.; Delahunt, B. Gene of the month: TMPRSS2 (transmembrane serine protease 2). J. Clin. Pathol. 202073, 773–776. [Google Scholar] [CrossRef]
  39. Shen, L.W.; Mao, H.J.; Wu, Y.L.; Tanaka, Y.; Zhang, W. TMPRSS2: A potential target for treatment of influenza virus and coronavirus infections. Biochimie 2017142, 1–10. [Google Scholar] [CrossRef]
  40. Taneera, J.; El-Huneidi, W.; Hamad, M.; Mohammed, A.K.; Elaraby, E.; Hachim, M.Y. Expression Profile of SARS-CoV-2 Host Receptors in Human Pancreatic Islets Revealed Upregulation of ACE2 in Diabetic Donors. Biology 20209, 215. [Google Scholar] [CrossRef]
  41. Cure, E.; Cure, M.C. COVID-19 may affect the endocrine pancreas by activating Na+/H+ exchanger 2 and increasing lactate levels. J. Endocrinol. Investig. 202043, 1167–1168. [Google Scholar] [CrossRef]
  42. Zippi, M.; Fiorino, S.; Occhigrossi, G.; Hong, W. Hypertransaminasemia in the course of infection with SARS-CoV-2: Incidence and pathogenetic hypothesis. World J. Clin. Cases 20208, 1385–1390. [Google Scholar] [CrossRef]
  43. Tisoncik, J.R.; Korth, M.J.; Simmons, C.P.; Farrar, J.; Martin, T.T.; Katze, M.G. Into the eye of the cytokine storm. Microbiol. Mol. Biol. Rev. 201276, 16–32. [Google Scholar] [CrossRef]
  44. Mehta, P.; McAuley, D.F.; Brown, M.; Sanchez, E.; Tattersall, R.S.; Manson, J.J. COVID-19: Consider cytokine storm syndromes and immunosuppression. Lancet 2020395, 1033–1034. [Google Scholar] [CrossRef]
  45. Zhou, F.; Yu, T.; Du, R.; Fan, G.; Liu, Y.; Liu, Z.; Xiang, J.; Wang, Y.; Song, B.; Gu, X.; et al. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: A retrospective cohort study. Lancet 2020395, 1054–1062. [Google Scholar] [CrossRef]
  46. Hojyo, S.; Uchida, M.; Tanaka, K.; Hasebe, R.; Tanaka, Y.; Murakami, M.; Hirano, T. How COVID-19 induces cytokine storm with high mortality. Inflamm. Regen. 202040, 37. [Google Scholar] [CrossRef]
  47. Liu, B.; Li, M.; Zhou, Z.; Guan, X.; Xiang, Y. Can we use interleukin-6 (IL-6) blockade for coronavirus disease 2019 (COVID-19)-induced cytokine release syndrome (CRS)? J. Autoimmun. 2020111, 102452. [Google Scholar] [CrossRef] [PubMed]
  48. Rao, S.A.; Kunte, A.R. Interleukin-6: An Early Predictive Marker for Severity of Acute Pancreatitis. Indian J. Crit. Care Med. 201721, 424–428. [Google Scholar] [CrossRef] [PubMed]
  49. Sathyanarayan, G.; Garg, P.K.; Prasad, H.; Tandon, R.K. Elevated level of interleukin-6 predicts organ failure and severe disease in patients with acute pancreatitis. J. Gastroenterol. Hepatol. 200722, 550–554. [Google Scholar] [CrossRef]
  50. Cao, Y.; Liu, X.; Xiong, L.; Cai, K. Imaging and clinical features of patients with 2019 novel coronavirus SARS-CoV-2: A systematic review and meta-analysis. J. Med. Virol. 202092, 1449–1459. [Google Scholar] [CrossRef]
  51. Assis, R.R.; de Jain, A.; Nakajima, R.; Jasinskas, A.; Felgner, J.; Obiero, J.M.; Norris, P.J.; Stone, M.; Simmons, G.; Bagri, A.; et al. Analysis of SARS-CoV-2 antibodies in COVID-19 convalescent blood using a coronavirus antigen microarray. Nat. Commun. 202112, 6. [Google Scholar] [CrossRef]
  52. Zhao, J.; Yuan, Q.; Wang, H.; Liu, W.; Liao, X.; Su, Y.; Wang, X.; Yuan, J.; Li, T.; Li, J.; et al. Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019. Clin. Infect. Dis. 202071, 2027–2034. [Google Scholar] [CrossRef]
  53. Iwasaki, A.; Yang, Y. The potential danger of suboptimal antibody responses in COVID-19. Nat. Rev. Immunol. 202020, 339–341. [Google Scholar] [CrossRef]
  54. Lee, N.; Chan, P.K.S.; Ip, M.; Wong, E.; Ho, J.; Ho, C.; Cockram, C.S.; Hui, D.S. Anti-SARS-CoV IgG response in relation to disease severity of severe acute respiratory syndrome. J. Clin. Virol. 200635, 179–184. [Google Scholar] [CrossRef]
  55. Yasui, F.; Kai, C.; Kitabatake, M.; Inoue, S.; Yoneda, M.; Yokochi, S.; Kase, R.; Sekiguchi, S.; Morita, K.; Hishima, T.; et al. Prior Immunization with Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus (SARS-CoV) Nucleocapsid Protein Causes Severe Pneumonia in Mice Infected with SARS-CoV. J. Immunol. 2008181, 6337–6348. [Google Scholar] [CrossRef]
  56. Caruso, P.; Longo, M.; Esposito, K.; Maiorino, M.I. Type 1 diabetes triggered by COVID19 pandemic: A potential outbreak? Diabetes Res. Clin. Pract. 2020164, 108219. [Google Scholar] [CrossRef]
  57. de-Madaria, E.; Capurso, G. COVID-19 and acute pancreatitis: Examining the causality. Nat. Rev. Gastroenterol. Hepatol. 202118, 3–4. [Google Scholar] [CrossRef] [PubMed]
  58. Group, W.; Apa, I.A.P.; Pancreatitis, A. IAP/APA evidence-based guidelines for the management of acute pancreatitis. Pancreatology 201313, e1–e15. [Google Scholar] [CrossRef]
  59. Goyal, H.; Sachdeva, S.; Perisetti, A.; Mann, R.; Inamdar, S.; Tharian, B. Hyperlipasemia and Potential Pancreatic Injury Patterns in COVID-19: A Marker of Severity or Innocent Bystander? Gastroenterology 2021160, 946–948. [Google Scholar] [CrossRef] [PubMed]
  60. Barlass, U.; Wiliams, B.; Dhana, K.; Adnan, D.; Khan, S.R.; Mahdavinia, M.; Bishehsari, F. Marked elevation of lipase in COVID-19 Disease: A cohort study. Clin. Transl. Gastroenterol. 202011, e00215. [Google Scholar] [CrossRef] [PubMed]
  61. Suchman, K.; Raphael, K.L.; Liu, Y.; Wee, D.; Trindade, A.J. Acute pancreatitis in children hospitalized with COVID-19. Pancreatology 202121, 31–33. [Google Scholar] [CrossRef]
  62. Müller, J.A.; Groß, R.; Conzelmann, C.; Krüger, J.; Merle, U.; Steinhart, J.; Weil, T.; Koepke, L.; Bozzo, C.P.; Read, C.; et al. SARS-CoV-2 infects and replicates in cells of the human endocrine and exocrine pancreas. Nat. Metab. 20213, 149–165. [Google Scholar] [CrossRef] [PubMed]
  63. Correia de Sá, T.; Soares, C.; Rocha, M. Acute pancreatitis and COVID-19: A literature review. World J. Gastrointest. Surg. 202113, 574–584. [Google Scholar] [CrossRef] [PubMed]
  64. Smatti, M.K.; Cyprian, F.S.; Nasrallah, G.K.; Al Thani, A.A.; Almishal, R.O.; Yassine, H.M. Viruses and Autoimmunity: A Review on the Potential Interaction and Molecular Mechanisms. Viruses 201911, 762. [Google Scholar] [CrossRef] [PubMed]
  65. Inamdar, S.; Benias, P.C.; Liu, Y.; Sejpal, D.V.; Satapathy, S.K.; Trindade, A.J.; Northwell COVID-19 Research Consortium. Prevalence, Risk Factors, and Outcomes of Hospitalized Patients with Coronavirus Disease 2019 Presenting as Acute Pancreatitis. Gastroenterology 2020159, 2226–2228.e2. [Google Scholar] [CrossRef] [PubMed]
  66. Hegyi, P.; Szakács, Z.; Sahin-Tóth, M. Lipotoxicity and Cytokine Storm in Severe Acute Pancreatitis and COVID-19. Gastroenterology 2020159, 824–827. [Google Scholar] [CrossRef] [PubMed]
  67. Hu, B.; Huang, S.; Lianghong, Y. Lianghong The cytokine storm and COVID-19. J. Med. Virol. 202193, 250–256. [Google Scholar] [CrossRef] [PubMed]
  68. Gullo, L.; Cavicchi, L.; Tomassetti, P.; Spagnolo, C.; Freyrie, A.; D’addato, M. Effects of Ischemia on the Human Pancreas. Gastroenterology 1996111, 1033–1038. [Google Scholar] [CrossRef]
  69. Lonardo, A.; Grisendi, A.; Bonilauri, S.; Rambaldi, M.; Selmi, I.; Tondelli, E. Ischaemic necrotizing pancreatitis after cardiac surgery. A case report and review of the literature. Ital. J. Gastroenterol. Hepatol. 199931, 872–875. [Google Scholar] [PubMed]
  70. Warzecha, Z.; Dembiński, A.; Ceranowicz, P.; Konturek, P.C.; Stachura, J.; Konturek, S.J.; Niemiec, J. Protective effect of calcitonin gene-related peptide against caerulein-induced pancreatitis in rats. J. Physiol. Pharmacol. 199748, 775–787. [Google Scholar]
  71. Esmon, C.T. Crosstalk between inflammation and thrombosis. Maturitas 200861, 122–131. [Google Scholar] [CrossRef]
  72. Wang, D.; Hu, B.; Hu, C.; Zhu, F.; Liu, X.; Zhang, J.; Wang, B.; Xiang, H.; Cheng, Z.; Xiong, Y.; et al. Clinical Characteristics of 138 Hospitalized Patients with 2019 NovelCoronavirus–Infected Pneumonia in Wuhan, China. JAMA 2020323, 1061–1069. [Google Scholar] [CrossRef] [PubMed]
  73. Lasson, Å.; Ohlsson, K. Consumptive coagulopathy, fibrinolysis and protease-antiprotease interactions during acute human pancreatitis. Thromb. Res. 198641, 167–183. [Google Scholar] [CrossRef]
  74. Du, J.D.; Zheng, X.; Huang, Z.Q.; Cai, S.W.; Tan, J.W.; Li, Z.L.; Yao, Y.M.; Jiao, H.B.; Yin, H.N.; Zhu, Z.M. Effects of intensive insulin therapy combined with low molecular weight heparin anticoagulant therapy on severe pancreatitis. Exp. Ther. Med. 20148, 141–146. [Google Scholar] [CrossRef] [PubMed]
  75. Maduzia, D.; Ceranowicz, P.; Cieszkowski, J.; Gałazka, K.; Kusnierz-Cabala, B.; Warzecha, Z. Pretreatment with Warfarin Attenuates the Development of Ischemia/Reperfusion-Induced Acute Pancreatitis in Rats. Molecules 202025, 2493. [Google Scholar] [CrossRef]
  76. Warzecha, Z.; Sendur, P.; Ceranowicz, P.; Dembinski, M.; Cieszkowski, J.; Kusnierz-Cabala, B.; Tomaszewska, R.; Dembinski, A. Pretreatment with low doses of acenocoumarol inhibits the development of acute ischemia/reperfusion-induced pancreatitis. J. Physiol. Pharmacol. 201566, 731–740. [Google Scholar] [PubMed]
  77. Warzecha, Z.; Sendur, P.; Ceranowicz, P.; Dembiński, M.; Cieszkowski, J.; Kuśnierz-Cabala, B.; Olszanecki, R.; Tomaszewska, R.; Ambroży, T.; Dembiński, A. Protective Effect of Pretreatment with Acenocoumarol in Cerulein-Induced Acute Pancreatitis. Int. J. Mol. Sci. 20167, 1709. [Google Scholar] [CrossRef] [PubMed]
  78. Maduzia, D.; Ceranowicz, P.; Cieszkowski, J.; Chmura, A.; Galazka, K.; Kusnierz-Cabala, B.; Warzecha, Z. Administration of warfarin accelerates the recovery in ischemia/reperfusion-induced acute pancreatitis. J. Physiol. Pharmacol. 202071, 417–427. [Google Scholar] [CrossRef]
  79. Ceranowicz, P.; Dembinski, A.; Warzecha, Z.; Dembinski, M.; Cieszkowski, J.; Rembiasz, K.; Konturek, S.J.; Kusnierz-Cabala, B.; Tomaszewska, R.; Pawlik, W.W. Protective and therapeutic effect of heparin in acute pancreatitis. J. Physiol. Pharmacol. 200859, 103–125. [Google Scholar]
  80. Xin-Sheng, L.; Fu, Q.; Jie-Qin, L.; Qin-Qiao, F.; Ri-Guang, Z.; Yu-Hang, A.; Kai-Cheng, Z.; Yi-Xiong, L. Low Molecular Weight Heparin in the Treatment of Severe Acute Pancreatitis: A Multiple Centre Prospective Clinical Study. Asian J. Surg. 200932, 89–94. [Google Scholar] [CrossRef]
  81. Tozlu, M.; Kayar, Y.; Ince, A.T.; Baysal, B.; Şenturk, H. Low molecular weight heparin treatment of acute moderate and severe pancreatitis: A randomized, controlled, open-label study. Turk. J. Gastroenterol. 201930, 81–87. [Google Scholar] [CrossRef]
  82. Agarwal, R.N.; Aggarwal, H.; Verma, A.; Tripathi, M.K. A case report of a patient on therapeutic warfarin who died of COVID19 infection with a sudden rise in d-dimer. Biomedicines 20219, 1382. [Google Scholar] [CrossRef]
  83. Di Micco, P.; Imbalzano, E.; Russo, V.; Attena, E.; Mandaliti, V.; Orlando, L.; Lombardi, M.; Micco, G.; Di Camporese, G.; Annunziata, S.; et al. Heparin and SARS-CoV-2: Multiple Pathophysiological Links. Viruses 202113, 2486. [Google Scholar] [CrossRef] [PubMed]
  84. Bukowczan, J.; Warzecha, Z.; Ceranowicz, P.; Kusnierz-Cabala, B.; Tomaszewska, R.; Dembinski, A. Therapeutic effect of ghrelin in the course of ischemia/reperfusion-induced acute pancreatitis. Curr. Pharm. Des. 201521, 2284–2290. [Google Scholar] [CrossRef]
  85. Warzecha, Z.; Dembiñski, A.; Ceranowicz, P.; Konturek, S.J.; Tomaszewska, R.; Stachura, J.; Konturek, P.C. IGF-1 stimulates production of interleukin-10 and inhibits development of caerulein-induced pancreatitis. J. Physiol. Pharmacol. 200354, 575–590. [Google Scholar] [PubMed]
  86. Nango, D.; Hirose, Y.; Goto, M.; Echizen, H. Analysis of the Association of Administration of various glucocorticoids with development of acute pancreatitis using US Food and Drug Administration adverse event reporting system (FAERS). J. Pharm. Healthc. Sci. 20195, 5. [Google Scholar] [CrossRef]
  87. Badalov, N.; Baradarian, R.; Iswara, K.; Li, J.; Steinberg, W.; Tenner, S. Drug-Induced Acute Pancreatitis: An Evidence-Based Review. Clin. Gastroenterol. Hepatol. 20075, 648–661. [Google Scholar] [CrossRef]
  88. Cienfuegos, J.A.; Almeida, A.; Aliseda, D. Pancreatic injury and acute pancreatitis in COVID-19 patients. Rev. Esp. Enferm. Dig. 2021113, 389. [Google Scholar] [CrossRef] [PubMed]
  89. Hwang, J.L.; Weiss, R.E. Steroid-induced diabetes: A clinical and molecular approach to understanding and treatment. Diabetes Metab. Res. Rev. 201430, 96–102. [Google Scholar] [CrossRef]
  90. Radhakutty, A.; Burt, M.G. Management of endocrine disease: Critical review of the evidence underlying management of glucocorticoid-induced hyperglycaemia. Eur. J. Endocrinol. 2018179, R207–R218. [Google Scholar] [CrossRef]
  91. Van Raalte, D.H.; Nofrate, V.; Bunck, M.C.; Van Iersel, T.; Schaap, J.E.; Nässander, U.K.; Heine, R.J.; Mari, A.; Dokter, W.H.A.; Diamant, M. Acute and 2-week exposure to prednisolone impair different aspects of β-cell function in healthy men. Eur. J. Endocrinol. 2010162, 729–735. [Google Scholar] [CrossRef] [PubMed]
  92. Klarskov, C.K.; Holm Schultz, H.; Wilbek Fabricius, T.; Persson, F.; Pedersen-Bjergaard, U.; Lommer Kristensen, P. Oral treatment of glucocorticoid-induced diabetes mellitus: A systematic review. Int. J. Clin. Pract. 202074, e13529. [Google Scholar] [CrossRef]
  93. Kaletra 200 mg/50 mg Film-Coated Tablets—Summary of Product Characteristics (SmPC)—(emc). Available online: https://www.medicines.org.uk/emc/product/221/smpc (accessed on 10 January 2022).
  94. Rimesh Pal, S.K.B. COVID-19 and diabetes mellitus: An unholy interaction of two pandemics. Diabetes Metab. Syndr. Clin. Res. Rev. 202014, 513–517. [Google Scholar] [CrossRef]
  95. Khadka, S.; Williams, K.; Solanki, S. Remdesivir-Associated Pancreatitis. Am. J. Ther. 2021. [Google Scholar] [CrossRef]
  96. Ehsan, P.; Haseeb, M.; Khan, Z.; Rehan, A.; Singh, R. Coronavirus Disease 2019 Pneumonia and Acute Pancreatitis in a Young Girl. Cureus 202113, e15374. [Google Scholar] [CrossRef]
  97. Jorgensen, S.C.J.; Kebriaei, R.; Dresser, L.D. Remdesivir: Review of Pharmacology, Pre-Clinical Data, and Emerging Clinical Experience for COVID-19. Pharmacotherapy 202040, 659–671. [Google Scholar] [CrossRef]
  98. Pan, H.; Peto, R.; Henao-Restrepo, A.; Preziosi, M.; Sathiyamoorthy, V.; Abdool Karim, Q.; Alejandria, M.; Hernández García, C.; Kieny, M.; Malekzadeh, R.; et al. Repurposed Antiviral Drugs for COVID19—Interim WHO Solidarity. N. Engl. J. Med. 2021384, 497–511. [Google Scholar] [CrossRef] [PubMed]
  99. Rahmani, H.; Davoudi-Monfared, E.; Nourian, A.; Khalili, H.; Hajizadeh, N.; Jalalabadi, N.Z.; Fazeli, M.R.; Ghazaeian, M.; Yekaninejad, M.S. Interferon β-1b in treatment of severe COVID-19: A randomized clinical trial. Int. Immunopharmacol. 202088, 106903. [Google Scholar] [CrossRef] [PubMed]
  100. Gonzalo-Voltas, A.; Fernández-Pérez-Torres, C.U.; Baena-Díez, J.M. Acute pancreatitis in a patient with COVID-19 infection. Med. Clin. 2020155, 183–184. [Google Scholar] [CrossRef] [PubMed]
  101. Díaz Lobato, S.; Carratalá Perales, J.M.; Alonso Íñigo, J.M. Can we use noninvasive respiratory therapies in COVID-19 pandemic? Med. Clin. 2020155, 183. [Google Scholar] [CrossRef] [PubMed]
  102. Sutera, L.; Dominguez, L.J.; Belvedere, M.; Putignano, E.; Vernuccio, L.; Ferlisi, A.; Fazio, G.; Costanza, G.; Barbagallo, M. Azithromycin in an older woman with diabetic gastroparesis. Am. J. Ther. 200815, 85–88. [Google Scholar] [CrossRef]
  103. Aspinall, S.L.; Good, C.B.; Jiang, R.; McCarren, M.; Dong, D.; Cunningham, F.E. Severe dysglycemia with the fluoroquinolones: A class effect? Clin. Infect. Dis. 200949, 402–408. [Google Scholar] [CrossRef] [PubMed]
  104. Zithromax Powder for Oral Suspension—Summary of Product Characteristics (SmPC)—(emc). Available online: https://www.medicines.org.uk/emc/product/3006/smpc#gref (accessed on 10 January 2022).
  105. Stoian, A.P.; Catrinoiu, D.; Rizzo, M.; Ceriello, A. Hydroxychloroquine, COVID-19 and diabetes. Why it is a different story. Diabetes Metab. Res. Rev. 202137, e3379. [Google Scholar] [CrossRef] [PubMed]
  106. Kuzan, A. Toxicity of advanced glycation end products (Review). Biomed. Rep. 202114, 46. [Google Scholar] [CrossRef] [PubMed]
  107. Kuzan, A.; Chwiłkowska, A.; Maksymowicz, K.; Bronowicka-Szydełko, A.; Stach, K.; Pezowicz, C.; Gamian, A. Advanced glycation end products as a source of artifacts in immunoenzymatic methods. Glycoconj. J. 201835, 95–103. [Google Scholar] [CrossRef]
  108. Liao, Y.-H.; Zheng, J.-Q.; Zheng, C.-M.; Lu, K.-C.; Chao, Y.-C. Novel Molecular Evidence Related to COVID-19 in Patients with Diabetes Mellitus. J. Clin. Med. 20209, 3962. [Google Scholar] [CrossRef]
  109. Sartore, G.; Ragazzi, E.; Faccin, L.; Lapolla, A. A role of glycation and methylation for SARS-CoV-2 infection in diabetes? Med. Hypotheses 2020144, 110247. [Google Scholar] [CrossRef]
  110. Kim, J.H.; Park, K.; Lee, S.B.; Kang, S.; Park, J.S.; Ahn, C.W.; Nam, J.S. Relationship between natural killer cell activity and glucose control in patients with type 2 diabetes and prediabetes. J. Diabetes Investig. 201910, 1223–1228. [Google Scholar] [CrossRef]
  111. Zhang, W.; Li, C.; Xu, Y.; He, B.; Hu, M.; Cao, G.; Li, L.; Wu, S.; Wang, X.; Zhang, C.; et al. Hyperglycemia and Correlated High Levels of Inflammation Have a Positive Relationship with the Severity of Coronavirus Disease 2019. Mediat. Inflamm. 20212021, 8812304. [Google Scholar] [CrossRef]
  112. Chen, J.; Wu, C.; Wang, X.; Yu, J.; Sun, Z. The Impact of COVID-19 on Blood Glucose: A Systematic Review and Meta-Analysis. Front. Endocrinol. 202011, 574541. [Google Scholar] [CrossRef]
  113. Arumugam, T.; Ramachandran, V.; Gomez, S.B.; Schmidt, A.M.; Logsdon, C.D. S100P-Derived RAGE Antagonistic Peptide Reduces Tumor Growth and Metastasis. Clin. Cancer Res. 201218, 4356–4364. [Google Scholar] [CrossRef]
  114. Chiappalupi, S.; Salvadori, L.; Vukasinovic, A.; Donato, R.; Sorci, G.; Riuzzi, F. Targeting RAGE to prevent SARS-CoV-2-mediated multiple organ failure: Hypotheses and perspectives. Life Sci. 2021272, 119251. [Google Scholar] [CrossRef]
  115. van Dam, P.A.; Huizing, M.; Mestach, G.; Dierckxsens, S.; Tjalma, W.; Trinh, X.B.; Papadimitriou, K.; Altintas, S.; Vermorken, J.; Vulsteke, C.; et al. SARS-CoV-2 and cancer: Are they really partners in crime? Cancer Treat. Rev. 202089, 102068. [Google Scholar] [CrossRef] [PubMed]
  116. Zhang, Z.; Li, L.; Li, M.; Wang, X. The SARS-CoV-2 host cell receptor ACE2 correlates positively with immunotherapy response and is a potential protective factor for cancer progression. Comput. Struct. Biotechnol. J. 202018, 2438–2444. [Google Scholar] [CrossRef] [PubMed]
  117. Boone, B.A.; Orlichenko, L.; Schapiro, N.E.; Loughran, P.; Gianfrate, G.C.; Ellis, J.T.; Singhi, A.D.; Kang, R.; Tang, D.; Lotze, M.T.; et al. The Receptor for Advanced Glycation End Products (RAGE) Enhances Autophagy and Neutrophil Extracellular Traps in Pancreatic Cancer. Cancer Gene Therapy 201522, 326–334. [Google Scholar] [CrossRef]
  118. Pergolini, I.; Demir, I.E.; Stöss, C.; Emmanuel, K.; Rosenberg, R.; Friess, H.; Novotny, A. Effects of COVID-19 Pandemic on the Treatment of Pancreatic Cancer: A Perspective from Central Europe. Dig. Surg. 202138, 158–165. [Google Scholar] [CrossRef] [PubMed]
  119. Bacalbasa, N.; Diaconu, C.; Savu, C.; Savu, C.; Stiru, O.; Balescu, I. The impact of COVID-19 infection on the postoperative outcomes in pancreatic cancer patients. In Vivo 202135, 1307–1311. [Google Scholar] [CrossRef]
  120. Nagai, K.; Kitamura, K.; Hirai, Y.; Nutahara, D.; Nakamura, H.; Taira, J.; Matsue, Y.; Abe, M.; Kikuchi, M.; Itoi, T. Successful and Safe Reinstitution of Chemotherapy for Pancreatic Cancer after COVID-19. Intern. Med. 202160, 231–234. [Google Scholar] [CrossRef] [PubMed]
  121. Catanese, S.; Pentheroudakis, G.; Douillard, J.Y.; Lordick, F. ESMO Management and treatment adapted recommendations in the COVID-19 era: Pancreatic Cancer. ESMO Open 20205, e000804. [Google Scholar] [CrossRef]
  122. Jones, C.M.; Radhakrishna, G.; Aitken, K.; Bridgewater, J.; Corrie, P.; Eatock, M.; Goody, R.; Ghaneh, P.; Good, J.; Grose, D.; et al. Considerations for the treatment of pancreatic cancer during the COVID-19 pandemic: The UK consensus position. Br. J. Cancer 2020123, 709–713. [Google Scholar] [CrossRef]
  123. Ugwueze, C.V.; Ezeokpo, B.C.; Nnolim, B.I.; Agim, E.A.; Anikpo, N.C.; Onyekachi, K.E. COVID-19 and Diabetes Mellitus: The Link and Clinical Implications. Dubai Diabetes Endocrinol. J. 202026, 69–77. [Google Scholar] [CrossRef]
  124. Al Mahmeed, W.; Al-Rasadi, K.; Banerjee, Y.; Ceriello, A.; Cosentino, F.; Galia, M.; Goh, S.-Y.; Kempler, P.; Lessan, N.; Papanas, N.; et al. Promoting a Syndemic Approach for Cardiometabolic Disease Management During COVID-19: The CAPISCO International Expert Panel. Front. Cardiovasc. Med. 20218, 787761. [Google Scholar] [CrossRef] [PubMed]
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Comparison between RT-qPCR for SARS-CoV-2 and expanded triage in sputum of symptomatic and asymptomatic COVID-19 subjects in Ecuador

BMC Infectious Diseases volume 21, Article number: 558 (2021) 

Abstract

Background

The quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) effectively detects the SARS-COV-2 virus. SARS-CoV-2 Nevertheless, some critical gaps remain in the identification and monitoring of asymptomatic people.

Methods

This retrospective study included 733 asymptomatic and symptomatic COVID-19 subjects, who were submitted to the RT-qPCR test. The objective was to assess the efficacy of an expanded triage of subjects undergoing the RT-qPCR test for SARS-COV-2 to identify the largest possible number of COVID-19 cases in a hospital setting in Ecuador. SARS-CoV-2 Firstly, the sensitivity and specificity as well as the predictive values of an expanded triage method were calculated. In addition, the Kappa coefficient was also determined to assess the concordance between laboratory test results and the expanded triage.

Results

Of a total of 733 sputum samples; 229 were RT-qPCR-positive (31.2%) and mortality rate reached 1.2%. Overall sensitivity and specificity were 86.0% (95% confidence interval: 81.0–90.0%) and 37.0% (95% confidence interval: 32.0–41.0%) respectively, with a diagnostic accuracy of 52.0% and a Kappa coefficient of 0.73. An association between the positivity of the test and its performance before 10 days was found.

Conclusions

The clinical sensitivity for COVID-19 detection was within acceptable standards, but the specificity still fell below the values of reference. The lack of symptoms did not always mean to have a negative SARS-COV-2 RT-qPCR test. The expanded triage identified a still unnoticed percentage of asymptomatic subjects showing positive results for the SARS-COV-2 RT-qPCR test. The study also revealed a significant relationship between the number of RT-qPCR-positive cases and the performance of the molecular diagnosis within the first 10 days of COVID-19 in the symptomatic group.

For More Information: https://bmcinfectdis.biomedcentral.com/articles/10.1186/s12879-021-06272-8

Analysis of the Study of the Expression of Apoptosis Markers (CD95) and Intercellular Adhesion Markers (CD54) in Healthy Individuals and Patients Who Underwent COVID-19 When Using the Drug Mercureid

Authors: Sergey N Gusev1*, Velichko LN2, Bogdanova AV2, Khramenko NI2, Konovalova NV2 Published Date: 26-08-2021

Abstract

SARS-CoV-2, the pathogen, which is responsible for coronavirus disease 2019 (COVID-19), has caused unprecedented morbidity and mortality worldwide. Scientific and clinical evidence testifies about long-term COVID-19 effects that can affect many organ systems. Cellular damage, overproduction of proinflammatory cytokines and procoagulant abnormalities caused by SARS-CoV-2 infection may lead to these consequences. After suffering from COVID-19, a negative PCR test is only the beginning of a difficult path to full recovery. 61 % of patients will continue to have the signs of post-covid syndrome with the risk of developing serious COVID-19 health complications for a long time. Post-COVID syndrome is an underestimated large-scale problem that can lead to the collapse of the healthcare system in the nearest future.

The treatment and prevention of post-covid syndrome require integrated rather than organ or disease specific approaches and there is an urgent need to conduct a special research to establish the risk factors.

For this purpose, we studied the expression of markers of apoptosis (CD95) and intercellular adhesion (CD54) in healthy individuals and patients who underwent COVID-19, as well as the efficacy of the drug Mercureid for the treatment of post-covid syndrome.

The expression level of the apoptosis marker CD95 in patients who underwent COVID-19 is 1.7-2.5 times higher than the norm and the intercellular adhesion marker CD54 is 2.9-4.4 times higher. This fact indicates a persistent high level of dysfunctional immune response in the short term after recovery. The severity of the expression of the intercellular adhesion molecule (ICAM-1, CD54) shows the involvement of the endothelium of the vascular wall in the inflammatory process as one of the mechanisms of the pathogenesis of post-covid syndrome.

The use of Mercureid made it possible to reduce the overexpression of CD95 in 73.4 % of patients that led to the restoration of the number of CD4+/CD8+ T-cells, which are crucial in the restoration of functionally active antiviral and antitumor immunity of patients. Also, the use of Mercureid led to a normalization of ICAM-1 (CD54) levels in 75.8 % of patients.

The pharmacological properties of the new targeted immunotherapy drug Mercureid provide new therapeutic opportunities for the physician to influence a number of therapeutic targets, such as CD95, ICAM-1 (CD54), to reduce the risk of post-COVID complications.

For More Information: https://athenaeumpub.com/analysis-of-the-study-of-the-expression-of-apoptosis-markers-cd95-and-intercellular-adhesion-markers-cd54-in-healthy-individuals-and-patients-who-underwent-covid-19-when-using-the-drug-mercureid/