COVID-19 mortality in Italy varies by patient age, sex and pandemic wave

SARS-CoV-2 has caused a worldwide epidemic of enormous proportions, which resulted in different mortality rates in different countries for unknown reasons. We analyzed factors associated with mortality using data from the Italian national database of more than 4 million SARS-CoV-2-positive cases diagnosed between January 2020 and July 2021, including > 415 thousand hospitalized for coronavirus disease-19 (COVID-19) and > 127 thousand deceased. For patients for whom age, sex and date of infection detection were available, we determined the impact of these variables on mortality 30 days after the date of diagnosis or hospitalization. Multivariable weighted Cox analysis showed that each of the analyzed variables independently affected COVID-19 mortality. Specifically, in the overall series, age was the main risk factor for mortality, with HR > 100 in the age groups older than 65 years compared with a reference group of 15–44 years. Male sex presented a two-fold higher risk of death than female sex. Patients infected after the first pandemic wave (i.e. after 30 June 2020) had an approximately threefold lower risk of death than those infected during the first wave. Thus, in a series of all confirmed SARS-CoV-2-infected cases in an entire European nation, elderly age was by far the most significant risk factor for COVID-19 mortality, confirming that protecting the elderly should be a priority in pandemic management. Male sex and being infected during the first wave were additional risk factors associated with COVID-19 mortality.


Results
The Italian cohort of SARS-CoV-2-positive individuals included 4,333,014 persons of median age 46 years, with a slight predominance of female cases (Table 1). Overall, 50.4% of the cases were symptomatic and 415,390 were hospitalized, with 13.7% of them requiring intensive care. The median age of non-hospitalized patients was much lower than that of hospitalized cases (44 vs. 70 years). Of the entire case series, 240,850 were diagnosed during the first wave, 1,907,690 became infected between July 1, 2020 and December 31, 2020, and 2,104,894 were diagnosed in the first seven months of 2021. At the end of the study, 127,524 subjects were dead, with 35,837 having been diagnosed as infected before June 30, whereas 49,120 and 40,755 dead patients had been diagnosed in the second and third semesters, respectively (for 1812 subjects, information about the date of infection detection was missing). Over 31 thousand non-hospitalized people had died by July 25, 2021, while the total number of deaths among the hospitalized people was 95,907, including 25,320 during the first wave. The median age at death of non-hospitalized patients was 86 years, with 94% of them being ≥ 65 years old (not shown). Instead, the median age of deceased hospitalized patients was 81 years, with 90% of them ≥ 65 years old (not shown). Surprisingly, more than 38 thousand people who were hospitalized were listed in the dataset as being asymptomatic.
To investigate the factors affecting mortality after SARS-CoV-2 infection, we first drew Kaplan-Meier curves for the whole series and, separately, for non-hospitalized and hospitalized patients. For the whole series, the analysis was limited to 4,224,698 persons, after eliminating 108,316 persons with incomplete data (including 1744 persons for whom the date of death was erroneously listed as being before the date of diagnosis or hospitalization). For non-hospitalized and hospitalized persons, these numbers were 3,816,311 and 412,942, respectively. We tested the effects of age, sex, and pandemic wave (first wave, before June 30, 2020, was taken as reference) on the risk of death 30 days after infection detection or hospitalization. Highly significant associations (log-rank test, P < 2 × 10 -16 ) for all three variables were observed, in the whole series and in the two subsets (Fig. 1). The probability of survival decreased with increasing age and was lower for males than females and for persons who were diagnosed in the first wave.
Since the proportionality hazard assumptions were not verified in our series (Schoenfeld residuals test, P < 2 × 10 -16 ), we carried out weighted multivariable Cox analyses to deal with non-proportional hazards. Multivariable analysis of mortality in the whole series demonstrated that age, male sex, and the first pandemic wave were highly significant, independent risk factors for death ( www.nature.com/scientificreports/ ratio (HR) = 691; 95% CI, 634 to 753] in the age ≥ 85 years group. Children (0-14 years old) showed a much lower risk of death (HR = 0.120) than both the reference age group and the older groups. Male sex was associated with a ~ twofold risk of death (HR = 2.05), compared to females. The first pandemic wave (before June 30, 2020) was associated with an excess risk of death of almost threefold, compared to the subsequent periods (HR = 0.379 and 0.346 in the second and third semesters, respectively). Multivariable analysis of non-hospitalized individuals, 30 days after diagnosis, also showed that age, sex, and pandemic wave were all independent poor prognostic factors (Table 3). Again, age conferred the highest risk of death, with an HR > 130 in the groups of subjects 65 years or older and HR > 1400 for those 85 years and older. Indeed, the vast majority of deaths (94%) among non-hospitalized patients regarded patients ≥ 65 years old. The risk estimates associated with male sex and pandemic wave are similar to those for the whole series, except for non-hospitalized patients of the third semester, who had an even lower risk of mortality (HR = 0.21).
Finally, multivariable Cox analyses of the smaller subgroup of hospitalized patients also showed that age, sex, and pandemic wave were significantly associated with the risk of death 30 days after hospitalization ( Table 4). The estimates of the risk of death in this subgroup were lower than in non-hospitalized patients, as evidenced by the smaller values of HR reaching 59 even in the oldest age group, when compared to the reference group. www.nature.com/scientificreports/ Finally, being hospitalized after the first wave was associated with a lower risk of death (HR was about 0.7 in both semesters after June 30, 2020), but this effect was less intense than that observed among non-hospitalized persons. Also, the poor prognostic role of male sex was confirmed in these hospitalized patients (HR = 1.44).

Discussion
The Italian COVID-19 epidemiological surveillance dataset analyzed here contained information on over 4 million persons molecularly diagnosed with a SARS-CoV-2 infection until July 25, 2021. The dataset included information about age, sex, date of diagnosis, presence vs. absence of symptoms, date of hospitalization (if pertinent), date of death (if pertinent), and a few other data. Survival analyses on the whole series and on subsets of non-hospitalized and hospitalized patients strongly confirmed the pivotal role of age in the probability of survival of COVID-19 patients. The analysis by age category, adjusted for sex and pandemic wave, showed that age groups  www.nature.com/scientificreports/ older than 65 had mortality risks that were hundreds of times greater than that of the 15-to 44-year-old reference class. The 0-14 years age group had a mortality risk that was about 10 times less than that of the reference class. Male sex was also confirmed to be a poor prognostic factor, but with a much smaller effect. Additionally, our analysis demonstrated that being diagnosed during the first pandemic wave (until June 2020) was associated with an approximately threefold higher mortality risk than being diagnosed later.
In non-hospitalized patients, the mortality risk associated with age was greater than that for the whole series. This difference might be explained by the observation that most deceased non-hospitalized patients were very old, with a median age of 86 years. As a possible interpretation, we suppose that some elderly persons deteriorated rapidly and died before they could be hospitalized.
In hospitalized patients, old age was associated with an excess risk of death, as in the whole series, although the statistical estimates were lower. For example, for the age group ≥ 85 years old, the HR was 58.7 for hospitalized patients and 687 for the whole series. The difference may be explained by the facts that hospitalized patients were much older than subjects of the whole series (median age, 70 vs. 46 years), and that hospitalization itself poses an excess risk of death, as age is a known risk factor for hospitalization 18,19 , including in our series (not shown).
Our finding of age being a risk factor for COVID-19 mortality is in agreement with that of a meta-analysis by Shi et al. 8 on 27 studies (including 24 from China, two from the United States, and one from Italy) and a Table 2. Factors associated with death in the whole Italian series of people infected by SARS-CoV-2. Of the entire series, 108,316 cases were excluded due to incomplete or erroneous data, for a total of 4,224,698 SARS-CoV-2-positive subjects analyzed and 109,605 deaths within 30 days of diagnosis. 1 Multivariable weighted Cox analysis (non-proportional hazards model).

Feature
Hazard ratio (95% confidence interval)   20 on 66 studies with > 17 million patients from 14 countries. Both meta-analyses found an association between old age and excess risk of mortality from COVID-19, although the quantitative risk estimates differ. Of note, these meta-analyses did not report HRs associated with survival, since no Cox analyses were done. To the best of our knowledge, only one other nation-wide study, conducted in France by Semenzato et al. 21 , used Cox models to analyze the effects of age on the risk of mortality in a large number of hospitalized COVID-19 patients. Although the age groups differ between the two studies, the risk estimates are similar, with HRs > 50 in elderly patients in both studies. Several immunological mechanisms responsible for the increased risk of death from COVID-19 in the elderly can be hypothesized. One study demonstrated that pre-existing T-cell immunity induced by circulating human alpha-and beta-coronaviruses is present in young adults but virtually absent in older adults 22 . Consequently, older adults had a minimal baseline frequency of cross-reactive T cells directed toward the novel SARS-CoV-2; for this reason, they may be at higher risk of severe COVID-19 disease and death. Moreover, the phenomenon of immunosenescence, which involves age-related changes in innate and adaptive immunity, has been imputed as being associated with the increased mortality of older adults infected with SARS-CoV-2 23 . The elderly exhibit a deficient immunologic response to SARS-CoV-2 infection, which may be another reason for their increased risk of severe disease and death 24 .
In the Italian nationwide COVID-19 series, male sex was an unfavorable prognostic factor for survival, with a risk that was twofold higher than for females in the whole series, and > 80% and ~ 50% higher in non-hospitalized and hospitalized patients, respectively. This result is in agreement with those of several other studies 8,20 , although the quantitative risk estimates differ. The HRs for male sex calculated in this study, which range from 1.46 to 2.05, are similar to those reported by Semenzato et al. 21 . Additionally, in an analysis of the excess number of deaths, standardized by age in 29 high-income countries, men were more affected than women in almost all countries 25 .
The mechanism by which sex is an unfavorable prognostic factor for COVID- 19 is not yet known. Most likely, several sex-related factors contribute to the higher risk of males for poorer COVID-19 outcomes. A study of 1683 Italian patients who underwent chest computed tomography at admission showed that men had a higher prevalence of cardiovascular comorbidities, more coronary calcifications, and a higher coronary calcium score than females 26 . Notably, the higher coronary calcific burden of men appeared to be associated with higher mortality. A study of about 3000 COVID-19 patients in a single center in China observed that the level of inflammatory cytokines in peripheral blood was higher in males than in females 27 . Also, the percentages of CD19 + B cells and CD4 + T cells were generally higher in female patients during the course of the disease. Overall, males had greater inflammation, lower lymphocyte counts, and lower and delayed antibody responses during SARS-CoV-2 infection and recovery than females. Finally, from the perspective of an immunological mechanism, it has been hypothesized that chronic, subclinical, systemic inflammation, characteristic of aging, and immunosenescence contribute to the excess risk of COVID-19 mortality in elderly men 28 .
Our multivariable analysis provides strong support for the hypothesis that mortality from COVID-19 was much greater during the first wave (January to June 30, 2020) than later. Indeed, taking the first wave as reference, in the subsequent periods we observed a ~ threefold reduced risk of death, both in the whole series and in non-hospitalized patients. In hospitalized patient, the excess risk of death was ~ 30% lower in the two semesters after the first wave. The excess risk of death associated with pandemic wave was first reported in an Italian study of hospitalized patients 12 , and then confirmed by studies of Massachusetts healthcare workers 13 , patients of the U.S. Veterans Affairs healthcare system 29 , and UK patients 30 . The reasons for this effect could include the initial lack of preparedness of national health systems for pandemic management, the lack of knowledge about the Table 4. Factors associated with death in hospitalized COVID-19 patients in the Italian national database. Among hospitalized persons, 2448 cases were excluded due to incomplete or erroneous data, for a total of 412,942 SARS-CoV-2-positive subjects analyzed and 85,503 deaths within 30 days of diagnosis. 1 Multivariable weighted Cox analysis (non-proportional hazards model).

Feature
Hazard ratio (95% confidence interval) www.nature.com/scientificreports/ most effective therapies for COVID-19 patients with severe disease, and the possibility that frailer people were more affected at the beginning of the pandemic than the rest of the population 31 . Another possible explanation for a lower risk of mortality after June 30, 2020, and in particular, during the third semester of the pandemic, compared with the first semester, may be mass vaccination, which began in January 2021; indeed, vaccines are associated with a reduced risk of severe COVID-19 and mortality 32 .
In the Italian COVID-19 epidemiological surveillance dataset, more than 2 million infected persons were symptomatic (50.4% of all cases). Modeling studies on the prevalence of infection in different populations suggested that the total number of SARS-CoV-2-positive individuals exceeds symptomatic cases by an order of magnitude or more [33][34][35] . If this holds true for the Italian population, then ~ 20 million people in Italy have been infected by SARS-CoV-2, i.e., 10 times the 2 million symptomatic cases. Why some infections are asymptomatic and others lead to severe COVID- 19 has not yet been elucidated. Cross-reactive immunity, pre-existing in individuals who had been exposed to other coronaviruses, could be one of the mechanisms for asymptomatic and moderate courses of SARS-CoV-2 infection in many individuals 36 .
A limitation of our study is the lack of data about COVID-19 patients' comorbidities, which are important risk factors for outcome 8 . Other possible confounders that we cannot consider in our model are, for instance, demographic, geographic and environmental factors 37,38 . This lack of information prevented us from analyzing other risk factors for death. Moreover, the reasons why some hospitalized patients were classified as asymptomatic are not known, but their hospitalization may have been due to reasons other than COVID-19. For example, in 5432 cases, the date of SARS-CoV-2 infection detection was after the date of hospitalization, and in 13,144 patients the diagnosis was on the same day. An additional limitation could be an underestimation of the number of cases and deaths during the first wave, due to initial unpreparedness of the health system to deal with the crisis caused by the pandemic and, thus, the initially limited testing capacity 39 . This might have determined a poorer data collection in the first wave as compared to the following periods.
Overall, this study confirms that age and male sex are independent risk factors for COVID-19 mortality for both hospitalized and non-hospitalized patients. Because age was found to be the most impactful negative prognostic factor, it should be considered in pandemic management, by giving priority to strategies aimed at protecting elderly people. Additionally, this is the first country-wide study to demonstrate a higher risk of death during the first pandemic wave than later. Similar nation-wide studies in different countries, to the best of our knowledge, have not been published. Thus, we cannot compare our study with those from other nations with different mortality rates, and we cannot exclude that such differences are due to unequal pandemic management in the first wave, considering that Italy was the first Western nation to be affected. Our study also suggests that the medical research that started with the pandemic onset and that led to the development of increasingly more effective clinical protocols contributed to improving COVID-19 patient survival 25 . Despite the limitations of this study, principally due to the lack of some clinical data (e.g. about comorbidities and environmental factors), this study demonstrates the usefulness of a national database for studying a new disease such as COVID-19. Efforts should be made in Italy to create a more detailed national database like those of the United Kingdom 40 and France 41 that collect more data on demographics, symptoms, diagnostic tests and treatments. National health databases, especially when accompanied by a national biobank of blood samples, offer great possibilities for biomedical research. They allow the construction of cohorts with unparalleled statistical power and help study risk factors for common diseases, rare diseases, and new emerging diseases such as COVID-19. Their availability could impact treatment and public health. Therefore, the creation of such databases in countries that do not yet have them and the creation of European databases are desirable.

Data availability
ISS data that have been used for the present study are available upon request at this web link: https:// www. iss. it/ richi esta-dati-covid 19.