Our research is the first population-based study that used direct laboratory measures of smoking exposure, aiming at refining the association between active smoking and SARS-CoV-2 infection susceptibility. In this study, we observed a lower proportion of positive SARS-CoV-2 serology in current smokers compared with non-smokers/ex-smokers. Similarly, current smokers were less likely to have received a diagnosis of SARS-CoV-2 infection. No evidence was found about the risk of hospitalization in COVID-19 patients, likely because of the small number of hospitalized cases in our sample. We also conducted logistic regression analyses and found that the association was persistently negative even after adjusting for sex, age, previous SARS-CoV-2 infection, presence of comorbidities, and group of enrolment (as a proxy of infection risk exposure). Furthermore, the point estimates based on the PS-matched models were consistent with those for the whole study population.
Total sero-prevalence of IgG antibodies in this study was 5.4%. This proportion is in line with the results gathered in other population-based sero-epidemiological surveys conducted after the first epidemic wave in the most affected areas in Italy and elsewhere (March–June 2020), also reflecting the significant SARS-CoV-2 circulation in the Troina area [26, 47,48,49,50]. In particular, our study revealed a proportion of subjects with circulating antibodies higher than that detected by the Italian Institute of Statistics (ISTAT) during the same period, although remained below the 7.5% registered in Lombardy region in the same survey, which was hit hardest in terms of cases and death toll during between March and June 2020 [10, 26, 37]. Stratifying by cohort, the rate of sero-prevalence among HCWs peaked at 14.8%, roughly in line with similar studies that surveyed HCWs [37, 51,52,53]. In this regard, it should be mentioned that antibody prevalence in HCWs showed a high variability, according to different aspects of surveys design and conduction, including magnitude of SARS-CoV-2 spread in study settings, type of healthcare facilities and workers enrolled, local availability of personal protective equipment .
The reduced risk for confirmed SARS-CoV-2 infection in tobacco users has been previously reported. For instance, Israel described a risk reduction in current smokers , and a study conducted in Lombardy region confirmed the proportion of 9.2% of IgM/IgG in current smokers, compared with 19.6% of non-smokers (former and never) . Compared with this previous evidence and other analogous studies [26,27,28], this survey allowed investigating the smoking status and history resolving problems related to self-reporting and, thus, eliminating possible bias. Furthermore, the use of community-based data also avoided selection bias associated with the use of case series, which raised questions about the representativeness of cases compared with the general population. It is worth also noting that previous research relayed on the assessment of smoking in hospitalized subjects, with a major limitation due to the lack of appropriate controls . Another similar weakness can be inferred from studies that enrolled patients with confirmed SARS-CoV-2 infection, leaving outside asymptomatic or pauci-symptomatic individuals. Indeed many studies included online surveys, which found that the burden of COVID-19-like symptoms and self-reported SARS-CoV-2 infection were significantly associated with smoking in syndromic surveillance data [24, 34, 46, 47, 54]—consistently with our findings—with a limitation on objective identifying and quantifying of attributable symptoms . In confirmation of this, Clift also highlighted that heavy smoking (i.e., above 20 cigarettes per day) was associated with a reduced risk of SARS-CoV-2 infection when weighting by the probability of having received a SARS-CoV-2 test, likely prescribed for the occurrence of overlapping symptoms [24, 46].
In contrast, current smoking has been identified as a possible risk factor for progression of the disease, and was associated with higher risks of severe COVID-19 outcomes and death in large population-based researches [24, 25].
More in general, as previously discussed, the research about the effects of active smoking on both infection and disease is still controversial [19, 21], with some methodological limitations and pitfalls found in the literature so far available, including the assessment of smoking status and history among study populations, and systematic ascertainment biases and confounders in case-series on hospitalized smokers which might have led to inaccurate determining the overall smoking-attributable risk across the research [20, 33]. Additionally, some research on the response to COVID-19 vaccines highlighted a link between smoking and the humoral response to COVID-19 vaccines with effects on IgG titre and kinetics, with smoking accelerating the decline in vaccine-induced antibodies titre [56,57,58]. If a similar smoking-attributable effect occurs with antibodies induced by natural SARS-CoV-2 infection, then a much lower prevalence of IgG positivity is to be expected in smokers. Taken together, these findings call for further research about the effect of smoking on COVID-19 and immunological response to both infection and vaccines [56,57,58].
The mechanisms by which tobacco use decreases the risk of SARS-CoV-2 infection (and increases the risk of severe prognosis in COVID-19 patients) are not fully understood. Anti-inflammatory properties mediated by α7 nicotinic acetylcholine receptors and reduction in membrane angiotensin-converting enzyme 2 (ACE-2) expression in bronchial cells—which could play a role in SARS-CoV-2 pathology—have been proposed [20, 26, 28, 59,60,61]. Coronaviruses bind the ACE-2 host cell receptors through homotrimeric spike protein (i.e., S1 and S2 subunit) of their envelope, and, therefore, ACE-2 expression on bronchial tissue is a strong determinant for coronaviruses infectivity. Some studies captured a significant decrease of membrane ACE-2 protein expression attributable to cigarette smoking [61,62,63,64,65]. Again, this effect on ACE-2 might be likely attributable to acute smoking exposure, and thus unlikely to be associated with smoking duration, as revealed by our analyses.
Furthermore, Farsalinos et al. speculated that an anti-inflammatory pathway induced by nicotinic acetylcholine receptor might modulate the immune response from hyper-inflammation stimulated in severe COVID-19 [20, 66]. The cholinergic anti-inflammatory pathway, mediated mainly through the vagus nerve, represents a reflex mechanism based on a bi-directional communication between the immune and nervous systems [67, 68].
It can restore immune homeostasis and prevent cytokine storm, a hallmark of severe COVID-19. This hypothesis warrants further study, but the authors also suggested that the cessation of nicotine intake in hospitalized smokers leads to dysregulation of the cholinergic anti-inflammatory pathway and uncontrolled immune response, and was thus responsible for higher risk for severe outcomes . Recently, a pharmaceutical company reported that α7 cholinergic agonists exhibit antiviral properties both in vitro and in vivo in experimental animals (macaques), but more clinical evidence is needed to verify or reject this hypothesis . Smoking is a leading cause of morbidity and mortality worldwide, and smokers should be encouraged to quit for reducing the heavy burden associated with tobacco use [15, 58]. Obviously, even if results of a low infection rate among smokers will be confirmed in further study, smoking must not be perceived as a protective measure for COVID-19, neither encouraged nor recommended. However, the possibility for therapeutic effects of nicotine or nicotinic-cholinergic agonists on COVID-19 warrants further investigation by the research community through experimental in vitro studies and in clinical trials [58, 61, 70].
This paper has a number of strengths and weaknesses. The study is the first one to use objective measure of the smoking status, thus avoiding reporting bias and allowing to precisely detecting active smoking among participants. It also uses a specific and sensitive antibody assay, which accurately correlate with SARS-CoV-2 infection. Moreover, the field collection of the samples was conducted well before the launch of the national mass vaccination campaign, an important confounder in sero-epidemiologic studies. This is a unique aspect and constitutes a non-replicable added value of this research, as future studies will not be able to discount the possible confounding role of vaccine-induced IgG.
Lastly, the cohort was carefully assembled and sample size satisfactory, being representative of the population aimed to study and thus providing reliable estimates of the association between SARS-CoV-2 infection risk and smoking.
Despite these strengths, some limitations should be acknowledged. First, the limited number of hospitalized subjects did not allow inferring conclusions on this sub-group, leaving outside important aspects related to association of smoking and COVID-19 outcomes. Second, possible recall and notoriety biases should be acknowledged regarding the self-reported COVID-19 related symptoms, for this reason we excluded some possible confounders that could have affected the reliability of the data (e.g., duration of symptoms, etc.) [46, 55]. Third, our analysis was designed to investigate sero-positivity and no relationship between IgG titres and COVID-19 outcomes or smoking could be inferred.
In conclusion, this study documents a lower proportion of positive SARS-CoV-2 serology among current smokers, using direct laboratory measures of tobacco exposure and thus avoiding possible bias associated with self-reported smoking status. As such, the research captures actionable metric on the role of smoking in SARS-CoV-2 infection and COVID-19 outcomes, and contributes to refine current epidemiological risk estimates. Results may also serve as a reference for future clinical research on potential pharmaceutical role of nicotine or nicotinic-cholinergic agonists in COVID-19.