In this study, we tested our hypothesis that serum BChE activity could serve as a predictor of clinical status progression and outcome in COVID-19 patients. To investigate this, we analyzed BChE activity in the sera of 462 patients with PCR-confirmed COVID-19 from the first epidemic wave in Spain. The cohort consisted of 78 asymptomatic, 200 mildly symptomatic, 122 severely symptomatic, and 62 critically ill patients. Twenty-six patients from the two most severe groups died within 30 days of diagnosis. The most common symptoms reported were cough and fever, observed in approximately half of the cohort. While most symptoms were reported in mildly symptomatic patients, pneumonia and pulmonary failure were more prevalent in severely symptomatic and critically ill patients, respectively.

Although both males and females were present in comparable numbers, women predominated in the asymptomatic and mildly symptomatic groups, whereas males were more common in the two more severe groups. Deaths were more frequent in males, accounting for 69% of the total mortality. Several COVID-19 studies have reported that males were more severely affected than females^36,37,38,39consistent with previous findings from epidemiological data of SARS-CoV and MERS-CoV that also highlighted sex differences in disease manifestation^39,40,41. There is a lack of understanding of how sex influences COVID-19 outcomes. However, observed genetic and behavioral differences, which are multifactorial in nature, may play a significant role in susceptibility to viral infections³⁶. For example, the effects of the X chromosome and sex hormones on immune responses have been reported, potentially providing females with greater plasticity and adaptability to infections⁴². Toll-like receptor 7 (TLR7), encoded on the X chromosome, plays a critical role in the immune response⁴³. Several studies have shown that plasmacytoid dendritic cells (pDCs) from females produce more IFNα/β than males following TLR7 activation by viral RNA^36,43,44potentially giving females an advantage in combating COVID-19. Additionally, males are more likely to engage in risky behaviors such as smoking and alcohol consumption and tend to have a wider range of underlying health conditions, which may increase their risk for severe COVID-19 outcomes^36,37.

Seventy-five percent of the patients in the study were unvaccinated, with half of whom were in the mildly symptomatic group. For vaccinated patients, 38% of the asymptomatic group, 21% of the mildly symptomatic group, and 37% of the severely symptomatic and critically ill groups had received either one or two doses of the vaccine. Among those who had received two doses of the vaccine, only 29% developed severe symptoms and 9% became critically ill. Additionally, 11% of the severely symptomatic and 34% of the critically ill vaccinated patients died, and all these individuals were aged ≥ 65 years. Hence, our results are consistent with other studies demonstrating the protective effect of COVID-19 vaccination, although factors such as age may influence the efficacy of the vaccine⁴⁵.

It is well established that advanced age is a recognized risk factor for severe COVID-19 outcomes and increased mortality^27,39. Consistent with previous findings by Markuskova et al. (2023), we confirmed this association in our study¹. The chronological age of 65 years and older is commonly used to define elderly individuals in clinical practice⁴⁶and was also used in this study. In the studied cohort, both age groups, < 65 years and ≥ 65 years, were well represented in all symptom groups. However, consistent with the warnings and recommendations regarding the risks associated with older age and COVID-19 issued by various health organizations, governments, and researchers^27,33,47our results revealed that younger patients (< 65 years) were more represented in the two less severe groups, whereas older patients (≥ 65 years) predominated in the two groups with more severe clinical status. Furthermore, mortality was almost exclusively observed in the elderly patients (96%).

Our previous analysis of 148 patients with severe COVID-19 revealed significantly lower serum BChE activity in patients who succumbed to the disease¹consistent with observations by other research groups^2,3,48,49,50. Furthermore, we showed dynamic changes in serum BChE activity corresponding to the progression of clinical status¹reflecting the severity of the disease. Here, we confirmed lower BChE activity in deceased patients. Moreover, we observed an alignment between serum BChE activity and COVID-19 severity and symptoms.

Serum BChE activity differed among the symptom groups, showing higher activities in the asymptomatic and mildly symptomatic patients as compared to the two more severe groups. The lowest serum BChE activities were observed in the critically ill patients. Despite the distribution of patients across the symptom groups, BChE activity did not appear to be influenced by sex, as no significant difference was observed between females and males. Nevertheless, the lower BChE activity observed in the sera of elderly patients, as reported both here and previously by us¹ and others^{2,3,4,15,16,51,52,53}prompted us to perform a follow-up age-sensitive analysis. While results from patients < 65 years were preserved and BChE activity was a function of severity, decreasing in severely symptomatic and even more so in critically ill groups, age ≥ 65 years flattened the effect with only a tendency for lower BChE in the critically ill group. The lack of significant variation in patients ≥ 65 years may be attributed to the overall lower BChE activity in elderly patients.

Patients who received two doses of the vaccine demonstrated reduced BChE activity. This unanticipated discovery is counterintuitive and not yet fully understood. One potential explanation for this phenomenon is that it may reflect immune system activation triggered by vaccination. Confounding factors, such as age, comorbidities, or the timing of sample collection relative to vaccination, may also influence the results. Given the complexity of immune and cholinergic interactions, further research is needed to clarify the underlying mechanisms and potential clinical implications.

To the best of our knowledge, this is the first study to investigate the relationship between BChE activity and the symptoms in COVID-19 patients. Our subgroup analysis revealed that BChE activity was significantly associated with specific COVID-19 symptoms.

Lower BChE activity was associated with respiratory symptoms such as pneumonia and dyspnea, but not with cough or pulmonary failure. Conversely, higher BChE activity was associated with neurological symptoms such as anosmia, ageusia, and headache, potentially reflecting lower systemic inflammation in patients with isolated neurological manifestations. However, this observational finding does not imply causality. BChE activity was not associated with gastrointestinal, algesic, musculoskeletal, or fever and chills.

Regarding respiratory symptoms, previous studies have reported a reduction in BChE activity in patients with chronic obstructive pulmonary disease (COPD), suggesting a possible mechanistic link, although the exact mechanism remains unclear^54,55,56. In contrast, increased BChE activity has been reported in the early stages of some neurological diseases associated with inflammation^13,57although the exact mechanism remains unclear due to the complexity of inflammatory responses involving multiple organs^58,59,60. In addition, factors such as the grade or severity of the disease and age may induce different inflammatory responses. Nevertheless, it is important to mention that the assessment of certain symptoms, particularly neurological and musculoskeletal symptoms, may not have been fully accurate in critically ill patients who required high-flow oxygen therapy, mechanical ventilation, or extracorporeal membrane oxygenation. Given that these patients were often unable to communicate effectively with medical staff, and considering the challenging conditions during the COVID-19 pandemic, some symptoms may have been underreported or overlooked.

Our findings support the potential role of BChE as a prognostic marker in patients with COVID-19, though causal mechanisms remain to be confirmed. The relationship between BChE activity and clinical status remains, however, unclear. Our previous study revealed a correlation between serum BChE activity and inflammatory markers, including C-reactive protein and interleukin-6¹. This is further supported by the work of others suggesting the prognostic potential of BChE in inflammation and sepsis^54,61,62,63. In recent years, a growing body of evidence has highlighted the role of acetylcholine in inflammatory processes^13,54,64. Several immune cells, including T cells, B cells, and NK cells, have been shown to produce acetylcholine, with its release being triggered by infection⁶⁵. Acetylcholine modulates the immune response by activating α7 nicotinic receptors on macrophages, inhibiting nuclear factor (NF-κβ) activation, preserving high mobility group box 1 protein (HMGB1) in its nuclear form, and reducing pro-inflammatory cytokine production^13,17,66. Immune cell-derived acetylcholine plays an important role in the response to sepsis, viral infections, and autoimmune diseases⁶⁷. BChE, by mediating the degradation of acetylcholine, may be indirectly involved in the regulation of pro-inflammatory factors as acetylcholine plays a crucial role in the cholinergic anti-inflammatory pathway^13,54. However, the activity of cholinesterases, specifically BChE, in metabolic homeostasis and inflammation is complex and appears to be even more so in various pathological situations. Hence, BChE activity likely follows different mechanisms in conditions caused by chronic or acute inflammation^5,50,54. In pathological conditions caused by low-grade systemic inflammation, such as type 1 or type 2 diabetes⁵⁷hypertension, hyperlipidemia⁵⁷Alzheimer’s disease⁵⁸Parkinson’s disease⁵⁹and multiple sclerosis, an increase in BChE has been observed in the early stages of these diseases^13,57,59. However, as the diseases progress to more advanced stages, a decrease in BChE activity has been reported, which has been associated with an increased risk of death in these diseases^15,57,61. Therefore, the reduction of BChE in severe and deceased COVID-19 patients, as similarly observed in diseases such as cancer^68,69chronic obstructive pulmonary disease⁷⁰cardiovascular disease^71,72HIV⁷³hemodialysis⁷⁴and stroke⁷⁵could be considered an indicator of disease progression and poor prognosis, further supporting its potential role as a biomarker for severe outcomes in various pathological conditions.

The association of BChE activity with COVID-19 severity, mortality, age, and specific symptoms underscores its potential as a multifaceted biomarker for COVID-19. Given the complexity of the cytokine storm and the intricate interplay of various factors, integrating BChE activity into a composite index with other biomarkers may improve diagnostic accuracy and prognostic assessment. Additionally, considering the upstream role of the cholinergic system in inflammation, BChE activity may provide insight into therapeutic targets aimed at modulating the inflammatory response in COVID-19 patients.

Our study has several limitations. First, its observational nature limits the ability to establish causality between BChE activity and COVID-19 outcomes. Second, as a monocentric study, the generalizability of the findings may be limited; however, it offers detailed patient phenotyping from a single institution. Third, the demographics of the study cohort may affect the broader applicability of the findings. Fourth, although we recognize that BChE activity can be influenced by medications and comorbidities such as diabetes, chronic obstructive pulmonary disease (COPD), and liver disease, and we were unable to comprehensively assess these potential confounding factors due to the emergency conditions of the COVID-19 pandemic, which limited the availability of detailed clinical records. This may have affected the observed associations between BChE and disease severity. Future studies should aim to incorporate detailed comorbidity profiles to better control for these variables. Fifth, due to the specific circumstances of the pandemic, it was not possible to collect blood samples from all patients at consistent time points, potentially introducing variability. Finally, in some cases, the assessment of patient symptoms and characteristics may have been hindered by the patients’ unconscious state and their inability to communicate, compounded by the constraints imposed by the emergency situation.

In summary, in this study, we confirmed our previous findings of significantly lower serum BChE activity in patients with severe COVID-19 and in critically ill COVID-19 patients. In addition, we demonstrated that BChE activity was associated with COVID-19 severity, mortality, specific symptoms, age, and vaccination status. While BChE activity was not influenced by sex, it decreased with age and disease severity. Respiratory symptoms, such as pneumonia and dyspnea, and neurological symptoms, such as anosmia, ageusia and headache, as well as vaccination status were associated with variation in BChE activity. Our findings suggest that BChE may serve as a valuable tool for assessing the severity of COVID-19, predicting outcomes, and informing therapeutic strategies.