The onset of LC has been mainly observed in COVID-19 survivors that experienced mild to severe infection. Although it is clear that individuals who experienced asymptomatic acute infection are not protected from developing LC (2, 25), the prevalence of LC in those with severe COVID has been estimated at around 70% (26–30). Overall, while there is a gradient of risk related to the severity of the acute infection, where patients with severe acute COVID-19 are at the greatest risk for developing LC, the sheer volume of asymptomatic, mild, and moderate cases of acute COVID-19 translates to their predominance in unselected LC cohorts (29, 31–36). Therefore, the most significant burden of LC may be expected to occur among these patients. Post‐intensive care syndrome (PICS) describes a collection of health disorders that are common among survivors of critical illness, encompassing new or worsening impairments in mental, cognitive, or physical health status occurring after critical illness and persisting beyond acute care hospitalization (36, 37). Eventually, this condition will impact the long‐term prognosis, affecting both the functional outcomes and survival of patients (37). As observed in the recent pandemic, survivors of COVID-19 seem to be at a particularly high risk of developing PICS (38).

A consistent feature of LC globally is its high prevalence in women, especially those aged 40-50 years, who are now considered highly susceptible to developing LC (26, 27). Collective data extrapolated from approximately 4,182 self-reported incident cases of COVID-19 revealed that 15% of women experienced symptoms persisting for 28 days or more, compared to 9.5% of men. In the group aged 40 to 50, women had double the risk of developing LC compared to men. A sex difference was not evident in those over 70 years (15). The immune landscape of COVID-19 patients may differ vastly between the sexes, which may underpin susceptibility to LC onset in at least some, perhaps a sizeable, fraction of women. This is because one of the leading hypotheses on LC pathogenesis posits that LC (or some of its phenotypes) is caused by immune dysregulation in the form of autoimmune disorders, which are known to be more prevalent in women than in men. Generally, more robust and rapid immune responses are seen in women, protecting them from initial infection and disease severity, including acute COVID. Contrastingly, men have a less robust immune system that dampens with age due to weakened T-cell responses (39); hence they are more likely to experience severe COVID-19 disease (30, 40). However, a robust immune response may not be beneficial in terms of LC risk and, in the long term, protective mechanisms may become overstimulated leading to immune dysregulation, ultimately resulting in the physical manifestations of LC symptoms in women (30). The molecular basis for some of this tendency towards stronger immunity and autoimmunity is now emerging. Specifically, TLR7, one of the key innate sensors of viral infections (including detection of SARS-CoV-2) is encoded on the X chromosome and is incompletely silenced in women, who then have more than one functional copy of this gene (41). Moreover, in women, increased oestrogen has been linked to decreased COVID-19 severity and mortality, presumably mediated by enhanced innate and humoral responses (42). Both of these mechanisms could contribute to autoimmune processes in LC. A study conducted in Jordan involving 495 non-hospitalised study participants between the ages of 14 and 70 revealed that female participants reported more LC symptoms than male participants. These included fatigue (61 vs 47%), anxiety (51 vs 40%), palpitations (40 vs 30%), weakness (36 vs 24%), breathlessness (33 vs 17%), and change in smell sensation (50 vs 30%) (43). In another meta-analysis of 20 studies by Maglietta et al. (2022), involving 13 340 adult patients that had been discharged after being hospitalised for COVID-19, statistically significant associations were found between female sex and any symptoms (OR 1.52, 95% [CI]: 1.27-1.82), mental health symptoms (OR 1.67, 95% [CI]: 1.21-2.29), and fatigue (OR 1.54, 95% [CI]: 1.32-1.79) (44). In a UK-based longitudinal study, meta-analysed associations from 10 cohorts encompassing 6907 LC patients revealed that women had a higher risk of LC outcomes than men (4+ weeks: OR 1.49; 95% [CI]: 1.24–1.79; 12+ weeks: OR 1.60; 95% [CI]: 1.23–2.07) (45).

Early reports suggested that older age correlated with an increased risk of LC. In a study by Sudre et al. (2021), data revealed that nearly 21.9% of patients over 70 years reported symptoms persisting for four weeks or more, compared to 9.9% aged 18 to 49 years (15). Similarly, in a multicenter prospective cohort study across Israel, Spain, Switzerland, and Italy, 20% of the patients attending COVID-19 recovery clinics were older than 65, and the older individuals were more likely to be symptomatic and more likely to experience cough and arthralgia (46). However, older age was not always found to be a risk factor for fatigue or dyspnoea, the most common LC symptoms. In contrast, a recent study determined that those aged between 30 to 39 years had a 6% lower risk, and those aged ≥70 years had a 25% lower risk compared to those aged 18–30 years, respectively (23). A recent meta-analysis, including 30 371 patients, found no significant association between age and the risk of Long COVID (OR 0.86, 95% CI 0.73 – 1.03) (47). The relationship between age and LC risk requires further investigation to characterize the complex association between advancing age, including aging of the immune system, specific biological mechanisms, and individual clinical phenotypes. Concerningly, LC may also target children, including those with asymptomatic acute infection, often resulting in extreme tiredness, shortness of breath, muscle pain, chest discomfort, palpitations, headaches and reduced cognitive abilities that often result in symptoms such as extreme tiredness, shortness of breath, last for beyond six months (30, 34, 35).

There is growing evidence that the COVID-19 pandemic has disproportionally impacted ethnic minorities and migrants in terms of cases, hospitalizations, deaths and mental health outcomes in many parts of the world. In a US-based study evaluating patients two months after COVID-19 diagnosis, Black outpatients were found to be at higher risk of experiencing LC symptoms, specifically shortness of breath, myalgia, or arthralgia, compared to ethnic minorities and Caucasian individuals (48). Heightened risks were also reported in Black Afro-Caribbean ethnic groups, mixed ethnicity and other minority ethnic groups containing patients of Polynesian, Middle Eastern or Native American origin compared to Caucasian groups (23). Similarly, in a UK-based study which evaluated patients 4-8 weeks after hospitalization for a severe COVID-19 infection, 42% of Black, Asian, and minority ethnic patients experienced shortness of breath compared to 25% of Caucasian patients. This trend was also observed in Post-Traumatic Stress Disorder (PTSD)-related LC complaints (47). A rapidly increasing concern is that vulnerable communities with partial or complete lack of quality healthcare access face challenges in convincing healthcare providers of their LC-related symptoms. Several LC-related epidemiological studies have been conducted, yet the black minority in non-African cohorts has been underrepresented, especially concerning registries, treatment regimen programs and clinical trials (23). Therefore, one critical and urgent need remains to disentangle the role of ethnicity (and genetics, see below) from the role of poor access to care in these highly vulnerable populations.

A remarkable finding by Zeberg and Pääbo was that a Neanderthal gene variant/haplotype on chromosome 3 significantly elevated the risk for severe COVID-19 symptoms. Possessing this specific variant conferred a 60% increased likelihood of being hospitalized (49). This variant can be found in 50% of South Asian and 16% of Europe inhabitants but is rare in African populations. In a subsequent study, utilizing data from the Genetics of Mortality in Critical Care consortium, Zeberg and Pääbo identified another Neanderthal haplotype in a region of chromosome 12 encoding oligoadenylate synthetases (OAS1-3), which, in contrast, appeared to protect against severe COVID-19 disease. Each copy of this haplotype lessened the need for intensive care-related hospitalization by 22% (50). Genes in this region encode enzymes essential to the body’s immune response to RNA viruses (51). These enzymes facilitate the breakdown of viral genomes. The genetic variability of human leukocyte antigen (HLA) molecules, which bind fragments of pathogenic microorganisms and present them to T lymphocytes to initiate or guide immune responses, can affect the susceptibility to and severity of SARS-CoV-2 infection. Severely ill patients were found to have decreased expression of HLA-DR molecules, which was associated with an immunosuppressed host response (52). Class I HLA molecules with a stronger predicted capacity to bind SARS-CoV-2 antigenic peptides were found in patients experiencing mild infection and exhibited an increased degree of heterozygosity compared to patients with moderate to severe COVID-19, consistent in their role in directing antiviral responses by CD8 T lymphocytes (53). It remains unclear if these factors are associated with an increased risk of Long COVID, independently of their association with severe disease and autoimmunity, where class II HLA molecules usually associate with susceptibility. Longitudinal studies incorporating host genetics will allow for the determination of genetic risk factors for LC.

A dampened or otherwise dysfunctional immunity is a crucial contributing risk factor for LC. This is often observed in individuals with malignancies or those undergoing chemotherapy, post-transplant treatment, and in conditions such as HIV/AIDS or other disorders that diminish the host immune system’s defence capabilities. Immunocompromised young adults, as well as children, are prone to prolonged viral infection, continued viral shedding, and subsequent accrual of viral variants, supporting the suspicion that weakened or inadequate host immune responses increase the possibility of SARS-CoV-2 variants emerging as a mechanism of escape to avoid immune detection, ultimately establishing conditions that favour LC onset (54–56). The prevalence of LC in PLWHA was estimated to be 43.6% (95% [CI], 33.4–54.2). Moderate to severe COVID‐19 disease was significantly associated with the post COVID condition (adjusted odds ratio, 4.7; 95% CI, 1.4–17.9; p = .016). Among PLWHA with LC, the most reported persistent symptoms were fatigue (19.1%) and cough (22.3%) (57). Elevated levels of persistent inflammation may be the underlying cause leading to the onset of LC in PLWHA. Generally, HIV is a chronic inflammatory disease associated with abnormal prolonged immune activation. Disturbances to the immune environment such as an encounter with COVID-19 may lead to a higher prevalence of LC among PLWHA. Other contributing factors that could predispose PLWHA to LC include dysfunction of the microvascular circulatory system (58), reactivation of human herpesviruses (10), and autoimmunity (59).

Another risk factor that may increase the probability of patients developing LC is the presence of lower-than-normal concentrations of specific immunoglobulin (Ig) neutralizing antibodies IgM and IgG3, in combination with other clinical risk factors such as the severity of initial acute COVID-19 infection, age and a history of asthma (60). IgM and IgG3 are the first antibodies produced in response to a new infection and are associated with boosted protection against viruses, bacteria and parasites. These antibodies were found more commonly amongst LC individuals than COVID-19 survivors without LC (10, 60, 61). Almost two-thirds of patients who develop LC have evidence of established autoantibody production at the time of COVID-19 diagnosis. The titer of autoantibodies negatively correlates with the titer of protective SARS CoV-2 antibodies such as IgM and IgG3. In combination with other clinical risk factors, an antibody-based signature may assist in determining a patient’s level of risk for LC (10, 60).

Asthma, Type 2 diabetes mellitus, hypertension, and psychological conditions such as anxiety and depression have been associated with an increased risk of developing LC. The persistence of symptoms following acute COVID-19 infection is frequently also observed in people with asthma. In a study by Buttery and colleagues (2022), 67.8% of COVID-19 asthmatic LC patients reported more frequent use of their inhaler therapy, 73.7% reported increased difficulty breathing and 59.6% reported decreased asthma control after initial COVID-19 infection. A significant proportion of patients experienced protracted asthmatic exacerbations, worsening of asthma symptoms, and the occurrence of additional symptoms besides their usual symptom profile following acute COVID-19 (62). Whether this can be explained simply by a hyperreactive airway, as is likely the case with other acute infections in asthmatics, remains to be seen.

In a study by Aminian and colleagues (2022) observed patients from day 30 to 8 months after testing positive for SARS-CoV-2, hospital admission rates were 28% higher in individuals with class II obesity (35-39 kg/m²) and 30% higher in class III obesity (≥40 kg/m²) when compared to a normal BMI range (18.5 to 24.9 kg/m²). This study proposed that people with moderate to severe obesity are at higher risk of LC, with an increased risk of requiring hospitalization (63). Elevated BMI is associated with greater risk of developing LC, patients are 1.031 times more likely to suffer from post-COVID-19 symptoms for each 1 kg/m² increase in BMI (RR 1.031, 95% [CI]: 1.016-1.047 (64). Poorly controlled type 2 diabetes increases the likelihood of severe COVID-19 infection and is associated with an increased risk of morbidity and mortality. The COVID-19 pandemic has led to a rise in poorly controlled diabetes, progression from prediabetes to diabetes, an increase in the number of new-onset diabetes, and an escalation of corticosteroid-induced diabetes. Prolonged states of uncontrolled hyperglycemia may result in organ damage, especially to the microvascular system, which can be worsened in SARS-CoV-2 infected patients (see coagulopathy below). Post-COVID pulmonary fibrosis is more common in patients with uncontrolled diabetes. Low-grade inflammation associated with Type 2 diabetes may be aggravated during acute SARS-CoV-2 infection, leading to heightened inflammation during the post-COVID-19 period resulting in the onset of several symptoms associated with LC (16).

Pre-COVID-19 psychological distress was associated with a 1.3 to 1.5-fold increase in risk for reporting LC associated symptoms. An analysis of individuals reporting two or more LC symptoms revealed that psychological distress preceding the acute COVID-19 infection increases the risk for LC by 50%. Common symptoms reported in such individuals included fatigue, digestive, neurological, cardiovascular, respiratory, and cognitive symptoms present for more than four weeks after acute infection. Psychological distress was linked to a 15-51% greater risk of daily life impairment due to LC consequences, with 56% of individuals reporting at least occasional daily life impairment relating to the condition (65).

Early analysis from the SARS-CoV-2 pandemic revealed that hypertension is the most shared pre-existing clinical condition observed amongst hospitalized COVID-19 patients, affecting 30-50% of global cohorts. Hypertension has now also been implicated in the onset of LC. A case-control study conducted by Fernández-de-las-Peñas (2022), a first of its kind to investigate the link between hypertension and LC, revealed that hypertensive participants demonstrated a substantially larger number of LC symptoms (66). The mechanism underlying LC development in hypertensive individuals is still not fully understood.

Since the start of the pandemic in 2020, there has been significant effort to provide information on potential biological mechanisms influencing the viral activity and overall pathogenicity of SARS-CoV-2. Persistence of the virus or its components is a highly plausible biological mechanism for LC. Studies have demonstrated the persistence of viral antigens, viral RNA and whole virus in the brain, sinus, adrenal glands, kidneys, gut, lymph nodes, spleen, lungs and heart (67). Viral persistence may result in symptoms through (i) direct viral cytopathic effects; (ii) regional inflammation; (iii) provoking a.n immune response causing an elevated and prolonged state of inflammation; and (iv) prompting autoimmunity (54, 68).

Viral persistence may be associated with a high degree of immune activation during acute SARS-CoV-2 infection (69). COVID-19 severity is influenced by dysfunction and dysregulation of the host immune response resulting from disturbance of the type I interferon pathway in acutely SARS-CoV-2 infected host cells. Reduced or delayed host immune signaling responses would be expected to favor viral persistence and the potential production of SARS-CoV-2 variants (54). Furthermore, SARS-CoV-2 has been shown to contain superantigen-like molecules that may overstimulate T cells, resulting in a cytokine storm, but eventually also in T cell exhaustion and a weakened capacity for viral clearance, inadvertently allowing SARS-CoV-2 to persist (70). Additionally, viral mimicry of self-proteins may enable it to evade immune surveillance and viral clearance. It should also be highlighted that LC exhibits autoimmune disease characteristics (see autoimmunity below); SARS-CoV-2 infected individuals may be more prone to the onset of LC owing to further activation of an already aggravated memory immune response that cannot be sufficiently modulated by mechanisms of homeostasis when the viral infection is finally cleared (71).

Autoimmunity is emerging as an increasingly plausible biological mechanism for at least some LC phenotypes. The prevalence of autoantibodies in LC patients is elevated compared to the general population (72). Severe COVID has shown striking similarity to systemic autoimmunity seen in systemic lupus erythematosus (SLE), with marked extrafollicular B cell maturation and autoimmune autoantibody production by double-negative (CD27- IgD-) B cells (73). The resultant autoantibodies target specific extractable nuclear antigens such as LA/SS-B, Ro/SS-A, Jo1, P1 and U1-SnRNP, further reminiscent of SLE (72, 74). Auto-antibodies to interferon type 1, in up to 10% of severe COVID cases, would also be expected to limit the host immune system’s ability to effect viral clearance appropriately, but their role in LC remains speculative at this time. More recently, multi-dimensional immune profiling of 99 LC patients by Rapid Extracellular Antigen Profiling, a high throughput method capable of measuring antibody reactivity to >6000 extracellular and secreted human proteins, failed to identify any stereotypical autoantibodies to distinguish LC patients from healthy controls (75). However, differential antigen targeting in the LC and control groups revealed shared patterns of autoreactivity in a subset of LC patients with tinnitus and nausea. Moreover, it remains unclear whether antibodies to intracellular autoantigens and non-protein autoantigens are implicated in the pathogenesis of Long COVID.

There is rapidly growing evidence to support the presence and persistence of microclots in individuals with LC relative to those without prior COVID-19 infection (58). A similar phenomenon has previously been demonstrated in individuals with type 2 diabetes (76). These LC-associated microclots are (i) resistant to fibrinolysis; (ii) harbour antitrypsin rendering them resistant to lysis processes; (iii) encompass inflammatory molecules, which may prompt inflammation upon lysis; and (iv) postulated to result in the occlusion of the microcirculation in various capillary beds triggering tissue dysfunction and ultimately resulting in organ dysregulation and dysfunction, culminating in clinical presentations related to an organ system (77, 78).

In COVID-19 patients, it was proposed that the SARS-CoV-2 virus enters the host’s upper respiratory tract causing damage to the olfactory mucosal cells and the loss of smell and taste. The virus would then travel toward the brain through axonal transport or via the lymphatic system. This event would be expected to induce an inflammatory glial reaction (79) that, with or without a CNS-targeted autoimmune response, could contribute to the overall CNS effects with symptoms such as extreme tiredness, headaches and cognitive impairments, including memory loss, lack of concentration and brain fog (80, 81). Tachycardia, palpitations and chest discomfort are common symptoms of LC in approximately 20 to 40% of COVID-19 survivors. The brain’s respiratory and cardiovascular control centres are closely intertwined in the brainstem, in regions such as the caudal ventrolateral medulla (CVLM), ventral respiratory column (VRC) and the pontine respiratory group (PRG). The CVLM is made up of neurons responsible for regulating heart rhythms. The VRC controls rhythmic breathing with coordinated cycling between inspiration and expiration. The PRG modulates the transition between expiration and inspiration. Thus, disturbances in this region can steadily result in dysautonomic phenotypes. SARS-CoV-2 may also enter the brainstem directly via the surface expression of ACE-2 (82) because the brainstem has a relatively high expression of ACE-2 receptors compared to other brain regions (79, 83). These events would lead to the generation of inflammatory mediators that damage vascular cells and the brainstem. Microthrombosis resulting from vascular injury may further potentiate neuroinflammation and brainstem dysfunction. Thus, continuous brainstem dysfunction at the level of CVLM, VRC and PRG may explain LC’s prominent cognitive and cardiorespiratory-related symptoms (82).

Thromboinflammation is mainly triggered by the interplay between SARS-CoV-2 and the humoral innate immune system, specifically by an interplay between coagulation, fibrinolytic and complement systems. Activation of and/or damage to endothelial cells, leukocytes and platelets all funnel into thrombotic and inflammatory reactions (84). During LC, the ongoing endothelial dysfunction combined with an impaired innate immune response may promote the onset of an acute inflammatory reaction of the endothelium, potentially leading to acute myocardial injury, myocarditis, and thromboembolic complications affecting both the venous and arterial circulation (36). SARS-CoV-2 mediated endothelial dysfunction may also induce excessive production of thrombin, fibrinolysis inhibition, and continuous activation of the complement pathways resulting in prolonged dysfunction of the microvascular system and microthrombosis (85). Microvascular thrombosis occurs systemically and can affect several organs, especially those with high capillary density, such as the lungs (86). The mechanisms underlying the association between LC and the onset of cardiovascular diseases are not yet fully understood. However, several mechanisms have been proposed, including continuous damage directly induced by viral invasion and subsequent death of cardiomyocytes, infection of endothelial cells, endothelialitis (87), transcriptional alteration of cardiomyocytes, complement activation and complement-mediated coagulopathy and microangiopathy (88), ACE-2 downregulation, renin–angiotensin–aldosterone system dysregulation (89), autonomic dysfunction (90), increased concentrations of pro-inflammatory cytokines (91) and transforming growth factor β (TGF-β)/SMAD signalling pathway activation inducing fibrosis of cardiac tissue (92).

Persistent symptoms in LC patients may arise because of tissue or organ injury induced by abnormal clotting or inflammatory during acute COVID-19 infection. In a Swiss-based prospective observational cohort study involving adult patients that survived acute COVID-19 following mild/moderate or severe/critical COVID-19, substantial functional and radiological abnormalities suggestive of lung parenchymal and small airway disease was observed 4 months after the initial infection (93). It has also been noted that more than one third of recovered COVID-19 patients develop lung abnormalities associated with fibrosis, following hospitalisation (94). Risk factors associated with the development of post-COVID-19 lung fibrosis include advancing age, prolonged ICU stay and duration of mechanical ventilation, excessive alcohol consumption, smoking, severe COVID-19, and a high burden of comorbidities including coronary artery disease, diabetes and hypertension (93–96). Mechanical ventilator-induced lung injury (VILI) poses an additional risk due to the abnormal alveolar pressure or volume dynamics that cause lung tissue injury leading to the generation of proinflammatory modulators thus exacerbating acute lung injury, facilitating increased pulmonary fibrosis in those that survive. Patients with a history of cigarette smoking exhibited a much higher incidence of post-COVID-19 lung fibrosis compared to non-smokers. In a cross-sectional prospective study conducted in Egypt, out of 30 smoking patients, 18 developed post-COVID-19 pulmonary fibrosis (60%) (95). Post-COVID lung fibrosis has been documented as a probable perturbing sequela among COVID-19 survivors, in which permanent structural distortion within the lung tissue environment may occur leading to irreversible dysfunction (95).

Biological mechanisms underlying cardiovascular injury in the onset of LC are still not yet full elucidated. Postulated mechanisms include persistent damage as a result of direct invasion of cardiomyocytes by SARS-CoV-2 followed by cell death, viral infection of endothelial cells and endotheliitis, transcriptional alteration of multiple cardiac cell types, complement activation and complement-mediated coagulopathy and microangiopathy, ACE2 downregulation and deregulation of the renin–angiotensin–aldosterone system, dysautonomia, increased pro-inflammatory cytokines production and release and activation of Transforming growth factor β (TGF-β) signaling via the Smad signalling pathway (91). TGF-β stimulates fibroblasts and accelerates extracellular matrix production within injured tissues. Disproportionately excessive aggregation of extracellular matrix, results in fibrosis and eventually scarring of cardiac tissue (91, 96). Another proposition is that a chronic inflammatory response is induced by prolonged viral reservoirs in the cardiomyocytes following acute infection. This condition may be further aggravated by obesity, which stimulates activation of the inflammatory signalling pathway mediated by c-Jun N-terminal Kinase (JNK) and nuclear factor-kappa B (NF-κB) (97). Downstream effects lead to the generation of multiple pro-inflammatory cytokines in adipocytes such as monocyte chemoattractant protein-1 and Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (RANTES), chemokines that exacerbate endothelial dysfunction via endothelial nitric oxide synthetase uncoupling and production of reactive oxygen species, which promotes insulin resistance and pro-inflammatory macrophages infiltration (97, 98). Subsequently, these processes culminate in the unsolicited detrimental damage of cardiac tissue, advancing to chronic fibrosis of the myocardium and compromised ventricular compliance, diminished myocardial perfusion, intensified myocardial stiffening, leading to decreased cardiac contractility and an increased likelihood of arrhythmias (98).

SARS-CoV-2 infection-induced reactivation of latent herpesviruses as a possible contributing factor to LC has been studied. In a study of 215 individuals with Long COVID, unexpected increases were observed in antibody responses directed Epstein-Barr virus (75). Also, in a cohort of 280 adults with prior SARS-CoV-2 infection, Peluso et al. observed that LC symptoms, such as fatigue and neurocognitive dysfunction, at a median of 4 months following initial diagnosis, were associated with serological evidence suggesting recent EBV reactivation (early antigen-diffuse IgG positivity) or high nuclear antigen (EBNA) IgG levels but not with ongoing EBV viremia. Serological evidence suggesting recent EBV reactivation (early antigen-diffuse IgG positivity) was most strongly associated with fatigue (OR = 2.12). In this study, underlying HIV infection was also associated with neurocognitive LC (OR = 2.5). However, those with serologic evidence of prior CMV infection were less likely to develop neurocognitive LC (OR = 0.52) (99). In another study the authors tested for SARS-CoV-2 RNA in stool and throat washings, and EBV DNA in stool, throat washings and blood by real-time PCR (EBV) and real-time RT-PCR (SARS-CoV-2) in 30 LC patients characterized by persistent fatigue, post-exertional malaise (PEM), autonomic dysfunction and/or orthostatic intolerance and who all had had a mild SARS-CoV-2 infection. Twenty age- and sex-matched patients who had fully recovered after the SARS-CoV-2 infection served as controls. SARS-CoV-2 RNA indicating persistent infection was not detected in throat washing nor stool. SARS-CoV-2 antibody titres (IgA and IgG) did not differ between the cohorts. EBV real-time PCR was negative in all blood or stool samples. However, EBV DNA was detected in throat washings in 15/30 (50%) of LC patients, while in only 4/20 (20%) of non-LC patients (p = .0411) (100). A study of patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and healthy donors as controls analyzed whether a mild/asymptomatic SARS-CoV-2 infection imposes latent EBV, HHV6 and HERV-K virus reactivation. At 3-6 months after infection, virus-specific antibodies in saliva were substantially induced, indicating a strong reactivation of all three viruses in both cohorts. In patients with ME/CFS, however, antibody responses were significantly stronger, in particular against EBV-encoded nuclear antigen-1 (EBNA1) than in the controls (101).

The role, if any, reactivated EBV infection may have on the development of LC remains to be fully elucidated.

The metabolic sequelae of SARS-CoV-2 infection are highly complex, involving the dysregulation and dysfunction of multiple organs. Metabolic dysregulation is thought to be driven by immunopathological events, including the overproduction of inflammatory molecules leading to persistent inflammation and incomplete systemic recovery. Closely related coronavirus diseases, such as Middle East Respiratory Syndrome (MERS) and SARS, have been shown to induce long-term metabolic modifications in convalescent patients resulting in liver disorders, altered glucose metabolism and hypertriglyceridemia. Several studies have demonstrated the presence of persistent metabolic derangement in SARS-CoV-2 infection, suggesting this mechanism is one of the underlying routes of LC pathogenesis (102). The earliest bioenergetic effects of SARS-CoV-2 infection occur through the binding of viral polypeptides to critical host cell mitochondrial proteins, retarding ATP production. Subsequently, SARS-CoV-2 produces a pervasive inhibition of the transcription of key mitochondrial genes central to cellular respiration (103). Mitochondrial dysfunction and resultant bioenergetic failure have been demonstrated during acute infection. The same is true of lipid metabolic dysbalance, including the production of phospholipases that were found to be predictive of poor outcomes (104). However, establishing impaired cellular respiration and/or lipid dysregulation in the context of LC requires further research (105, 106).

Recently, evidence has emerged to propose the role of the lung‐gut axis in increasing the risk for severe COVID-19 (107). Gut microbiota dysbiosis correlates with poor clinical outcomes in mechanically ventilated COVID‐19 patients. These findings indicate a strong interrelation between SARS‐CoV‐2 consequences and host gut microbiota, which may also be associated with an increased risk of LC. The substantial rise in pathogenic bacteria and simultaneous reduction in anti‐inflammatory microbiota promotes sustained intestinal inflammation during COVID‐19, subsequently leading to prolonged recovery (108). Su and colleagues (2022) followed 155 patients for 14 months after being diagnosed with acute COVID-19 infection; 76.4% were diagnosed with LC at six months and 78.7% at 14 months. In LC-associated dysbiosis three most common symptoms were disrupted sleep (35.5%), cognitive difficulties relating to memory (44.5%) and fatigue (50.9%). It was found that the gut microbiota of the LC patients did not fully recover at one year in after COVID-19 diagnosis (109); genera Prevotella and Veillonella dominated the oral microbiome of COVID-19 patients. The Prevotella genus produces proteins that can promote SARS-CoV-2 infection and can increase disease severity. These genera have also been associated with an elevated risk of pneumonia-induced mortality in frail, older patients. Both genera are known inducers of inflammatory responses. Prevotella strains mainly activate TLR-2 and boost the expression of inflammatory cytokines IL-1 and IL-23. Veillonella species is a potent inducer of IL-6. Several other pro-inflammatory microbiotas may be responsible for prolonged disease symptoms, such as multiple Actinomyces species, S. moorei and L. wadei. In LC patients, the study demonstrated that metabolic pathways accompanying the generation of pro-inflammatory molecules were abundantly increased. In contrast, anti-inflammatory pathways were downregulated, which may be one of the main attributes underlying the persistence of LC and associated symptoms (110). Beyond direct disturbance of the microbiome, evidence is accumulating to suggest that mucosal barrier breach and microbial component/product translocation into the systemic circulation may occur in LC (“leaky gut”). This includes specific evidence of fungal translocation as measured by systemic beta-glucan increase, which has been detected in 74% of LC cases by a recent study (111).

Amyloidogenesis is a speculative mechanism for some LC symptoms (112), including cognitive, behavioral, and psychiatric symptoms resembling neurodegenerative disorders. Many neurodegenerative diseases, such as Parkinson’s and Alzheimer’s are distinguished by the abundance of amyloid or amyloid plaques (113). Amyloid fibrils of the SARS-CoV-2 spike protein have been localized in nerve tissues with heterologous seeding promoting the accumulation of endogenous proteins, which may lead to neurodegeneration (114). In COVID-19 patients, blood clotting is related to extracellular amyloidotic fibrillar assemblages. Hence, when plasma from healthy donors was incubated with the SARS-CoV-2 spike protein, hypercoagulation and impaired fibrinolysis were demonstrated. Previous studies have linked amyloidosis to thromboinflammation, activation of surface-activated coagulation system (FXII Kallikrein/Kinin), disruption of the fibrinolytic system, dysregulation of blood coagulation and cerebral amyloid angiopathy; collectively, suggesting associations between COVID-19 phenotypes and S-protein amyloidogenesis. A study conducted by Nystrom and Hammarstrom (2022) found amyloidal sequences in SARS-CoV-2 spike protein and that endoproteolytic cleavage of the protein resulted in amyloid formation (114). Amyloidosis, resulting from the systemic deposition of amyloid proteins, may present as localized or systemic conditions with many generalized or organ-specific phenotypes overlapping with the reported LC clinical phenotypes (114)

Some symptoms observed in LC patients suggest immune or virus-induced autonomic nervous system (ANS) disruption, resulting in temporary or possibly long-term orthostatic intolerance. Many have hypothesized that COVID-19 infection affects the ANS (see the above discussion of autonomous centers in the brain stem and accessibility to SARS-CoV-2) and that the SARS-CoV-2 virus itself may mediate the resultant autonomic dysfunction. POTS involves many symptoms such as nausea, heart palpitations, blurry vision, headaches, tiredness, cognitive impairments and lightheadedness. The exact cause underlying the development of POTS in LC remains unknown, but dysautonomia has been commonly implicated. Autonomic disorders like POTS are often associated with abnormal activity of muscarinic receptors and alpha and beta (α/β) adrenoreceptors. Usually, when a healthy individual stands, blood accumulates in the pelvis and limbs, which causes a reduction in venous return to the heart. Cardiac baroreceptors sensing the volume change, stimulate an increase in sympathetic adrenergic and neural tone modulated by epinephrine and norepinephrine, respectively. Consequently, these events lead to tachycardia and splanchnic vascular bed vasoconstriction for elevated venous cardiac return. During orthostatic intolerance, norepinephrine and epinephrine release induces distinct tachycardia, manifesting as chest pain, shortness of breath and palpitations; symptoms frequently associated with LC Extremely high catecholamine levels can cause inconsistent vasodilatation, withdrawal of sympathetic activity and vagus nerve activation, ultimately resulting in dizziness, blood pressure instability and fainting (115–117).

Individuals previously hospitalized with severe COVID-19 infection are more likely to develop type 2 diabetes mellitus, chronic renal disease (CRD), chronic liver disease, coronary artery disease or organ failure due to SARS-CoV-2 activity within host tissues. Interestingly, organ impairment was also observed in non-hospitalized COVID-19 infected patients that did not require supplemented oxygen (160, 161). Individuals with Long COVID exhibited an increased risk and excess burden for diabetes in the 12 months following acute infection. Risks and burdens of post-COVID outcomes increased in a graded fashion correlating with acute disease severity (162). Patients at high risk for developing CRD included those with frailty, chronic illnesses, disability, and immune senescence (163). Liver injury in COVID-19 patients is associated with prolonged hospitalization and acute severe disease. Among critically ill adult COVID-19 patients, the incidence of hyperbilirubinemia is increased by 1.7-fold, and indirect markers of liver injury, including hypoalbuminemia, are raised by 7-fold (164). Recovered COVID-19 patients with critical cardiopulmonary involvement may show clinical and histological features of severe cholangitis and intrahepatic microangiopathy. Notably, severe LC-associated cholangiopathy increases the risk for long-term hepatic morbidity (165). Patients surviving beyond the first 30 days after infection have an increased incidence risk of cardiovascular disease across several categories, including thromboembolic, heart failure, myocarditis, pericarditis, non-ischemic and ischemic heart disease, dysrhythmias and cerebrovascular events. Overall, the risk and 1-year burden of cardiovascular disease in survivors of acute COVID-19 are substantial for those presenting with COVID-19 beyond the first 30 days of infection (137).

In vaccinated patients experiencing a breakthrough episode of COVID-19, first and second dose vaccination was associated with reduced likelihoods of requiring hospitalization or developing more than five symptoms in the first week of infection. For those who had two vaccine doses, the odds of long-duration symptoms (≥28 days) was significantly reduced compared to unvaccinated individuals. In a study by Antonelli et al. (2022), most persistent symptoms following acute COVID-19 were reported less often in vaccinated individuals than in unvaccinated individuals (166). At-risk individuals remain highly susceptible to infection with SARS-CoV-2 despite vaccination due to immune-senescence, multimorbidity (diabetes, hypertension, and obesity) and poor socioeconomic status. Nonetheless, vaccination in this vulnerable population is associated with lower mortality risk (HR = 0.66, 95% CI: 0.58, 0.74) and a significantly lower risk of Long COVID (HR = 0.85, 95% CI: 0.82,0.89) (4, 167). Notably, vaccination was observed to have reduced the risk of Long COVID symptoms such as sleeping disturbances, myalgia, renal injury and cognitive deficits, in individuals vaccinated either before or after acute infection (168). The probability of persisting Long COVID symptoms decreases steadily following vaccination, with a growing body of evidence suggesting that lasting improvements are observed after a second vaccine dose (169). Azzolini et al. (2022) revealed the association between the number of BNT162b2 vaccine doses and Long COVID in healthcare workers who did not require hospitalization, providing evidence that the number of vaccine doses received was inversely related to the incidence of Long COVID: unvaccinated 41.8% (95%CI, 37.0%-46.7%), one dose vaccination 30.0% (95% CI, 6.7%-65.2%), two-dose vaccination 17.4% (95%CI, 7.8%-31.4%), and three-dose vaccination 16.0%(95%CI, 11.8%-21.0%) (167). The impact of the COVID-19 vaccination on people presenting with Long-COVID still remains incompletely understood, with conflicting reports from existing studies.

Our proposed visualization of Long COVID and its pathogenesis presents a dynamic, modular and systems-level approach to the condition ( Figure 1 ). The visualization provides an overview of the current thinking and evidence establishing the multi-nodal connection/s between acute SARS-CoV-2 infection and clinical phenotypes. It incorporates risk factors and biological mechanisms as essential nodes in the development of the Long COVID. The strength of the association between any two nodes, based on a qualitative synthesis of the current evidence by the authors, is represented by the thickness of the connecting lines. It is apparent that a single pathway is unlikely to explain the genesis and evolution of this complex post-viral phenomenon and that multi-hit, multi-mechanistic pathways are more likely. Patients may present with distinct and easily identifiable phenotypes or overlapping/multi-system phenotypes, with the evolution of the clinical phenotypes over time. The visualization is capable of expressing this complexity by demonstrating that pathways may evolve or may emerge and abate dynamically in an individual patient. The propensity of a particular pathway to alter with time is the subject of research in several Long COVID cohorts. It is also apparent that clinical phenotypes may result from a particular biological mechanism or as a result of multiple biological mechanisms acting in parallel or in series over time. It is equally apparent that a single biological mechanism may result in diverse clinical phenotypes, especially when the mechanism is active systemically and does not demonstrate organ-tropic effects or is capable of disrupting the physiology of more than one organ system. For example, autoimmunity may affect coagulation, the central nervous system, the cardiovascular system, and the musculoskeletal system ( Figure 2 ). We do not have sufficient evidence yet on how individual biological mechanisms interact or whether one mechanism may propagate or spur the development of other mechanisms. It is plausible, for example, that anti-interferon antibodies may result in ineffective viral clearance and viral persistence, which may result in dysregulated immunity and autoantibody production, which may, in turn, result in neutrophil extracellular trap persistence and micro clotting. Each of these mechanisms may give rise to clinical sequelae and diverse phenotypes. The specific epidemiological and biological risk factors for the development of the putative biological mechanisms have not been fully elucidated but likely act through a complex interplay of genetic, host, pathogen, and acute infection factors with temporal evolution. There is a high likelihood that Long COVID represents a constellation of diverse clinical phenotypes resulting from complex pathways described by the interaction between multiple risk factors driving the development of single or multiple biological mechanisms capable of generating a diversity of clinical sequelae.

As scientific, clinical, and public health efforts for Long COVID escalate, an increasing level of interdisciplinary, inter-mural, and international coordination will be necessary. Highly defined, focused, and niche studies are helpful but they need to advance the field as a whole. The complexity of Long COVID demands systems biology as well as machine learning approaches and a constant awareness of the entire network of pathways implicated in the genesis and evolution of Long COVID. The visualization is a valuable reference tool to 1) locate and contextualize research efforts, 2) identify potential confounding mechanisms and pathways, 3) direct efforts for collaboration, and 4) provide a high-level perspective for individual scientific efforts. Research in this field will require well-characterized cohorts with longitudinal sampling and follow-up. Such cohorts represent global, and arguably communal, resources for understanding the fundamental building blocks of Long COVID and should be evaluated with due regard to the complexity of the condition, the vast network of pathways which may be implicated, and the dynamic nature of the interactions. The historical dependence on the follow-up of hospitalized, acute severe COVID-19 cohorts may not produce broadly generalizable information on Long COVID. The visualization is a call to step away from the simplified approach of siloed, highly defined research questions, toward sustained and collaborative efforts for a systems medicine and systems biology approach characterized by depth, breadth, and tight integration of the research components. Strengths of the associations in the visualization can be updated and monitored using natural language reading to broadly evaluate literature support or lack thereof for different connections and interactions, and with the use of machine learning algorithms, to distill the model into a more specific and actionable tool.

Furthermore, the visualization represents the entire cascade of events from acute infection to clinical phenotypes, including the phenotypic evolution and the risk for discrete clinical events. A clinical service for patients with post-viral fatigue would benefit from recognizing the biological mechanisms which could have led to the development of this phenotype and the risk factors that could contribute to the development of the biological mechanism/s in individual patients ( Figure 3 ). Similarly, a scientist focused on understanding the role of autoimmunity should be aware of the competing explanatory biological mechanisms in the development of Long COVID and should be mindful of the Long COVID clinical and laboratory-based phenotypes which are under study (and equally so, those which are not). The visualization should guide existing and future opportunities for scientific and clinical development at each node and for the system overall. As a dynamic and iterative dashboard, the visualization should evolve alongside the evidence, incorporating new nodes and pathways as they become illuminated, and be developed into a framework for Long COVID clinical care and research. Lastly, the visualization is a reminder that the diverse clinical phenotypes of Long COVID, emanating from pathways of near-irreducible complexity, will require a holistic, multi-disciplinary, and comprehensive clinical and public health approach, that should further incorporate socioeconomic factors in the near future.

Future iterations of this visualization would benefit from incorporating kinetic aspects of each element, the impact of variants on driving particular pathways, the impact of vaccines and therapeutics interventions for acute COVID-19, and ultimately potential therapeutic interventions directed at specific biological mechanisms or clinical phenotypes.